key: cord-352988-9ey3ir5e authors: xiang, yang-fei; ju, huai-qiang; li, shen; zhang, ying-jun; yang, chong-ren; wang, yi-fei title: effects of 1,2,4,6-tetra-o-galloyl-β-d-glucose from p. emblica on hbsag and hbeag secretion in hepg2.2.15 cell culture date: 2010-10-08 journal: virol sin doi: 10.1007/s12250-010-3144-y sha: doc_id: 352988 cord_uid: 9ey3ir5e a polyphenolic compound, 1,2,4,6-tetra-o-galloyl-β-d-glucose (1246tgg), was isolated from the traditional chinese medicine phyllanthus emblica l. (euphorbiaceae) and assayed for its potential as an anti-hepatitis b virus (hbv) agent. the cytotoxicity of 1246tgg on hepg2.2.15 as well as hepg2 cells was determined by observing cytopathic effects, and the effects of 1246tgg on secretion of hbsag and hbeag in hepg2.2.15 cells were assayed by enzyme immunoassay. results indicates that treatment with 1246tgg (6.25 μg/ml, 3.13 μg/ml), reduced both hbsag and hbeag levels in culture supernatant, yet the inhibitory effects tend to decline with the assay time. this study provides a basis for further investigation of the anti-hbv activity and possible mechanism of action of 1246tgg. hepatitis b virus (hbv), an important causative pathogen of cirrhosis-related liver failure and hepatocellular carcinoma (hcc), is a public health problem of worldwide concern, and is responsible for one million deaths each year worldwide [7] . china has the biggest hbsag carrier population with more than one-third of the world's 350-400 million chronic hbv carriers [11] . though and hepatitis b e antigen (hbeag) [12] . currently, two therapies, conventional interferon alfa (ifnα) and used in the southwest of china for treating eczema, wart, diarrhea, and headache after a fever [13, 18] . acyl glucoses have been shown to be potent antiviral agents against herpes simplex virus (hsv) [3, 9] , human immunodeficiency virus (hiv) [2, 10] , severe acute respiratory syndrome coronavirus (sars-cov) [15] as well as other viruses. here, we investigated the anti-hbv activity of 1246tgg by detecting the hbsag and hbeag secretion levels in hepg2.2.15 cell culture, a cell line derived by transfection of cloned hbv dna into human hepatoblastoma cell line hepg2 and used to assay for anti-hbv agents [4] . compound 1246tgg was isolated and its structure was identified by the state key laboratory of phytochemistry and plant resources in west china in the kunming institute of botany, chinese academy of sciences, using procedures as described in a previous paper [18] . briefly, the ethanol extract of the fresh leaves and branches of p. emblica was suspended into water and then extracted with diethyl ether. the diethyl ether layer was partitioned between hexane and methanol, and the methanol layer was further chromatographed successively over sephadex lh-20, silica gel, mci-gel chp 20p and chromatorex ods to obtain the desired compound (purity > 95%) in the form of a pale amorphous powder. its structure was identified by comparison of the physical and spectral data with literature values and the 1h-1h cosy spectrum (fig. 1) . the isolated compound was then dissolved in dimethyl sulfoxide (dmso) before use. the final concentration of dmso was less than 0.2%. hepg2 and hepg2. only 5% fbs was added. cells were cultured at 37°c in a humid atmosphere with 5% co 2 . the cytotoxicity assay was performed by observing cytopathic effect (cpe). hepg2 or hepg2. day. cytopathic effects were classified into five levels as follows: >75%, between 75% and 50%, between 50% and 20%, <25% and no cytopathic effect. the assay was performed in four parallel wells. concentrations without cytotoxicity were used for hbsag and hbeag inhibition assay. for hbsag and hbeag secretion assay, hepg2.2.15 cells were seeded onto 24-well tissue culture plates (corning) 3×10 4 cells/well and incubated at 37℃ in a humid atmosphere with 5% co 2 for 24 h before the test. similarly, 1246tgg at two concentrations (6.25 µg/ml and 3.13µg/ml) were diluted in maintenance media and added every 3 d during the 10 d treatment period, namely, on the 1st, 4th and 7th day. before the second treatment of 1246tgg (on the 4th and 7th day) and at the end of the treatment period (on the 10th day), culture media of each compound concentration was collected and stored at -20℃. hbsag and hbeag levels in culture media were measured using an enzyme immunoassay kit (intec) according to the manufacturer's instructions and absorbance at 450nm was measured using an elisa reader (bio-rad). the assay was performed in four parallel wells. results were expressed as  or ±s.d. of four parallel wells. statistical calculations were carried out with the spss 13.0 for windows software package (statistica). one-way anova was used for statistical analyses; p values < 0.05 were considered to be significant. the results of cytotoxicity assay are listed in table 1 . on the 10th day (after the third treatment), 1246 tgg at concentrations ranging from 200µg/ml to 12.5µg/ml all induced cytophathic effects to different extents. to confirm whether the cytotoxicity caused by 1246tgg was specific to hbv dna transfected to determine the inhibitory effects of 1246tgg on hbv antigen secretion, cells were treated with 1246tgg at concentrations of 6.25µg/ml and 3.13µg/ml every 3 d during the 10 d treatment period. as shown in polyphenols, especially flavonoid, phenolic acids and other derivates might be potential antiviral agents [14] . among these, galloyl glucoses, with various number of galloyl groups in the glucose core structure, penta-o-galloyl-β-d-glucose (pgg), was found to be efficient in inhibiting the ns3 protease of hcv [1] . pgg also decreased extracellular hbv in a dosedependent manner in hepg2.2.15 cell culture [8] . in this study, 1246tgg was isolated from traditional chinese medicine p. emblica and its activity in affecting hbv antigen secretion was reported for the first time. 1246tgg showed cytotoxicity towards hepg antiviral compounds from traditional chinese medicines galla chinese as inhibitors of hcv ns3 protease inhibitory effects of egyptian folk medicines on human immunodeficiency virus (hiv) reverse transcriptase inhibition of herpes simplex virus infection by tannins and related compounds a cell culture assay for compounds which inhibit hepatitis b virus replication human hepatic cell uptake of resveratrol: involvement of both passive diffusion and carrier-mediated process resveratrol in human hepatoma hepg2 cells: metabolism and inducibility of detoxifying enzymes hepatitis b virus infection in vitro antiviral activity of 1,2,3,4,6-penta-o-galloyl-beta-dglucose against hepatitis b virus studies on antiviral activity of several hydrolyzable tannins sulfated pentagalloyl glucose (y-art-3) inhibits hiv replication and cytopathic effects in vitro, and reduces hiv infection in hu-pbl-scid mice management of chronic hepatitis b: experience from china the hepatitis b virus ethnopharmacology of phyllanthus emblica l current status of natural products from plants as anti-herpes simplex virus 1 agents respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose contemporary clinical research of traditional chinese medicines for chronic hepatitis b in china: an analytical review phyllanemblinins a-f, new ellagitannins from phyllanthus emblica key: cord-276565-vkbu581j authors: he, qing; zhang, guo; gu, ye; wang, jitao; tang, qiyuan; jiang, zicheng; shao, chuxiao; zhang, hongguang; chen, zhenhuai; ma, baoyi; liu, dengxiang; xie, guanghang; xu, dan; huang, yifei; zhang, haijun; liang, mingkai; huang, huihong; wang, yan; liu, hongyan; yang, jie; pan, hongqiu; zou, shengqiang; li, fujian; wang, fang; liu, chuan; wang, wenjuan; xiong, bin; li, xun; liu, lei; yang, jianrong; qi, xiaolong title: clinical characteristics of covid-19 patients with pre-existing hepatitis b virus infection: a multicenter report date: 2020-09-11 journal: am j gastroenterol doi: 10.14309/ajg.0000000000000924 sha: doc_id: 276565 cord_uid: vkbu581j nan coronavirus disease 2019 (covid-19) has become a global challenge since december 2019 (1) . of the 99 patients with covid-19 in wuhan, 43 (43.4%) had differing degrees of liver function abnormality (1) . therefore, liver disease in covid-19 attracted widespread concern (2). there are no data yet focusing on the impact of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in patients with underlying liver disease, such as hepatitis b virus (hbv) infection. to date, 257 million people are living with hbv infection worldwide (3) . thus, it is indispensable to study the clinical characteristics of covid-19 patients with preexisting hbv infection. data were obtained from a cohort (coronavirus disease 2019-hepatitis b virus-chinese portal hypertension diagnosis and monitoring study group, covid-hbv-chess) to consecutively monitor covid-19 patients in 10 designed hospitals of 8 provincial administrative regions in china ( figure 1a ). patients were hospitalized from january 10, 2020, to february 20, 2020, with a final follow-up on april 2, 2020. this study was approved by all ethics commissions, with a waiver of written informed consent. as of april 2, we have collected and analyzed 571 cases diagnosed with laboratoryconfirmed sars-cov-2 infection by real-time fluorescence polymerase chain reaction. fifteen (2.63%) of 571 patients had a history of hbv infection that seems to be lower than the incidence of hbv infection in the overall chinese population (5.7%) (3). there were no cases with cirrhosis diagnosed by either clinical findings and/or liver biopsy in the cohort. of them, 3 (20.00%) of 15 patients had a history of antiviral treatment (entecavir), and all had suppression of the hbv. the mean age of covid-19 positive patients with pre-existing hbv infection was 45.80 years (sd, 11.06), and 10 (66.67%) were men. the common symptoms at onset of illness were fever (9 [60.00%]), dry cough (7 [46.67%]), and diarrhea (2 [ figure 1b) . in the covid-hbv-chess study, we analyzed the clinical characteristics of covid-19 patients with pre-existing hbv infection for the first time, to our best knowledge; only by multicenter analysis can we follow-up covid-19 with underlying liver disease, such as hbv infection. we found that patients with pre-existing hbv infection might have a lower incidence of intensive care unit admission or death, and similar findings were reported in severe acute respiratory syndrome (sars) coronavirus with hbv coinfection during the outbreak of sars in 2003 (4). our hypothesis of the mechanism of this protective effect might be mediated by host immune responses (5) on the indirect interplay between hbv and sars-cov-2. however, the study was limited with a epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study liver injury in covid-19: management and challenges accelerating the elimination of viral hepatitis: a lancet gastroenterology & hepatology commission clinical significance of hepatic derangement in severe acute respiratory syndrome dominance of hepatitis c virus (hcv) is associated with lower quantitative hepatitis b surface antigen and higher serum interferong-induced protein 10 levels in hbv/hcvcoinfected patients national clinical research center for infectious diseases, the third people's hospital of shenzhen, the second affiliated hospital of department of infectious diseases and critical care medicine, the affiliated third hospital of jiangsu university, zhenjiang, china; 8 department of respiratory medicine, the people's hospital of baoding, baoding, china; 9 department of respiratory medicine, the people's hospital of linxia hui prefecture correspondence: xiaolong qi, md. e-mail: qixiaolong@vip.163.com key: cord-004605-gsi4yxzj authors: nan title: prüfung und deklaration der wirksamkeit von desinfektionsmitteln gegen viren: stellungnahme des arbeitskreises viruzidie* beim robert koch-institut (rki) sowie des fachausschusses „virusdesinfektion“ der deutschen gesellschaft zur bekämpfung der viruskrankheiten (dvv) und der desinfektionsmittelkommission der deutschen gesellschaft für hygiene und mikrobiologie (dghm) date: 2004 journal: bundesgesundheitsblatt gesundheitsforschung gesundheitsschutz doi: 10.1007/s00103-003-0754-7 sha: doc_id: 4605 cord_uid: gsi4yxzj nan der nachweis der wirksamkeit von desinfektionsmitteln ist die grundlegende voraussetzung für ihre erfolgreiche und sinnvolle anwendung. diesbezügliche anforderungen an das desinfektionsmittel leiten sich dabei aus den eigenschaften der zu inaktivierenden erreger und den bestimmungsgemäßen anwendungsbedingungen ab. gegenwärtig verwendete deklarationen wie "virusinaktivierend", "viruzid" oder "wirksam gegen" bestimmte viren wurden bisher nicht einheitlich angewendet und interpretiert. die vorliegende stellungnahme gibt das ergebnis der diskussion des arbeitskreises viruzidie am rki sowie des fachausschusses "virusdesinfektion" der dvv und der desinfektionsmittelkommission der dghm wieder. ziel dieses arbeitskreises war es, wissenschaftlich begründete anforderungen an die prüfung der wirksamkeit von desinfektionsmitteln gegen viren und die entsprechenden prüfmethoden als voraussetzung für eine sachgerechte deklaration zusammenzustellen. das spektrum humanmedizinisch relevanter viren ist im anhang dargestellt. hinsichtlich der widerstandsfähigkeit gegen desinfektionsmittel lassen sich aufgrund der struktur 2 gruppen -die behüllten und die unbehüllten virenunterscheiden [1] . entsprechend werden die begriffe ◗ "begrenzt viruzid" als wirksam gegen behüllte viren und ◗ "viruzid" als zusätzlich wirksam gegen unbehüllte viren verwendet. diese unterscheidung ist zweckmäßig, da die "viruzide" wirksamkeit schwieriger zu erzielen, jedoch auch nicht in allen fällen erforderlich ist. in abhängigkeit vom anwendungsbereich muss deshalb zunächst entschieden werden, welches wirkungsspektrum die desinfektionsmaßnahmen umfassen sollen. durch dieses abgestufte vorgehen soll eine sachgerechte und angemessene desinfektion unter berücksichtigung von umweltbelastung und verträglichkeit erzielt werden. unter diesem gesichtspunkt kann zukünftig auch die deklaration einer wirksamkeit gegen ausgewählte unbehüllte viren, die häufig umfangreiche ausbrüche verursachen, wie z. b. rotaviren [2, 3, 4, 5, 6] , insbesondere bei händedesinfektionsmitteln sinnvoll sein. die deklaration "begrenzt viruzid" würde in diesem fall mit einem entsprechenden zusatz versehen werden. ähn-liche überlegungen könnten auch für noroviren (norwalk-like-viren) [7, 8] sinnvoll erscheinen. nicht zuletzt wegen der geringen infektionsdosis dieser unbehüllten viren sollten hier jedoch bis auf weiteres nur "viruzide" desinfektionsmittel eingesetzt werden. unter praktischen gesichtspunkten lassen sich 3 anwendungsbereiche unterscheiden: ◗ für die abschließende instrumentendesinfektion 1 gibt die anlage "anforderungen an die hygiene bei der aufbereitung von medizinprodukten" der richtlinie für krankenhaus-hygiene und infektionsprävention [9] vor, dass hierfür nur desinfektionsmittel mit "viruzider" wirksamkeit anzuwenden sind. die bisher vorliegenden prüfmethoden (s. 5) verwenden bestimmte testviren stellvertretend für das bekannte spektrum der viralen krankheitserreger. im unterschied zur künftigen europäischen norm verlangt die gemeinsame prüfrichtlinie von dvv und bga (jetzt rki) [11, 12, 13] zusätzlich zur testung von polio-und adenoviren auch die prüfung von papova-und vakziniaviren. da sich papovaviren in einigen untersuchungen resistenter als poliound adenoviren erwiesen haben [1] , kann insbesondere im hinblick auf papillomaviren auf die testung dieser viren nicht verzichtet werden, wenn eine "viruzide" wirksamkeit deklariert werden soll. die von der who ergriffenen maßnahmen zur eradikation der poliomyelitis machen es erforderlich, dass für die desinfektionsmittelprüfung nur der polio-impfstamm typ i, stamm lsc-2ab verwendet werden darf [14, 15] . er erfüllt die kriterien der who für die bisherige polio-lebendimpfstoffherstellung [16] . diese setzen auch voraus, dass nicht mehr als 10 passagen kultiviert werden und eine exakte dokumentation dazu vorliegt. der bislang in prüflaboratorien verwendete stamm gleichen namens erfüllt diese kriterien in der regel nicht. in folge der eradikationsmaßnahmen wird es weltweit zu einer einstufung von polioviren in eine höhere risikogruppe (schutzstufe 3 oder 4 gemäß biostoffverordnung) kommen, was eine weitere verwendung als prüfvirus weitestgehend ausschließen wird und somit die festlegung eines anderen geeigneten virus notwendig macht. das hepatitis-a-virus (hav) als ebenfalls unbehülltes virus hat sich in vergleichenden untersuchungen mit poliovirus zum teil als resistenter erwiesen [17] . besonderes interesse gilt hier den klinisch relevanten durch blut, gewebe und körperflüssigkeiten übertragbaren behüllten viren, wie z. b. dem human-immunodeficiency-virus (hiv), hepatitis-c-virus (hcv) und hepatitis-b-virus (hbv). die deklaration "begrenzt viruzid" erfolgt künftig, wie im folgenden begründet, auf der basis von prüfungen unter verwendung relevanter testviren, die den rückschluss auf die wirksamkeit auch gegen hiv, hcv und hbv zulassen. für die deklaration "begrenzt viruzid" stehen gegenwärtig vakziniavirus und bvdv (bovine viral diarrhea virus) als testviren zur verfügung. das vakziniavirus [18] wurde bereits in der bga/dvv-richtlinie [11, 12] als vertreter für die behüllten viren aufgeführt. hcv lässt sich nicht in vitro kultivieren. für das in vielen eigenschaften vergleichbare bvdv liegen jedoch aus der validierung von inaktivierungsverfahren bei der herstellung von blut und blutprodukten umfangreiche erfahrungen vor, die seine verwendung als testvirus auch für die desinfektionsmittelprüfung nahe legen [19] . eine ausdrückliche deklaration der wirkung gegen hcv allein auf dieser basis ist jedoch nicht gerechtfertigt. auch die auslobung der wirksamkeit gegen hiv setzt eine prüfung unter verwendung von hiv in zellkulturen voraus, welche jedoch aufgrund der gefährlichkeit des virus (schutzstufe 3) nicht erstrebenswert ist. eine spezielle problematik stellen aussagen zur hbv-wirksamkeit dar [20] . hbv ist für die virusdesinfektion von besonderer bedeutung, da bereits geringste blutmengen bei hoher viruslast zu infektionen führen können und der erreger weit verbreitet ist (weltweit sind schätzungsweise 350 millionen menschen, d. h. 5-7% der gesamtbevölkerung chronisch infiziert [21] ). die bewertung der wirksamkeit von desinfektionsmitteln gegenüber hbv war in der vergangenen zeit auch unter experten anlass für kontroverse diskussionen bezüglich der aussagekraft der durchgeführten prüfverfahren. unstrittig ist, dass derzeit kein zellkultursystem zum empfindlichen nachweis von hbv und daher keine geeignete, va-lidierbare prüfmethode für die bestimmung der infektiosität von hbv zur verfügung steht. somit ist die bezeichnung "wirksam gegen hbv" auf der basis der gegenwärtig verfügbaren tests nicht in jedem falle valide, da der verlust der infektiosität nur in begrenztem umfang in einem biologischen system nachgewiesen wurde. bisher wurden 2 surrogatteste, der madt (morphologic alteration and desintegration test) [22] und die reduktion von hbsag (hepatitis surface antigen) [23] zur bestimmung der wirkung von desinfektionsmitteln gegenüber hbv durchgeführt. der madt bestimmt die morphologische veränderung von hbv-partikeln im elektronenmikroskop nach desinfektionsmittelbehandlung, während der hbsag-reduktions-test die antigenität von hbsag nach desinfektionsmittelbehandlung im kommerziellen festphasen-radio-immuntest bestimmt. der madt erfordert eine visuelle beurteilung der viruspartikel, die nur sehr erfahrenen betrachtern möglich und schwierig zu standardisieren ist. der hbsag-reduktionstest mit käuflichen testkits arbeitet mit antikörpern, deren epitope von den herstellern nicht bekannt gegeben werden und deren spezifität nicht in einem klaren zusammenhang zu neutralisierenden epitopen steht. der besseren standardisierbarkeit dient die weiterentwicklung der o. a. surrogatmethoden hinsichtlich molekularbiologischer alteration der hbv-dna und desintegration von hbv-epitopen [24, 25] . die neutralisierende wirkung einiger weniger monoklonaler antikörper (z. b. ma18/7) gegen bestimmte sequenzen von hbv ist in zellkulturversuchen nachgewiesen worden [25] . werden die zugehörigen epitope dieser antikörper durch ein desinfektionsmittel zerstört, ist es plausibel anzunehmen, dass auch die infektiosität zerstört ist. in suspensionsversuchen mit peressigsäurehaltigen desinfektionsmitteln erwies sich das präs1-epitop des antikörpers ma18/7 als das resistenteste. diese befunde lassen einen hochempfindlichen hbsag-nachweis mittels des antikörpers ma18/7 als validierungsmethode geeignet erscheinen. es ist jedoch wahrscheinlich, dass eine reihe wirksamer desinfektionsmittel aufgrund ihrer wirkungsmechanismen dieses epitop nicht zerstört. ähnlich verhält es sich mit der hbv-dna als zielstruktur für die validierung von desinfektions-bzw. virusinaktivierungsverfahren. hbv-dna kann mittels quantitativer pcr (bevorzugt realtime-pcr) hochempfindlich nachgewiesen werden. die zerstörung der primärstruktur der dna bedeutet zerstörung der infektiosität und zugleich blockierung der amplifikation in der pcr [25] . die resistenz der dna gegen ein mittel schließt seine wirksamkeit jedoch nicht aus, wie vergleiche mit schimpansenversuchen zeigten [26, 27] . für die validierung der hbv-inaktivierung stehen heute jedoch schimpansen de facto nicht mehr zur verfügung. auch primäre humane hepatozyten sind schwierig zu erhalten, wenig suszeptibel und von wechselnder qualität. dem menschlichen hbv ähnlich sind die hepadnaviren von gibbons, wollaffen und woodchucks. jedoch sind diese tiere schwierig zu halten und die primäre hepatozytenkulturen daraus ähnlich problematisch wie die des menschen. eine denkbare alternative sind primäre hepatozytenkulturen aus tupaialeber (tupaias -spitzhörnchen). einfacher hingegen ist die zucht von enten. entenhepatitis-b-virus (duck hepatitis-b-virus -dhbv) gehört wie das hbv des menschen zur familie der hepadnaviridae. der infektiositätstest mit dhbv ist in den usa und australien für die prüfung der hbv-wirksamkeit von desinfektionsmitteln anerkannt [28] . auch dhbv lässt sich, ähnlich wie hbv, nur in einem lebenden tier bzw. in primären hepatozytenkulturen vermehren. dhbv ist gut erforscht und hat den vorteil, weder human-noch tierpathogen zu sein und kann somit in der biologischen schutzstufe 1 gehandhabt werden. zum nachweis der erfolgten infektion der hepatozyten kann die immunfluoreszenz mit einem anti-dhbv-serum oder eine geeignete pcr dienen. die dvv organisiert gegenwärtig untersuchungen zur etablierung entsprechender prüfmethoden. als konsequenz aus der vorstehenden darlegung ergibt sich die forderung, auf die ausdrückliche deklaration "wirksam gegen hbv" künftig zugunsten der deklaration "begrenzt viruzid" zu verzichten, bis auf der grundlage der hier geschilderten untersuchungen eine aussagekräftige methode zur prüfung der wirksamkeit von desinfektionsmitteln gegen hbv etabliert ist. nach abschluss dieser untersuchungen ist eine überarbeitung der anforderungen für die deklaration "begrenzt viruzid" vorgesehen. derzeit liegen die folgenden prüfmethoden vor: 1. richtlinie des bundesgesundheitsamtes und der deutschen vereinigung zur bekämpfung der viruskrankheiten (dvv) zur prüfung von chemischen desinfektionsmitteln auf wirksamkeit gegen viren [11] die europäische norm ist im bereich der viruzidietestungen noch lückenhaft, sodass der hier publizierte vorschlag geeignet ist, impulse auch für die europäische viruzidienormung zu geben. die prüfung der wirksamkeit von für den humanmedizinischen bereich vorgesehenen desinfektionsmitteln ist weiterhin gemäß der gemeinsamen richtlinie des bga (jetzt rki) und der dvv [11, 12, 13] zu prüfen. die tabelle 1 enthält die für die jeweilige deklaration zu verwendenden testviren. handbuch der virusdesinfektion a test for the assessment of hygienic hand disinfection using rotavirus a study on the sensitivity of bovine rotavirus to some chemical agents the action of alcohols on rotavirus, astrovirus and enterovirus chemical disinfection of human rotaviruses: efficacy of commerciallyavailable products in suspension tests inactivation of a rotavirus by disinfectants inactivation of feline calicivirus, a surrogate of srsvs, by different types of alcohol. poster presented at the 5th int.conference of the hospital infection society inactivation of feline calicivirus, a norwalk virus surrogate anforderungen an die hygiene bei der aufbereitung von medizinprodukten anforderungen an die hygiene bei der reinigung und desinfektion von flächen richtlinie des bundesgesundheitsamtes und der deutschen vereinigung zur bekämpfung der viruskrankheiten zur prüfung von chemischen desinfektionsmitteln auf wirksamkeit gegen viren kommentar zur richtlinie des bundesgesundheitsamtes und der deutschen vereinigung zur bekämpfung des viruskrankheiten zur prüfung von chemischen desinfektionsmitteln auf wirksamkeit gegen viren bekanntmachung des fachausschusses "virusdesinfektion" der deutschen vereinigung zur bekämpfung der viruskrankheiten und des robert koch-institutes who containment of wild poliovirus stocks. who/polio/0202.www.who.org 15. who global action plan for laboratory containment of wild poliovirus annex 1 requirements for poliomyelitis vaccine (oral) (requirements for biological substances no hepatitis a virus: a test method for virucidal activity how contagious is vaccinia? cpmp/bwp/269/95 note for guidance on plasma-derived medical products measures for disinfection and control of viral hepatitis.in: block s (ed) disinfection, sterilisation and preservation, chapter 30 epidemiologischer bericht zur situation in deutschland bis zum jahr influence of different disinfection conditions on the structure of the hepatitis b virus (dane particle) as evaluated in the morphological alteration and disintergation test (madt) zerstörung der antigenität und beeinflussung der immunochemischen reaktivität von antigenen des hepatitis-b-virus (hbsag, hbcag und hbeag) durch desinfektionsmittel -ein prüfmodell methodological approaches to disinfection of human hepatitis b virus molecular approaches to validate disinfectants against human hepatis b virus comparision of the morphological alteration and disintegration test (madt) and the chimpanze infectvity test for determination of hepatitis b virucidual activity of chemical disinfectants inactivation of hepatitis b virus by intermediate-to-high-level disinfectant chemicals richtlinie des robert koch-institutes zur prüfung der viruzidie von chemischen flächendesinfektionsmitteln und instrumentendesinfektionsmitteln, die in die liste gemäß § 10c des bundes-seuchengesetzes aufgenommen werden sollen quantitativer suspensionsversuch auf viruzidie für in der humanmedizin verwendete chemische desinfektionsmittel und antiseptika, prüfverfahren und anforderungen richtlinien für die prüfung chemischer desinfektionsmittel,3.aufl.verlag der deutschen veterinärmedizinischen gesellschaft e be key: cord-007562-4hcs0z65 authors: bijlenga, g. title: proposal for vaccination against sars coronavirus using avian infectious bronchitis virus strain h from the netherlands date: 2005-07-19 journal: j infect doi: 10.1016/j.jinf.2005.04.010 sha: doc_id: 7562 cord_uid: 4hcs0z65 nan occult hepatitis b virus infection among anti-hbc positive blood donors: necessitates substitution of screening by hbv nat safe blood transfusion still remains a major concern and so far all the efforts in this direction have failed to achieve zero residual risk of transfusion transmitted hepatitis b virus (hbv) infection. in this direction the recently published work by silva et al. in journal of infection has revealed remarkable observations. 1 this report shows 3.3% hbv dna positivity of the blood donor's samples that were anti-hbc positive, more enlightening finding is the hbv dna positivity among the high level anti-hbs positive donors. at this tertiary care centre of saudi arabia out of 26 606 blood units collected during 2000-2003, isolated anti-hbc positivity was 3.2% and hbsag positivity 1.9%, where as 10.1% of the blood units were anti-hbc and anti-hbs positive. as per policy of health ministry, the anti-hbc and anti-hbs positive blood units were utilized and the isolated anti-hbc blood units were rejected. 2 the blood units which are anti-hbc and anti-hbs positive do not appear to transmit hbv infection and there is inverse correlation between anti-hbs level and infectivity, only 10% of the blood units with low level (!0.1 iu/ml) anti-hbs are infectious. 3 the observation by silva et al. that hbv dna positivity among anti-hbc and high level anti-hbs positive blood donors is a pointer towards the transfusion transmitted risk involved by transfusion of anti-hbc and anti-hbs positive blood units. though the viral load in these samples was low (!1000 copies/ml) but this can be highly infectious if transfused to an immunocomprised patient. considering the volume of infectious blood transfused any amount of hbv dna will be infectious as the minimum infecting dose of hbv in chimpanzees is only 100 virus particles. 4 in many of the developed countries and most of the developing countries the blood units collected are still being screened for hbsag, anti-hbc and anti-hbs by enzyme immuno assay. on many occasions the results are indeterminate and has to be repeated leading to higher per unit cost of blood screening and lot of rejection of the invaluable units of collected blood or exclusion of the generous donor because of isolated anti-hbc positivity and still the safety of transfusion transmitted hbv is compromised. this high rate of rejection of collected blood units and the exclusion of the anti-hbc positive blood donors leads to the unceasing blood shortage in the blood banks. the hbv screening policy for the collected units of blood needs reassessment in light of the present report 1 and hbv dna testing should be preferred instead of three enzyme immuno assay tests. hbv dna testing by nat of all the collected units of blood should be adopted by all the blood banks, in order to possibly achieve zero risk of transfusion transmitted hbv infection and also to reduce the rejection rate of the precious units of collected blood by testing for anti hbc. the outbreak of severe acute respiratory syndrome (sars) in 2003 has resulted in a number of infections and deaths among healthcare workers (hcws) and those in contact with sars-infected persons. the virus, now classified provisionally as a coronavirus in group 4, is highly contagious and treatment of infected persons has so far been disappointing. the first evidence of successful treatment in monkeys (cynomolgus macaques) was reported recently using alpha-interferon (ifn-alpha) 4 administered from 1 to 3 days after experimental exposure. this gave only some success, whereas the drug given at 3 days before experimental infection significantly reduced viral replication and excretion from their throats. lung damage was also reduced by 80% as compared with non-treated monkeys. in a review article on avian infectious bronchitis (ib) vaccine strain h, 1 various characteristics of this vaccine were outlined. here i shall mention the most valuable properties of this ib vaccine so far known to underline the hypothesis that it may be beneficial in people at risk from sars coronavirus. (1) it has been observed that the ib vaccine h is able to protect against a broad spectrum of different heterologous serotypes of ib challenge viruses. 12 these serotypes differ in their surface proteins (spikes-s1) which are responsible for the induction of neutralizing antibody. differences in s1 of only 2-3% can change the serotype of an ib virus. 3 therefore, it can be concluded that the protection provided by the vaccine strain h is not only dependant on the production of neutralizing antibody, but is also due to the induction of other immunological reactions. (2) the role of the nucleocapsid protein (n) is still not well understood but it may play an important role in protection, inducing specific cytotoxic t lymphocytes. 2,7-10 thus, the vaccine strain h may be responsible for the induction of protection through its nucleocapsid protein. 13 in order to evaluate the importance of cellular mediated immunity (cmi) in protecting against ibv infections more studies would be necessary to explain all the mechanisms of cross-protection of the vaccine strain h, for instance the induction of interleukine 2 (il 2). (3) the observation that interferon (if) is poorly induced by ibv and may not be induced by the vaccine strain at passage level 52, could be an indication that if plays a limited role in heterologous protection. 5 (4) in a study by marra et al. 6 it was concluded that the sars coronavirus is a novel coronavirus. stavrinides and guttman 11 concluded recently that the sars coronavirus is mammalian-like through the replicase protein, and avian-like through the m and n proteins. they also observed a mammalian-avian mosaic in the s protein. these observations are of extreme importance to the consideration of an avian coronavirus as a possible candidate for a vaccine against sars coronavirus. in adequately equipped laboratory facilities (p4): (a) it is proposed to use passage 52 of the h strain of vaccine in preliminary experimental studies in monkeys. this passage level has been chosen for its retention of cross-protective characteristics. the vaccine strain h at passage 120 induces only a low level of interferon 5 but has lost its heterologous protection characteristics due to the attenuation of the virus. (b) in order to produce a valuable immunological reaction in monkeys with the ib h52 vaccine it will be necessary to inoculate a high dose of live virus vaccine, for example 10 8 median embryo infectious doses (100.000.000 eid 50 ) intranasally, intramuscularly and/or subcutaneously. it is not expected that the virus will be infectious for macaques, therefore, a high dose will be required in order to achieve an adequate response of the immune system. for more than 50 years avian ib infections have occurred worldwide and there are no reports of infection among human beings, including in poultry farmers or other people who have had direct contact with highly contagious ib viruses of chickens. (c) in the study using alpha if in macaques the amount of sars coronavirus virus (scv) used for challenge was 1!10 6 median tissue culture infectious dose (tcid 50 ) in 5 ml of pbs administered intratracheally. 4 however, it was not mentioned in that publication whether or not a prechallenge titration of this virus was performed. it is very important to establish the amount of challenge virus, which will provoke disease and eventually death. therefore, before starting the experiment titration of the challenge virus in these monkeys should be performed in order to determine the amount of virus, which will produce clinical symptoms in not more than 90% of the infected animals. if an overdose is applied no real effect of the treatment will be demonstrable and if insufficient challenge virus is used no results will become available. (d) it is of extreme importance that the h52 vaccine virus should be free of all microorganisms other than ib live vaccine virus, therefore, its production and passage in specific pathogen free (spf) embryonated eggs is a prerequisite. (e) it is proposed to challenge the vaccinated monkeys at 2 and 14 days after vaccination with a challenge scv which has been titrated in macaques (see point c). this proposal is based on the likely immediate effect of the vaccine at 2 days through immunostimulation mechanisms and at 2 weeks, if protection is observed, through the heterologous cross-protective activity of the vaccine virus. it is without question that careful consideration by the relevant official health authorities must be given before an animal live virus vaccine is applied to human beings. the application of the ib vaccine strain h in humans should be restricted and only hcws and other persons at risk but not yet showing any signs of the disease will be considered as candidates for vaccination. if clinical symptoms are observed other methods of treatment, such as administration of alpha if are recommended. low rate of occult hepatitis b virus infection among anti-hbc positive blood donors living in a low prevalence region in brazil epidemiology of antibody to hepatitis b core antigen screening among blood donors in eastern saudi arabia: need to replace the test by hbv dna testing occult hepatitis b virus infection: implications in transfusion b-propiolactone irradiation: a review of its effectiveness for inactivation of viruses in blood derivatives development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review induction of anti-viral immune responses by immunization with recombinant-dna encoded avian coronavirus nucleocapsid protein review article. severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques induction of chicken interferon by avian infectious bronchitis virus the genome sequence of the sarsassociated coronavirus specific cytotoxic t lymphocytes are involved in in vitro clearance of infectious bronchitis virus the carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protect chickens from acute infection cytotoxic t lymphocytes responses to infectious bronchitis virus infection adoptive transfer of infectious bronchitis virus primed alphabeta t cells bearing cd8 antigen protects chicks from acute infection mosaic evolution of the severe acute respiratory syndrome coronavirus potential for polyvalent infectious bronchitis vaccines study of protection by recombinant fowlpox expressing c-terminal nucleocapsid protein of infectious bronchitis virus against challenge 74250 la tour-en-faucigny, france q 2005 the british infection society key: cord-307044-4czeehkq authors: liu, jiaye; wang, tingyan; cai, qingxian; sun, liqin; huang, deliang; zhou, guangde; he, qing; wang, fu‐sheng; liu, lei; chen, jun title: longitudinal changes of liver function and hepatitis b reactivation in covid‐19 patients with pre‐existing chronic hbv infection date: 2020-08-06 journal: hepatol res doi: 10.1111/hepr.13553 sha: doc_id: 307044 cord_uid: 4czeehkq aim: with pandemic of covid‐19 currently and high endemic of chronic hbv infection worldwide, it is quite urgent to investigate liver function changes of covid‐19 patients with chronic hbv infection, and how sars‐cov‐2 infection in turn affects the course of chronic hbv infection. method: we conducted a retrospective study based on 347 covid‐19 patients (21 vs. 326 with vs. without chronic hbv infection). with the psm method, we yielded 20 and 51 matched patients for hbv group and non‐hbv group, respectively. results: at the end of follow‐up, all these 71 patients achieved sars‐cov‐2 clearance (p=0.1). during the follow‐up, 30% vs. 31.4% in hbv group vs. non‐hbv group progressed to severe covid‐19 (p=0.97). after psm, the longitudinal changes of median values for liver biochemistries were no significant difference between two groups. in hbv group vs. non‐hbv‐group, 35% (7/20) vs. 37.25% (19/51) (p = 0.86) had abnormal alt at least once during hospitalization, while 30% (6/20) vs. 31.37% (16/51) for abnormal ast (p = 0.91), 40% (8/20) vs. 37.25% (19/51) for abnormal ggt (p = 0.83), and 45% (9/20) vs. 39.22% (20/51) for abnormal tbil (p = 0.91). moreover, 3 patients in hbv group had hepatitis b reactivation. conclusions: liver dysfunction presented in covid‐19 patients with/without chronic hbv. moreover, those covid‐19 patients coinfected with chronic hbv could had a risk of hepatitis b reactivation. it is necessary to monitor liver function of covid‐19 patients, as well as hbv dna levels for those coinfected with hbv during the whole disease course. coronavirus disease 2019 (covid-19), an emerging respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has recently become a pandemic. a total of 14,348,858 confirmed cases and 603,691 deaths were reported globally as of july 20, 2020. 1 unfortunately, neither targeted drugs nor vaccines are available to date, and the number of infections is growing around the world. for the foreseeable future, covid-19 could constantly pose a great threat to the people of the world. covid-19 is typically characterized by the symptoms of viral pneumonia such as fever, fatigue, dry cough and anosmia, which may evolve to respiratory failure. unexpectedly, there is increasing evidence that some individuals with covid-19 have frequent abnormal liver function. [2] [3] [4] it was observed that the elevated liver biochemistries were more common in severe covid-19 cases than in mild cases. 5 the limited autopsy results of liver of covid-19 cases showed moderate microvascular steatosis, 6 or hepatocyte degeneration accompanied by lobular focal necrosis and neutrophil infiltration. 7 these histological characteristics of liver were not specific manifestations of liver damage caused by sars-cov-2. therefore, it is unclear currently whether liver damage/dysfunction of covid-19 patients is mainly due to the sars-cov-2 infection or other coexisting conditions. hepatitis b remains another major worldwide public health problem, with approximately 257 million individuals infected with hepatitis b virus (hbv), and more than 94 million suffer from chronic hepatitis b (chb). 8 a large cohort study from china reported that 2.1% (21/1 099) of enrolled covid-19 cases had pre-existing hepatitis b. 9 given the endemic and high burden of hbv infection, chb may be one important comorbidity of pre-existing liver diseases affecting the outcome of covid-19. it is confirmed that hbv infection can cause the damage of innate immune responses and imbalance of adaptive immune responses. 10 meanwhile, uncontrolled inflammatory innate responses and impaired adaptive immune responses causing by sars-cov-2 may lead to harmful tissue damage, both locally and systemically. 11 therefore, coinfection of sars-cov-2 and hbv may increase the damage of immune function and liver. however, to the best of our knowledge, no studies had been carried out on the impact of chronic hbv infection on the disease progression and liver function changes of covid-19 patients, and how the sars-cov-2 infection in turn affects the course of chronic hbv infection. more evidence is urgently needed to guide the screening of hbv coinfection and management of comorbidity of chb during the pandemic of covid-19. hence, in this study we aimed to assess the independent effect of hbv infection on the outcomes of covid-19 as well as the progression of hbv infection. all diagnosed covid-19 patients according to who interim guidance, with or without chronic hbv infection, who admitted to shenzhen third people's hospital during january 1 to march 1, 2020, were enrolled. chronic hbv infection was determined on the basis of testing positive for hbsag and/or hbv dna at hospital admission and medical history of chronic hbv infection. the clinical outcomes of covid-19 and dynamics of liver biochemistries were monitored up to april 12, 2020, the final date of follow-up. the inclusion criteria were as follows: (1) subjects diagnosed with covid-19, (2) the records were well documented, (3) subjects with longitudinal follow-up, i.e., liver function testing, chest computed tomography (ct) scan, or blood gas assay with at least across two days. the exclusion criteria were: (1) subjects without data available at baseline, i.e., blood routine examinations, liver biochemistries, ct score, 12 blood gas assay, (2) subjects coinfected with human immunodeficiency virus, (3) subjects coinfected with hepatitis virus other than hbv, or had liver diseases other than chb. the process of patients' enrolment was presented in baseline was defined as the first time of hospital admission due to covid-19. at baseline and during follow-up, all subjects included in this study underwent routine examination, monitoring of liver biochemistries, and sars-cov-2 nucleic acid testing with a median follow-up interval of 3 days. the primary outcome was progression to severe covid-19, and the secondary outcomes included clearance of sars-cov-2, liver injury, and hepatitis b reactivation. the time point of covid-19 onset was defined as the day of symptoms presence self-reported by patients who were further confirmed with sars-cov-2 after hospital admission. the virus clearance was defined by the presence of two consecutive negative results in quantitative pcr detection for sars-cov-2 rna at an interval of 24 hours, and the day of the first one of these two tests were considered as the clearance day. patients were discharged from hospital after the clearance of sars-cov-2. hepatitis b reactivation was defined as the abrupt reappearance of hbv dna viremia in a patient with previously inactive or resolved hbv infection, or an sudden and rapid rise of hbv dna level by at least 2 log10 in those with previously detectable. 13 according to the national guidelines for community-acquired pneumonia, and the diagnosis and treatment plan for the new coronavirus in china, all covid-19 patients were classified into severe or mild cases based on chest radiography, clinical examinations, and symptoms. 14, 15 we analyzed the dynamics of liver biochemistry indicators (i.e., alt, ast, total bilirubin [tbil], gamma-glutamyl transferase [ggt]) to investigate the liver function changes. the normal range of alt, ast, ggt and tbil in this study was 0-45 u/l, 0-45 u/l, 0-49 u/l, and 1.7-21 umol/l, respectively. as one patient coinfected with hbv was underwent liver biopsy, we investigated the pathological characteristics of liver injury of this patient. all statistical analysis was conducted using r 3.6.1. we performed propensity score matching (psm) on the selected 347 subjects so that the group coinfected with hbv is comparable to the group without hbv coinfection in terms of observed covariates at baseline. 16 the factors for propensity score calculation include age, gender, body mass index (bmi), time intervals between covid-19 onset to hospital admission, number of comorbidities except for chb, liver biochemistries (alt, ast, ggt, tbil), pao2/fio2 ratio, chest ct score, crp, lymphocyte count, and platelet count at baseline. the psm process was conducted by using r package matchit, with nearest-neighbour method, 1:3 matching ratio, and a caliper size of 0.1. for baseline characteristics, we used median (interquartile range [iqr]) for continuous variables and wilcoxon test for comparison, while we reported count (percentage) for categorical variables and fisher exact test for comparison. we used kaplan-meier (k-m) method to estimate cumulative probabilities for the clearance of sars-cov-2 and progression to severe covid-19 and compared the probabilities between hbv group and non-hbv group using a log-rank test. we use multivariable cox proportionalhazards model to compare the risk of progression to severe covid-19. to investigate the longitudinal changes over time, we first performed comparison of the median values of liver biochemistries (alt, ast, ggt, tbil) over time between groups using wilcoxon signed-rank test. then we compared the values of these indicators between groups at each time point (to examine difference between groups) and compared the values of these indicators at each time point to their baseline values within each group (to examine difference within groups) using wilcoxon test or kruskal−wallis test. in addition, we also reported the proportion of patients with abnormal values for liver biochemistries over time to examine the liver function changes, with using χ 2 test or fisher exact test to compare the proportions between groups. all significance tests performed were two-sided. p values less than 0.05 were deemed statistically significant and 95% confidence intervals (cis) were calculated for point estimates. a total of 347 covid-19 patients with/without hbv coinfection (21 vs. 326) were analysed before matching. in hbv coinfection group, 20 were diagnosed as hbeag-negative chronic hbv infection or hbeag-negative chb, and one patient had a pre-existing cirrhosis while did not receive any imaging examinations during the hospitalization of covid-19. we described the details of history of hbv infection and antiviral treatment, virological and serological testing at baseline in supplementary the propensity score matching of entire study population yielded 20 and 51 matched patients for hbv group and non-hbv group, and the covariates used for matching and not used for matching were comparable between the two groups after matching (table 1 , all p > 0.1). at the end of follow-up, all the patients in both groups achieved sars-cov-2 clearance and none of them died. the median time to sars-cov-2 clearance (21 days, 95% ci: 19-29) in hbv group was longer than that in non-hbv group (14 days, 95%ci: 13-21), however, no significant difference was observed regarding the probability of sars-cov-2 clearance over time between the two groups (p=0.1, figure 1a ). during the follow-up period, 30% (6/20) and 31.4% (16/51) of patients in hbv group and non-hbv group progressed to severe covid-19, respectively, and there was no difference between the two groups in the probability of progression to severe covid-19 over time (p=0.97, figure 1b ). by multivariate analysis, the risk of progression to severe covid-19 was not statistically table 3 ). in hbv group vs. non-hbv group, 35% (7/20) vs. 37.25% (19/51) had abnormal alt at least once during hospitalization, respectively (p = 0.86). the proportion of abnormal alt had a rise-fall trend in hbv group while a constantly increasing in non-hbv group since admission to hospital (figure 2a) , which was 18.18% and 19.23% at 15 days in the two groups, respectively (p = 0.94). figure 2d ). as the median of testing/assessing time intervals and follow-up durations were 3 days and 14 days for liver biochemistries (alt, ast, ggt, tbil), we compared the dynamic levels of these indicators within/between the two groups at baseline, 3, 6, 9, 12, 15 days during hospitalization. the median levels of liver biochemistries over time were no significant difference between two groups ( figure 3 ; wilcoxon signed-rank test, alt: p=0.56, ast: p=0.58, ggt: p=0.43, tbil: p=0. 16 ). in addition, we found no significant difference in the alt, ast, ggt and tbil levels between the two groups at each time point (supplementary figure 2a-2d) . for the 20 covid-19 patients with chronic hbv infection, 19 of whom had hbv dna viral load testing at least twice during hospitalization, however, one patient had not any test of hbv dna viral load. of the 19 patients, three patients had hbv reactivation, 15 patients had the hbv dna viral loads maintained at low levels (<300 iu/ml) or undetectable, and two patients' hbv dna viral loads were at high levels throughout the hospitalization. all the three patients with hepatitis b reactivation were hbeag negative and did not received any antiviral treatment for hbv before admission. we further described the dynamics of hbv dna viral load and liver biochemistries, and treatment information in figure 4 :  case 1: received methylprednisolone therapies during 3 to 6 days after admission, and interferon α-1b treatment (atomized inhalation) during 5 to 9 days after admission; this article is protected by copyright. all rights reserved. hbv dna viremia was undetectable on 9 days, but abruptly measured as 3.30 log10 iu/ml on 30 days, and finally as 4.05 log10 iu/ml when discharged; alt and ast respectively increased to 387 iu/l (8.6 xuln) and 497 iu/l (11 xuln) on 8 days after admission.  case 2: received methylprednisolone therapy during 2 to 5 days after admission, and interferon α-1b treatment (atomized inhalation) during 1 to 25 days after admission; hbv dna levels were at 3.5-5 log10 iu/ml in early time of hospitalization but had a rapid increase from 2.26 log10 iu/ml on 29 days to 6.65 log10 iu/ml on 31 days; alt and ast were mildly high (< 2xuln) at admission and then restored to normal levels since 3 days after admission.  case 3: received interferon α-1b treatment (atomized inhalation) during 1 to 53 days after admission; hbv dna viremia was undetectable at admission but hepatitis b reactivation (detectable as 1.30 log10 iu/ml) at 9 days after admission; alt and ast were persistently normal during hospitalization. one female patient who was 33 years old and whose bmi was 17.8 kg/m 2 at the admission to hospital, had a history of hbv infection over than 20 years but had not received antiviral treatment for chb previously. in addition, this patient had not any history of alcohol use. the maximum level of alt and ast of this patient was 31.8 u/l and 35.6 u/l during hospitalization, respectively. the patients discharged from hospital after sars-cov-2 clearance but readmitted to the hospital due to hepatalgia and had a liver biopsy 40 days after the clearance of sars-cov-2. the detailed information regarding her treatment course and laboratory tests was provided in supplementary figure 3 . the results of liver needle biopsy for this patient showed the structure of the hepatic lobules was clear, and the hepatocytes in the interlobular were arranged orderly, with some hepatocytes diffuse swelling (ballooning degeneration), and necrosis of isolated hepatocytes. no canalicular bile plugs and interface hepatitis were seen. reticulin staining and sirius red staining indicated periportal fibrosis. the portal tracts were infiltrated with few inflammatory cells. the immunohistochemical analysis showed positive for hbsag and negative for hbcag ( figure 5 ). this observational study found no significant difference in probability of sars-cov-2 clearance and progression to severe covid-19 over time in covid-19 patients with vs. without chronic hbv infection. we observed similar dynamics and non-significant difference at each time point on liver biochemistries (alt, ast, ggt, tbil) between the two groups. however, we observed a continuous abnormality of alt and ggt for both groups, which may be due to sars-cov-2 infection. more importantly, we identified three patients who underwent hepatitis b reactivation. this study provided preliminary evidence concerning the effect of chronic hbv infection on the outcomes and liver function of covid-19 patients and added important data to support the management of covid-19 patients with chronic hbv infection for physicians. investigation of sars-cov-2 shedding will be the key for determining the risk of transmission and formulating the criteria of releasing from quarantine. for the non-hbv covid-19 patients, we found the median time of sars-cov-2 clearance was 12 days after the onset of covid-19 symptoms, which was consistent with a study on eight discharged covid-19 patients from singapore (median, 14 days). 17 . despite the confirmed host immune dysfunction resulting from chronic hbv infection, our results reveal that chronic hbv infection could not delay the sars-cov-2 shedding for covid-19 patients, compared to those without chronic hbv. after excluding non-hbv related chronic liver diseases, we explored the independent impact of chronic hbv infection on the progression to severe covid-19 and found that chronic hbv infection did not increase the risk of progression to severe covid-19. together with these comparisons, we tend to conclude that the comorbidity of chronic hbv infection would not increase the risk of poor outcomes related to sars-cov-2. it is worth mentioning that only one patient in hbv group were cirrhotic in this study. patients with hbv related cirrhosis typically have poor immune function compared to those who had chronic hbv infection but without cirrhosis. therefore, further studies are necessary to examine the impact of hbv related cirrhosis on the outcomes of covid-19. previous studies have found that both hepatocytes and bile duct epithelial cells may also express the angiotensin-converting enzyme 2 (ace2) receptor, while the latter has a higher concentration. it suggests sars-cov-2 might cause the damage of both hepatocytes and bile duct epithelial cells. current studies showed that 6.2% -36.6% of patients had increased serum ast levels, while 21.3% -28.1% had elevated serum alt levels. 18 a few studies reported the abnormal proportions of tbil (4.9% to 10.53%) 4,19,20 and ggt (6.5% to14.8%) 4,19 at baseline. our study also identified the abnormality of the above-mentioned liver laboratory tests. moreover, with further analysis of longitudinal patterns, we found that the abnormality of ast and tbil manifested as transient elevation, while high proportions of patients with abnormal alt and ggt did not achieve the normalization of these two indicators. the dynamics of alt and ggt suggested that sars-cov-2 possibly caused a continuous damage of bile duct epithelial cells and hepatocytes during the disease course. although chronic hbv infection would not increase the injury of liver compared to non-hbv covid-19 patients as suggested in this study, liver function monitoring is still essential for both covid-19 patients with and without chronic hbv infection during the whole disease course. glucocorticoids have powerful anti-inflammatory effects, and have been confirmed to alleviate clinical symptoms, shorten treatment course, and improve the absorption of lung infiltrates for severe acute respiratory syndrome (sars) patients. 21, 22 in our study, six covid-19 patients with hbv received methylprednisolone, one type of corticosteroids. it is well known that moderate to high dose (≥10 mg) of methylprednisolone can lead to a high risk of hepatitis b reactivation. 23, 24 in our study, two of the three patients developed hepatitis b reactivation, which was possibly caused by methylprednisolone. however, one of the three patients who did not receive any corticosteroid also developed hepatitis b reactivation. our results suggested that for covid-19 patients who had chronic hbv infection, whether or not corticosteroids were used, they could have a risk of hepatitis b reactivation, therefore it is necessary to monitor the hbv dna levels for these patients, and for them physicians should take precautions to hepatitis b reactivation. this study is no without limitations. firstly, the number of patients in hbv group was small, although we expected to increase the test efficiency using psm designed with 1:3 matching ratio, and we validated our results by multivariable cox proportional-hazards model. secondly, we were unable to explore if the liver abnormality associated with covid-19 treatment drugs, as all the 71 patients in two groups had received them (supplementary table 2 clinical characteristics of non-icu hospitalized patients with coronavirus disease 2019 and liver injury: a retrospective study liver injury during highly pathogenic human coronavirus infections liver impairment in covid-19 patients: a retrospective analysis of 115 cases from a single center in wuhan city, china covid-19 and the liver: little cause for concern pathological findings of covid-19 associated with acute respiratory distress syndrome general anatomy report of novel coronavirus pneumonia patients polaris observatory collaborators. global prevalence, treatment, and prevention of hepatitis b virus infection in 2016: a modelling study clinical characteristics of coronavirus disease 2019 in china diagnosis and treatment of adults with community-acquired pneumonia. an official clinical practice guideline of the american thoracic society and infectious diseases society of america clinical epidemiology: the essentials epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore covid-19 and liver dysfunction: current insights and emergent therapeutic strategies exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study use of glucocorticoid in treatment of severe acute respiratory syndrome cases american gastroenterological association institute technical review on prevention and treatment of hepatitis b virus reactivation during immunosuppressive drug therapy cord blood transplantation rescued by medical treatment key: cord-293646-d4qcckh1 authors: meanwell, nicholas a.; serrano-wu, michael h.; snyder, lawrence b. title: chapter 22. non-hiv antiviral agents date: 2003-12-31 journal: annual reports in medicinal chemistry doi: 10.1016/s0065-7743(03)38023-6 sha: doc_id: 293646 cord_uid: d4qcckh1 publisher summary this chapter focuses on non-hiv antiviral agents. the development of antiviral agents to treat non-hiv infections is largely focused on therapies for the treatment of chronic hepatitis infections b and c. nucleoside analog continue to be the mainstay of hepatitis b virus (hbv) therapeutics. the first small molecule inhibitor of hepatitis c virus (hcv), the ns3 protease inhibitor biln-2061, entered phase 2 clinical trials, producing a striking reduction in viral load in treated individuals. the development of the hcv replicon system and its application to screening for antiviral agents provided tangible benefit with the disclosure of mechanistically and structurally diverse hcv inhibitors. adefovir dipivoxil has been approved in the united states and the european union for the treatment of hbv, providing a second small molecule antiviral to add to lamivudine (3tc) and the injectable protein ifnα as the only approved agents for treating hbv infection. the chapter also provides details of the inhibitors of hepatitis b and c virus, the inhibitors of simplex virus and human cytomegalovirus, the inhibitors of respiratory viruses and the inhibitors of west nile virus and papilloma virus. introduction -the development of antiviral agents to treat non-hiv infections is largely focussed on therapies for the treatment of the chronic hepatitis infections b and c (1). nucleoside analogues continue to be the mainstay of hbv therapeutics and clinical development of several continued during 2002. the last year has seen the first small molecule inhibitor of hcv, the ns3 protease inhibitor biln-2061, enter phase 2 (p2) clinical trials, producing a striking reduction in viral load in treated individuals. the development of the first hcv replicon system in 1998 and its application to screening for antiviral agents is beginning to provide tangible benefit with the disclosure of mechanistically and structurally diverse hcv inhibitors. there remains considerable interest in inhibitors of herpes simplex and human cytomegalovirus viruses, particularly non-nucleoside compounds. developments in the area of respiratory virus inhibitors have focussed more on respiratory syncytial virus with a description of the first antiviral active in animal models following oral administration. the west nile virus outbreak in the us, originally confined to the east coast, broadened considerably during the summer of 2002, claiming 230 lives. in the wake of the events of september ilth, 2001, smallpox was a prominent concern as a potential agent of bioterrorism. developments in each of these areas will be reviewed. inhibitors of hepatitis b virus (hbv) -adefovir dipivoxil ihepsera'") (1) was approved in the us for the treatment of hbv on september 20', 2002 and in the european union on march 1 lth, 2003, providing a second small molecule antiviral to add to lamivudine (3tc) and the injectable protein ifna as the only approved agents for treating hbv infection. adefovir is an effective inhibitor of 3tc-resistant hbv caused by the rtm2041 and rtll8om + rtm204v mutations in the reverse transcriptase (2) and resistance to adefovir has not been seen after 48 weeks of monotherapy (3, 4) . a clinical study with entecavir (z), currently undergoing p3 trials, compared a dose of 0.1-i mg/day of 2 to 3tc (100 mg/day) for 48 days in 181 patients previously unresponsive to 3tc treatment (5). compared to 3tc, treatment with 2 resulted in lower overall viral loads, lower alt levels and a higher proportion of patients with undetectable hbv dna levels. moreover, adverse events for 2 were less than those observed with 3tc. a separate p2 trial of 2. in 177 treatment-naive patients for 22 weeks at a dose of 0.5 mglday showed that reduction in viral dna levels was independent of baseline alt levels, with treatment resulting in a 4.7-4.8 loglo reduction in hbv dna (6). l-nucleosides represent a promising area of antiviral research (7). ldt (telbivudine, 3) is currently in p3 clinical trials designed to evaluate 1200 patients for safety and efficacy compared with standard treatment in hbeag+ and . recent p2b data was released from a trial involving 104 adults randomized to receive 3tc plus 2 or 3tc monotherapy once daily for 1 year. viral load reductions of greater than 6 logto were seen for all patients in the study arms containing 3 and no treatment-limiting or dose-related adverse events were reported. the mechanism of action of l-fmau (clevudine) (fi), currently in pi/p2 trials, is not firmly understood but recent molecular dynamics simulation experiments have suggested that the triphosphate derivative of 4 may act as a competitive inhibitor rather than as a substrate of hbv polymerase (9,lo). emtricitabine (coviraciltm) (5) is also currently in p3 clinical trials for the treatment of hbv. an nda seeking approval to market 5 for the treatment of hiv was filed in september 2002 (9). results from a pi/p2 cli%zal trial with ach-126443 (elvucitabine, 5) have been released (11-13). in 36 hbv treatment-naive patients receiving single daily doses of l-100 mg of 5, mean declines in plasma hbv dna of up to 2.5 loglo were observed after 14 days of treatment. furthermore, plasma levels in excess of the i&o values for wild type and ymdd mutants were achieved in the low dose arms. it is therefore anticipated that s will be efficacious against 3tc resistant infections in an ongoing p2 trial. this mutation, which arises in response to 3tc therapy, is found in the precore region and confers hbeag negativity. the major metabolite of 7 formed in rat or human serum is the mono-ester, which is a more potent hbv inhibitor, ec% = 70 nm, than 3tc. it is postulated that the arylthio moiety is responsible for the specificity towards hbv and lower cytotoxicity than pmea, the active component of adefovir dipivoxil. additional analogs in this structural class have been prepared with the phenylthio-and 3-methoxyphenylthio ethers showing the most promise whilst other aromatic thioethers exhibited higher cytotoxicity (14, 151) . non-nucleoside inhibitors of hbv are beginning to emerge that are anticipated to show reduced cross-resistance with nucleoside analogues. at130 (b) and its close analog at61 are active against wild type hbv and the rtll80m, rtm2041, and rtll80m+rtl204v mutants (ec50 = 2-5 pm) in hepg2-derived cells (17, 18) . it has been postulated that 8 interferes with the packaging of pregenomic viral rna resulting in inhibition of viral reverse transcription. pyridinedicarboxamide s represents the first report of a non-nucleoside inhibitor of hbv reverse transcriptase enantiomer of q is active in cell culture and appears to prevent proper formation of the viral nucleocapsrd. inhibitors of hepatitis c virus (hcv) -nearly 170 million individuals are infected with hepatitis c virus (hcv) worldwide and hcv infection is responsible for 8,000-10,000 deaths annually in the united states, a burden expected to increase significantly (22, 23) . a second pegylated interferon-a (ifn), roche's pegasys, was approved in 2003, both as mono therapy and in conjunction with ribavirin (11) (copegustm) (24). however, safety concerns with combination therapy remain, as the accumulation of 11 in erythrocytes can lead to hemolytic anemia. this has prompted a search for safer interferon co-therapies which include the active enantiomer of ij, levovirin, and the prodrug viramidine (12), which improves liver at the expense of erythrocyte exposure (2526). the development of safe, efficacious, and hcv-specific antiviral agents remains an important goal and the development of subgenomic hcv replicons has dramatically enhanced the potential to identify inhibitors (27, 28) . a significant advance towards establishing a correlation between replicon inhibition and clinical efficacy was recently accomplished with the disclosure of preliminary clinical data for biln-2061, a selective inhibitor of the ns3 serine protease of hcv that is structurally related to 13. this highly modified macrocylic tripeptide derivative is extremely potent in vitro, with k, values of 0.3 nm and 0.66 nm towards hcv-la and hcv-1 b ns3 proteases, respectively (29). these figures are similar to the potency observed in cell culture in the cognate hcv replicons, ec50 = 4 nm and 3 nm, respectively (29). antiviral efficacy was established in a study conducted with 10 patients with chronic hcv and significant liver fibrosis, where all patients treated with biln-2061 (200 mg p.o. b.i.d.) displayed a decrease in serum hcv rna levels of at least 2.0 log,, copies/ml after two days of treatment (30). four of these patients recorded a reduction in viral load of more than 3 orders of magnitude and viral titers returned to baseline following cessation of therapy, with no drug-related safety issues identified (31). the intensity of effort devoted towards the discovery of inhibitors of hcv ns3 has continued, with the focus largely on peptide-based molecules that are required to effectively complement the active site and proximal regions of the protease (32-34). the crystal structure of a macrocyclic inhibitor related to 13 bound to hcv protease has been disclosed and an acyclic tripeptidic inhibitor has been used to generate resistant subgenomic replicons in which mutations mapped to the protease (3836). amongst several strategies disclosed, the c-terminal carboxylate of peptide inhibitors may be replaced with an n-acyl sulfonamide moiety, as exemplified by 14 (37). novel approaches to constrain or mimic the peptidic backbone are represented by macrocycle l5, the tetrahydroindolizine b (ic 5~ = 0.12 pm), the imidazolone 17 and the bicyclic proline derivative 18 (k, = 0.042 pm, ks0 = 0.251 pm) (38-46). these inhibitors are constructed around either an a-keto amide or a boronic acid moiety, wellprecedented as serine protease inhibitor motifs that engage the catalytic serine residue in a covalent but reversible interaction. ph 1 i however, chemical reactivity is not a prerequisite for potent inhibition in a peptidic background since phenethylamide 19 and the azapeptide 20 inhibit hcv ns3 with ki values of 0.6 and 0.2 pm, respectively (47,48). the more active diastereomer of the ahydroxy amide p3 element explored in the context of 21 was found to possess the (i?)configuration, unanticipated and explained by the presence of an intramolecular hydrogen bond that orients the lipophilic moiety of this isomer into s3, as depicted (49). non-peptidic inhibitors of ns31ns4a protease are much less common but some progress has been made in this direction. the bicyclic lactam 22 is a mechanismbased inhibitor of hcv ns3 whilst additional examples of bis-benzimidazole derivatives that rely upon zr?' to consolidate the enzyme-inhibitor complex have been described (50,51). the ns5b rna polymerase is another structurally-characterized viral protein that is an attractive target for therapeutic intervention (52-55). inhibitors of hcv polymerase can be broadly divided into nucleoside and non-nucleoside derivatives. the nucleoside analog ribavirin (ii) has been suggested to interfere with both the initiation and elongation steps of hcv rna replication (52). several hcv ns5b inhibitors incorporate modified d-ribose elements and include the 2'-me derivative 23, ec50 = 0.25 pm, the 4'-azido analog 24, ec 50 = 1.2 pm, and the 2'-deoxy-2'-fluoro cytidine derivative 25, ego = 0.74 pm (56-59). several non-nucleoside inhibitors of hcv ns5b have been reported, including a series of phenylalanine derivatives of which compound 28, k, = 2.2 fm, is representative (60,61). this compound has been co-crystallized with ns56 and appears to bind to the inactive, open conformation of the polymerase almost 35 a from the active site (62). other scaffolds with which hcv polymerase inhibitors have been discovered include an amino thiophene, represented by 27, the enolic rhodanine 28 (i&o = 1 .o pm), and structural variations of previously disclosed benzimidazole derivatives 29 (go c 0.5 fm) claimed to be active in replicons (61,63-67). mechanistic studies with the benzo[l,2,4]thiadiazine polymerase inhibitor 30 suggest interference with the initiation step of viral rna synthesis, allowing for a potential synergy with existing elongation inhibitors (68). to hcv therapy include blocking the viral rna internal ribosomal entry site (ires), binding of the viral e2 envelope glycoprotein or attachement (69-72). the highly conserved ires has been targeted by oligonucleotides and artificial ribozymes, but little progress has been made in the development of small molecule inhibitors (70,71). p2 clinical evaluation of the antisense 20-mer oligonucleotide isis-14803 revealed a l-2 logto reduction in plasma hcv rna levels in approximately 30% of the patients after 4 weeks of treatment (73). another approach, which may prove complementary to virus-specific hcv therapy, is the induction of interferon production in host cells. small molecules that act via toll-like receptor activation have been identified as activators of an immune response (74). rna interference (rnai) is a rapidly emerging technology that has proven to be a powerful means of selectively controlling protein production in cell culture. inhibition of hcv replication in replicons has been accomplished using this procedure whilst the demonstration of selective targeting of the liver protein fas in vivo using rnai holds promise for the treatment of hcv (75-78). maribavir demonstrated in viva anti-hcmv activity in all of the dosage regimens tested (100, 200, and 400 mg tid, and 600 mg bid), with mean reductions in semen hcmv titers of 2.9 to 3.7 loglo pfulml (92). the hcmv serine protease has been perceived as an attractive antiviral target. recent crystal structure data demonstrated significant conformational flexibility in the s3 binding pocket of protein complexed with two peptidomimetic inhibtors (94). a series of frans-lactams, represented by 35 and 36, 1% = 0.54 and 0.34 pm, respectively, are derived from the same structural platform as the hcv ns3 inhibitor 22 but inhibit the hcmv serine protease with excellent selectivity (9597). mechanistic studies are consistent with acylation of the active site serlz of hcmv protease in a reversible and time-dependent manner. chap. 22 non-hiv antiviral agents meanwell et al. 219 all but one of the currently licensed drugs available to treat hsv act by inhibiting the viral dna polymerase, providing a suitable backdrop for the emergence of resistant virus and a rationale for identifying inhibitors of other viral proteins (98,99). two groups have reported novel thiazole-containing inhibitors of the hsv helicase-primase. bay 57-1293 (37) is a leading pre-clinical candidate that is more potent (ec50 = 12 nm) than any anti-herpetic currently used to treat hsv infections (100,101). in a murine lethal challenge model of hsv-1 and hsv-2, 37 was protective with an edso value of 0.5 mglkg, which compares with the much higher doses of 22 and 16 mglkg for hsv-1 and hsv-2, respectively, required for acyclovir to show efficacy. additional patent applications that extend this promising chemotype have appeared (102,103). a second series of hsv helicase-primase inhibitors, of which bils-179 bs (38) is representative, has been disclosed (104). bils 179 bs inhibits viral growth with an ec50 of 27 nm, displays an excellent therapeutic index of >2000 and reduces cutaneous hsv-1 and genital hsv-2 disease in a murine model when treatment is initiated 3 hours post-infection. interestingly, when treatment was initiated 65 hours after infection, 38 reduced hsv-1 pathology by 75% and hsv-2 mortality by 75% (200 mg/kglday) when compared to acyclovir or untreated animals (104). a series of 4-oxo-dihydroquinolone derivatives that are potent and broad spectrum non-nucleoside inhibitors of dna polymerases of the herpesvirus family, including hcmv, hsv-1, hhv-8 and vzv, have been the subject of a number of recent disclosures (105). these compounds exhibit no significant inhibitory activity towards human ci-, y-, or 6-polymerases. pnu-183792 (39) is an effective antiviral in cell culture, potently inhibiting hcmv (ec 50 = 0.69 pm), vzv (ec% = 0.37 pm) and hsv (ec50 = 0.58 pm), that is active towards ganciclovir-and cidofovir-resistant hcmv and acyclovir-resistant hsv (106). excellent oral bioavailability and a protective effect in a murine cmv animal model were also reported. a series of related analogs have been reported in the recent patent literature (107,108). pyrazolopyridine derivatives have been reported to possess activity against hsv-1 in vero 76 cells with 40 having an ec50 of 0.12 fm (109-i 12). structurally related irnidazopyridine derivatives are active against hsv types l-8 with an e&o of 0.14 pm (113). cmv-423 (41) is a potent inhibitor of hcmv, ec50 = 4-7 nm, that appears to act at a step in viral replrcation preceding dna polymerization (114). overview -respiratory viruses continue to be a significant source of mortality and morbidity. the annual death rates due to influenza in the us are estimated to have doubled over the last 20 years, attributed to an aging of the population (115, 116) . this study also revealed a greater appreciation of the contribution of respiratory syncytial virus to mortality. the recently discovered human metapneumovirus (hmpv) was identified as a significant cause of wheezing in infants (117,118). the influenza inhibitor oseltamivir (tamiflutm) was approved for the treatment of influenza in adults and children and for prevention in adults and adolescents by the european community in june 2002, consolidating its position as the market leading neuraminidase (na) inhibitor. however, development of the third neuraminidase inhibitor peramivir was terminated by biocryst in june after disappointing p3 results in which the orally bioavailable compound failed to meet the key efficacy endpoint of reducing the time to onset of relief of symptoms. in march, 2003 an outbreak of severe acute respiratory syndrome (sars) emerged in southeast asia for which the culprit was quickly identified as a new coronavirus distinct from any previously identified human coronavirus. of the 1918 influenza continues and chimeric viruses containing the 1918 na or ml ion channel showed susceptibility to oseltamivir and amantadine or rimantadine, respectively, both in vitro an in viva. (119,120). the role of na inhibitors in pandemic influenza has been reviewed and the identification of structurally novel na inhibitors has continued (121). substitution of the primary amine moiety of oseltamivir with a vinyl group afforded the potent influenza b na inhibitor 42, ki = 45 nm. (122) additional sar studies around zanamivir have focussed on the c-7 hydroxyl where replacement by f or methylation affords the potent na inhibitors 43 and 44, icso values of 0.8 and 6.1 nm, respectively, which compares favorably with an i&o of 5-10 nm for the prototype (123,124). polymeric analogues of zanamivir linked to a polyglutamate backbone via the 7 position showed enhanced influenza inhibitory activity compared to monovalent analogues (125 the o-methyl analogue of zanamivir is claimed to protect mice against a lethal influenza infection following oral administration of the prodrug g whilst the bicyclic ether 48 is the first zanamivir derivative to demonstrate oral efficacy in the mouse model (127, 128) . the optimization of a screening lead into potent, cyclopentanebased inhibitors of neuraminidase using a combination of structure-based design and combinatorial chemistry has been described in detail (129). mechanism-based inhibitors of the hrv 3c cysteine protease have been probed using a combination of structure-based design principles and parallel synthesis methods in an effort to find less peptidic inhibitors (132). the chroman a emerged as an inhibitor of hrv-14 replication in cell culture, ec50 = 160 nm; however, the serotype coverage of this compound was poor with much reduced potency against other subtypes, an observation rationalized in the context of structural data (132). the hrv 2a cysteine protease releases itself from the viral p2 polyprotein, cleaves the p2 in both a cis and tram fashion to release the 2b and 2c proteins and also proteolyzes the host cap binding complex in order to compromise host cell transcription. a series of n-phenylated pyrazole derivatives have been claimed as inhibitors of this essential enzyme with 52 an effective antiviral agent in cell culture, ec50 = 1.8 pm, cc50 = 388 i.im (133). the hrv rna-dependent rna polymerase from hrv-16, a potential drug discovery target, has been cloned, expressed and purified from e. co/i and shown to be enzymatically active (134). inhibitors of resdiratotv svncvtial virus (rsv) -the role of rsv in morbidity and mortality continues to be a focus of research designed to provide a more accurate perspective of this virus as a mediator of significant disease burden, (115, 116, 135, 136) . the enhanced awareness of rsv has stimulated interest in this virus as a therapeutic target with the recent emergence of new structural classes of inhibitor. however, clinical proof-of-principle remains to be established for rsv antivirals. viropharma has suspended development of vp-14637, an rsv fusion inhibitor under examination as a topically administered agent. the benzimidazole bms-433771 (53) has been described as the first rsv inhibitor to demonstrate antiviral activity in animal models following oral administration (137, 138) . mechanism of action studies indicate that g is an inhibitor of the fusion of viral and host cell membranes and targets the rsv f (fusion) protein (137). analogues of 53 form the basis of proprietary claims (139, 140) whilst trimeris has disclosed a series of benzimidazole-based inhibitors of rsv fusion (141). representative of this series is 54, which is active in cell culture, ego = 10 ng/ml. -coronaviruses are enveloped, single-strand, positive-sense rna viruses most commonly associated with respiratory infections in man (142). two coronaviruses are the underlying cause of approximately 30% of upper respiratory tract infections, usually mild to moderate in severity and generally recognized as the common cold, although in the immunocompromised population coronavirus infections can lead to pneumonia. however, in march, 2003 an outbreak of severe acute respiratory syndrome (sars) emerged in southeast asia that very quickly spread to 27 countries, carried largely by air travelers (143-145). more than 6500 sars infections were documented worldwide within the following 2 months that were associated with substantial morbidity, including fever, non-productive cough, malaise, chills, headache and dyspnea, and significant mortality, with over 460 deaths reported (146). whilst sars appears to have originated in southern china in november, 2002, reports of major outbreaks in hong kong and toronto, canada in march 2003 brought the disease to world prominence (143-145). the attributes of modern technology, communication and international teamwork led to the rapid isolation and sequencing of the virus causing sars, identified as a novel coronavirus with little similarity to previously known human coronaviruses (147-153). final confirmation came with the demonstration that infection of monkeys with sars produced a syndrome identical to that seen in man and that virus could subsequently recovered (154). these experiments were rapidly followed by the development of diagnostic assays based on a real-time quantitative pcr method and a taqman protocol (148, 155). transmission of sars appears to be via person-person contact with infected droplets rather than airborne and, possibly, a fecal-oral route. inhibitors of coronaviruses are largely unknown although treatment with ribavirin (11) and oseltamivir have been examined in an empirical fashion (143, 156). scope of the mosquito-borne west nile virus outbreak in the us broadened considerably in the summer of 2003 extending to almost all states and causing over 2,500 cases of encephalitis and 230 deaths (157,158) . the virus presents several conventional proteins as targets suitable for intervention including the ns3 protein, which expresses serine protease, helicase and nucleoside triphosphatase activities, and the ns5 rna-dependent rna polymerase (159). moreover, screening using a cell culture assay provides a panoply of less well understood targets. however, few potent and selective wnv inhibitors have been described to date. ribavirin (ii) inhibits wnv rna production in oligodendroglial cells, a human neural cell line, with an ec50 of -60 fm (160). the nucleoside analogue hmc-ho4 (55) interferes with the helicase activity of wnv ns3 with an i& of 30 pm and is a similarly potent antiviral in cell culture (161). inhibitors of vaccinia virus (smallpox) -smallpox has aroused considerable concern with discussion prominently focussed on its potential as an agent of bioterrorism (162) (163) (164) (165) (166) (167) . many inhibitors of vaccinia virus replication interfere with host cell pathways and include 11 (inosine monophosphate dehydrogenase), the cyclopentenyl nucleoside analogue neplanocin a (s-adenosylhomocysteine hydrolase) and pyrazofurin (omp decarboxylase) (168, 169) . the @dine and 5-f cytidine analogues of neplanocin a are equally potent inhibitors of several orthopox viruses, including smallpox (170) ori-1001, which complements the el mrna start codon of hpv and has demonstrated efficacy in two animal models, is currently undergoing pi12 clinical evaluation. the fused tetracyclic amide .g is claimed to inhibit the e2-dependent binding of papilloma virus el to dna, a cntrcal step in viral replication, with an i&j of ~500 nm (175) . a vaccine derived from an hpv-16 virus-like particle, completely prevented the incidence of cervical neoplasias in hpv-16naite young women (176) . this impressive result provides strong encouragement to the development of a vaccine with a spectrum that, in addition, encompasses the broader range of hpvs (18, 31, 33 and 35) responsible for the majority of cervical cancers (177) . a combined, multivalent vaccine based on self-assembling virus-like particles and directed towards hpv-16 and hpv-18, recognized as medi-517, is entering p2 trials (178) . ----2'deoxynucleotides ";$~rn$~~ychem., 9, 899 (2002 145. nln (7~7) september section iv-cancer and infectious diseases plattner key: cord-275104-imqmyqhz authors: fioravanti, jessica; di lucia, pietro; magini, diletta; moalli, federica; boni, carolina; benechet, alexandre pierre; fumagalli, valeria; inverso, donato; vecchi, andrea; fiocchi, amleto; wieland, stefan; purcell, robert; ferrari, carlo; chisari, francis v.; guidotti, luca g.; iannacone, matteo title: effector cd8+ t cell-derived interleukin-10 enhances acute liver immunopathology date: 2017-09-30 journal: journal of hepatology doi: 10.1016/j.jhep.2017.04.020 sha: doc_id: 275104 cord_uid: imqmyqhz background & aims besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector cd8+ t cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (il)-10 and, therefore, contain immunopathology. whether the same occurs during acute hepatitis b virus (hbv) infection and role that il-10 might play in liver disease is currently unknown. methods mouse models of acute hbv pathogenesis, as well as chimpanzees and patients acutely infected with hbv, were used to analyse the role of cd8+ t cell-derived il-10 in liver immunopathology. results mouse hbv-specific effector cd8+ t cells produce significant amounts of il-10 upon in vivo antigen encounter. this is corroborated by longitudinal data in a chimpanzee acutely infected with hbv, where serum il-10 was readily detectable and correlated with intrahepatic cd8+ t cell infiltration and liver disease severity. unexpectedly, mouse and human cd8+ t cell-derived il-10 was found to act in an autocrine/paracrine fashion to enhance il-2 responsiveness, thus preventing antigen-induced hbv-specific effector cd8+ t cell apoptosis. accordingly, the use of mouse models of hbv pathogenesis revealed that the il-10 produced by effector cd8+ t cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. conclusion effector cd8+ t cell-derived il-10 enhances acute liver immunopathology. altogether, these results extend our understanding of the celland tissue-specific role that il-10 exerts in immune regulation. lay summary: interleukin-10 is mostly regarded as an immunosuppressive cytokine. we show here that hbv-specific cd8+ t cells produce il-10 upon antigen recognition and that this cytokine enhances cd8+ t cell survival. as such, il-10 paradoxically promotes rather than suppresses liver disease. it is widely recognized that hepatitis b virus (hbv) replicates non-cytopathically in the hepatocyte. most of the clinical complications related to this infection is reflected in the adaptive immune response, particularly the virus-specific effector cd8 + t cell response. [1] [2] [3] [4] [5] indeed, by killing infected cells and secreting antiviral cytokines, effector cd8 + t cells (cd8 t e ) reaching the infection sites are major contributors to viral clearance as well as tissue immunopathology. [1] [2] [3] [4] [5] paradoxically, during some acute viral infections (e.g. those caused by influenza virus, respiratory syncytial virus, coronavirus and vaccinia virus), cd8 t e have been shown to produce the regulatory cytokine interleukin (il)-10, [6] [7] [8] [9] [10] [11] in addition to pro-inflammatory cytokines, chemokines and effector molecules. cd8 t e -derived il-10 is generally thought to limit tissue damage, 6, 7, 11 however the net effect induced by this cytokine might depend on the pathophysiological context. 12 whether hbv-specific cd8 t e produce il-10 upon hepatocellular antigen (ag) recognition and the role that this cytokine plays in liver immunopathology are currently unknown. we show here that serum il-10 is readily detectable in an acutely hbv-infected adult chimpanzee in a manner that coincides with intrahepatic cd8 + t cell infiltration. using mouse models of acute hbv pathogenesis, we confirmed that il-10 is indeed produced by hbv-specific cd8 t e upon hepatocellular ag recognition in vivo. to our surprise, il-10 blockade ameliorated rather than worsened liver disease. ex vivo analyses of hbv-specific cd8 t e isolated from the livers of hbv replication-competent transgenic mice or from the blood of acutely infected patients revealed that il-10 acts in an autocrine/paracrine fashion to increase il-2 responsiveness and rescue cd8 t e from ag-induced apoptosis. chimpanzees chimpanzee a0a006 has already been described. 13 the animal was handled according to humane use and care guidelines specified by animal research committees at the national institutes of health, the scripps research institute, and bioqual laboratories. the chimpanzee was individually housed at bioqual laboratories (rockville, md), an american association for accreditation of laboratory animal care international-accredited institution under contract to the national institute of allergy and infectious diseases. chimpanzee a0a006 was inoculated with 10 10 genome equivalents of hbv obtained from an hbv-positive serum of chimpanzee 5835 that was previously inoculated with a monoclonal hbv isolate (genotype d, ayw subtype; genbank accession no. v01460) 25 contained in hbv transgenic mouse serum, 14 as described. blood was obtained by venipuncture and analyzed for serum il-10 (see below). mice c57bl/6, cd45.1 (inbred c57bl/6), and balb/c mice were purchased from charles river. il-10 à/à mice (b6.129p2-il10 tm1cgn /j) were purchased from the jackson laboratory. hbv replication-competent transgenic mice (lineage 1.3.32, inbred c57bl/6, h-2 b ), that express all of the hbv antigens and replicate hbv in the liver at high levels without any evidence of cytopathology, were previously described. 13, 14 in indicated experiments, these mice were used as c57bl/6 x balb/c h-2 bxd f1 hybrids. hbv nucleocapsid (cor)-specific (referred to as cor93 cells) t cell receptor (tcr) transgenic mice (lineage bc10.3, inbred cd45.1), in which >98% of the splenic cd8 + t cells recognize a k b -restricted epitope located between residues 93-100 in the hbv core protein (mglkfrql), were previously described. 17 env28 (envelope) tcr transgenic mice (lineage 6c2.36, inbred balb/ c), in which $83% of the splenic cd8 + t cells recognize a l d -restricted epitope located between residues 28-39 of hbsag (ipqsldswwtsl), were previously described. 17 in indicated experiments, lineage bc10.3 or lineage 1.3.32 were crossed with il-10 à/à mice. mice were housed under specific pathogen-free conditions and used at 8-10 weeks of age. in all experiments, mice were matched for age, sex and (for the 1.3.32 animals) serum hbeag levels before experimental manipulations. all experimental animal procedures were approved by the institutional animal committee of san raffaele scientific institute. five patients with acute self-limited hbv infection were enrolled at the unit of infectious diseases and hepatology in parma, italy. patients had clinical, biochemical, and virological evidence of acute hbv infection (aminotransferase levels at least 10 times the upper normal limit and detection of hbsag and igm anti-hbcag ab in the serum). patients were negative for anti-hcv, anti-delta virus, anti-hiv-1 and anti-hiv-2 ab and for other markers of viral or autoimmune hepatitis. t cell response was tested one month from the time of acute illness. the study was approved by the ethical committee of the azienda ospedaliero-universitaria of parma, and all subjects gave written informed consent. in vitro generation of cd8 t e was performed as described. 16, 26 briefly, splenocytes from cor93 or env28 tcr transgenic mice were incubated with 10 lg/ml of cor93-100 (k b ; mglkfrql) or env28-39 (l d ; ipqsldswwtsl) peptides (primm), respectively, at 37°c for 1 h, washed, and cultured in complete rpmi 1640 (10% fbs, 2 mm l-glutamine, 50 lm 2-mercaptoethanol, hepes 10 mm, non essential amino acid 100 lm and penicillin plus streptomycin). two days later, cells were cultured in fresh medium supplemented with 2.5% el-4 supernatant. media sup-plemented with cytokines were replaced every 2 days. after 8 or 9 days of culture, cells were tested for the expression of cd8, cd69, cd25, cd44, cd62l, ccr7, ifnc and granzyme b by facs prior to subsequent use, as described. 16 10 7 cells of each cell type were injected intravenously into recipient animals. total rna was isolated from cultured cells or frozen livers (left lobe) with relia-prep rna miniprep system (promega) following the manufacturer's instructions. for quantitative rt-pcr, 1 lg of total rna was reverse transcribed prior to qpcr analysis for mouse il10 and ifng (taqman mm01288386 and mm01168134 probes, applied biosystems) in an abi 7900ht fast real-time pcr system (applied biosystems). all experiments were performed in triplicate and normalized to the reference gene gapdh. enzyme-linked immunosorbent assays il-10 and ifn-c in mouse sera or cell supernatants were measured by elisa (biolegend and r&d, respectively), according to the manufacturer's instructions. hbeag in mouse sera was measured by elisa (diapro) following the manufacturer's instructions. il-10 in chimpanzee sera was measured using a human il-10 elisa (r&d systems) according to the manufacturer's instructions. the extent of hepatocellular injury was monitored by measuring serum alanine aminotransferase (alt) activity at multiple time points after treatment, as previously described. 16 in vivo il-10r-specific antibody treatment primary hepatocytes were isolated from wild-type or il-10 à/à hbv replicationcompetent transgenic mice (inbred c57bl/6) exactly as described. 16 hepatocyte purity (assessed by flow cytometry-based parameters of size) and viability (assessed by light microscopy-based morphology and trypan blue dye exclusion) were routinely greater than 70% and 80%, respectively. hepatocytes (10 6 cells/ml) were incubated at a 1:2 ratio with cor93 cd8 t e for 4 h in the presence of 10 lg/ml brefeldin a (bfa, sigma) prior to intracellular ifn-c staining. single-cell suspensions of livers were generated as described. 18 for analysis of ex vivo intracellular cytokine production, cell suspensions of livers were obtained as described above except that 1 lg/ml of bfa (sigma) was included in the digestion buffer. all flow cytometry stainings of surface-expressed and intracellular molecules were performed as described. 27 antibodies (abs) used included pb-and pe-conjugated anti-cd8a (53-6.7), alexa fluor 488-, percp-, and apc-cy7-conjugated anti-cd45.1 (a20), alexa fluor 488-, and alexa fluor 647-conjugated anti-ifn-c (xmg1.2), pe-and pb-conjugated anti-cd25 (pc61), apc-conjugated annexin v (eos9.1), 7aad (bd pharmingen). all abs were purchased from biolegend, unless otherwise indicated. for phosphorylated stat5 analysis, cells were fixed with 4% paraformaldehyde, permeabilized with absolute methanol and stained with pe-cy7-conjugated anti-phosphostat5 (47/stat5py694, ebioscience). all flow cytometry analyses were performed in facs buffer containing pbs with 2 mm edta and 2% fbs on a facs canto (bd pharmingen) and analyzed with flowjo software (treestar). for haemotoxylin and eosin staining, livers were perfused with pbs, harvested in zn-formalin and transferred into 70% ethanol 24 h later. tissue was then processed, embedded in paraffin and stained as previously described. 16 bright-field images were acquired through an aperio scanscope system cs2 microscope and an imagescope program (leica biosystem) following the manufacturer's instructions. the extent of hepatocellular injury was monitored by histopathological and quantitative morphometric analyses as described. 18 the number of injured hepatocytes (identified as either apoptotic or necrotic based on standard cytopathological criteria) and intrahepatic inflammatory cells (mononuclear and polymorphonuclear) were counted in at least 50 high power fields of liver tissue (corresponding to about 2 mm 2 ). results are expressed as number of cells per mm 2 . in vitro cell culture assays with murine cd8 t e to test the expression and production of il-10 and ifn-c by cor93 t e , cells were incubated in complete rpmi 1640 media with 2 lg/ml of rhil-2 (roche) at 5â10 6 cells/ml for 4 h at 37°c in the presence or absence of the cor93 peptide. to assess the effect of il-10 on ag-induced apoptosis, cor93 cd8 t e (10 7 cells/ ml) were incubated for 1 h at 37°c with 18 lg/ml of anti-il-10r ab (bioxcell), 400 ng/ml of recombinant mil-10 (biolegend) or left untreated prior to the addition of cor93 peptide (1 mg/ml) and human il-2 (1 mg/ml). for the assessment of cell viability, cells were harvested 8 h later and stained with annexin v and 7aad, as described above. for the assessment of cd25 expression, cells were harvested 24 h after peptide stimulation. for stat5 phosphorylation, cor93 cd8 t e were cultured overnight in serum-free rpmi complete medium (gibco, life technologies), prior to treating them as described above. cells were harvested 15 min after peptide stimulation. to measure the effect of il-10 on ag-induced apoptosis, peripheral blood mononuclear cells (pbmc) from five hla-a201 + patients with acute self-limited hepatitis b (concentration of 2â10 6 /ml) were incubated for 1 h at 37°c with 20 lg/ml of anti-il-10r ab (bd pharmingen), 200 ng/ml of recombinant human il-10 (biolegend) or left untreated prior to the addition of core 18-27 peptide (1 lg/ml) and human il-2 (100 iu/ml). after a 5 h incubation, cells were extensively washed, stained with core 18-27 dextramer, anti-cd8 and anti-cd3 mouse abs for 15 min in the dark, then stained with annexin v and 7aad (bd pharmingen), according to the annexin v staining protocol (bd pharmingen). the cells were acquired immediately on a facscanto ii multicolor flow cytometer and were analyzed with the diva software (bd biosciences, immunocytometry systems, ca, usa). total dna was isolated from frozen livers (left lobe) for southern blot analysis, as previously described. 18 results are expressed as mean + sem. all statistical analyses were performed in prism 5 (graphpad software). means between two groups were compared with two-tailed t test. means among three or more groups were compared with oneway or two-way analysis of variance (anova) with bonferroni's post-hoc test. some data were analyzed using fisher's least significant difference (lsd) test we first assessed whether il-10 is produced upon acute hbv infection. we longitudinally analyzed the sera of a chimpanzee (a0a006) that had been inoculated with a monoclonal hbv inoculum of 10 10 ge of hbv dna, as described. 13 serum il-10 was not detectable until cd8 + t cells accumulated in the liver and its appearance coincided with the onset of a necroinflammatory liver disease (fig. 1a) , suggesting that this cytokine might have been produced by virus-specific cd8 t e upon hepatocellular ag recognition. to test this hypothesis and to assess the role of il-10 in liver immunopathology, we employed a well-established model of acute hbv pathogenesis, i.e. the adoptive transfer of hbv-specific cd8 + t e into hbv replication-competent transgenic mice. 14-16 naïve cd8 + t cells from hbv nucleocapsid (cor)specific tcr transgenic mice 17 (referred to as cor93 cells) were differentiated in vitro into bona fide cd8 t e . upon intravenous injection of 10 7 cor93 cd8 t e into hbv replication-competent transgenic mice, the hepatic mrna expression of the il10 gene increased sharply, reaching peak levels at 4-8 h after t cell transfer and mirroring the kinetics of the prototypical cd8 t e -derived pro-inflammatory and antiviral cytokine gene ifng (fig. 1b) . accordingly, il-10 was also detected in the serum of these mice between 4 and 8 h after t cell transfer, again echoing the ifn-c kinetics (fig. 1c) . consistent with the hypothesis that il-10 is produced by cd8 t e upon hepatocellular antigen recognition, we found that cor93 t e cells stimulated for 4 h in vitro with the cognate cor93 peptide produced both cytokines (fig. 1d-g; fig. s1 ). finally, to unambiguously identify the cellular source of the il-10 detected in the liver of hbv replication-competent transgenic mice upon adoptive transfer of cor93 cd8 t e , we genetically deleted the il10 gene in hbv replication-competent recipient mice, in cor93 cd8 t e or in both. by using this approach, we could demonstrate that the transferred cd8 t e cells are the unique source of il-10 in this experimental setting, as il10 expression was only detected when the transferred t cells were il10-competent, regardless of the recipient genotype (fig. 1h) . to gain insight into the role of cd8 t e -derived il-10 expression in liver immunopathology, we selectively blocked il-10 receptor signaling by injecting hbv replication-competent transgenic mice with an anti-il-10ra ab 2 h prior to cor93 t e transfer. to our surprise, we found that il-10 blockade decreased, rather than increased, liver damage by about 3-fold at the peak of the disease ( fig. 2a) . of note, anti-il-10ra ab injection did not affect the total number of circulating cor93 t e cells (data not shown), indicating that this treatment did not deplete the transferred t cells. also, anti-il-10ra ab treatment significantly reduced liver disease when hbv envelope-specific tcr transgenic 16, 17 or polyclonal cd8 t e 18 were injected into hbv replication-competent transgenic mice instead of cor93 t e (data not shown). moreover, consistent with il-10 being produced exclusively by the transferred cd8 t e in this model, deletion of the il10 gene in hbv replication-competent recipient mice -which did not alter the ag presentation capacity of hepatocytes (fig. s2 ) -did not affect the severity of liver disease induced by cor93 t e transfer (fig. 2b) . to explore the mechanisms underlying this il-10-mediated increase in liver immunopathology, we quantified the number, function and viability of intrahepatic cor93 cd8 t e 2, 8, and 24 h after adoptive transfer into hbv replication-competent transgenic mice that were or were not subjected to anti-il-10ra ab treatment. il-10r blockade significantly decreased the number of total and ifn-c + intrahepatic cor93 cd8 t e cells recovered 24 h after injection ( fig. 2c and d) . we next addressed if this reflected an increase in cell death. indeed, the percentage of intrahepatic cor93 cd8 t e that underwent apoptosis at 8 and 24 h after transfer was significantly increased by il-10r blockade ( fig. 2e; fig. s3 ), and this was reflected by a lower disease severity ( fig. 2f and g) . although the number of intrahepatic cor93 t e was reduced upon il-10r blockade, these cells probably produced enough ifn-c to abolish viral replication (fig. s4 ). to explore if cd8 t e -derived il-10 directly decreased ag-induced apoptosis, we exposed purified cor93 cd8 t e to ag in the presence or absence of il-10r blockade or exogenous il-10. as shown in fig. 3a , ag exposure for 8 h triggered cor93 cd8 t e apoptosis. notably, il-10r blockade increased the percentage of cor93 cd8 t e that became apoptotic, whereas the addition of exogenous recombinant il-10 decreased cor93 cd8 t e apoptosis ( fig. 3a and fig. s5a) . we next set out to determine the mechanism whereby il-10 prevents cd8 t e from dying. since il-10 has been proposed to enhance the growth of activated cd8 + t cells in the presence of il-2, [19] [20] [21] we reasoned that cd8 t e -derived il-10 might increase il-2 responsiveness by, for instance, modulating il-2 receptor expression. indeed, ag-induced il-2ra (cd25) upregulation on cd8 t e was lower upon il-10r blockade, whereas the addition of exogenous recombinant il-10 increased cd25 upregulation (fig. 3b and fig. s5b ). accordingly, blocking il-10r signaling decreased the capacity of cd8 t e to respond to il-2 (as assessed by stat5 phosphorylation), and il-10 treatment increased il-2 sensitivity ( fig. 3c; fig. s5c ). finally, we explored whether the capacity of cd8 t e -derived il-10 to act in an autocrine/paracrine fashion to rescue cd8 t e from ag-triggered apoptosis was restricted to murine cd8 t e or it extended to hbv-specific cd8 t e isolated from acutely infected patients. to this end, pbmcs from 5 hla-a201 + patients with acute hepatitis b, in whom hbv-specific cd8 + t cells can be specifically visualized by hbv-specific dextramers 22 (fig. s6 ), were stimulated with the cognate cor18 peptide in the presence or absence of anti-il-10ra abs or exogenous il-10. the results mirrored those obtained with murine cd8 t e , in that il-10r blockade increased ag-induced apoptosis, whereas the addition of recombinant il-10 partially rescued cd8 t e from cell death (fig. 3d ). in conclusion, our results indicate that cd8 t e -derived il-10 acts in an autocrine/paracrine fashion to increase il-2 responsiveness, rescuing cd8 t e from ag-induced apoptosis. although the net contribution that il-10 plays during a natural hbv infection -where this cytokine may be produced by additional cell types 23,24 -remains to be determined, the results described herein suggest that il-10 may promote rather than suppress liver immunopathology. with medium (white), anti-il-10ra (20 lg/ml, light blue), or ril-10 (200 ng/ml, dark blue). after 1 h cor18-27 peptide (1 lg/ml) was added (+cor18) or not (ctrl). the percentage of annexin v+ (apoptotic) cells was determined on core 18-27 dextramer + cells 5 h later. results are representative of 2 independent experiments. results are expressed as mean + sem. *p .05, **p .01, ***p .001. means between two groups were compared with two-tailed t test. means among three or more groups were compared with one-way or two-way analysis of variance with bonferroni's post-hoc test. data in fig. 3d were analyzed using fisher's least significant difference (lsd) test. european molecular biology organization (embo) young investigator program (to m.i.), embo long-term fellowships altf 694-2016 (to a.b.) and altf 1018-2016 (to d.i.), a marie curie intra-european fellowship (ief) for career development 327336 immunobiology and pathogenesis of viral hepatitis mouse models of hepatitis b virus pathogenesis immune outcomes in the liver: is cd8 t cell fate determined by the environment? cd8(+) t cells mediate viral clearance and disease pathogenesis during acute hepatitis b virus infection cd8(+) t cell control of hepatitis b virus replication: direct comparison between cytolytic and noncytolytic functions effector t cells control lung inflammation during acute influenza virus infection by producing il-10 autocrine regulation of pulmonary inflammation by effector t-cell derived il-10 during infection with respiratory syncytial virus shifting hierarchies of interleukin-10-producing t cell populations in the central nervous system during acute and persistent viral encephalomyelitis locally produced il-10 limits cutaneous vaccinia virus spread cd8+ treg cells suppress cd8 + t cell-responses by il-10-dependent mechanism during h5n1 influenza virus infection highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirus-induced encephalitis hepatoprotective and anti-inflammatory cytokines in alcoholic liver disease the size of the viral inoculum contributes to the outcome of hepatitis b virus infection high-level hepatitis b virus replication in transgenic mice intracellular inactivation of the hepatitis b virus by cytotoxic t lymphocytes immunosurveillance of the liver by intravascular effector cd8(+) t cells cd40 activation rescues antiviral cd8 + t cells from pd-1-mediated exhaustion platelets mediate cytotoxic t lymphocyte-induced liver damage il-10: a novel cytotoxic t cell differentiation factor inhibitory and stimulatory effects of il-10 on human cd8+ t cells the light and the dark sides of interleukin-10 in immune-mediated diseases and cancer characterization of hepatitis b virus (hbv)-specific t-cell dysfunction in chronic hbv infection interleukin-10 and the interleukin-10 receptor the regulation of il-10 expression nucleotide sequence of the hepatitis b virus genome (subtype ayw) cloned in e. coli effector differentiation is not prerequisite for generation of memory cytotoxic t lymphocytes bisphosphonates target b cells to enhance humoral immune responses we thank l. giustini, m. mainetti and m. raso for technical support; m. silva for secretarial assistance and the members of the iannacone laboratory for helpful discussions. flow cytometry was carried out at fractal, a flow cytometry resource and advanced cytometry technical applications laboratory established by the san raffaele scientific institute. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhep.2017.04. 020. the authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.please refer to the accompanying icmje disclosure forms for further details. key: cord-300642-c7adeis1 authors: lai, andrew sh; lai, kar neng title: viral nephropathy date: 2006 journal: nat clin pract nephrol doi: 10.1038/ncpneph0166 sha: doc_id: 300642 cord_uid: c7adeis1 viral infections can cause many glomerular diseases. the diagnostic criteria for virus-related nephropathy include detailed clinical and laboratory data, and tissue molecular analysis. several mechanisms are involved in the pathogenesis of virus-related nephropathy, including tropism of the virus in the kidney, induction of abnormal immune complexes, direct cytopathogenic effects, and multiorgan failure. hepatitis b virus is associated with membranous nephropathy and mesangiocapillary glomerulonephritis in endemic areas. hepatitis c virus causes various forms of glomerulonephritis, including cryoglobulinemia-mediated glomerulonephritis. infection with hiv is associated with a collapsing focal segmental glomerulosclerosis, a distinctive disease that affects mainly africans and african americans. in the course of hiv infection, other types of immune complex glomerulonephritis can occur, most frequently in whites. recent reports indicate a role for parvovirus b19 in 'idiopathic' collapsing focal segmental glomerulosclerosis. both hantaviruses, and coronaviruses associated with severe acute respiratory syndrome, can lead to acute renal failure. renal biopsy followed by appropriate serological and molecular testing is essential for defining virus-related glomerular lesions and guiding prognostic and therapeutic evaluation. the pathogenetic links between viral infection and renal disease are often difficult to establish. the criteria for proving causality are complex and include (besides recognition of the clinical syndrome) serological diagnosis, identification of specific viral antigenemia, and detection in glomerular structures of viral antigens and host antibodies. according to koch's postulates, the etiological link should be confirmed by complete cure following eradication of the virus, but this is not always possible. the concentration of viral antigens is higher in tissue than in the circulation, where antigens are complexed with specific autoantibodies. virological and molecular analy sis of pathologic tissues by in situ hybridiza tion, polymerase chain reaction and ultra structural analysis led to successful detection and identifica tion of the virus. it should be noted that tubular uptake of viral particles is common and does not necessarily establish an etiological link with renal disease. improvement of the renal disease concomitant with clearance of the suspected antigen, or recurrence of glomerulonephritis following reinfection, are additional clinical criteria. different mechanisms are operative in different viral nephropathies (box 1). in acute glomerulonephritis, direct viral infection of the glomerulus induces proliferative changes following release of cytokines. 1 the nephropathy is reversible in most cases if the virus is rapidly cleared. in chronic forms of glomerulonephritis, persistent viral infection provides continuous antigenic stimulation, resulting in antibody production and formation of immune complexes. studies indicate a role in the disease pathogenesis for these immune complexes, which can be derived from the circulation or formed in situ. 2,3 viral proteins cause inflammatory renal diseases via synthesis of various mediators that can cause sclerosis and worsen glomerulopathy. 4, 5 viral infections can cause many glomerular diseases. the diagnostic criteria for virus-related nephropathy include detailed clinical and laboratory data, and tissue molecular analysis. several mechanisms are involved in the pathogenesis of virus-related nephropathy, including tropism of the virus in the kidney, induction of abnormal immune complexes, direct cytopathogenic effects, and multiorgan failure. hepatitis b virus is associated with membranous nephropathy and mesangiocapillary glomerulonephritis in endemic areas. hepatitis c virus causes various forms of glomerulonephritis, including cryoglobulinemia-mediated glomerulonephritis. infection with hiv is associated with a collapsing focal segmental glomerulosclerosis, a distinctive disease that affects mainly africans and african americans. in the course of hiv infection, other types of immune complex glomerulonephritis can occur, most frequently in whites. recent reports indicate a role for parvovirus b19 in 'idiopathic' collapsing focal segmental glomerulosclerosis. both hantaviruses, and coronaviruses associated with severe acute respiratory syndrome, can lead to acute renal failure. renal biopsy followed by appropriate serological and molecular testing is essential for defining virus-related glomerular lesions and guiding prognostic and therapeutic evaluation. a direct cytopathic effect of viral proteins has also been postulated. 6 in hepatitis c virus (hcv)-induced mesangiocapillary glomerulonephritis (mcgn), production of circulating cryo globulins is induced as an abnormal host response to infection. cryoglobulins are either type ii or type iii. at least two classes of immuno globulins are involved, one of which is polyclonal. 7 in acute renal failure associated with infection by hantavirus or severe acute respiratory syndrome coronavirus, the pathogenetic mechanisms of interstitial nephritis, disseminated intravascular coagulopathy, and multiorgan failure-rather than formation of immune complexes-are predominant. box 2 lists the viruses that are known to induce renal diseases. globally, the most frequent and well recognized virus-related glomerulonephropathies are those associated with hepatitis b virus (hbv), in which formation of immune complexes is important. hcv is the etiological agent of cryoglobulinemiarelated mcgn in most cases. infection with hiv can induce a broad spectrum of glomerular lesions via multiple pathogenic mechanisms. parvovirus b19 (pvb19) is associated with non-hiv collapsing glomerulopathy, 8 idiopathic focal segmental glomerulosclerosis (fsgs), 9 and immune complex glomerulonephritis. 10 polyoma bk virus and hantavirus most frequently cause tubulointerstitial damage; occasionally, virus is simultaneously localized to the glomerulus. a rare or speculative role in glomerulonephritides is currently attributed to other viruses, such as those causing yellow fever, 11 mumps, 12 measles, 13 varicella, 14 and herpes. 15 hbv is a hepatotropic, double-stranded dna virus of the hepadnaviridae family. hbv itself is not cytopathic; hepatitis develops as a result of the host's immune reaction to infected hepatocytes. hbv uses reverse transcriptase to transcribe rna into dna. unlike retroviruses, however, hbv dna is not integrated into host cell dna during replication. after an hbv particle binds to and enters a hepatocyte, hbv dna enters the cell's nucleus and is converted into covalently closed circular dna. this highly stable genetic material acts as the intermediate template for transcription of rna copies. this pregenomic messenger rna is transported to the cytoplasm. it has dual functions: acting as a template for synthesis of new hbv dna, and carrying genetic information to direct synthesis of viral proteins. today, an estimated 350-400 million people worldwide are infected with hbv. in endemic areas, transmission is usually vertical-that is, from infected mother to child. horizontal transmission occurs via direct contact with blood (e.g. during blood transfusions) or mucous membranes (e.g. during sexual contact), or via the percutaneous route upon contact with blood or body fluids (e.g. during intravenous drug use and needle sharing). familial clustering of the virus occurs in some regions. the reported prevalence of hbv-associated nephropathy closely parallels the geographic patterns of prevalence of hbv. 16 frequently reported in asian populations 17 and in children, 18 particularly male children. 16 by contrast, mesangial proliferative forms with iga deposits seem to be most common in adults. 19 three types of glomerulonephritis with pathologic characteristics similar to the human subtypes have been described in woodchucks chronically infected with hepatitis virus. 20 as in humans, the membranous pattern of injury most frequently affects young woodchucks, whereas the mesangial proliferative pattern of injury tends to affect older animals. the male : female ratio of affected woodchucks was significantly greater than that of the chronic carrier population. 20 in most reports, diagnosis of hbv-asso ciated glomerulonephritis has been based on persistence of circulating hbv or hbv dna, absence of other causative agents, and presence of hbvspecific antigen(s) or viral genome in the glomerulus. one major difference between the human and woodchuck studies is that the hepatitis b e antigen (hbeag) system has not been characterized in the latter. in clinical practice, regression of pathology following viral eradication is not easy to demonstrate because of ethical concerns relating to repeat renal biopsies in humans subsequent to clinical remission. as such, the diagnosis of hbv-associated renal disease usually relies heavily upon detection of hbv-specific antigen(s) in glomeruli. laboratory testing for diagnosis and assessment of response to treatment should include standard liver biochemistries (serum alanine aminotransferase, γ-glutamyltransferase, and bilirubin levels), and hbv serologies (hepatitis b surface antigen, hbeag, anti-hepatitis b e, and anti-hepatitis b core antigen antibodies). hbeag is present in 80% of patients, who might also have high titers of anti-hepatitis b core antigen. 21 subjects with biochemical hepatitis should be tested for circulating hbv dna 22 and undergo liver biopsy. an α-fetoprotein assay could be an important adjunct. 23 serum c3 and c4 levels can be low in 20-50% of patients. hbv-related membranous nephropathy tends to manifest slightly differently in pediatric and adult patients. in children, there is a strong male preponderance, and the most frequent presentation is nephrotic syndrome, microscopic hematuria, and normal or mildly impaired renal function. 24 pediatric chronic hbv carriers often do not have overt liver disease, and transaminase levels are usually normal. in adults, proteinuria or the nephrotic syndrome are the most common manifestations. adult male predominance is less obvious than in pediatric populations. adults are more likely than children to have hypertension, renal dysfunction, and clinical evidence of liver disease. the prognosis of hbv-associated membranous nephropathy in children is favorable. stable renal function and high rates of spontaneous remission have been reported in several geographical areas in which disease prevalence is high. by contrast, adults with hbvassociated membranous nephro pathy typically develop progressive disease. in hong kong, up to 29% of patients had progressive renal failure, and another 10% developed terminal uremia over 5 years. 21 the prognosis is even worse for patients with nephrotic-range proteinuria and abnormal liver function tests at presentation. over 50% of these patients require renal replacement therapy within 3 years. 25 vertical transmission is associated with poorer outcomes than horizontal transmission, as is endemic versus sporadic infection. 21,26 box 2 viral infections that cause nephropathy. as mentioned above, hbv infection can also cause mcgn (with or without cryoglobulinemia), mesangial proliferative glomerulonephritis, and igan. 19,27 polyarteritis nodosa has been reported in some patients with hbv and might respond to treatment with corticosteroids and interferon-α. 28 occasional concomitance of the pathologic subtypes can lead to 'double' glomerulopathies. for instance, membranous nephropathy and igan have been reported to coexist in an hbv carrier. 29 unlike affected children, who have a high rate of spontaneous remission, 30 adults with hbvassociated membranous nephropathy typically develop progressive disease. 21 various management strategies have been tried, but an ideal agent is yet to be found. treatment for hbvassociated renal disease should ideally achieve the following objectives: (i) amelioration of nephrotic syndrome and its complications; (ii) preservation of renal function; (iii) normalization of liver function and prevention of hepatic complications of hbv; and (iv) permanent eradication of hbv. because of the involvement of immune complexes in the disease, immunosuppressive therapy-similar to that used in the idiopathic form of the disease-was once fashionable. corticosteroids were reported to provide symptomatic relief in isolated cases. the contemporary view, however, is that steroid and cytotoxic agents can cause deleterious hepatic flares or even fatal decompensation by enhancing viral replication when the drugs are withdrawn. 31 another approach is treatment with an antiviral agent. interferon-α is a naturally occurring cytokine produced by b lymphocytes, null lymphocytes, and macrophages that exerts antiviral, antiproliferative and immuno modulatory effects. while reportedly useful in children, 32 interferon-α has produced mixed results in adults with hbv-associated membranous nephropathy. 21, 26 introduction of the nucleoside analog lamivudine has revolutionized the treatment of chronic hbv infection. 33 lamivudine is the (-)-enantiomer of 3'-thiacytidine. this analog inhibits dna synthesis by terminating the nascent proviral dna chain through inter ference with the reverse transcriptase activity of hbv. in children and adults with hbv-asso ciated membranous nephropathy, lamivudine has been anecdotally reported to induce remission of nephrotic syndrome and to suppress viral replication. 24, 34 in a recent analysis comparing 10 adult nephrotic patients with hbv-related membranous nephropathy who received lamivudine with 12 matched historical control subjects who presented in the pre-lamivudine era, lamivudine significantly improved proteinuria, aminotransferase levels, and renal outcome over a 3-year period. 25 randomized studies in a larger cohort of patients are needed to prove this effect. a potential limitation of prolonged treatment with lamivudine is emergence of drugresistant virus strains resulting from induction and selection of hbv variants with mutations at the tyrosine-methionine-aspartate-aspartate (ymdd) motif of dna polymerase. one agent that might be useful in lamivudine-resistant cases is adefovir dipivoxil, an acyclic nucleotide analog that is effective against both lamivudineresistant hbv mutants and wild-type hbv. 35 this agent does have nephrotoxic potential, and there are no clinical data on its efficacy in hbvrelated membranous nephro pathy that does not respond to lamivudine treatment. data do indicate, however, that the recommended dose of 10 mg adefovir dipivoxil is associated with a relatively low risk of nephro toxicity. 36 while awaiting an ideal agent for treatment of hbv-associated glomerulopathy, active immuniza tion remains the most effective means of immunoprophylaxis. 37 in taiwan, active immunization of all newborns since 1984 has led to a dramatic (10-fold) decline in the incidence of neonatal hbv infection and its sequelae. 38 in the us, universal vaccination of infants began in 1991, and a 67% reduction in hbv infection was recorded 10 years later. in 2003, the who recommended that all countries establish universal hbv immunization programs for infants and adolescents. 39 hcv is a small rna virus in the flaviviridae family. evolution of hcv has been characterized by the emergence of six major genotypes (based on sequence homology) and more than 50 subtypes. to date, around 170-200 million individuals worldwide are estimated by the who to be chronically infected with hcv. although viral replication is primarily confined to the liver, a variety of extrahepatic disease manifestations are associated with hcv infection. the principal renal manifestation of hcv infection is mcgn type i, usually in the context of type ii (mixed) cryoglobulinemia. 40 the prevalence of mcgn in hcv type ii cryoglobulinemia is approximately 30%. mcgn is occasionally observed in patients with hepatitis c in the absence of cryoglobulinemia. 40 type ii mcgn (e.g. dense deposit disease) has not been described in association with hcv infection. two immunologic features of hcv might underlie predisposition to extrahepatic disease manifestations. first, hcv is known to evade immune elimination, leading to chronic infection and accumulation of circulating immune complexes. mcgn associated with hcv infection might result from this phenomenon. second, hcv stimulates production of monoclonal rheumatoid factors. this feature causes type ii cryoglobulinemia, which accounts for most symptomatic cryoglobulinemic vasculitis. although this manifestation occurs relatively infrequently, as do all the extrahepatic disease manifestations, it accounts for much of the increased morbidity and mortality that accompanies the disease. between 35% and 90% of hcv-infected patients have been reported to have mixed cryoglobulinemia. 41, 42 prevalence increases with duration of hepatitis. it should be noted, however, that the prevalence of mixed cryo globulinemia has not been determined in popula tions of unselected hcv-infected patients. frank symptomatic cryoglobulinemia affects 1% or less of patients and is usually associated with high levels of rheumatoid factor and cryo globulins. testing of unselected patients with cryo globulinemia has shown that up to 90% have anti-hcv antibody. type i mcgn has long been regarded as idiopathic, but a consider able proportion of patients has concomitant chronic hcv infection. the exact proportion of patients with type i mcgn who are anti-hcv-antibody-positive is unknown. the true prevalence of mcgn without detectable cryoglobulinemia is difficult to assess. such cases might represent a subclinical form of cryoglobulinemia in which circulating cryoglobulins have not been detected by standard laboratory techniques. further, production of igm antibodies with anti-igg activity might induce formation of immune complexes that lack cryoprecipitable properties. 43 finally, these patients might develop detectable circulating cryoglobulinemia only later in the course of the disease. 44 laboratory testing coupled with renal biopsy establishes the diagnosis of hcv-related mcgn. most patients will have anti-hcv antibody, as well as hcv rna, in serum. serum transaminase levels are elevated in 70% of patients. cryoglobulins are detected in 50-70% of patients. serum electrophoresis and immunofixation detects type ii (mixed) cryo globulins. monoclonal rheumatoid factor, almost in variably an igmκ, is a distinguishing feature of cryo globulinemic glomerulonephritis. the amount of cryoglobulins, usually measured as a cryocrit, varies between patients and with time in a given patient (range 2-70%). κ light chains are also commonly present in the urine. the serum complement pattern, which does not change greatly with clinical disease activity, is also discriminative. characteristically, early complement components (c4 and c1q) and ch50 are present at very low or undetectable levels in these patients, whereas the c3 level tends to remain normal or is only slightly depressed. renal disease associated with hcv is rare in children. the typical age of disease onset is the fifth or sixth decade of life after longstanding infection, often in association with mild subclinical liver disease. patients might have other symptoms of cryoglobulinemia, such as palpable purpura and arthralgias. renal manifestations include nephrotic (20%) or non-nephrotic proteinuria and microscopic hematuria. 45 acute nephritic syndrome is the presenting feature in about a quarter of cases. renal insufficiency, frequently of mild severity, occurs in about half of patients. over 80% of patients have refractory hypertension at presentation, which might be responsible for a considerable number of cardiovascular deaths. the clinical course of hcv-associated renal disease can vary dramatically. this disease does not frequently progress to uremia, despite the persistence of urinary abnormalities in the majority of patients. when such progression does occur, it tends to be in males and those of older age. according to an italian series, around 15% of patients eventually require dialysis. 46 other forms of glomerular injury, including membranous nephropathy, fsgs, mesangial proliferative glomerulonephritis, and crescentic glomerulonephritis, have been reported in hcv carriers as individual case reports and small series. notably, membranous nephropathy in hcv carriers is characterized by the absence of cryoglobulin and male predominance. 47 in general, therapy can be directed at two levels: removal of cryoglobulins by plasmapheresis; and inhibition of cryoglobulin synthesis by attenuating immune responses (using steroids or cytotoxic agents) or suppressing viral replication (using interferon and ribavirin). before the association between hcv and cryo globulinemic mcgn was established, steroids and cyclophosphamide were the mainstays of treatment. our awareness of this link has facilitated a more rational approach to management of this condition. controlled trials have shown that antiviral therapy with interferon-α is asso ciated with improvements in systemic symptoms of immune complex disease. unfortunately, posttherapy relapse occurs in a large pro portion of patients, particularly when interferon monotherapy is administered in short courses. introduction of combination therapy with interferon-α2b plus ribavirin was an important milestone in the treatment of chronic hepatitis c. 48 this cocktail has also produced favorable results in mixed cryo globulinemia, although non-responses and relapses after initial improvements still occur. 49 the introduction of pegylated forms of interferon (peginterferon) in 2000 was another breakthrough in treatment of chronic hepatitis c. recent data on peginterferon and ribavirin combination therapy are encouraging. 50 an increased rate of treatment failure in carriers of hcv genotype 1 has been recognized. 51 observational studies support the effectiveness of peginterferon and ribavirin combination therapy in hcv-associated cryoglobulinemic mcgn. one therapeutic drawback is the hemolytic effect that complicates ribavirin therapy, particularly in patients with functional renal impairment. this difficulty has been overcome by adjusting the dose according to glomerular filtration rate instead of body weight alone, and utilizing recombinant erythropoietin to combat anemia. post-treatment renal biopsy showed histological improvement in two of three patients who received combination therapy for 12 months. 52 the wide spectrum of glomerulopathies occurring in the course of hiv infection can be classified into four groups. the first group is the classical hiv-associated nephropathy (hivan), a distinct entity with histological features of fsgs with tuft collapse or, more rarely, mesangial hyperplasia. hivan seems to be related to a direct effect of hiv or viral proteins on renal epithelium. 53 the second group is a diffuse proliferative-mesangiocapillary or lupuslike glomerulonephritis, with predominantly mesangial immune deposits, also known as hiv immune-complex-mediated disease. 54 this group also includes other immune-complex-mediated glomerulonephritides with more-heterogeneous histological features. the third group (which includes immunotactoid glomerulonephritis) is heterogeneous with regard to glomerular lesions and pathogenic mechanisms, some of which are still undefined. the true role of hiv infection in glomerulopathies of this type is also uncertain. the final group includes hiv-associated thrombotic microangiopathy/hemolytic uremic syndrome, in which hiv is the main, but not sole, etiological factor. ethnic/geographic background is an important determinant of the type of glomerulopathy associated with hiv; for example, collapsing fsgs is prevalent in patients of african descent. the fsgs variant of hivan is the most commonly reported chronic renal disease associated with hiv infection. hivan affects up to 10% of hiv-infected patients of african descent-mainly males at risk of drug abuse, and often african americans. glomerular changes associated with this variant are capillary wall collapse of varying severity, with widening of bowman's space. visceral epi thelial cells undergo hyperplasia and hypertrophy, and develop protein inclusions in their swollen cytoplasm surrounding the collapsed lobules. sclerosis affects segments of capillary tuft or the whole glomerular surface. tubular cells might undergo degenerative changes, necrosis or flattening. large dense casts can develop in dilated tubules. detection of hiv rna in renal tissue from patients with hivan, and of hiv dna in patients with and without nephropathy, 55 raises the question of whether renal cells can be infected in vivo (tubular epithelial cells can be infected in vitro). 4 renal uptake of viral gene products might induce transactivation of host lai and lai may 2006 vol 2 no 5 www.nature.com/clinicalpractice/neph genes. in renal cells, hiv proteins can cause apoptosis, 4 phenotypic modifications, 56 and subsequent tubulointerstitial fibrosis. clinical manifestations of hivan with fsgs are nephrotic-range proteinuria and renal insufficiency. hypertension and edema are uncommon. in overt cases, ultrasonography typically reveals enlarged, highly echogenic kidneys, which probably develop in response to microcystic tubular dilatation. before effective antiretroviral treatment was available, clinical progression was rapid. intensive antiretroviral treatment delays progression. 57 pathology of the hiv-associated disease mediated by immune complexes resembles lupus nephropathy. the clinical presentation is nephrotic syndrome with microscopic hematuria. progression to renal failure occurs, but more slowly than in hivan. patients often have hcv coinfection, but hiv seems to have the prevailing role. viral antigen has been detected in glomeruli, and antibodies eluted from the kidney react with hiv antigens in circulating immune complexes (iga-p24 antigen, igg-p24 and igg-gp120). in cases of hiv-related igan, circulating immune complexes containing iga idiotype antibodies have been detected. 58 most patients with hiv-associated thrombotic microangiopathy/hemolytic uremic syndrome present with acute renal failure, microscopic hematuria, and non-nephrotic proteinuria. multiorgan involvement is frequent and prognosis is poor, with a high rate of mortality. multifactorial etiologies encompass drugs, neoplasia, lymphoma and infection. pvb19 has been associated with acute glomerulonephritis. typical life stage of onset is the second or third decade. patients present with mild proteinuria and microhematuria, with low levels of serum complement 3. the pathology of this disease is characterized by endo capillary glomerulonephritis, mcgn, or both, with sub endothelial deposits. pvb19 capsid protein is found in the glomeruli. 10 spontaneous recovery is the norm. pvb19 is also associated with collapsing glomerulopathy. prevalence of pvb19 dna in renal biopsies (78%) and peripheral blood (87%) is significantly higher in patients with collapsing glomerulopathy than in those with other nephropathies. 10 glomerular and tubular infection with pvb19 might trigger collapsing glomerulopathy, but only in patients with immune defects and a racial pre disposition (african descent). 8 most intriguingly, tanawattanacharoen and coworkers 9 detected pvb19 dna in 80% of patients with 'idiopathic' fsgs, and frequently also in controls. these results possibly reflect the presence of latent dna from past infection. failure to localize pvb19 nucleic acid within kidney is evidence against ongoing, high-level viral replication. hantaviruses are responsible for 'hemorrhagic fever with renal syndrome' , an acute inter stitial nephritis resulting from direct vascular injury of renal tissue. 59 the severe form leads to acute renal failure in 50% of cases. less severe forms occur in nonendemic areas, and present primarily as fever, hepatitis, and mild renal impairment. in most cases, glomeruli are not affected and the pathology is tubulointerstitial. isolated cases with immune complex glomerular disease have been described, in association with diffuse proliferative glomerulonephritis and complete recovery after remission of the systemic clinical syndrome. 60 six percent of patients suffering from severe acute respiratory distress syndrome had acute renal impairment. 61 despite detection of viral dna in the urine, there was no evidence of viral tropism of the kidney. the pathology is exclusively tubulointerstitial nephritis. the mechanism of disease is probably related to multiorgan failure, rhabdomyolysis, and hemodynamic disturbance. renal infection with bk virus affects kidney allograft recipients, leading to renal dysfunction and sometimes graft loss. 62 more rarely, acute interstitial nephritis is observed in immunocompromised patients. 63 hepatitis a virus infection can present as acute post-infectious glomerulopnephritis with pathology resembling that of igan. 64 more commonly, hepatitis a virus induces acute renal failure secondary to acute fulminant hepatitis. 65 acute renal failure complicating other viral infections, such as epstein-barr virus 66 and dengue fever, 67 is related to multiorgan failure, rhabdomyolysis, and hepatorenal syndrome. diverse mechanisms of glomerular and tubulointerstitial injury and heterogeneous clinicopathologic patterns underlie the relationship between viral infection and glomerular disease. the etiological role of some viruses is still un defined. molecular biology techniques are vital in elucidating the precise location and role of viruses in the pathogenesis of virus-related nephropathy. treatment of hepatitis b virus-associated membranous nephropathy with recombinant alphainterferon a one-year trial of lamivudine for chronic hepatitis b: asia hepatitis lamivudine study group hbv associated nephrotic syndrome: resolution with oral lamivudine adefovir dipivoxil for the treatment of lamivudine-resistant hepatitis b mutants adefovir dipivoxil in chronic hepatitis b infection clinical practice: prevention of hepatitis b with the hepatitis b vaccine universal hepatitis b vaccination in taiwan and the incidence of hepatocellular carcinoma in children: taiwan childhood hepatoma study group safety of immunisation and adverse events following vaccination against hepatitis b membranoproliferative glomerulonephritis associated with hepatitis c virus infection immunological disorders in c virus chronic active hepatitis: a prospective casecontrol study hepatitis c, cryoglobulinemia, and cirrhosis: a meta-analysis secondary membranoproliferative glomerulonephritis hepatitis c virus infection and acute or chronic glomerulonephritis: an epidemiological and clinical appraisal renal involvement in hepatitis c infection: cryoglobulinemic glomerulonephritis long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis membranous glomerulonephritis associated with hepatitis c virus infection: case report and literature review randomised trial of interferon α2b plus ribavirin for 48 weeks or for 24 weeks versus interferon α2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis c virus: international hepatitis interventional therapy group (ihit) treatment of refractory, symptomatic, hepatitis c virus related mixed cryoglobulinemia with ribavirin and interferon-alpha peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection peginterferon-α2a and ribavirin combination therapy in chronic hepatitis c: a randomized study of treatment duration and ribavirin dose hepatitis c virus-related cryoglobulinemic glomerulonephritis: long-term remission after antiviral therapy renal complications of human immunodeficiency virus type 1 hiv-associated immunemediated renal disease renal epithelium is a previously unrecognized site of hiv-1 infection the dysregulated podocyte phenotype: a novel concept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis and hiv-associated nephropathy cohort study of the treatment of severe hiv-associated nephropathy with corticosteroids brief report: idiotypic iga nephropathy in patients with human immunodeficiency virus infection mechanism of disease in hemorragic fever with renal syndrome different pathohistological presentations of acute renal involvement in hantan virus infection: report of two cases acute renal impairment in coronavirus-associated severe acute respiratory syndrome bk-virus nephropathy in renal transplants-tubular necrosis, mhc-class ii expression and rejection in a puzzling game bk virus renal infection in a patient with the acquired immunodeficiency syndrome iga-dominant glomerulonephritis associated with hepatitis a renal failure in otherwise uncomplicated acute viral hepatitis epstein-barr virus-associated acute renal failure: diagnosis, treatment, and followup clinical characteristics and risk factors for concurrent bacteremia in adults with dengue hemorrhagic fever some of the authors' work cited in this review was supported by the l & t charitable fund and indocafe. the authors declared they have no competing interests. key: cord-025634-31n5fvex authors: zhuge, shurui; ge, congcong; yang, yuting; cui, yuxia; yue, xiaomei; zhang, zhenzhen; xu, hongmei; huang, ailong; zhao, yao title: the prevalence of occult hbv infection in immunized children with hbsag-positive parents: a hospital-based analysis date: 2020-05-29 journal: hepatol int doi: 10.1007/s12072-020-10055-9 sha: doc_id: 25634 cord_uid: 31n5fvex background and object: the risk of occult hbv infection (obi) in children whose mothers are hbv carriers has received more widespread attention, but there were few reports to focus on the children with hbsag-positive parents. in this study, we aimed to investigate the prevalence of obi in immunized children with hbsag-positive parents. methods: hbv-vaccinated chinese hospitalized children with hbsag-positive parents were analyzed in our investigation. eligible subjects were tested using a standard nested pcr for all hbv genes, and analyzed by direct sequencing. results: there were 327 hbsag-negative children included in the study out of about 9800 involved hbv-vaccinated hospitalized children. the positive rate of obi was 3.1% (10/327) in the eligible children and 14.1% (46/327) with hbv dna detectable. no significant differences were found between one and at least two regions positive groups (p > 0.05). the proportions of hbv dna detectable in children with hbv father-carriers and mother-carriers were similar. the risk factors for hbv dna-positive children could be male, anti-hbs levels, and anti-hbc positive. conclusion: there are 3.1% of obis and 14.1% of suspected obi in vaccinated children with hbsag-positive parents. the potential risk of suspected obi in children with hbsag-positive father should not be ignored. anti-hbc positivity may be a useful seromarker for suspected obi screening in vaccinated children. to prevent hbv breakthrough infection, accurate and convenient method is needed to detect obi timely and exhaustively. electronic supplementary material: the online version of this article (10.1007/s12072-020-10055-9) contains supplementary material, which is available to authorized users. chronic hepatitis b virus (hbv) infection is a major global health concern. occult hbv infection (obi) is regarded as the fifth phase of the natural history of chronic hbv infection [1] , which is defined as the presence of hbv dna in the liver (either with or without detectable hbv dna in the serum) of people who test negative for hepatitis b surface shurui zhuge, congcong ge, yuting yang and yuxia cui contributed equally to this work. the online version of this article (https ://doi.org/10.1007/s1207 2-020-10055 -9) contains supplementary material, which is available to authorized users. antigen (hbsag). on the basis of the hbv antibody profile, obi distinguished as: seropositive-obi, (hepatitis b core antigen antibodies [anti-hbc] and/or hepatitis b surface antigen antibodies [anti-hbs] positive) and seronegative-obi (anti-hbc and anti-hbs negative) [2] . the amount of hbv dna in serum is usually very low (< 200 iu/ml). nowadays, the available assays for occult hbv testing is the analysis of dna extracts from liver as well as blood, and samples amplify in two subsequent rounds of pcr ("nested" pcr) or by a "real-time pcr" technique. different clinical conditions have been involved in occult hbv infection: some studies suggest that obi has the potential to reactivate and cause severe acute disease under immune suppression; transmission of the infection by blood transfusion or organ transplantation; and contribute to the development of cirrhosis, hcc [1, 3] . the majority of hbv infections in children are contracted either during perinatal period or early childhood, and individuals from hbv hyper-endemic areas may be more likely with occult hbv infections [4] . in china, hepatitis b routine immunization began in 1992 and is free for all newborn babies since 2002. the national serosurvey (carried out in 2006) showed that the prevalence of hbsag fell from 9.8 to 7.2% for people aged 1-59 years between 1992 and 2006, and the infection in children under 5 years of age is only 1.0% [5] . currently the first vaccine dose was administered within 24 h of birth and subsequent doses at 1 and 6 months, and newborns with hbsag-positive mothers were recommended to receive hepatitis b immunoglobulin (hbig) within 24 h. in recent years, occult hbv infection was presented worldwide despite immunization against hbv, and varying proportions of infants born to hbsag-positive mothers have been reported with obi [6] [7] [8] . data are scanty on the risk of obi in children with hbv-positive father. in this study, we aimed at exploring the prevalence of obi in hepatitis b-vaccinated children with hbv-positive mothers and/or fathers, trying to identify the risk factors of obi. we had taken about 2.5 years to ask hospitalized children and their parents one by one, from april 2013 to november 2015. the inclusion criteria: (1) negative for hbsag, (2) from hbsag-positive parents (mother and/or father), (3) 3-dose hepatitis b vaccination immunized after birth, (4) other factors that may get infections such as blood transfusions, (5)with other pathogen infections, e.g., hepatitis c virus (hcv) and human immunodeficiency virus (hiv). serological markers (hbsag, anti-hbs, hepatitis b e antigen [hbeag] , hepatitis b e antibody [anti-hbe], and anti-hbc) were detected using commercial chemiluminescence microparticle immuno assay (cmia) kits (abbott gmbh & co. kg, wiesbaden, germany). subjects were considered hbsag-positive at values > 0.05 iu/ml, anti-hbs-positive or seroprotected at values ≥ 10 miu/ml, hbeag-positive at values ≥ 1 s/co (sample rate/cut off rate), anti-hbe-positive at values ≤ 1 s/co, and anti-hbcpositive at values ≥ 1 s/co. viral dna was extracted from 200 µl of serum using the qiaamp dna blood mini kit (qiagen inc., germany), according to the manufacturer's instructions. all dna samples were aliquoted and kept at − 20 °c prior to amplification and sequencing. specific primers were designed to target the x and c regions of the hbv genome using primer premier 5. primers targeting the s and pre-s regions were designed by shahmoradi et al. [9] . all primers (supplement table 1 ) were synthesized by sangon biotech co., ltd (shanghai, china). the sensitivity of the pcr assay was determined by serial dilutions of serum samples containing known concentrations of the hbv genome: 1 × 10 7 , 1 × 10 6 , 1 × 10 5 , 1 × 10 4 , 1 × 10 3 , 1 × 10 2 , 1 × 10 1 , iu/ml. the limit of detection for the nested pcr assay was approximately 10 iu/ml. the pcr mix was the same for all reactions and comprised 12.5 µl of 2 × taq pcr master mix (tiagen, beijing, china), and the first and the second-round primers (10 pm). five microliters of hbv dna were used in the first round pcr, and 5 µl of the first round pcr product was used as the template for the second round. amplification was performed for 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, and elongation at 72 ℃ for 1 min, followed by a final extension at 72 ℃ for 5 min. finally, 5 µl of pcr product was analyzed by electrophoresis in 1% agarose gel. precautions were taken to avoid cross-contamination during sample collection, dna extraction, pcr, and gel electrophoresis. to avoid the effect of cross-contamination on the results, negative and blank controls were included in each assay. only reproducible data from assays with "clean" negative controls were analyzed. the positive test result was repeated three times. pcr products were directly sequenced in an automated dna sequencer (abi 3730) and the data were analyzed using chromas 2.4.1 software (technelysium pty ltd., south brisbane, australia). sequences were analyzed by the blast tool of ncbi and molecular evolutionary genetics analysis (mega, version 6.0). hbv sequences from obi children were aligned and compared with genbank reference sequences (genotypes a-h). phylogenetic analysis was performed using the neighbor-joining method based on the nucleotide sequence of the amplified s, c, and pre-s region. bootstrap resampling and reconstruction were performed 1000 times to confirm the reliability of the phylogenetic analysis. genetic distances were evaluated using kimura 2-parameter corrections. the accession numbers for the reference sequences are as follows: af090842, d00330, ab033556, x65259, x75657, x69798, af160501, ay090454. all experiments were performed in accordance with relevant guidelines and regulations. two forms of informed consent were prepared, one was for parents when the child is less than 8 years, and another was to obtain both the children's and parents' informed information when the child is older than 8 years. the parents of the patients with obi were informed when their children's results were positive. statistical analyses were performed using the statistical package for social science (spss) for windows, version 20.0 (spss inc., chicago, usa). anti-hbs values ≥ 1000 miu/ ml were calculated as 1000 miu/ml. non-normal variables were expressed as median (interquartile range, iqr) and analyzed using mann-whitney u test. categorical variables were analyzed using the chi-squared (χ 2 ) test or fisher's exact test when the expected count in one cell was less than 5; yates correction was applied when appropriate. candidate variables with a p value < 0.25 on univariate analysis were included in multivariate logistic regression model. all statistical tests were two-tailed. a value of p < 0.05 was considered statistically significant. the sample selection and diagnostic workflow of tests in hepatitis b-vaccinated children were shown in fig. 1 . from april 2013 to november 2015, 9800 hospitalized children were given hbv seromarker test, and 400 hbv-vaccinated children whose mother, father, or both were hbsag-positive met. there are 21 children with blood transfusion were excluded, and 49 children could not involve in the research due to the rejection of their parents. finally, a total of 327 hbsag-negative children were involved in the study. within the 327 samples, there were 52.60% of children with hbsag-positive mothers, 44.95% with hbsagpositive fathers, and 2.45% with parents-carriers. all of these children received three doses of hepatitis b vaccine as planned and most (70.03%) received full prophylactic coverage (vaccine plus hbig). hbv seromarkers were identified in the children, and positive rate was 74.62% (244/351) in anti-hbs (≥ 10 miu/ml), 7.12% (25/351) in anti-hbc (s/co ≥ 1), and 0.85% (7/351) in anti-hbe (s/ co ≤ 1), respectively. all 327 samples were analyzed to determine the existence of hbv dna by nested pcr, and hbv dna was detected in 46 [14.1%; 95% confidence interval (ci) 10.3-17.9%] children ( fig. 1, table 1 ); using nested pcr, 20 (43.5%; 95% ci: 28.6-58.4%), 23 (50.0%; 95% ci 35.0-65.0%), and 16 (34.8%; 95% ci 20.5-49.1%) children were found positive for surface, core/pre-core, and pre-s regions (supplement fig. 1 ), none of sample amplified positive for hbv complete genome and x region. overall, three (6.5%) samples were positive for three regions, seven (15.2%) samples were positive for two regions, 36 (78.3%) were positive for one region ( table 2) . the nested pcr amplification products of hbv s, and c, pre-s gene fragments in the 46 serum samples were successfully sequenced. all sequence information had been retrieved on national center for biotechnology information (ncbi), and accession numbers were from mg738731 to mg738789. different fragments of sequence information were used to construct the phylogenetic tree separately, and compared with standard sequences of hbv genotypes a-h (supplement fig. 2 ). there were 22 (47.8%) genotype b and 24 (52.2%) genotype c. samples with sufficient blood were quantified for hbv-dna using commercially real-time pcr-based detection kit, and mutation analysis at amino acid levels with the s, c, pre-s gene sequencing information was done. one 'a' determinant (amino acids 124-147) variant m133l was detected in isolate p77, which was associated with vaccine escape. p77 harbored an 'a' determinant variant and with a high level of hbv dna (11,800 miu/ml) was categorized as "false" obi. demographic, epidemiological data, serological markers, and parent-carrier status identification within the 46 children were shown in table 2 , 87.0% (40/46) in anti-hbs positive, 13.0% (6/46) in anti-hbc positive, and no children were anti-hbc positive alone. comparison was done between one region positive group (n = 10) and ≥ two regions positive group (n = 36) detected by nested pcr, and no significant differences were found including age, gender, hbig usage, anti-hbs titer, positive rate of alt, ast and anti-hbc, maternal and paternal factors in those children ( forty-six [14.10% (95% ci 10.3-17.9%)] hbsag-negative children were detected hbv dna positive by nested pcr, which were confirmed through sequencing analysis. there were 5 (5/9, 55.6%), 15 (15/35, 42 .9%), and 20 (20/44, 45 .5%) children with hbv carrier fathers detected ≥ two regions positive, one region positive, and ≥ one region positive by nested pcr, respectively (fig. 2) . the proportions of hbv dna detectable in children with hbv father-carriers and mother-carriers were similar, and no statistical difference were found (p > 0.05). for hbv dna detectable children, 20 (13.6%, 95% ci 8.0-19.2%), 24 (14.0%, 95% ci 8.7-19.2%) were found with hbv father-carriers, mother-carriers. univariate statistical analysis was done between the hbv dna detectable and undetectable children, and no statistically significant difference was found in the basic characteristics (gender, age, hbig usage, serum anti-hbc, ast, alt, maternal and paternal factors) (p > 0.05), except for anti-hbs titer. (supplement table 2 ). . 1 the diagnostic workflow of tests in hbsag-negative children. serum samples were obtained from 327 hbsagnegative children whose mother, father, or both were hbsagpositive. children not accord with the criteria were excluded. the remaining samples were examined by nested pcr to identify the risk factors that may affect hbv dnapositive children, variables were explored with multivariate logistic regression model, and the dependent variable being the weighted in the hbv dna detectable children. independent variables included age, gender, anti-hbs, anti-hbc (variables for inclusion were carefully chosen, to ensure parsimony of the final model). results showed that male [odds ratio (or) 4.24], anti-hbs titer (or 3.67), and anti-hbc-positive (or 4.81) had higher risk with hbv dna detectable than females (p < 0.05) ( table 4 ). firstly, identified in the 1970s, more and more evidence has suggested that the clinical significance of obi, which has become a major health issue attracting much attention. in recent years, variable proportions of obi had been reported in immunized children with hbv-positive mothers. to our acknowledgement, this is the first study to explore the prevalence of obi among hepatitis b vaccinated children with hbsag-positive parents lived in hbv highly endemic areas. in this study, results of nested pcr amplification show 14.10% (46/327; 95% ci 10.3-17.9%) of hbsag-negative children with ≥ one gene fragment positive. detection ≥ two regions of hbv genome by nested pcr is considered as the standard for hbv infection, only ten children (3.1%) would be considered as having obi if definition is followed. recently, the results of the quantitative rt-pcr for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) nucleic acid show that 87.5% of patients were finally defined as sars-cov-2 positive, in whom originally had only one positive target [10] . thus, to examine the possibility of whether one hbv gene fragment detectable could indicate the existing occult hbv infection, we did the following analysis. first, there were 36 (11.0%; 95% ci 7.6-14.4%) and 10 (3.10%; 95% ci 1.2-4.9%) patients tested positive for one and ≥ two hbv gene fragments with strict quality control during the experiment, and no statistical differences were found between the two groups. second, results of previous studies had found that one hepatitis b fragment positive also could indicate occult hbv infection: jazayeri et al. detected hbv dna in 28% (21/75) children by real-time pcr quantitatively, while some of the samples only amplified one segment [9] ; for other researches amplified one hbv fragment with the samples, and the positive amplicons were confirmed from hbv genome by sequencing analysis [11, 12] . the sensitivity of nested-pcr assays may not be consistent, and thus there could be 14.1% of suspected obis in children with hbsag-positive parents. the risk of suspected obi in children with hbsag-positive parents should not be ignored. considering the significant reservoir of hbv infections, the status of hbv infection in many countries is still not optimistic. relatively high prevalence of obi was found previously, range from 7.7 to 28% [6, 9, 13] . in china, hbv infection is still a severe public health burden with 97 million hbv carriers, and 14.1% of suspected obi may exist in immunized children with hbsag-positive parents. obi may be involved in different clinical contexts, the development of cirrhosis and hepatocellular carcinoma [1] . therefore, it could be important to have long term follow-up in children after uncovering obi despite vaccination to prevent chronic complication in adulthood. there is an equal potential risk of occult hbv infection in children with the hbsag-positive father and mother. the positive rate of suspected obi in father carriers was equal to mother carriers (13.6% vs. 14.0%), and 25% in both parents were hbv carriers. in china, it has been reported that 23.2% of hbsag-positive families contained more than two hbv carriers [14] . the pattern of father-to-child transmission may not be ignored [15] [16] [17] [18] . it is not uncommon that hbv integration was detected in pbmcs and cellular genes in hcc cases [19, 20] . hbv dna integrates into the paternal dna and causes the neonatal be infected through the sperm is possible. many independent factors had been analyzed between the suspected obi positive and negative children in the study. similar to hbv highly endemic areas, obi in children living in hbv low prevalence regions are also more common than "overt" hbv infection, and the risk of obi could be closely related to the parents' infection status [21, 22] . results in previous study conducted in hbv low prevalence regions have shown that the risk factors for obi could include whether to get hepatitis b vaccine, the genotype of hbv, while none of those found significant difference in this study [21] . the titer of anti-hbs was higher in the suspected obipositive than obi-negative (p < 0.05). the escape mutations in hbv s gene needs to take attention [7, 9, 13, 23] . hbcag is the most immunogenic hbv component during infection [24] . in recent years, some researches have suggested that anti-hbc was a very useful marker for obi screening in hbsag-negative subjects [25] [26] [27] . the risk of suspected obi in anti-hbc-positive children may need to pay more attention, while it has to be stressed that not all anti-hbc positive individuals are found to be hbv dna positive (anti-hbc negative also does not exclude obi), and that anti-hbc tests may provide false-positive results. there are some limitations exist in the study. first, this study is related to the hospital-based study design and children who are hospitalized may not represent children in the general population. anyway, we had tried to avoid other factors that might get infections such as blood transfusions. second, exploring the risk of obi with one region positive and sequence analysis may be insufficient in this study, while it may be the more convenient and accurate method for suspected obi detection. three, in this study, although the transmission of hbv was mainly from parental carriers, the influence of other persons could not exclude, e.g., family members other than parents. nevertheless, most chinese children live with their parents and vertical transmission is the main way in asian areas [28] . thus, the children in this study with occult hbv infection were more likely from parent-to-child transmission. in conclusion, a relatively high prevalence of suspected obi may exist in hepatitis b-vaccinated chinese children with hbsag-positive parents, and the importance of monitoring obi should be taken into account, especially for hbv hypo-endemic areas. paternal factors should not be ignored with an equal potential risk of suspected obi in children with hbsag-positive father and (or) mother. anti-hbc seropositivity may be a useful marker for suspected obi screening in vaccinated children. diagnosis of obi with one hbv region amplifying positive using nested pcr may be reliable. to prevent hbv breakthrough infection, accurate and convenient method is needed to detect obi timely and exhaustively. the clinical significance of occult hbv infection statements from the taormina expert meeting on occult hepatitis b virus infection association between occult hepatitis b infection and the risk of hepatocellular carcinoma: a metaanalysis occult hepatitis b epidemiological serosurvey of hepatitis b in china-declining hbv prevalence due to hepatitis b vaccination occult hbv infection in immunized neonates born to hbsag-positive mothers: a prospective and follow-up study occult hepatitis b virus infection in anti-hbs-positive infants born to hbsag-positive mothers in china occult hepatitis b virus infection in children born to hbsag-positive mothers after neonatal passive-active immunoprophylaxis high prevalence of occult hepatitis b virus infection in children born to hbsagpositive mothers despite prophylaxis with hepatitis b vaccination and hbig epidemiological and clinical features of the 2019 novel coronavirus outbreak in china occult hbv infection status among chronic hepatitis c and hemodialysis patients in northeastern egypt: regional and national overview characterization of occult hepatitis b virus infection among hiv positive patients in cameroon occult hepatitis b virus infection in hepatitis b vaccinated children in taiwan the global burden of liver disease: the major impact of china hepatitis b virus infection among children born in the united states to southeast asian refugees the analysis of s gene phylogenetic tree of hbv in transmission from father to infant molecular evidence of father-to-child transmission of hepatitis b virus hepatitis b virus infections in families in which the mothers are negative but the fathers are positive for hbsag detection and sequence analysis of hepatitis b virus integration in peripheral blood mononuclear cells large scaled analysis of hepatitis b virus (hbv) dna integration in hbv related hepatocellular carcinomas high prevalence of occult hepatitis b virus genotype h infection among children with clinical hepatitis in west mexico risk factors associated with horizontal transmission of hepatitis b viral infection from parents to children in mexico impact of hepatitis b virus surface protein mutations on the diagnosis of occult hepatitis b virus infection the nucleocapsid of hepatitis b virus is both a t-cell-independent and a t-cell-dependent antigen universal infant immunization and occult hepatitis b virus infection in children and adolescents: a population-based study transmission of hepatitis b by transplantation of livers from donors positive for antibody to hepatitis b core antigen hepatitis b reactivation in occult viral carriers undergoing hematopoietic stem cell transplantation: a prospective study natural history of chronic hepatitis b in euro-mediterranean and african countries acknowledgements we wish to thank yu shi for sample collection and junjie tan for assisting with the quantification of hbv. we also thank professor xiaodong zhao for helpful discussion. conflict of interest shurui zhuge, congcong ge, yuting yang, yuxia cui, xiaomei yue, zhenzhen zhang, hongmei xu, ailong huang, yao zhao declare no competing interests.ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. all included subjects gave informed consent for their participation in the study, and we checked hbv infection of the mothers and/or fathers again. the study was approved by the ethics committee of the children's hospital of chongqing medical university.informed consent informed consent was obtained from all individual participants included in the study. all authors reviewed and approved the final version of the manuscript. key: cord-287151-4hlvrfeh authors: steinmann, j title: surrogate viruses for testing virucidal efficacy of chemical disinfectants date: 2004-04-30 journal: journal of hospital infection doi: 10.1016/j.jhin.2003.12.030 sha: doc_id: 287151 cord_uid: 4hlvrfeh abstract since important agents of viral nosocomial infections like hepatitis b and c viruses and norovirus do not replicate sufficiently in cell culture systems, disinfectants with suspected efficacy against these viruses must be evaluated by different methods. besides molecular approaches and indirect tests, the use of surrogate viruses with similar biophysical properties and genomic structure allows the assessment of virucidal efficacy of chemical disinfectants in quantitative suspension tests. furthermore, insights into the survival of these viruses in the environment are possible. in recent years, duck hepatitis b virus and bovine viral diarrhoea virus have been tested as surrogates for hepatitis b and c viruses. feline calicivirus serves as a surrogate for the group of norovirus. by including these viruses in inactivation experiments, valuable data from suspension tests can be derived on the virucidal efficacy of chemical disinfectants. even in vivo tests using fingerpads of adult volunteers can be performed with these animal viruses without risk of infection. in contrast to in vitro examinations, the results of these tests allow use recommendations of chemical disinfectants for outbreak situations and daily routine disinfection. the importance of viruses in causing nosocomial infections is well recognized, and the incidence of such infections continues to increase. the risk is particularly high in departments carrying out invasive procedures as well as in departments with young, elderly and immunosuppressed patients. there are many anecdotal reports on nosocomial infections, but no recent epidemiological studies. disinfection is one of the most effective measures for prevention of nosocomial virus infections. therefore, there is an urgent need to assess the virucidal efficacy of hand-, surface-and instrument disinfectants in order to interrupt chains of infection in hospitals and other medical areas. since viruses are quite different from bacteria, inactivation results based upon bacteriological studies cannot be applied to viruses. furthermore, the great heterogenicity of human pathogenic viruses makes it difficult to choose the most suitable viruses for assessing virucidal efficacy in vitro and in vivo. in europe, virucidal testing is performed in a stepwise procedure. as screening examinations of single compounds are not as relevant as in bacteriology, no phase 1 test (basic test) exists. most of the experiments are performed with a quantitative suspension test (phase 2, step 1) allowing the demonstration of a virucidal efficacy with constant parameters such as temperature, volume ratios and a defined soil load. phase 2, step 2 methods describe procedures simulating practical conditions. according to the german guideline of the federal health office (bundesgesundheitsamt ¼ bga, now robert koch-institute, berlin, germany) and of the german association for the control of virus diseases e.v. (dvv) a titre reduction of 10 4fold is necessary for demonstrating efficacy in suspension tests. 1 the european draft of a guideline also requires the same reduction with identical volume ratios for confirming the virus-inactivating properties of a chemical disinfectant. 2 besides volume ratios and soil load, the choice of test viruses is one of the most important question in addressing inactivation experiments. in north america, each virus which can cause nosocomial infection is regarded as a test virus. in europe, model viruses have been chosen that are representative of a wide range of virus families. in germany, poliovirus type 1 (vaccine strain lsc 2ab, formerly strain mahoney/pette), adenovirus type 5 (formerly adenovirus type 2), papovavirus strain 777 and vaccinia virus strain elstree are test viruses for hand-, surface-and instrument disinfectant. 1 in europe (pren 14476), only the polio-and the adenoviruses are chosen. 2 additionally, bovine parvovirus is incorporated into the guideline for the evaluation of instrument disinfectant due to its heat stability. in germany, the robert koch-institute and the dvv plan to divide the requirements of disinfection into high level (polio-and adenovirus inactivation necessary) and low level (only inactivation of bovine viral diarrhea virus and vaccinia virus necessary). besides the viruses mentioned in the guidelines, there are other important pathogens such as hepatitis b virus (hbv), hepatitis c virus (hcv) and norovirus which cause nosocomial infections but cannot be propagated sufficiently by cell culture techniques. due to their importance, these agents would be part of the guidelines if culture were possible. because they are not culturable, surrogate viruses have been introduced into virucidal testing. hbv and hcv are the most prevalent bloodborne pathogens. noroviruses, first detected in faecal specimens, are now responsible for many outbreaks of gastro-enteritis following the introduction of molecular techniques into virus detection. a further use of surrogate viruses is when the virus needs a level of containment which is not readily available. for example, the recently detected virus causing severe acute respiratory syndrome (sars), which belongs to the family coronaviridae and requires a high level of laboratory safety. 3 both bovine coronavirus (bcv) and the avian infectious bronchitis virus (ibv) resemble this new virus and may serve as surrogates in the future. table i lists the four important surrogate viruses. the importance of surrogates for virucidal testing of hbv, hcv and norovirus will be discussed in detail. human hbv belongs to the family of hepadnviridae and is a serious viral pathogen in man that is highly contagious and can spread through blood, saliva and semen. hbv is a small, enveloped dna virus (40 -48 nm in diameter) that replicates its partially double-stranded dna genome through reverse transcription of an rna intermediate. the total genome is 3020 -3320 nucleotides for the full, and 1700 -2880 nucleotides for the short, length strand, respectively. hepadnaviruses employ an episomal covalently closed circular dna as a nuclear transcription template and establish a dna pool to regulate gene expression by a copy number. they are noncytopathic viruses and often establish a long-term persistent infection. in the past, a variety of approaches to study inactvation of hbv were utilised due to the lack of sufficient cell culture replication (table ii) . the chimpanzee test system allows the use of human hbv itself in the test, but the small number of available animals limits its relevance for proper testing of chemical disinfectants. the so-called indirect tests like the morphologic alteration and disintegration test (madt), dna polymerase inactivation and the hbsag test do not reflect our increased knowledge of hbv. adaption experiments with a cell culture of the hepatoma cell line hepg2 showed only a small effect. 4 a molecular approach based upon the destruction of sequential epitopes and the inability to amplify the target sequence with a polymerase chain reaction (pcr) needs further investigations. 5 recently, the asian tree shrew, tupaia belangeri, was introduced as a novel animal model. 6 the primary tupaia hepatocytes are susceptible to infection with hbv and woolly monkey hepatitis b virus (wmhbv). inactivation assays with hbv and these hepatocytes may allow disinfection experiments in the future. in addition to hbv, hepadnaviruses include a growing number of viruses that infect selected hosts in the wild (table iii) . the ground squirrel, the woodchuck hepatitis virus and the duck hepatitis b virus (dhbv) are best studied. non-human hepadnaviruses are useful not only for studying antiviral drug development and evaluation of antiviral compounds mainly in the blood product industry but also for inactivation experiments with disinfectants. dhbv (genus avihepadnavirus) shares many physical properties with the closely related hbv and the mechanism for initiating and inducing a long-term infection is similar. however, dhbv lacks the open reading frame for a multifunctional protein termed hbx. furthermore, its genome and the single-stranded gap of the partially doublestranded dna is slightly smaller. however, dhbv has often been used as an in-vivo model for the preclinical evaluation of nucleoside analogs and in blood product industry and therefore this virus was also introduced into inactivation studies by murray and co-workers. 7 two methods of laboratory testing based on the susceptibility of hepatocytes of the pekin duck exist. first, titration can be performed in day-old ducklings followed by bleeding nine weeks after infection and detection of dhbv dna by southern blotting or by pcr. however, as a large number of animals and special housing are required and the time for results is considerable, in vitro systems have been developed. thus, hepatocytes can be prepared from embryonated eggs (day 17 -21) and after infection the virus can be detected by immunofluorescence or quantitative pcr. the susceptibility of dhbv to sodium hypochlorite and sodium dichloroisocyanate was compared with the susceptibility of hbv to these substances. 8 the results demonstrate a good correlation between inactivation of infectivity in vivo and the total inhibition of the in vitro hepadnavirus dna polymerase activity. with the dhbv model it has been possible to study the capability of a hydrogen peroxide gas plasma sterilizer. 9 by assessing the efficacy of two quaternary ammonium chloride disinfectants with a pcr for dna detection, concentrations of 1200 and 1800 ppm were found to be effective against dhbv. 10 later on, a quantitative pcr was developed based on sybr green dye. 11 the dhbv model clearly demonstrated the importance of cleaning angioscopes before disinfection and sterilisation. 12 further experiments will be needed to study the hbv inactivating properties of many disinfectants which have not been evaluated by the chimpanzee model in the past. hcv is a member of the family flaviviridae containing three genera. 13 the genus hepacivirus contains exclusively hcv. flavivirus as another genus contains among others the yellow fever virus group, the dengue group and the tick-born encephalitis virus group. the border disease virus, the hog cholera virus and the bovine viral diarrhea virus (bvdv) belong to the third genus pestivirus. the characteristics of the family flaviviridae are shown in table iv . there are no in-vitro tests with hcv. recently, the ability of a vero cell clone to bind hcv to cell surface receptors was introduced in disinfectant testing. 14 phenolics and a chlorine-based compound were active, indicating that this enveloped virus is not unduly resistant. 15 additionally, a rt-pcr was described for testing antiseptic/disinfectant in contrast to hbv, there is no closely-related animal virus for hcv. bvdv, like hcv, is a small, enveloped, positive-stranded rna virus. the bvdv genome is approximately 12.3 kb in length with a 5 0 nontranslated region (ntr), a single large open reading frame (orf), and a 3 0 -ntr lacking a poly(a)tail. the orf is translated into a single polyprotein that is co-and post-translationally cleaved into 11 or 12 mature proteins by a combination of viral and host proteases. the bvdv was chosen as a surrogate because there are similarities in terms of genome structure and mode of replication. data with bvdv inactivation in a quantitative suspension test by chemical germicides are not available, since the great susceptibility of the virus to these substances has not initiated such studies in the past. norovirus (formerly norwalk-like viruses, norwalk viruses or small round structured viruses) is a genus within the family caliciviridae and causes acute gastro-enteritis in humans. 17 norovirus infections are typically mild and self-limited. the disease is characterized by an abrupt onset, a short duration and a high proportion of those infected having diarrhoea, abdominal cramps and vomiting. people become infected by faecal-oral transmission via contaminated water and food, by hand-to-mouth transfer from contaminated surfaces, by ingestion of aerosolised vomit and by secondary person-toperson transmission. outbreaks have been reported in many places e.g. in hospitals, residential homes, recreational camps, schools and hotels, and on cruise ships. these outbreaks often seem to be the result of more than one mode of transmission. criteria for suspected outbreaks are vomiting in . 50% of cases, short duration of illness, an incubation period of 15 -48 h and involvement of staff and patients. control measurements consist of cohorting of staff and individuals, wearing gloves, hand disinfection and washing, excluding affected staff, cleaning and disinfecting vomit and faecal spillages promptly. 18 in contrast to both hepatitis viruses, noroviruses are non-enveloped. the viruses contain one molecule of linear positive-sense rna. the virion is 27 -40 nm in diameter. the genome encodes the non-structural proteins at the 5 0 end and a single major capsid protein towards the 3 0 end. the family caliciviridae contains four genera: norovirus, sapovirus, lagovirus and vesivirus. the public health impact of norovirus infections is increasingly recognised. furthermore, the contamination of the environment and the importance of human hands as vehicles for virus transmission have focussed the interest on adequate virus inactivation by hand-and surface disinfectants. like hbv and hcv, there is no suitable cell culture system to support the replication of noroviruses. the use of feline calicivirus (fcv) which shares many similarities with noroviruses 19 was introduced as a surrogate by slomka and appleton. 20 recently, a plaque assay with crandell rees feline kidney cells was established. 21 this method will provide an alternative to end-point titration assays for quantitative experiments with fcv in inactivation studies. there are few published inactivation experiments with fcv. scott and co-workers studied 35 products mainly used in veterinary medicine; 11 were virucidal for fcv after an exposure time of 10 min. 22 alcohols such as 50% propan-2-ol, 50% ethanol and 35% methyl alcohol were not effective, whereas phenolics, clorox, aldehydes and creolin inactivated the test virus. in another study, 0.5% glutaraldehyde and 1000 ppm hypochlorite were effective within one minute, whereas 1:10 quaternary ammonium, 75% ethanol and 1% anionic detergents failed to demonstrate virucidal efficacy within this exposure time. 23 our own experiments with various 70% alcoholic solutions (ethanol, propan-1-ol, propan-2-ol) on artifically contaminated fingerpads according to e 1838-02 of american society for testing and material (astm) 24 showed that ethanol with 30 s exposure time was superior to the other types of alcohol. among different soil loads 5% foetal calf serum had no inhibitory effect on the inactivation process, whereas a tripartite soil load according to the introduction of surrogate viruses has provided important information on the behaviour of certain important human pathogenic viruses in the past. although they are not incorporated into official guidelines by standard-setting organisations, the us environmental protection agency (epa) has allowed label claims for human hbv after testing with dhbv since august 2000 (virucides dis/tss-7/1981). even the labeling disclaimer to indicate that the disinfectant has been tested against dhbv is unnecessary. this shows the important role of animal virus testing and the use of appropiate surrogates in disinfection experiments. the world health organisation has recently declared that there are no new infections with the virus of severe acute respiratory syndrome (sars), but the possibility of its remergence still exists. epidemiological data seem to suggest that the virus is spread by droplets or by direct and indirect contact, although airborne spread cannot be excluded. if transmission by indirect contact plays an important role, disinfection with products of proven efficacy is critical. due to the high risk of working in the laboratory with this new coronavirus, surrogates such as bovine coronavirus or avian infectious bronchitis virus could provide acceptable replacements for the sars virus in chemical disinfectant testing. at present, our inactivation data with dhbv, bvdv and fcv are limited, but established cell culture systems with these viruses will allow more information on the behaviour and inactivation of important viruses causing nosocomial infections. the introduction of dhbv by the epa shows that these surrogate viruses can be incorporated into official guidelines. federal health office) and deutsche vereinigung zur bekämpfung der viruskrankheiten e.v. (dvv; german society for the control of virus diseases) for testing the effectiveness of chemical disinfectants against viruses identification of a novel coronavirus in patients with severe acute respiratory syndrome inactivation of hepatitis b virus in plasma by hospital in-use chemical disinfectant assessed by a modified hepg2 cell culture molecular approaches to validate disinfectants against human hepatitis b virus efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis b virus duck hepatitis b virus: a model to assess efficacy of disinfectants against hepadnavirus infectivity chemical disinfection of duck hepatitis b virus: a model for inactivation of hepatitis b virus inactivation of duck hepatitis b virus by a hydrogen peroxide gas plasma sterilization system: laboratory and 'in use' testing development of viral disinfectant assays for duck hepatitis b virus using cell culture/pcr comparison of cell culture systems for duck hepatitis b virus using sybr green quantitative pcr evaluation of disinfection and sterilization of reusable angioscopes with the duck hepatitis b model evolutionary relationship of hepatitis c, pest-flavi-, plant viruses and newly discovered gb hepatitis agents hepatitis c virus infection of a vero cell clone displaying efficient virus-cell binding effect of phenolic and chlorine disinfectants on hepatitis c binding and infectivity evaluation of disinfectant efficacy against hepatitis c virus using a rt-pcr-based method human calicivirus in europe management of hospital outbreaks of gastro-enteritis due to small roundstructured viruses sequence and genomic organization of norwalk virus feline calicivirus as a model system for heat inactivation studies of small round structured viruses in shellfish a feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for norwalk virus virucidal disinfectants and feline viruses inactivation of feline calicivirus, a norwalk virus surrogate standard test method for determinating the virus-eliminating effectiveness of liquid hygienic handwash and handrub agents using the fingerpads of adult volunteers. designation: e-1838-02 key: cord-022607-34hj17sn authors: wain‐hobson, simon; vartanian, jean‐pierre title: editing hbv into oblivion date: 2004-11-24 journal: hepatology doi: 10.1002/hep.20499 sha: doc_id: 22607 cord_uid: 34hj17sn nan hepatitis c virus (hcv) readily sets up persistent infection, and in doing so must evade both innate and adaptive immune responses. cellular (t-cell) immune responses are thought to play a significant role in determining clinical outcome, with strong and sustained responses typically associated with viral control. 1,2 cellular immune responses are also potentially involved in immunomediated pathogenesis, both directly and through recruitment or modulation of other inflammatory cells in the liver. consequently, much recent effort has been expended to attempt to analyze how the virus evades both cd8 ϩ and cd4 ϩ t cells and define the differences between "successful" and "unsuccessful" outcomes (i.e., spontaneous control of viremia vs. chronicity) . relatively fewer data are available describing how intrahepatic t-cell responses might be linked to pathology. a general consensus is emerging that in acute infection, regardless of outcome, cd8 ϩ t-cell responses can be readily detected; however, in those cases in which the virus is not controlled, such responses are not sustained at high levels in blood beyond a few weeks. 3 this could be explained partly by viral escape through mutation, as has been elegantly shown in animal models. 4 however, even in cases when epitopes appear to be intact, responses typically are weak or absent in blood ex vivo once chronicity is established. they do not appear to be entirely deleted, because they can be reconstituted in vitro through restimulation with antigen, or even fished out from blood directly using ultrasensitive detection techniques. 5 thus various groups have proposed that functional alterations in cd8 ϩ t cells may be associated with persistent infection. those include relatively weak interferon gamma (ifn-␥) production, 6,7 impaired proliferative capacity, 8 and a "stunted" maturation state. 6, 9, 10 it is not clear whether the lack of mature effector cells seen in the circulation is as a result of compartmentalization in the liver, (where they may be deleted) or failure to generate such cells in the first place-potentially through defects in antigen-presenting cells, inhibitory effects of viral gene products (such as core) 11 or failure of cd4 ϩ t-cell help. 1 things look slightly different in the liver itself. early studies showed it is possible to clone out cd8 ϩ t cells of diverse specificities from the liver of infected patients. 12, 13 it appears that antigen-specific t cells exist at higher frequencies infected livers-probably a relative increase of approximately 10-fold as a proportion of cd8 ϩ t cells. 14, 15 (it should be noted that enrichment of virusspecific memory t cells-for example, those specific for cytomegalovirus and epstein-barr virus-is seen even in normal livers in human and murine models. 16 ) interestingly, such t cells in both infected and normal livers appear to be activated, as judged by expression of cd69, although this effect is not antigen specific. 15 the questions that emerge then are: are such t cells fully functional in the intrahepatic environment, and how does such activity relate to disease progression? the study by accepezzato et al. is a large-scale analysis of intrahepatic cd8 ϩ t-cell populations using class i tetramers and intracellular cytokine staining to better understand exactly the functionality of the intrahepatic virus-specific t cells. these analyses are technically very demanding because of the very low cell yields available, and previously have only examined very limited patient numbers, compared with nearly 50 in this study. 14, 15 as previously, accepezzato et al. found low frequencies of virus-specific t cells in blood in most cases but found generally higher overall frequencies in liver. the maturation state in the liver appears to be more advanced than in blood, reflecting accumulation of "effector memory" (ccr7 ϫ ) cells in the liver. for example, some cells appear to be high in perforin, a situation that is very rarely found in blood among hcv-specific populations. 6, 10 the most interesting results, however, relate to the functionality of the cells. in addition to tetramer staining, the authors stimulated the t cells in vitro with peptide and analyzed the release of ifn-␥ or interleukin 10 (il-10). ifn-␥ is a classical antiviral cytokine that is believed to be of special relevance in the clearance of infected hepatocytes. 17 il-10 is a multifunctional cytokine associated with suppression of t helper 1-type responses. in the present study, three secretion patterns seemed to emerge. in some patients, there appeared to be very weak secretion of either cytokine, while in a few patients there appeared to be a dominance of ifn-␥; several other patients exhibited a dominant il-10 secretory response. interestingly, the overall proportion of cells secreting ifn-␥ in response to viral peptides correlated positively with the level of intrahepatic inflammation (histological activity index score); conversely, the frequency of il-10 secreting cells correlated inversely with the histological activity index score. what is the role of intrahepatic virus-specific cd8ϩ t cells in determining acute viral clearance and immunopathology? with regard to acute viral clearance, this is still very unclear and unlikely to be easily addressed in human studies. it would, for example, be very interesting to know whether the early emergence of il-10 -secreting populations in the liver was associated with failure to initially control the virus. as with many studies, it is very difficult to disentangle cause and effect-il-10 -secreting cells might reasonably emerge as a consequence of long-term inflammation. the observation that even in these chronically infected patients, several had intrahepatic t-cell populations that expressed either no cytokine or largely ifn-␥ suggests that induction of il-10 secretion cannot alone explain the propensity of hcv to evade cd8ϩ t cells. regarding pathology, il-10 -secreting cd8ϩ t cells may play an important role in suppression or regulation of inflammation-a feature that actually might represent an appropriate adaptation of t-cell responses to chronic antigen exposure. in this respect, they may be akin to the more classical cd4 ϩ t regulatory cells. such populations may have anti-self specificities; however, persistent stimulation may induce similar regulatory activity even in cd4 ϩ t cells specific for foreign antigens. a role for cd4 ϩ cd25 ϩ t cells in regulation of peripheral cd8 ϩ t-cell responses has recently been proposed. 18 why, then, might some patients generate il-10 -secreting t-cell populations and others not? one question arising from the study is whether the phenotype observed reflects an adaptation only of hcv-specific t cells, or whether it is a feature of the overall infiltrate. hcv-specific t cells appear to represent only a relatively small fraction of the overall intrahepatic cd8 ϩ t-cell response (about 1% in this study, although perhaps slightly higher than this if other epitopes were included). simply analyzing the overall cytokine preferences of intrahepatic t-cell infiltrates might be very informative. there are suggestions that polymorphisms in chemokine and chemokine receptor genes might influence intrahepatic pathology, so a genetic basis (e.g., in cytokine/receptor genes) for these distinct responses might be relevant. 19 alternatively, viral factors might lead to diverse outcomes; genotype did not appear to play an obvious role, although this issue is confounded by the fact that some of the peptides used are poorly cross-reactive. viral mutants (altered peptide ligands) emerging in vivo have been associated with modulation of cytokine secretion of t cells. 20 finally-looking forward-could the cytokine secretion profiles of intrahepatic t cells be potentially linked to treatment response? this is an intriguing question, given that it is observed that combination therapy for chronic intrahepatic t-cell decision-making. cd8 ϩ t-cell responses found in blood are usually weak and are low in markers of activation and maturation. in the liver, cd69 expression-indicating recent activation-is observed, and in the present study, perforin expression is also described. cytokine secretion appears to take one of two main paths: predominantly ifn-␥ or predominantly il-10. although the molecular and cellular pathways are not understood in detail, it is likely that ifn-␥ secretion has antiviral activity but is also proinflammatory, while il-10 is anti-inflammatory. the long-term effect on fibrosis (which was generally fairly mild in the present study) requires further analysis. disease boosts previously weak or undetectable t-cell responses in the periphery. 21, 22 it is still not clear to what extent such responses are reflected in the liver and how they link ultimately with sustained virological responses. nevertheless, it has been shown that the addition of ribavirin to treatment regimens reduces the il-10 secretion of recovered antiviral t cells. 22 thus, it seems plausible that treatment will shift the cytokine balance in the liver, which could influence significantly the overall success of therapy. one possible message from this study is that t cells in hcv appear to be able to adapt in the face of a persisting virus, and in doing so modulate the intrahepatic environment to one that is less inflammatory (fig. 1) . the relationship between host and virus in hcv infection is potentially a long-term one. as in human relationships, each partner is capable of making compromises to minimize confrontation. in some cases this behavior modification is more successful than others. it remains to be seen whether-in cases where the relationship is breaking down-we can but watch from the sidelines, or could possibly intervene to re-educate t cells into a more appropriate response. of polymerase errors. accordingly, their coding capacity is limited by the probability of generating a lethal mutation. genome sizes range from the 3 kb of hepatitis b virus (hbv) up to the 27 to 32 kb typical of the coronaviruses. the error threshold is that mutation rate just compatible with viable replication. chemical mutagenesis or the incorporation of ambiguous bases can displace mutation rates beyond the error threshold so resulting in the collapse of information. 1,2 given this, many have wondered whether nature has not seized upon this singular vulnerability of rna viruses and retroviruses to "going over the edge." for nearly 20 years it has been known that negative stranded genomes, particularly those of measles virus, may undergo genetic editing of adenosine in the context of double stranded rna. multiple adenosine residues would be deaminated, resulting in inosine. as inosine base pairs as guanosine, adenosine editing generated a3 g hypermutants. 3 some years later another form of hypermutation cropped up among the classical retroviruses. 4 massive and monotonous substitution of g for a, involving up to 60% of g residues, was distributed across the entire 10-kb hiv-1 genome. although the frequency and degree of g3 a hypermutation are most striking for the lentiviral subgroup of retroviruses, which includes human immunodeficiency virus (hiv), elsewhere g3 a hypermutants have been described for only a handful of retroviruses including the "other" human retrovirus, human t-cell leukemia virus (htlv). the situation took a fascinating turn when will's group sequenced a couple of subgenomic hbv dna molecules from the serum of a single patient. 5 the genomes showed signs of extensive g3 a substitution at a frequency typical of hiv g3 a hypermutants. since then, these two hypermutants have remained the only such examples despite a burgeoning hbv database. the fact that g3 a hypermutation is found among viruses with obligatory reverse transcription steps, notably hbv and the primate lentiviruses, suggests a common mechanism occurring in the cytoplasm. the conceptual break leading to an understanding of retroviral g3 a hypermutation has come recently in two stunning punches. first, the vif gene is conserved among all the primate lentiviruses, where vif is an abbreviation for "viral infectivity factor." some established t-cell lines are permissive for the replication of hiv-1 ⌬vif viruses while others are not, suggesting restriction by a host cell protein. using a subtractive screen malim's group in london showed that a single gene product, cem15, was responsible for restricted hiv replication. 6 when screened against the databases, cem15 proved to be identical to apobec3g, which is part of a 7-gene cluster that mapped to chromosome 22q13. 7 what are these genes, denoted apobec3a-g? the sequences of all seven show clear amino acid homology to cytidine deaminases, including that of escherichia coli, but particularly the mammalian enzyme apobec1. this name is derived from the fact that the protein is the catalytic subunit of the "apolipoprotein b editing complex" that specifically deaminates cytidine c6666 to uracil (u) in apolipoprotein b messenger rna (mrna). 8 second, a crop of five papers showed that when a hiv-1⌬vif virus was cotransfected along with a human apobec3g complementary dna (cdna) clone, the molecule was incorporated into budding virions. upon infection of a susceptible target cell, g3 a hypermutants were recovered with alacrity. as only g3 a substitutions were found, even though reverse transcription results in double strand (ds) dna formation, this suggested that only one strand was being edited. 9 -13 this was only (bio)logical if the nascent minus dna strand was being edited, which was rapidly confirmed. all groups showed that viral genomic rna was not edited. deamination of cytidine residues in neosynthesized minus strand dna yields uracil and occurs post-cdna synthesis in a manner independent of reverse transcriptase. 14 now as u base pairs with adenosine, when apobec3g-edited minus strand dna is copied into plus strand dna by reverse transcriptase, the multiple us are copied into a. although referred to as g3 a hypermutants the "action" concerns c residues on the minus dna strand. upon this vibrant stage, turelli et al. have come forth with an intriguing study of the effect of apobec3g expression on hbv replication. 15 they assayed core-associated hbv dna resulting from transfection of human hepatoma huh7 cells with a hbv-producing plasmid. when cotransfected with human apobec3g, hbv dna synthesis was strongly curtailed. important controls showed that apobec3g was incorporated into the core particles yet did not affect hepatitis b c antigen (hbcag) production. however, three findings suggest that hbv does not parallel the hiv hypermutation paradigm. firstly, g3 a hypermutated hbv dna was not found despite searching. secondly, core-associated hbv rna was reduced more than 10-fold, suggesting that the block in hbv dna synthesis results primarily from an inhibition of viral pregenomic rna packaging. finally and remarkably, serine substitutions of functionally critical cysteine residues in apobec3g failed to abrogate the antiviral activity for hbv but did so in the hiv control-so controls are useful! for the purist, a negative result, the inability to find hypermutated hbv dna-remains just that. however, the antiviral activity of the apobec3g serine mutants and reduced rna packaging represent positive results, which are challenging, to say the least. certainly the lack of hypermutants is coherent if the cytosine deamination activity of apobec3g is not involved in hbv restriction. the vexing point is that the experiments were driven with single strand (ss) dna cytosine deamination as the working hypothesis, even though hbv g3 a hypermutants do exist, albeit rarely. commenting on the science paper in the form of a "technical comment," rösler et al. identified bona fide g3 a hypermutants at low frequency in an analogous transfection protocol, albeit using the widely known cell line hepg2. 16 to complicate matters, they failed to identify hyermutants using huh7 cells, which led them to postulate a cell line effect. commenting on rösler et al., turelli et al. refuted this idea because they could achieve apobec3g restriction of hiv using huh-7 as a transfection support. 17 the latter comment is especially interesting in that it shows that hbv replication can be restricted by apobec3f. 17 now, among the apobec3 cluster of gene products, only apobec3f and 3g can restrict hiv replication. each has a subtle sequence bias in the way it deaminates dna. apobec3f shows a preference for cytidine in the context of tpc, while apobec3g prefers the cpc. interestingly, the two naturally hypermutated subgenomes showed an overall bias for tpc, indicating that they were probably edited more by apobec3f than by apobec3g. 5 this nicely fits with the new finding. as for hiv, the same two apobec3 members are involved in hbv restriction. at the low resolution of whole-liver mrna profiling, only apobec3c is strongly expressed, while apobec3f and apobec3g are expressed at borderline levels (http://genecards.bcgsc.bc.ca/). by contrast, immune cells express copious amounts of most apo-bec3 molecules. hence, apobec profiling of liver tissue might well reflect circulating lymphoid cells. if hepatocytes expressed little or no apobec3 molecules this would help explain the dearth of naturally observed hbv g3 a hypermutants in the databases. how can one square hbv restriction by apobec3g when there is little expression of apobec3g in the normal liver? perhaps the mrna profiling is too macroscopic, too low-resolution to be of much use. alternatively, the inflammatory response to hbv might upregulate apobec3g. certainly the pkca/␤i / mek / erk pathway has been shown control basal levels of apobec3g mrna in some t-cell lines, 18 which consequently declined when cells were treated with inhibitors or arrested in the g 0 state of the cell cycle by serum starvation. alternatively, given the expression of most apobec3 molecules in lymphoid tissue, rare and abortive infection of cd4 ϩ and cd8 ϩ t lymphocytes by hbv is another working hypothesis. 19 could apobec3g function by simply binding to c residues in hbv genomic rna so precluding it from becoming packaged? but if so, why should this not occur for hiv replication? we do not know. yet the parallel with human apobec1 is striking: when expressed alone in e. coli, it is highly mutagenic for dna, 20 yet if incorporated into an editing complex it edits a single c residue in the apolipoprotein b mrna. could it be that just beneath the plasma membrane apobec3g acts nonspecifically as a ssdna cytosine deaminase, whereas deep down in the endoplasmic reticulum where hbcag particles are assembled, it is part of a multiprotein complex that modulates the activity of the apobec3g subunit? an analysis of primate apobec3g gene sequences indicates that they are evolving under positive selection although selection is present in lineages for which there is no natural simian immunodeficiency virus (siv), such as orangutans and macaques. 21 these primates are of asian origin, whereas all naturally occurring sivs are found in equatorial africa. hbv has arguably been in primates for a longer period of time than has siv-the presence of hbv-like viruses in gibbon and orangutan are cases in point. 22, 23 the absence of hbv and siv in the macaque lineage suggests that selection on apobec3g is probably unrelated to retroviruses. the lack of restriction of single mouse homologue of apobec3g on murine retroviral vectors 24 suggests that a blanket interpretation of these molecules as part of innate antiretroviral immunity is too simplistic, at least for the moment. while no cellular function has been ascribed to apobec3g and its immediate paralogues, once in place some of them represent formidable barriers to retroviral infection. retroviruses have either to avoid cells in which apobec3 molecules are abundantly expressed or escalate and overcome the obstacle via the acquisition of some novel gene product. otherwise their genomes will be edited beyond the error threshold and into oblivion. the lentiviral vif gene fits the latter scenario. the paper by turelli et al. shows that there is far more to apobec3 genes than initially thought. these are exciting times with much to be done. it will be fascinating to see how the picture develops. different clinical behaviours of acute hcv infection are associated with different vigor of the anti-viral t cell response analysis of a successful immune response against hepatitis c virus ctl responses are induced during acute hcv infection but are not sustained the outcome of hepatitis c virus infection is predicted by escape mutations in epitopes targeted by cytotoxic t lymphocytes ultra-sensitive class i tetramer analysis reveals previously undetectable populations of antiviral cd8ϩ t cells sustained dysfunction of antiviral cd8ϩ t lymphocytes after infection with hepatitis c virus determinants of viral clearance and persistence during acute hepatitis c virus infection impaired effector function of hepatitis c virus-specific cd8ϩ t cells in chronic hepatitis c virus infection memory cd8ϩ t cells vary in differentiation phenotype in different persistent virus infections pervasive influence of hepatitis c virus on the phenotype of antiviral cd8ϩ t cells suppression of host immune response by the core protein of hepatitis c virus: possible implications for hepatitis c virus persistence hla class i-restricted cytotoxic t lymphocytes specific for hepatitis c virus. identification of multiple epitopes and characterization of patterns of cytokine release liver derived ctl in hcv infection; breadth and specificity of responses in a cohort of patients with chronic infection quantitative analysis of hcv-specific cd8ϩ t cells in peripheral blood and liver using peptide-mhc tetramers direct ex vivo comparison of the breadth and specificity of the t cells in the liver and peripheral blood of patients with chronic hcv infection virus-specific cd8 ϩ t lymphocytes within the normal human liver viral clearance without destruction of infected cells during acute hbv infection suppression of hcv-specific t cells without differential hierarchy demonstrated ex vivo in persistent hcv infection association of genetic variants of the chemokine receptor ccr5 and its ligands, rantes and mcp-2, with outcome of hcv infection immunobiology of hepatitis c virus (hcv) infection: the role of cd4 t cells in hcv infection the dynamics of t-lymphocyte responses during combination therapy for chronic hepatitis c virus infection hepatitis c virus-specific t-cell reactivity during interferon and ribavirin treatment in chronic hepatitis c the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen lethal mutagenesis of hiv with mutagenic nucleoside analogs biased hypermutation and other genetic changes in defective measles virus in human brain infections wain-hobson s. selection, recombination, and g3 a hypermutation of human immunodeficiency virus type 1 genomes naturally occurring hepatitis b virus genomes bearing the hallmarks of retroviral g3 a hypermutation isolation of a human gene that inhibits hiv-1 infection and is suppressed by the viral vif protein an anthropoid-specific locus of orphan c to u rna-editing enzymes on chromosome 22 molecular cloning of an apolipoprotein b messenger rna editing protein hypermutation of hiv-1 dna in the absence of the vif protein the cytidine deaminase cem15 induces hypermutation in newly synthesized hiv-1 dna broad antiretroviral defence by human apobec3g through lethal editing of nascent reverse transcripts dna deamination mediates innate immunity to retroviral infection species-specific exclusion of apobec3g from hiv-1 virions by vif apobec3g is a single stranded dna cytidine deaminase and functions independently of hiv reverse transcriptase inhibition of hepatitis b virus replication by apobec3g comment on "inhibition of hepatitis b virus replication by apobec3g author reply to comment on "inhibition of hepatitis b virus replication by apobec3g transcriptional regulation of apobec3g, a cytidine deaminase that hypermutates human immunodeficiency virus distribution of hepatitis b virus dna sequences in different peripheral blood mononuclear cell subsets in hbs antigen-positive and negative patients rna editing enzyme apobec1 and some of its homologs can act as dna mutators rapid evolution of primate antiviral enzyme apobec3g a new group of hepadnaviruses naturally infecting orangutans (pongo pygmaeus) complete sequencing of a gibbon hepatitis b virus genome reveals a unique genotype distantly related to the chimpanzee hepatitis b virus apobec3g targets specific virus species key: cord-258665-8q3tsggm authors: aydın, hakan berk; cheema, jamal ahmed; ammanath, gopal; toklucu, cihan; yucel, muge; özenler, sezer; palaniappan, alagappan; liedberg, bo; yildiz, umit hakan title: pixelated colorimetric nucleic acid assay date: 2020-03-01 journal: talanta doi: 10.1016/j.talanta.2019.120581 sha: doc_id: 258665 cord_uid: 8q3tsggm abstract conjugated polyelectrolytes (cpes) have been widely used as reporters in colorimetric assays targeting nucleic acids. cpes provide naked eye detection possibility by their superior optical properties however, as concentration of target analytes decrease, trace amounts of nucleic acid typically yield colorimetric responses that are not readily perceivable by naked eye. herein, we report a pixelated analysis approach for correlating colorimetric responses of cpe with nucleic acid concentrations down to 1 nm, in plasma samples, utilizing a smart phone with an algorithm that can perform analytical testing and data processing. the detection strategy employed relies on conformational transitions between single stranded nucleic acid-cationic cpe duplexes and double stranded nucleic acid-cpe triplexes that yield distinct colorimetric responses for enabling naked eye detection of nucleic acids. cationic poly[n,n,n-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide] is utilized as the cpe reporter deposited on a polyvinylidene fluoride (pvdf) membrane for nucleic acid assay. a smart phone application is developed to capture and digitize the colorimetric response of the individual pixels of the digital images of cpe on the pvdf membrane, followed by an analysis using the algorithm. the proposed pixelated approach enables precise quantification of nucleic acid assay concentrations, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses, for instance, rgb analysis. the obtained results illustrate that a ubiquitous smart phone could be utilized for point of care colorimetric nucleic acids assays in complex matrices without requiring sophisticated software or instrumentation. bio-sensors play an essential role in medical diagnostics, environmental monitoring and food safety analysis [1, 2] . nucleic acid assays are key targets in biosensing that enables viral load monitoring and disease diagnosis [3, 4] . conventional assays such as polymerase chain reaction (pcr), phenol-chloroform extraction and electrophoresis are reliable, but requires laboratory techniques that are time consuming [5] . rapid disease diagnostic tests are however required for monitoring infections such as hepatitis, dengue, etc. [6] [7] [8] [9] furthermore, developing countries often lack infrastructure as well as properly trained personnel to provide accurate diagnosis [10, 11] . recently, the concept of "lab on paper" has generated significant interest for nucleic acid assaying due to its ease of use and cost effectiveness, especially for applications in resource limited settings [12] [13] [14] [15] [16] [17] . conjugated polyelectrolytes such as cationic polythiophene have been explored for biosensing applications due to its unique optical and electronic properties [18, 19] . the colorimetric response of cpe relies on conformational alternations in the backbone of the polymer [20, 21] . the conformational transitions are usually followed by changes in fluorescence intensity and the extent of the conformational alternations in backbone depend on the energy required for interring twisting. xia et al. [22] . suggested a strategy for colorimetric detection of dna, protein and small molecules by incorporating water-soluble conjugated polyelectrolyte and gold nanoparticle with the target molecules. although the target analytes could be colorimetrically ascertained for subnm concentrations, assaying in solution state is tedious and yields optimal responses only in a controlled laboratory condition [22] . therefore, polythiophene-mirna complexation on a paper-based platform for detection of mir21 sequence was first evaluated by yildiz et al. [23] . the complementary peptide nucleic acid (pna) sequence to mir21 was deposited on the cpe impregnated pvdf paper. naked eye perceivable colorimetric responses were obtained at clinically relevant concentration ranges upon addition of mir21 in buffer solutions [23] . in a subsequent study, the colorimetric responses were evaluated by an image processing software that enabled quantification of nucleic acids in plasma samples [24] . other reports for detection of oligonucleotide which are related to middle east respiratory syndrome coronavirus (mers-cov), mycobacterium tuberculosis (mtb) and human papillomavirus (hpv) also indicate the potential applications for paper based sensors [25] . however, most of the current paper-based assays are dependent on either naked eye interpretation of colorimetric responses or an image processing software for analysis, which may introduce artefacts thereby inhibiting accurate quantification of colorimetric responses. furthermore, conventional image processing software requires a server or a computer as well as several intermediate data transfer steps for diagnosis [26, 27] . recently, smart phone based detection methodologies have attracted significant attention [28] [29] [30] [31] . herein, we propose a smart phone application algorithm that evaluates the colorimetric responses based on a pixel level analysis approach, enabling quantification of nucleic acids with high precision at clinically relevant concentration ranges. the algorithm analyzes color differences and retrieves the color profiles of the pixels of the sample droplet pattern of the cpe on pvdf membrane for reliable and sensitive interpretation of colorimetric responses. the cpe employed herein is cationic poly [n,n,n-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide], referred to as pt. pt as an active layer on pvdf membrane incorporated within a cartridge (scheme 1) generates two distinct optical signals with a color transition from orange to pink/grey, in case of hbv dna-pt duplex formation or intact orange color in case of hbv dna-pna-pt triplex formation (pna, complementary to target hepatitis b viral dna is used as a model system). hence, the orange color (hbv dna-pna-pt) and the significantly different pink/grey color (pna-pt) enables colorimetric or naked eye detection of target hbv dna. however, trace amounts of nucleic acids often yield weak colorimetric responses that are not perceivable by naked eye indicating the requirement of additional image processing protocols. in this study, an algorithm has been developed for precise interpretation of colorimetric response of pt on pvdf membranes. experimental results suggest that the developed algorithm enables reliable determination of colorimetric responses that are not readily perceivable by the naked eye nor by conventional rgb analysis. the smart phone application based on the described algorithm has been developed and validated for the detection of hbv dna. the algorithm could be installed in an application format on any smartphone with a camera for capture and analysis of colorimetric responses, and for enabling point of care nucleic acids assays in complex matrices. furthermore, the proposed cartridge-based assay and smart phone application offer great potential for rapid and point of care screening of infectious diseases. pt was synthesized as described elsewhere [23] . all the chemicals for pt synthesis were purchased from sigma-aldrich and used without further purification. deionized (di) milliq water (resistivity of 18 mω cm) was used for buffer preparation and pt synthesis. the following hbv dna and pna sequences were purchased from idt and panagene, respectively. hbv pvdf filter centrifugal columns were purchased from merck millipore. sony xperia x compact (android 7.1.1 nougat, chipset: qualcomm msm8956 snapdragon 650, cpu: hexa-core (4 × 1.4 ghz cortex-a53 & 2 × 1.8 ghz cortex-a72), gpu: adreno 510, ram: 3gb) camera was utilized for capturing digital images of the samples. the uv chamber and cartridges were fabricated by using 2 mm thick black poly (methylmethacrylate) panels that were laser cut by epilog laser cutter. the pvdf membranes were then mounted on top of the cartridge and stored at 4°c. human plasma was obtained from genetex, taiwan. pt was coated onto pvdf centrifugal tubes by adding 200 μl of varying concentrations of pt. tubes were centrifuged at 8000 rpm for 3 min. the filtrate was again added to the tube and centrifuged to obtain a homogeneous deposition of pt on pvdf membrane. this process was repeated several times to optimize pt coating on pvdf membrane based on the fluorescence intensities. subsequently, the deposited films were washed 5 times with di water by centrifuging at 8000 rpm for 3 min 2 μl of hbv dna at varying concentrations in plasma samples were then dropped on pt incorporated pvdf membrane to serve as control spots. pvdf membranes with 2 μl of prehybridized hbv dna and pna was also prepared as a reference sample to serve as sample spots. as illustrated in scheme 1, the sample is dropped on to the center of control and sample spots of the pmma cartridge and placed in the uv chamber prior mounting the smart phone. the developed smartphone application named biorgb is then activated for recording the images for pixelated analysis. all the images are captured by a digital camera with a resolution of 2000 × 2000 pixels. scheme 1 illustrates the proposed detection methodology, utilizing a smart phone attached to a uv-led chamber and a cartridge consisting of pmma cover and pt impregnated pvdf membrane. the cartridge has three spots: reference spot, control spot, (hbv dna) and sample spot (hbv dna + pna). an optical transition occurs upon dropping the sample on the cartridge, and then it is subsequently quantified by mobile application. unlike previous report that utilize an image processing software, for instance imagej software, to quantify optical transition of pt at nm concentration levels of nucleic acids based on selected regions of interest, the proposed mobile application scans the individual pixels of the sample droplet pattern. the pixelated analysis of droplet pattern yields high statistical precision to set up corroborating correlation between control and sample spots. analysis of individual pixels is of utmost importance in colorimetric assaying as the color transitions are usually weak at low analyte concentrations. the process of color analysis by the smart phone application is illustrated in fig. 1a . the application is designed to run on a three-step operation modes. in the first step, the algorithm locates the vertical centroid of the digital image via analysis of rgb components of individual pixels along the vertical direction. a horizontal red line along the vertical centroid is illustrated in fig. 1a fig. 1a , are recorded for the pixels corresponding to the pt deposited on pvdf membrane. upon recoding rgb color codes of all the pixels along the vertical centroid, the algorithm records additional nine sets of horizontal axis pixel color codes, that correspond to four pixels above and five pixels below the vertical centroid in order to ascertain the colorimetric responses. the algorithm then constructs a 10 × 2000 matrix to determine red, green blue component of all the individual pixels along the scanning direction (detailed description of image processing is provided in the supporting information; fig. s1 (a)-(e)). finally, the algorithm executes an averaging operation to yield a pixelated color code that is displayed at the user interface in form of a bar chart. in order to test the microstain recognition performance of the smart phone application, 4x4 pixel microstain has been created artificially on the image shown in fig. 1b (25x magnification is shown in fig. 1c) . rgb color codes were found to be as [221, 165, 79] on microstain region while it was [250, 187, 90] on the rest of the image. as shown in fig. 1c , the red intensity profile centered at 250 drops significantly to 220 for the microstain region, assuring that the microstain with a slight color difference with respect to the background can be recognized by the smart phone application. the next validation experiments for the smart phone applications was conducted by creating an artificial droplet pattern as shown in fig. 1d . the red, green and blue intensity profiles of the artificial droplet pattern exhibit two symmetrical minima corresponding to the outer and inner edges of the rim of the droplet pattern. these results show that the smart phone application is capable of recognition of both microstains and droplet patterns and might be subsequently utilized for colorimetric analysis of pt droplets on pvdf membranes. the red component exhibits the steepest change as compared to blue and green since the optical transition of pt is in the range of 500-600 nm. the algorithm is therefore set to utilize the red components for analysis and quantification of nucleic acids. in next step, we conducted experiments by using hbv dna to validate smart phone application for pixelated nucleic acid assay. fig. 2a shows a typical image analysis of reference, control, and sample spots (from left to right) in which hbv dna concentration is 1 μμ. the red intensity of control spot (pt + hbv dna), is substantially lower than reference (pt) and sample (pt + hbv dna + pna) spots, indicating significant quenching of pt by hbv dna at a concentration of 1 μμ. the red intensity variation of control spot from 240 (pt) to 160 (pt + hbv dna), as evaluated by the algorithm, concurs with the dark red color of the droplet profile observed from the corresponding digital images. however, at lower hbv dna concentrations shown in fig. 2b , naked eye interpretation of colorimetric response of the control spots may not be readily feasible. therefore, in order to maximize the colorimetric responses, the stoichiometric ratio of pt with respect to hbv dna concentration to be evaluated (pt concentration on pvdf) and the homogeneity of pt on pvdf membrane is optimized by adopting a multi-coating approach. fig. 2b shows the images of 5, 6, 7 and 8 times pt centrifuged pvdf membranes with varying concentration of hbv dna between 0 and 50 nm. the red intensity profile of 5 and 6 times pt centrifuged pvdf membranes changes from 189 to 182 and 188 to 186, respectively, upon consecutive addition of hbv dna, whereas 7 and 8 times pt centrifuged pvdf membrane yield significantly larger changes in red intensity with increasing hbv dna concentrations, from 189 to 154 and 188 to 155, respectively. fig. 2c illustrates that there is no significant difference in red intensity changes upon addition of hbv dna between 7 and 8 times pt centrifuged pvdf membrane. fig. 2d scheme 1. schematic illustration of cartridge and smart phone-assisted nucleic acid assay. step 1 illustrates the assembly of plastic cartridge consisting of three layers; step 2 shows the transfer of hbv dna sample to the cartridge and step 3 shows the mounting cartridge on the uv-led chamber that serves as an accessory for mounting the smart phone. step 4 illustrates the smart-phone assisted biorgb analysis yielding bar graphs that represents the pixelated (and averaged) red intensities of the three sample spots. indicates that the total color response or hue of 5 and 6 times pt deposited pvdf membrane is lower than 7 and 8 times pt centrifuged pvdf membrane, further ascertaining that there is no substantial difference in colorimetric response upon hbv dna addition between 7 and 8 times pt centrifuged pvdf membrane. therefore, 7 times pt centrifuged pvdf membranes are utilized for the proposed pixelated nucleic acid assay. fig. 3a shows the biorgb output of the 7 times pt centrifuged pvdf membranes in the form of bar graphs that correspond to averaged red intensities of the individual pixels of the digital images of the membranes. as for sample and control membrane, the algorithm is set to yield a color code of 10 × 1000 pixels (starting from the edge of the droplet pattern, fig. s1 (e) , as a droplet of sample typically spreads to over 1000 pixel diameter area). the algorithm eventually displays the averaged values of 500 pixels along the horizontal axis as 20 bar graphs for visual interpretation of colorimetric responses, as illustrated in fig. 3a. δ red values (fig. 3b shows the illustration of algebraic derivation of color matrix for obtaining δ red values by subtracting rgb color codes of control from that of sample) increases with hbv dna concentration providing a linear correlation between hbv dna concentration and δ red values. fig. 3c illustrates the residual plots that show a δ red scattering of less than 1% over the concentration range of interest. the residual plot ascertains that biorgb application is reproducible, with responses varying within 1% for the 1 nm to 1 μm concentration range of hbv dna. the calibration curve illustrated in fig. 3d , obtained from the average δ red values of corresponding concentrations shown in fig. 3a , indicates good linearity within the concentration ranges of interest. the final validation of proposed methodology was conducted using the hbv dna spiked into plasma. fig. 4a illustrates that the digital image does not reveal a definite droplet shaped pattern for membrane upon addition of hbv dna, and that visual identification of hbv dna is not feasible for lower concentrations (around 1 nm). however, biorgb analysis yields distinguishable δ red signals for 1, 10, 100 and 1000 nm as 10, 25, 50 and 60, respectively (fig. 4b , difference between control and sample). it is to be noted that the algorithm analyzes the response from all the individual pixels rather than by the averaging regions of interest, an approach adopted by typical image analysis software. as concentration increases from 1 nm to 1 μm, the color saturation and darkening occurs, and therefore the control spot (hbv dna) and sample spot (hbv dna + pna) are distinguishable by biorgb. as shown in the residual plot in fig. 4c , the scattering of δ red values are 2.5%, which is larger than for the responses obtained in di. the major reason of the increase in scattering may due to increase in refractive index (turbidity) in plasma samples. however, the scattering observed in plasma samples does not deteriorate the data-driven decision of biorgb. fig. 4b illustrates that the proposed biorgb smartphone application possesses a scattering of less than 2.5% (reproducibility of over 97.5%) within the hbv dna concentration range of 1 nm to 1 μm. in the next step control experiments have been performed. the first control experiment perfomed with dna1 (dna1: 5' -ttt ata gaa gta gtg gta cc-3′ non complementary sequence to pna) shows that the responses of dna1-pna are much lower than hbv dna-pna (see s2 ). this result demonstrates that pna is specific to hbv dna sequence. the interference experiment has performed in the co-existence of target hbv dna sequence and dna1. the result shown in s3 reveals that the non-complementary dna1 does not associating complementary pna as compared to target hbv dna and the effect of noncomplementary dna1 on red intensity value is marginal. the response of the proposed assay to longer dna sequence (dna 31 = 31 base pairs) has been also tested since the real samples may contain longer sequence. the result shown in fig. s4 demonstrated that red intensity for hbv dna (shorter sequence) and dna 31-pna 31 (longer sequence) are nearly identical and this assures the proposed assay is applicable for the longer dna sequences as well. the colorimetric responses shown in fig. 4a are then analyzed by imagej software in order to validate the biorgb analysis. rgb analysis yielded red intensity variations of 15, 25, 45 and 65 for 1, 10, 100 and 1000 nm of dna, respectively, which are comparable to those obtained using biorgb. however, these values tend to vary depending on the regions of interest that are subjective to human judgement. furthermore, scattering intensities in rgb based colorimetric analysis are prone to be higher owing to manual section of pixels (as observed from fig. s5 , for concentration of 10 nm and higher), which compromises the overall sensitivity and robustness of the assay. in contrary, the proposed methodology eliminates the error involved in defining the regions of interest for image analysis approach and also yields a sensitive approach for analyzing colorimetric responses. a cartridge-based point of care assay for colorimetric detection of nucleic acids has been developed using a smart phone algorithm. the sensor approach is ideal for use in resource limited settings, where colorimetric response can be captured and interpreted using a simple smartphone. the smartphone application is capable of capturing and digitizing the colorimetric responses followed by an analysis of the individual pixels using an in-built algorithm. the proposed pixelated approach yields quantitative dna concentration read outs, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses. the developed algorithm can be installed in an application format in any smartphone with a camera, for capture and analysis of colorimetric responses, enabling point of care nucleic acids assays in complex matrices. the obtained results illustrate that a ubiquitous smartphone can be utilized for colorimetric nucleic acids assays in complex matrices via the developed application that perform point of care without requiring sophisticated software nor instrumentation. successful detection of hbv dna was demonstrated in plasma, mimicking clinical samples, with a detection limit of 1 nm. the recent hbv detection methodologies in table 1 demonstrate a comparison between present study and others. the major advantage of our method appears as instrument-free detection at nm to μm., our strategy can be expanded for detection of other nucleic acid sequences of interest by incorporating appropriate complementary pna probes on the cartridges. overall, the proposed methodology significantly accelerates diagnosis at near patient location within seconds making them facile, inexpensive and ideal for applications in point of care diagnosis. the manuscript describes a smart phone based methodology for point-of-care nucleic acid assay without laborious lab settings. the methodology suggested here is suitable for cost-effective, rapid and facile screening of infectious diseases at clinical levels. the authors declare no conflict of interest. biosensors and their applications -a review critical overview on the application of sensors and biosensors for clinical analysis advances in biosensing strategies for hiv-1 detection, diagnosis, and therapeutic monitoring biosensor for dengue virus detection: sensitive, rapid, and serotype specific paperbased sample-to-answer molecular diagnostic platform for point-of-care diagnostics advances and challenges in biosensorbased diagnosis of infectious diseases diagnosing dengue virus infection: rapid tests and the role of micro/nanotechnologies recent advances in micro/nanotechnologies for global control of hepatitis b infection point-of-care nucleic acid testing for infectious diseases diagnostics for developing countries nucleic acid testing-benefits and constraints 3d capillary-driven paper-based sequential microfluidic device for electrochemical sensing applications diagnostics for the developing world: microfluidic paper-based analytical devices equipment-free detection of k+ on microfluidic paper-based analytical devices based on exhaustive replacement with ionic dye in ion-selective capillary sensors a fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection single-step recombinase polymerase amplification assay based on a paper chip for simultaneous detection of multiple foodborne pathogens ultrasensitive paper based nucleic acid detection realized by three-dimensional dna-aunps network amplification conjugated polyelectrolytes: synthesis, photophysics, and applications optical detection of dna and proteins with cationic polythiophenes recent advances in fluorescent and colorimetric conjugated polymer-based biosensors twisting of conjugated oligomers and polymers: case study of oligo-and polythiophene colorimetric detection of dna, small molecules, proteins, and ions using unmodified gold nanoparticles and conjugated polyelectrolytes naked eye detection of lung cancer associated mirna by paper based biosensing platform tailoring conformation-induced chromism of polythiophene copolymers for nucleic acid assay at resource limited settings multiplex paper-based colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides flow-through colorimetric assay for detection of nucleic acids in plasma luminescent device for the detection of oxidative stress biomarkers in artificial urine smart phone assisted detection and quantification of cyanide in drinking water by paper based sensing platform smartphone instrument for portable enzymelinked immunosorbent assays smartphone spectrometer for colorimetric biosensing smartphone-based food diagnostic technologies: a review detection of hepatitis b virus dna with a paper electrochemical sensor flow-through colorimetric assay for detection of nucleic acids in plasma clinical evaluation of micro-scale chip-based pcr system for rapid detection of hepatitis b virus ultrasensitive paper based nucleic acid detection realized by three-dimensional dna-aunps network amplification hepatitis b plasmonic biosensor for the analysis of clinical serum samples colorimetric detection of hepatitis b virus (hbv) dna based on dna-templated copper nanoclusters gold aggregating gold: a novel nanoparticle biosensor approach for the direct quantification of hepatitis c virus rna in clinical samples this work has been supported by the the scientific and technological research council of turkey tubi̇tak project no:116z547. authors hba, sö, my, and uhy also acknowledge to material research center iyte-mam for imaging facility services. authors sö, my are yök 100/2000 scholarship holders. this research was also supported by the nithm interdisciplinary diabetes and metabolic diseases grant (2016), nanyang technological university, singapore. supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120581. key: cord-297077-p604vvbi authors: tai, dar‐in; jeng, wen‐juei; lin, chun‐yen title: a global perspective on hepatitis b‐related single nucleotide polymorphisms and evolution during human migration date: 2017-11-06 journal: hepatol commun doi: 10.1002/hep4.1113 sha: doc_id: 297077 cord_uid: p604vvbi genome‐wide association studies have indicated that human leukocyte antigen (hla)‐dp and hla‐dq play roles in persistent hepatitis b virus (hbv) infection in asia. to understand the evolution of hbv‐related single nucleotide polymorphisms (snps) and to correlate these snps with chronic hbv infection among different populations, we conducted a global perspective study on hepatitis‐related snps. we selected 12 hbv‐related snps on the hla locus and two hbv and three hepatitis c virus immune‐related snps for analysis. five nasopharyngeal carcinoma‐related snps served as controls. all snp data worldwide from 26 populations were downloaded from 1,000 genomes. we found a dramatic difference in the allele frequency in most of the hbv‐ and hla‐related snps in east asia compared to the other continents. a sharp change in allele frequency in 8 of 12 snps was found between bengali populations in bangladesh and chinese dai populations in xishuangbanna, china (p < 0.001); these areas represent the junction of south and east asia. for the immune‐related snps, significant changes were found after leaving africa. most of these genes shifted from higher expression genotypes in africa to lower expression genotypes in either europe or south asia (p < 0.001). during this two‐stage adaptation, immunity adjusted toward a weak immune response, which could have been a survival strategy during human migration to east asia. the prevalence of chronic hbv infection in africa is as high as in asia; however, the hbv‐related snp genotypes are not present in africa, and so the genetic mechanism of chronic hbv infection in africa needs further exploration. conclusion: two stages of genetic changes toward a weak immune response occurred when humans migrated out of africa. these changes could be a survival strategy for avoiding cytokine storms and surviving in new environments. (hepatology communications 2017;1:1005–1013) c hronic hepatitis b virus (hbv) is a global disease. the majority of carriers of hepatitis b surface antigen (hbsag) are inhabitants of africa and asia. (1, 2) immune tolerance is a hallmark of persistent hbv infection. (3) typically, patients with chronic hepatitis b are infected through their parents in the early stage of life. (4) remarkably, the immune system of the host may respond to the hbv (5) but does not produce the immune clearance of hbv. hbv may replicate in host cells peacefully until they enter immune clearance phases 2-4 decades later. (3) if the hbv can be eradicated, hbv replication will be terminated, and ultimately 50% of hosts may clear hbsag by 80 years of age. (6) genome-wide association studies from asia have revealed that the human leukocyte antigen (hla)-dp and hla-dq loci play roles in persistent hbv infection. (7) (8) (9) (10) (11) (12) (13) our objective is to understand the evolution of the single nucleotide polymorphisms (snps) that were responsible for hbv-related immune tolerance during human migration and to correlate the hbv-related snps with a prevalence of chronic hbv infection among global populations. based on the data from 1,000 genomes collected worldwide, we conducted a global perspective study on the allele frequency of hepatitis-related snps. based on a literature review, 12 hbv-and hlarelated snps, (7) (8) (9) (10) (11) (12) (13) five hepatitis-and immune-related snps in complement factor b (cfb), clusters of differentiation molecule 40 (cd40), and interferon lambda 4 (ifnl4) loci (14) (15) (16) (17) (18) , and five nasopharyngeal carcinoma (npc)-related snps in hla regions (19) (20) (21) were selected for this analysis (tables 1 and 2 ). these snp data from around the world were downloaded from the phase 3 data of 1,000 genomes (http://www. 1000genomes.org/). (22) the subjects participating in the 1,000 genome project were older than 18 years and had three out of four grandparents who identified themselves as members of the group. the location of the 26 populations evaluated in the 1,000 genomes are shown by abbreviation on a global hbsag prevalence map reported by hou et al. (2) (fig. 1) . the allele frequencies of different geographic groups in viral hepatitis-related snps and npc-related snps are illustrated in fig. 2 . the snp genotype differences between groups are listed in tables 1 and 2 . we used interactive chi-square tests to calculate the difference in genotypes between groups (http://quantpsy.org). among two hbv-and immune-related snps in the cfb and cd40 regions (14, 15) and three hepatitis c virus-related snps in the ifnl4 regions, (16) (17) (18) allele type differences can be found between africa and europe or between africa and south asia ( fig. 2a ). all these immune-related snp genotypes differed significantly between esan in nigeria and toscani in italy and between luhya in webuye, kenya (lwk) and gujarati in india (gih) ( table 1 ; p < 0.001). among 12 hbv-and hla-related snps, (7) (8) (9) (10) (11) (12) (13) the allele frequency showed marked differences between south and east asian genome samples (fig. 2b) . eight of the 12 snps differed significantly between bengali in bangladesh (beb) and chinese dai in xishuangbanna, china (cdx); these areas represent the junction of south and east asia ( table 2 ; p < 0.001). three of the 12 hbv-and hla-related snps (fig. 2b , dotted lines; rs9276370, rs3128917, and rs9380343) also showed significant differences between lwk in africa and gih in south asia ( table 2 ; p < 0.001). in contrast, we found the allele frequency of npc-related snps (18) (19) (20) to be relatively stable among different populations (fig. 2c ). based on the well-known human migration pathways (23, 24) and the recent data from 1,000 genomes, (22) our analysis of hepatitis-and immune-related snps demonstrate a significant change in allele frequency shortly after the migration out of africa ( fig. 2a) . all genotypes of five immune-related snps differed significantly between esan in nigeria in africa and toscani in italy in europe and between lwk in africa and gih in south asia (table 1 ; p < 0.001). in addition, both cfb and cd40 shifted from a higher expression in african genotypes (rs12614:tt; rs1883832:cc) to a lower expression in european and south asian genotypes (rs12614:cc; rs1883832: tt). (14, 15) these changes conferred a decrease in the strength of immune responses. the cc genotype of rs12979860 (ifnl4), which is more prevalent in east asia, is associated with a lower baseline ifnl3 (interleukin-28b) expression. (16, 17) the ifnl4 open reading frame is truncated by a polymorphic frame-shift insertion (rs368234815), which turns ifnl4 into a polymorphic pseudogene in east asian populations. (18) because the prevalence of hbsag is higher in africa than in europe or south asia, these trends of decreased immune protein expression are not related to hbv-specific immune tolerance. although it is clear that europeans and south asians are two different races, they showed similar genetic adaptions when they migrated out of africa. these changes suggest that the decreased expression of immune-related genes might have been an important survival strategy when humans migrated into new territories and faced new pathogens. the contact between different races of humans may induce devastating diseases, for example, when the new world was discovered by christopher columbus in 1492. (25) a similar situation was well documented when japan sent troops to taiwan in 1874 and 1895; only 0.1% to 0.3% of soldiers died in battle, while around 10% died of diseases in a short period of time after arrival. (26) our second principal result is that the allele frequency of hbv-and hla-related snps show marked differences between south and east asian genome samples (fig. 2b) . eight of the 12 snps differed significantly between beb and cdx ( table 2 ; p < 0.001). these two populations are located at the junction of south and east asia. the unique allele types of hbv-related snps in east asian populations are different from those of other geographic populations. these genotypic changes could be related to antigen presentation and could be associated with persistent hbv infection. (7) (8) (9) (10) (11) (12) (13) our findings are in agreement with a higher prevalence of hbsag in east asia than in south asia (fig. 1) . these genotypic populations are generally overlapped in the y chromosome haplogroup o1-o3 distribution map (https:// en.wikipedia.org/wiki/human_y-chromosome_dna_ haplogroup) as they started in the indo-china peninsula and travelled to northern china and japan. given the results, we theorized on the reason behind the dramatic allele differences in hbv-related snps between beb in south asia and cdx in east asia. one possible explanation for this variation involves the consideration of environmental landscape factors. (27) for example, bangladesh is a predominately rich, fertile, and flat land, with many areas situated less than 12 m above sea level. on the other hand, xishuangbanna is situated in a mountainous and forested area that has the largest diversity of plants and animals in china. regions with higher plant and animal biodiversity are often accompanied by an increased range and abundance of vector-borne or nonvector-borne diseases. (28) (29) (30) (31) (32) (33) (34) accordingly, the inhabitants of these areas should be able to tolerate an increased number of unfamiliar microorganisms. we speculated that the subjects who demonstrate direct and strong immune responses may die of a cytokine storm in fulminant hepatitis, severe acute respiratory syndrome, influenza, and other infections. (30) (31) (32) (33) (34) this concept is supported by a lower mortality rate from influenza h1n1 in asia than in australia, new zealand, and north america. (35) cytokine storm was first described in graft-versushost disease and was soon also identified in many infectious diseases (36) ; many cytokines, chemokines, and complements are involved. (37) (38) (39) the immunerelated snps selected in this study that included ifn (ifnl4), tumor necrosis factor-receptor (cd40), and complements (cfb) are all participants in cytokine storms. hla class ii molecules are associated with antigen presentation and are also modulated by cytokines. (40) a cytokine storm is considered to be a hyperreaction of the immune response to a pathogen that may cause fulminant disease and mortality. (36) (37) (38) (39) when humans migrate to a new territory, they face many unfamiliar pathogens. those subjects with a strong immune response will die of disease, but those subjects with a weak immune response to the pathogens may survive. chronic hbv infection with an immune tolerance stage is an example of a weak immune response. (3) (4) (5) east asian populations carry similar allele types of hbv-related snps (fig. 2b) , although the environments of northern china and japan differ substantially from those of southern china and the indo-china peninsula. (41) we therefore propose that there was a significant physical block to gene flow on the indo-china peninsula. most of the survivors in east asia exhibit delayed hbv-related immune clearance genotypes. this could have been a survival strategy to pass through the indo-china peninsula and southern china during human migration. such hla class ii genotypes are aimed toward an immune tolerance strategy. (7) (8) (9) (10) (11) (12) (13) these changes were successful because this group of people spread to northern china and japan and have become the largest population in the world numerically. however, such a survival benefit may have been a trade-off with cold tolerance as these populations were unable to cross the bering strait in large numbers. indigenous americans do not show the same hbv-related allele pattern; they have a low prevalence of chronic hbv infection and high influenzarelated mortality rates. (1, 2, 35) overall, we identified two genetic adaptations that occurred during human migration. the first was the decreased expression of immune-related genes after leaving africa; the second was the evolution of an hla system with migration into the indo-china peninsula. both events may have aimed to decrease the strength of the immune response and avoid cytokine storms when facing different types of pathogens. the high prevalence of chronic hbv infection in east asia could be a consequence of such a strategy. however, persistent hbv infection-related hla genotypes are not present in the african population (fig. 2b) and cannot be responsible for the high prevalence of hbsag in africa. different genetic and nongenetic mechanisms of chronic hbv infection are presented between east asian and african populations. (4, (42) (43) (44) we summarize the differences on hbsag carriers between east asia and africa in table 3 . these differences may provide a clue for the mechanism of the function of snps in the persistent hbv infection. the high prevalence of lowexpression-type immune-related snps and chronic hbv infection-related snps on the hla locus may be a reason for a longer hepatitis b e antigen (hbeag)-positive phase in east asia. ifn-alpha has been recommended for treatment of hbeag-positive chronic hepatitis b. in a larger series from pediatric patients, ifn-alpha was found to be an effective therapy in chronic hepatitis b with severe inflammation that facilitates hbeag seroconversion in earlier life. (45) in addition, hbv-and hla-related snps are also associated with spontaneous hbeag seroconversion. (46) (47) (48) these genetic polymorphisms could be a reason for an early hbeag seroconversion and a lower vertical transmission in africa compared to east asia. it is well known that hbv genotypes a, b, and d show an earlier hbeag seroconversion compared to genotype c. (42, 44) this early hbeag seroconversion was suggested to be the reason of low vertical transmission in africa. (49) however, hbv genotype b also had an early hbeag seroconversion but had a high vertical transmission rate in east asia. (4) therefore, host factors rather than hbv genotypes alone should be considered for the high vertical transmission rate in east asia. most hbv-related genome-wide association studies were done in east asia. we need studies to understand the genetic roles in persistent hbv infection in african populations. our study found two stages of genetic changes toward a weak immune response when humans migrated out of africa. these changes could be a survival strategy for avoiding cytokine storms and surviving in new environments. estimations of worldwide prevalence of chronic hepatitis b virus infection: a systematic review of data published between epidemiology and prevention of hepatitis b virus infection natural history of chronic hepatitis b virus infection in taiwan: studies of hepatitis b virus dna in serum effects of sex and generation on hepatitis b viral load in families with hepatocellular carcinoma trained immunity in newborn infants of hbv-infected mothers relative roles of hbsag seroclearance and mortality in the decline of hbsag prevalence with increasing age a genome-wide association study identifies variants in the hla-dp locus associated with chronic hepatitis b in asians a genome-wide association study of chronic hepatitis b identified novel risk locus in a japanese population new loci associated with chronic hepatitis b virus infection in han chinese genome-wide association study confirming association of hla-dp with protection against chronic hepatitis b and viral clearance in japanese and korean a genome-wide association study identified new variants associated with the risk of chronic hepatitis b association between hla variations and chronic hepatitis b virus infection in saudi arabian patients a genome-wide association study on chronic hbv infection and its clinical progression in male han-taiwanese genetic variants in five novel loci including cfb and cd40 predispose to chronic hepatitis b association of cd40 -1c/t polymorphism in the 5 0 -untranslated region with chronic hbv infection genome-wide association of il28b with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis c impaired induction of interleukin 28b and expression of interferon k 4 associated with nonresponse to interferon-based therapy in chronic hepatitis c selection on a variant associated with improved viral clearance drives local, adaptive pseudogenization of interferon lambda 4 (ifnl4) genome-wide association study reveals multiple nasopharyngeal carcinoma-associated loci within the hla region at chromosome 6p21.3 evaluation of human leukocyte antigen-a (hla-a), other non-hla markers on chromosome 6p21 and risk of nasopharyngeal carcinoma the principal genetic determinants for nasopharyngeal carcinoma in china involve the hla class i antigen recognition groove genomes project consortium a global reference for human genetic variation timing the first human migration into eastern asia late pleistocene climate drivers of early human migration the role of epidemic infectious diseases in the discovery of america germs of disaster: the impact of epidemics on japanese military campaigns in taiwan, 1874 and 1895 evolution and dispersal of the genus homo: a landscape approach vectors vs. humans in australia--who is on top down under? an update on vector-borne disease and research on vectors in australia influence of vectors' risk-spreading strategies and environmental stochasticity on the epidemiology and evolution of vector-borne diseases: the example of chagas' disease interleukin-17-producing cd4(1) t cells increase with severity of liver damage in patients with chronic hepatitis b serum interleukin (il)-9 and il-10, but not t-helper 9 (th9) cells, are associated with survival of patients with acute-on-chronic hepatitis b liver failure molecular pathology of emerging coronavirus infections a question of self-preservation: immunopathology in influenza virus infection cytokine storm plays a direct role in the morbidity and mortality from influenza virus infection and is chemically treatable with a single sphingosine-1-phosphate agonist molecule hospitalization fatality risk of influenza a(h1n1)pdm09: a systematic review and meta-analysis into the eye of the cytokine storm magnitude and quality of cytokine and chemokine storm during acute infection distinguish nonprogressive and progressive simian immunodeficiency virus infections of nonhuman primates influenza virus pathogenicity regulated by host cellular proteases, cytokines and metabolites, and its therapeutic options combined inhibition of complement and cd14 efficiently attenuated the inflammatory response induced by staphylococcus aureus in a human whole blood model antigen presentation and mhc class ii expression by human esophageal epithelial cells: role in eosinophilic esophagitis identification of spatial genetic boundaries using a multifractal model in human population genetics clearance of hepatitis b e antigen in patients with chronic hepatitis b and genotypes hepatitis b virus infection during pregnancy: transmission and prevention the risk of perinatal hepatitis b virus transmission: hepatitis b e antigen (hbeag) prevalence estimates for all world regions predictors of hepatitis b e antigen-negative hepatitis in chronic hepatitis b virus-infected patients from childhood to adulthood association between single-nucleotide polymorphisms and early spontaneous hepatitis b virus e antigen seroconversion in children effect of host and viral factors on hepatitis b e antigen-positive chronic hepatitis b patients receiving pegylated interferon-a-2a therapy effect of hla-dp and il28b gene polymorphisms on response to interferon treatment in hepatitis b e-antigen seropositive chronic hepatitis b patients epidemiology of hepatitis b virus and genotype author names in bold designate shared co-first authorship. key: cord-002253-ll5a0urm authors: kunanopparat, areerat; kimkong, ingorn; palaga, tanapat; tangkijvanich, pisit; sirichindakul, boonchoo; hirankarn, nattiya title: increased atg5-atg12 in hepatitis b virus-associated hepatocellular carcinoma and their role in apoptosis date: 2016-10-07 journal: world j gastroenterol doi: 10.3748/wjg.v22.i37.8361 sha: doc_id: 2253 cord_uid: ll5a0urm aim: to investigate autophagy-related genes, particularly atg12, in apoptosis and cell cycle in hepatitis b virus (hbv)-associated hepatocellular carcinoma (hcc) and non-hbv-hcc cell lines. methods: the expression of autophagy-related genes in hbv-associated hepatocellular carcinoma and non-hbv-hcc cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction (qrt-pcr) and western blotting. the silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression. results: the expression of autophagy related genes atg5, atg12, atg9a and atg4b expression was analyzed in hepg2.2.15 cells and compared with hepg2 and thle cells. we found that atg5 and atg12 mrna expression was significantly increased in hepg2.2.15 cells compared to hepg2 cells (p < 0.005). moreover, atg5-atg12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from hcc patients with hbv infection. we also analyzed the function of atg12 in cell apoptosis and cell cycle progression. the percentage of apoptotic cells increased by 11.4% in atg12-silenced hepg2.2.15 cells (p < 0.005) but did not change in atg12-silenced hepg2 cells under starvation with earle’s balanced salt solution. however, the combination blockade of notch signaling and atg12 decreased the apoptotic rate of hepg2.2.15 cells from 55.6% to 50.4% (p < 0.05). conclusion: atg12 is important for hbv-associated apoptosis and a potential drug target for hbv-hcc. combination inhibition of atg12/notch signaling had no additional effect on hepg2.2.15 apoptosis. previous studies have reported that hbv expression is correlated with autophagy induction [1] [2] [3] . hbv enhances and uses autophagy for its replication via the hbx protein, which binds and activates phosphatidylinositol-3-kinase class 3 (pik3c3), an enzyme important for the initiation of autophagy. autophagy inhibitors or the silencing of enzymes essential for the formation of autophagosomes suppresses hbv dna synthesis with a minimal effect on the hbv mrna levels [2] . the role of autophagy in the production of hbv virions was demonstrated in hbv transgenic mice with a liverspecific deficiency of atg5 [4] . we recently confirmed that atg12 knock down reduced hbv dna levels in hepg2.2.15 cells and induced the interferon signaling pathway, suggesting that autophagy machinery may aid hbv survival by reducing antiviral innate immunity [5] . many studies have provided evidence to support the role of autophagy in human cancer. beclin-1 was the first mammalian autophagy gene to be identified. the monoallelic deletion of beclin-1 at chromosome 17q21 is sporadically observed in approximately 75% of ovarian cancers [6, 7] , 50% of breast cancers [8] , and 40% of prostate cancers [9] . other mutations in autophagy genes such as atg5, atg12, atg9b are frequent in gastric and colon cancers [10] . uvrag, a beclin1-interacting protein [10, 11] and atg4c were also shown to suppress tumor gene activity [12] . liverspecific beclin-1 knockout heterozygous mice showed increased rates of hepatocellular carcinoma in old aged mice [13, 14] . furthermore, mosaic atg5 -/mice developed benign liver tumors at 6-mo of age [15] and atg7 hepatocyte-specific knockout mice also developed liver tumors later in life [15] . these data support the idea that autophagy defects contribute to tumorigenesis. however, autophagy deficient cells can occur via cellular damage caused by dysfunctional mitochondria, oxidative stress, endoplasmic reticulum stress, necrosis and p62 accumulation [16] . the accumulation of cell damage can lead to chromosome instability [17] and inflammatory responses [18] , resulting in tumor development. although, autophagy functions as a tumor suppressor in primary cells, it is important for cancer cell survival. interestingly, spontaneously occurring liver tumors did not progress in chimeric mice with atg5 or atg7 loss [15] . this finding implies that autophagy is required for tumor progression. additionally, autophagy is required for cancer progression of other types of cancers. for example, an atg3 deletion in hematopoietic cells prevents bcr-abl-mediated leukemia [19] . some tumor cells are susceptible to growth inhibition or death when autophagy is inhibited. guo et al [20] , found that ras-driven tumors required autophagy for tumor cell survival upon starvation. therefore, autophagy has a dual-function in cancer. it functions as a tumor suppressor during cancer initiation, but also functions to promote tumor progression and metastasis later in the development process. because hbv infection is associated with hepatocellular carcinoma (hcc) and requires the induction of autophagy for its survival, we investigated the involvement of autophagic genes in cancer cell survival using hbv-associated hcc and non hbv-hcc cell lines and liver tissues. to induce starvation conditions, the cells were incubated in serum-free earle's balanced salt solution (ebss; starvation medium; invitrogen, united states) for the indicated number of hours (between 4-8 h). total cellular rna was extracted using real genomics total rna extraction kit (rbc bioscience, taiwan). the quantity of rna was measured using a spectrophotometer. rna concentrations in a solution were read and a 260 nm absorbance reading of 1.0 was equivalent to about 40 µg/ml of rna and the ratio of absorbance at 260 and 280 nm was used to assess the rna purity of rna preparations. then 1 µg of total rna was converted to cdna using high-capacity cdna reverse transcription kits (applied biosystems, united states). pcr reactions were performed in a 20 µl reaction volume in a 96-well plate (applied biosystems, united states). the pcr mixture contained 2 µl of cdna template, 10 µl of commercial (2 ×) power sybr ® green pcr master mix (applied biosystems), and each primer at a final concentration of 0.5 µmol/l. real-time pcr runs were carried out using an abi thermal cycler 7500 real-time pcr instrument (applied biosystems). the conditions for thermal cycling were as follows: initial denaturation at 95 ℃ for 10 min, followed by 40 amplification cycles at 95 ℃ for 15 s and then 60 ℃ for 1 min. for each pcr run, a negative (notemplate) control was used to test for false-positive results or contamination. the absence of nonspecific amplification was confirmed by generating a melt curve using the applied biosystems real-time pcr system software. the primers utilized in this study are summarized in table 1 . to analyze the relative gene expression data, we used real-time quantitative pcr and the 2 -δδc t method [21] . the ct values were obtained from real-time pcr instrumentation. the quantity of mrna relative to a reference gene was calculated using the formula 2 -δc t , october 7, 2016|volume 22|issue 37| wjg|www.wjgnet.com beclin-1 becn1-f ggatcaggaggaagc becn1-r gatgtggaaggttgc atg5 atg5-f gcttcgagatgtgtggtttgg atg5-r actttgtcagttaccaacgtca atg12 atg12-f ttgtggcctcagaacagttg atg12-r gagagttccaacttcttggtctg atg9a atg9a-f cgtgtgggaaggacag atg9a-r ggcgctttctccactc atg4b atg4b-f tccataggccagtggtacg atg4b-r tgcacaaccttctgatttcc b-actin b-actin-f accaactgggacgacatggagaa bsignificant differences were determined by unpaired t test with graphpad prism software, version 5.0 (san diego, ca, united states). statistical significance was set at a p value of < 0.05. to investigate the involvement of autophagy in hcc, we used beclin-1 as a marker for hbv-induced autophagy under starvation conditions. we analyzed beclin-1 mrna expression in hbv-transfected hepg2.2.15 cells and the parental cell line, hepg2. at 4 h post-starvation, beclin-1 was up regulated in both cell lines and was then down regulated at 8 h (data not shown). thus, we selected autophagy induction with starvation in ebss for 4 h for all subsequent experiments. autophagy related genes that represent each major part of the autophagy machinery including (1) induction step (beclin1); (2) the first ubiquitin-like conjugation molecules (atg5 and atg12); (3) the cysteine protease that catalyzes the second ubiquitinlike conjugation molecules (atg4b); and (4) the transporter membrane required for autophagosome where δc t = (ct target rna -ct reference rna). comparison of gene expression was based on a comparative ct method (δδc t ), and the relative rna expression was quantified according to the formula of 2 -δδc t , where δδc t = (ct target rna (experimental group) -ct reference rna (experimental group)) -(ct target rna (control group) -ct reference rna (control group)). genes with high expression levels were selected for the functional study. all cell lines and human liver tissues samples were lysed with ripa buffer (25 mmol/l tris-hcl, ph 7.6, 150 mmol/l nacl, 1% np-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate). after sonication, protein concentrations of cell lysates were measured using bca assay (thermo scientific, united states). then 20 µg of protein from each cell line was loaded onto a 12% polyacrylamide gel and run at 130 v for 80 min. the proteins were transferred onto nitrocellulose membranes and were immunoblotted with primary mab against atg9a, atg12, cnotch1, gapdh (dilution, 1:1000; rabbit mab; cell signaling technologies, united states) and atg4b (dilution, 1:1000; mouse mab; abcam, united states). after washing, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (dilution 1:2000) and were analyzed with enhanced chemiluminescence (ecl) substrate (supersignal west femto chemiluminescent substrate; thermo scientific). images were quantified using a licor-odyssey image system (li-cor ® , united states). hepg2 cells were grown in dmem supplemented with 10% fbs, antibiotic-free at 37 ℃ in 5% co2 for 24 h. hepg2 cells were transfected with 500-1000 ng of pgfp-hbx plasmids (addgene, ma, united states) or pgfp (negative control) using 1-3 µl of lipofectamine hepg2 and hepg2.2.15 cells were transiently transfected with 500 ng of short hairpin rna (shrna) formation (atg9a) were tested. the mrna expression levels of these selected molecules were determined at baseline and at 4 h post-culture in ebss. there were no significant differences at baseline (data not shown). at 4 h post starvation, beclin1 mrna was down regulated in hepg2.2.15 compared to thle2 and hepg2 (p = 0.0213 and p = 0.0236, respectively) ( figure 1a ). no significant difference was observed for atg4b expression between hbv-transfected hepg2.2.15 and hepg2, but it was significantly increased in hepg2 and hepg2.2.15 compared with thle-2 (p = 0.0002 and p = 0.0007, respectively) ( figure 1e ). atg9a mrna expression was higher in hepg2 and hepg2.2.15 compared to thle2 but significantly down regulated in hbv-transfected hepg2.2.15 compared with hepg2 (p = 0.0013) ( figure 1d ). only the mrna expression of atg5 and atg12 was significantly increased in hbv-transfected hepg2.2.15 compared with hepg2 cells (p = 0.0027 ( figure 1b) and p = 0.0039 ( figure 1c ), respectively). the results of mrna levels were confirmed by western blot analysis. atg5-atg12 proteins were up regulated in hbv-transfected hepg2.2.15 whereas atg9a were down regulated compared to hepg2 cells. there was no difference in atg4b protein expression between hbv-transfected hepg2.2.15 and hepg2 ( figure 1f ). next, we analyzed atg5-atg12 levels in tumor tissues compared to adjacent non-tumor tissues from hcc patients with or without hbv infection. in hbvassociated hcc tissues, atg5-atg12 protein levels were increased in the majority of tumor liver tissues compared to adjacent non-tumor tissues (9/10 sample pairs) (figure 2a and c). in non-hbv hcc, atg5-atg12 protein levels were only increased in 3/8 tumor liver tissue samples ( figure 2b and c). these results suggested that atg5-atg12 proteins might have a selective advantage in hbv-associated hcc compared to non-hbv hcc. the x protein was shown to induce hbv replication by increasing beclin-1 transcription leading to the induction of autophagy [1] . in this study, we analyzed the effect of hbx on beclin-1 and on atg12 by transfection of hepg2 cells with the pgfp-hbx plasmid. then, we analyzed beclin-1 and atg12 mrna expressions using quantitative real-time rt-pcr. we found that beclin-1 (p = 0.0027) ( figure 3a ) and atg12 (p = 0.0139) ( figure 3b ) mrna expression were significantly increased in hepg2-gfp-hbx compared with hepg2-gfp after 48 h of transfection. these results suggested that hbx plays a role in atg12 induction, either directly or indirectly through beclin1. next, we studied the functional role of the atg12 autophagic gene by monitoring the biological effect after knockdown of its expression. cells containing plasmids with the greatest silencing efficiency were subjected to western blot analysis for protein expression. the transfection efficiency was estimated by monitoring gfp expression under inverted fluorescence microscope (data not shown). the protein level of atg12 in transfected hepg2 and hepg2.2.15 cells was decreased compared with mock controls ( figure 4a ). we investigated apoptosis and cell cycle progression in hepg2.2.15 cells compare to hepg2 cells after the silencing of atg12. the percentage of apoptotic cells was slightly increased (8.3%) in atg12-silenced hepg2 but unchanged in hepg2.2.15 under normal conditions, which is similar to what we have previously reported [5] . under starvation conditions with ebss, the percentage of apoptotic cells increased 11.4% in atg12-silenced hepg2.2.15 but did not change in atg12-silenced hepg2 ( figure 4b ). no significant changes in cell cycle progression were observed in either cell line ( figure 5 ). these results suggest that atg5-atg12 proteins are important for the survival of hbv-associated hcc during states of limited tumor nutrients. we recently reported that notch signaling played a role in cell cycle progression and apoptosis, particularly in hbv-associated hcc [22] . because atg12 was up regulated in hepg2.2.15 compared to hepg2, and plays a role in cell apoptosis, we assessed the effect of atg12 silencing in combination with a notch inhibitor in the hepg2.2.15 cell line. gamma-secretase inhibitors, also known as n-[n-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycine t-butyl ester; dapt, is used to block the notch pathway [23] . when using dapt combined with gene silencing of atg12 under starvation conditions, hepg2 cell apoptosis was increased from 38.5% to 48.3%, compared to dapt treatment alone, although there was no synergistic effect ( figure 6) . interestingly, the combination blockade of dapt and atg12 gave the opposite result by decreasing the apoptosis rate in hepg2.2.15 from 55.6% to 50.4% ( figure 6 ). no significant changes in cell cycle progression were observed for the combination treatment of atg12 sirna and dapt (figure 7) . these results suggested that autophagy and notch signaling are independent from each other in hbv-expressing cells compared to non-hbv hcc. finally, we analyzed atg12 expression after treatment with dapt and detected cleaved notch1 expression after silencing atg12 genes at baseline. atg12 mrna was decreased by dapt treatment in hepg2.2.15 ( figure 8a ). moreover, cleaved notch1 was diminished in atg12-silenced hepg2.2.15 under autophagy has been reported to be associated with hbv and hcc [13, 15, 24] . however, the mechanism is still poorly understood. moreover, recent evidence suggests that specific atg genes might contribute differently to viral infection and cancer development [25] [26] [27] [28] . some atg genes have autophagy independent functions [29] [30] [31] [32] . in this study, we focused on the atg5-atg12 complex that was up regulated in hepg2.2.15 and were highly expressed in hbv-associated hcc compared to non-hbv cell lines and hcc. hbx appears to play a role in atg12 induction, probably indirectly through the stimulation of beclin-1 and pik3c3 [1, 2] . we showed that silencing of atg12, which represented the elimination of the atg5-atg12 western blotting with specific antibodies was used to analyze atg12 protein expression in hbv-associated hcc and non-hbv hcc. gapdh was used as a protein loading control. graphs showing the intensity band ratio (tumor tissue/adjacent non-tumor tissue) quantified using the li-cor ® image system from western blot analysis were shown in a and b. representative western blot results were shown in c. hbv: hepatitis b virus; hcc: hepatocellular carcinoma. [34] they showed that hepg2 and huh6 induced autophagy (beclin-1, atg5 and lc3b expression) for cell survival under starvation conditions with serum-free medium or chemotherapy. the inhibition of autophagy via the silencing of autophagic genes (beclin-1 and atg5) or 3-ma treatment induced huh6 cell apoptosis under starvation conditions [34] . a study by liu et al [35] demonstrated that starvation with serum-free medium for 48 h induced autophagy although atg12 has been demonstrated to have pro-apoptotic activity through the atg12-atg3 conjugate [36] , atg12 also has anti-apoptotic activity. in mammalian hela cells, disruption of autophagy by silencing atg12 promotes cells to die via apoptosis under starvation conditions [37] . atg12 and other autophagic proteins (lc3 and atg5) are associated with mitochondrial quality control of human umbilical vein endothelial cells. atg12, atg5 and lc3b were up-regulated after mitochondrial damage, leading to increased anti-apoptotic effects and increased life span in an in vitro aging model [38] . the combination of trastuzumab with silencing of atg12 reduces cell viability and tumor growth in nude mice [39] . therefore, we suggest that the inhibition of atg12 might be a good target for the treatment of hbv-associated hcc. many cancer drug treatments increase tumor cell autophagy to protect cancer cells from apoptosis. autophagy inhibitors in combination with chemotherapy are designed to induce apoptosis in human cancers. for example, autophagy inhibition was previously shown to enhance the growth inhibitory effects of sorafenib [40] as well as the combination of vorinostat with sorafenib in hcc cell lines [41] . in this study, we examined apoptosis cell death after the inhibition of atg12 in combination with notch signaling in hepg2 and hepg2.2.15 cell lines. the combination treatment increased cell apoptosis in hepg2 cells. however, when we blocked both notch signaling and atg12 under starvation conditions, cell apoptosis did not increase. there appears to be some interaction between atg12 and notch signaling, specifically in hepg2.2.12, because the notch inhibitor dapt caused the down-regulation of atg12 mrna but this was not observed in hepg2. in addition, the inhibition of atg12 impaired notch activation in hepg2.2.15 but increased notch activation in hepg2. this observation regarding the regulation between autophagy and notch signaling in hepg2.2.15 was unexpected. both autophagy and notch signaling are highly conserved signaling pathways in eukaryotic cells. the core notch signaling pathway is through the binding of notch ligand to notch receptor which induces two proteolytic cleavages, metalloprotease and g-secretase, to release the notch intracellular domain, which translocates to the nucleus and binds to dna to regulate the transcription of target genes [42] . the notch pathway is associated with both tumor suppression and tumorigenesis in hcc [43, 44] . the connection of autophagy and notch signaling was demonstrated in drosophila where the atg4 mutation enhanced the notched-wing phenotype resulting in defective notch signaling [45] . in contrast, the overexpression of atg1 enhanced the notchedwing phenotype [46] , suggesting that some autophagic proteins have additional functions independent of autophagy that are related to the enhancement or suppression of notch signaling. mammalian target of rapamycin (mtor) is a positive regulator of notch signaling [47] and a negative regulator of autophagy [48] . previous reports have shown that silencing of notch1 activated phosphorylated akt and decreased mtor to induce glioblastoma cell apoptosis [49] . the inhibition of notch signaling induced autophagy via pten-pi3k/akt/mtor pathway to promote the adipogenic differentiation of bm-mscs [50] . other studies have demonstrated that autophagy regulates notch signaling by reducing the impact of notch1 on stem cell differentiation. the silencing of autophagy (atg7 or atg16l1) induced notch1 and cleaved notch1 in hek cells [51] . the unexpected relationship between autophagy and notch signaling in hepg2.2.15 was different from hepg2 and might be explained by the hbv genome. the hbv x protein activates the nf-κb pathway (p65 and p50) via notch signaling through a specific ligand and receptor to promote cell survival [52] . after treatment with dapt, nf-κb expression was decreased [53] . thus, the activation of nf-κb can either stimulate or inhibit autophagy via the upregulation of beclin-1 in t-cells [54] , whereas prolonged nf-κb activation suppresses atg5 and beclin-1 expression in macrophages [55] . however, the connection between hbv and notch related autophagy needs further study. in conclusion, our previous study demonstrated the role of autophagy machinery in hbv replication. we found that atg12-knockdown reduced hbv dna . moreover, this study demonstrated that autophagy is related to hbv-associated cell death responses. atg12 silenced hepg2.2.15 showed increased apoptosis under starvation conditions. the function of atg12 seems to be dominated in hbvassociated hcc. these results suggested that atg5-atg12 is important for the survival of hbv-associated hcc during states of limited tumor nutrients. however, as mentioned previously that autophagy has a dualfunction in cancer. despite its role as tumor promotion in the later phase, it can act as a tumor suppressor during cancer initiation. therefore, the therapeutic intervention that target autophagy has to take this information into consideration too. moreover, our result showed that the use of autophagy inhibition in combination with other anti-tumor therapies such as notch inhibitor, might not be a good strategy. further characterization of hbv and notch related autophagy is needed. hepatitis b virus (hbv) infection is associated with hepatocellular carcinoma (hcc) and has been proved to induce autophagy for its replication and survival. the involvement of some autophagic genes on cancer cell survival is still unknown. therefore, we investigated the role of atg genes in hbv-associated hcc and non hbv-hcc in this study. autophagy is a catabolic process for cell survival under nutrient limitation or stress conditions. however, autophagy can act as either an oncogene or tumor suppressor gene. a recent report showed that hbv used the autophagic pathway for its own benefit; however the molecular mechanism is still unclear. atg5-atg12 protein was up regulated in hepg2.2.15 and expressed in a high percentage of hbv-associated hcc compared to non-hbv cell lines and hcc. the atg12 silenced hepg2.2.15 cell line had increased apoptosis under starvation conditions. this is the first study to show a function of atg12 in apoptosis in hbv-associated hcc. these findings raise the possibility of targeting the autophagic pathway for the treatment of hbv-associated hcc patients. this paper reported that atg5-atg12 protein expression was increased in hbv-transfected hepg2.2.15 cells compared to hepg2 cells and was increased in tumor liver tissues compared to adjacent non-tumor tissues from hcc patients with hbv infection. the silencing of atg12 increased cell apoptosis under starvation conditions in hepg2.2.15 cells but not in hepg2 cells. their results suggest that atg5-atg12 is important for the survival of hbv-associated hcc in the state of tumor nutrient limitation. the inhibition of atg12 might be a good target for hbv-associated hcc. hepatitis b virus x protein sensitizes cells to starvation-induced autophagy via up-regulation of beclin 1 expression the early autophagic pathway is activated by hepatitis b virus and required for viral dna replication subversion of cellular autophagy machinery by hepatitis b virus for viral envelopment autophagy required for hepatitis b virus replication in transgenic mice autophagy machinery impaired interferon signalling pathways to benefit hepatitis b virus replication cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21 early loss of heterozygosity on 17q in ovarian cancer. the abe ovarian cancer genetics group detailed deletion mapping of chromosome 17q in ovarian and breast cancers: 2-cm region on 17q21.3 often and commonly deleted in tumors loss of heterozygosity of the brca1 and other loci on chromosome 17q in human prostate cancer here, there be dragons: charting autophagy-related alterations in human tumors manipulation of nonsense mediated decay identifies gene mutations in colon cancer cells with microsatellite instability tissue-specific autophagy alterations and increased tumorigenesis in mice promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor autophagy-deficient mice develop multiple liver tumors autophagy and tumorigenesis autophagy suppresses tumor progression by limiting chromosomal instability growth factor regulation of autophagy and cell survival in the absence of apoptosis autophagy is essential to suppress cell stress and to allow bcr-abl-mediated leukemogenesis activated ras requires autophagy to maintain oxidative metabolism and tumorigenesis analysis of relative gene expression data using real-time quantitative pcr and the 2 hepatitis b virus hbx activates notch signaling via delta-like 4/notch1 in hepatocellular carcinoma notch inhibition as a promising new approach to cancer therapy autophagy and apoptosis-related genes in chronic liver disease and hepatocellular carcinoma wipi-1alpha (wipi49), a member of the novel 7-bladed wipi protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy radiation-induced autophagy is associated with lc3 and its inhibition sensitizes malignant glioma cells coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication impact of the autophagy machinery on hepatitis c virus infection the atg5 atg12 conjugate associates with innate antiviral immune responses the non-canonical role of atg family members as suppressors of innate antiviral immune signaling nondegradative role of atg5-atg12/ atg16l1 autophagy protein complex in antiviral activity of interferon gamma autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (idisc)-mediated caspase-8 activation and apoptosis hepatitis b virus x protein reduces starvation-induced cell death through activation of autophagy and inhibition of mitochondrial apoptotic pathway inhibition of autophagy may suppress the development of hepatoblastoma phosphorylated akt inhibits the apoptosis induced by dram-mediated mitophagy in hepatocellular carcinoma by preventing the translocation of dram to mitochondria atg12 conjugation to atg3 regulates mitochondrial homeostasis and cell death inhibition of macroautophagy triggers apoptosis autophagy proteins lc3b, atg5 and atg12 participate in quality control after mitochondrial damage and influence lifespan autophagyrelated gene 12 (atg12) is a novel determinant of primary resistance to her2-targeted therapies: utility of transcriptome analysis of the autophagy interactome to guide breast cancer treatment targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via er stress-related apoptosis inhibition of autophagy significantly enhances combination therapy with sorafenib and hdac inhibitors for human hepatoma cells notch signalling: a simple pathway becomes complex notch signaling inhibits hepatocellular carcinoma following inactivation of the rb pathway notch signaling in hepatocellular carcinoma: guilty in association the loss of drosophila apg4/aut2 function modifies the phenotypes of cut and notch signaling pathway mutants a targeted genetic modifier screen links the swi2/snf2 protein domino to growth and autophagy genes in drosophila melanogaster mammalian target of rapamycin regulates murine and human cell differentiation through stat3/p63/jagged/notch cascade amp-activated protein kinase and the regulation of autophagic proteolysis akt-mtor signaling is involved in notch-1-mediated glioma cell survival and proliferation inhibition of notch signaling promotes the adipogenic differentiation of mesenchymal stem cells through autophagy activation and pten-pi3k/akt/mtor pathway autophagy regulates notch degradation and modulates stem cell development and neurogenesis hepatitis b virus x protein promotes the growth of hepatocellular carcinoma by modulation of the notch signaling pathway the hepatitis b virus x protein downregulates nf-κb signaling pathways through decreasing the notch signaling pathway in hbxtransformed l02 cells p65/ rela modulates becn1 transcription and autophagy prolonged classical nf-kappab activation prevents autophagy upon e. coli stimulation in vitro: a potential resolving mechanism of inflammation. mediators infla m m 20 08 key: cord-026112-58sa5z03 authors: dehghani-dehej, farzaneh; hosseini, zinat; mortazkar, poupak; khanaliha, khadijeh; esghaei, maryam; fakhim, atousa; bokharaei-salim, farah title: prevalence of hcv and/or hbv coinfection in iranian hiv-infected patients date: 2020-04-24 journal: nan doi: 10.2217/fvl-2019-0066 sha: doc_id: 26112 cord_uid: 58sa5z03 aim: hiv-infected patients risk coinfection with hbv and hcv. this study aimed to investigate molecular epidemiology of hbv and hcv coinfection in iranian hiv-infected individuals. materials & methods: in this cross-sectional study, serological markers of hbv and hcv infection (hepatitis b surface antigen [hbsag], hepatitis b e-antigen [hbeag], hepatitis b e-antibody [hbeab] and hepatitis b core antibody [hbcab]) and anti-hcv antibodies [anti-hcv abs] were tested in 198 iranian hiv-infected patients. from plasma, hbv viral load was determined using cobas taqman 48, and hcv-rna was detected by reverse transcriptase-nested pcr. results: 85 out of 198 (42.9%) patients were anti-hcv ab positive and 42/198 (21.2%) had detectable hcv-rna. eight (4.0%) had traceable hbv-dna. all these patients were infected by hbv genotype d. 55 (27.8%) were hbcab positive. nine (4.4%) were hbsag and anti-hcv ab positive. conclusion: none were hiv-rna/hcv-rna/hbv-dna positive, 21.2% were hiv-rna/hcv-rna positive and 4.0% were hiv-rna/hbv-dna positive. therefore, studies on diagnosing these infections in hiv-infected individuals may be valuable. epidemiology of the infection [11, 12] . hepatitis c has a global impact in terms of mortality and morbidity with over 70 million people infected all around the world [13] . according to studies in iran, the prevalence of hcv infection is nearly 0.5% (1.0% in men and 0.1% in women) [14, 15] . for hcv infection and the liver damage associated with it, the leading cause of mortality and morbidity is among hiv-infected patients. according to available evidence, hiv/hcv-coinfected patients are at higher risk for liver cirrhosis and hepatocellular carcinoma (hcc) [16, 17] . given that the transmission routes of hiv, hbv and hcv viruses are common, these infections can occur simultaneously. worldwide, nearly 40 million people are living with hiv, about 2.6 million people are infected with hbv and about 2.8 million people are hcv-infected [2] . hiv infection intensifies natural history of hbv infection, which can lead to an increase in rates of hbv persistence, relapse of hbv (resurgence of hepatitis b surface antigen [hbsag] , hepatitis b e-antigen [hbeag] or hbv-dna) and considerable clinical disease. previous studies of the hbv/hiv coinfection have shown that hiv leads to a lack of protective immunity against hbv, increased risk of cirrhosis and hcc and liver-related mortality [18, 19] . the effect of antiretroviral therapies (arts) on the natural history of hbv-related disease have been different, in some studies, it leads to recovery from hbv infection and in other studies, with relapse of hepatitis b [18, 20] . the death rate in hiv-positive patients decreased after taking combination arts, but only in those with hiv/hbv or hiv/hcv coinfection. the mortality rate is high due to liver damage. hiv/hbv-coinfected people have a higher rate of progression to liver fibrosis, cirrhosis, hcc, less clearance of hbsag and occult hbv infections (obi) are more frequent in these patients [13] . therefore, it seems that screening for hbv infection in the hiv-infected individuals should be done. testing hbsag, hbeag and ab and determining hbv viral load are an essential part of hbv infection assessment in hiv/hbv-coinfected patients. obviously, determination of cd4 counts and hiv viral load are necessary along with the antiretroviral drug-resistant response [21] . according to evidence, hcv/hiv-coinfected patients are at higher risk for cirrhosis and hcc [22] . hiv infection exacerbates natural history of hcv infection. hcv-rna loads in these patients are higher and clearance of hepatitis c viremia after acute infection in hiv-positive patients are less, and liver diseases in these patients show more progress than patients with hiv infection alone [20] . it is known that hbv and hcv infections have been associated with various clinical manifestations in people with hiv infection including impaired immune response during arts, and also increased susceptibility to artsrelated liver toxicity [23] . therefore, prior to the administration of art, patients should be tested for the presence of these infections. the aim for this study is to investigate the prevalence of hcv and/or hbv coinfection in iranian hiv-infected individuals. from september 2015 to june 2018, 198 consecutive iranian hiv-positive individuals who were referred to hospitals affiliated with iran university of medical sciences (iums), tehran, iran, were entered to this study. the research was approved by the iums' ethical committee, and all of the studied population were informed about this survey, and a written informed consent was obtained from all the subjects and also from parents of hiv-infected children in this cross-sectional study. collection of the specimens 5 ml of the patient's blood was taken from each participant into an edta-containing vacutainer tube. after separation of the plasma by centrifugation (5 min at 3000 rpm), plasma was stored at -80 • c until analysis. plasma specimens from ten individuals who were infected with hcv, and ten subjects who were infected with hbv were used as positive controls, and also plasma samples from ten healthy blood donors were used as negative controls for the experiments. serologic tests by enzyme immunoassay serological markers of hbv and hcv infection such as hbsag, hbeag, hepatitis b e-antibody (hbeab), hepatitis b core antibody (hbcab) and anti-hepatitis c virus antibodies (anti-hcv) abs were tested by the commercial enzyme immunoassay kits (dia. pro, milano, italy), according to the manufacturer's protocols. hbv viral load was assessed in 500 μl of the studied subjects plasma specimens using the high pure dna extraction kit and cobas taqman 48 kit (roche diagnostics, ca, usa) according to the manufacturer's procedure [24] . this test is a real-time pcr assay that is based on dual-labeled hybridization probe that targets two regions (precore and core) of hbv. the detection limit of the cobas taqman 48 kit is 6 to >1 × 10 8 iu/ml [25] . the hbv genotyping was determined in hbv-dna-positive specimens using the inno-lipa™ hbv kit (innogenetics, ghent, belgium) according to the manufacturer's protocols [26] . hcv detection by reverse transcriptase-nested pcr method & hcv genotyping with restriction fragment length polymorphism assay to detect genomic hcv-rna in the plasma samples of studied subjects, the viral rna was isolated from 140 μl of plasma using the qiaamp viral rna isolation kit (qiagen gmbh, hilden, germany) based on the manufacturer's procedure. the quantity and quality of the extracted rna was evaluated using the nanodrop™ spectrophotometer (thermo fisher scientific, wilmington, nc, usa) [27] . the hcv-rna was detected in extracted rna of plasma samples by the reverse transcriptase-nested pcr (rt-nested pcr) assay using two sets of primers for the 5non-translated region (5 -ntr) of hcv, as previously described in detail [28, 29] . the amplified pcr products of subjects' samples, negative and positive control specimens, and 100 bp dna size marker were electrophoresed on a 2.2% gel agarose and then stained with syber green and visualized by a uv transilluminator. the genotyping of hcv was determined in hcv-positive samples with restriction fragment length polymorphism (rflp) assay, based on a protocol that previously described in detail [28, 29] . the statistical analysis was performed using spss software version 20 (spss inc., il, usa). the kolmogorov-smirnov test was conducted to determine the quantitative variables' normality. the analysis of continuous variables was done using kruskal-wallis and one-way analysis of variance (anova) tests. the statistical differences between the two groups were evaluated by fisher's exact test and chi-square test when appropriate. p-values <0.05 were considered statistically significant. from september 2015 to june 2018, a total of 198 hiv-infected individuals (anti-hiv abs and hiv-rna positive) were enrolled in this cross-sectional study. the mean age of subjects was 35.3 ± 13.5 years (a range of 1-68 years old). out of 198 studied individuals, 126 (63.6%) were male. complete information of the demographic, laboratory and epidemiological characteristics were presented in table 1 . a significant association was observed between the sex of the participants and alanine aminotransferase (alt), aspartate aminotransferase (ast) level, anti-hcv abs, hcv-rna (p < 0.001) in plasma samples, and also in epidemiological parameters such as history of having unprotected sex, history of imprisonment, injection drug users (idus), idu sexual partners, history of tattooing, history of needle stick (p < 0.001) and history of transfusion (p = 0.034) ( table 1) . a significant relationship was observed between coinfection with hcv or hbv in hiv-infected patients and cd4 + t-cell count (p = 0.044), in other words, cd4 + t-cell count was very low in patients with these coinfections. a strong association was observed between the sex of the participants and level of education (p = 0.042) and marital status (p < 0.001) ( table 2) . 85 (42.9%) of studied subjects were positive for anti-hcv abs in plasma samples; and 42 (21.2%) had detectable hcv-rna in the plasma ( table 1 ). the hcv genotyping was performed using rflp assay for hcv-rna-positive specimens, and the results of hcv genotyping are presented in table 3 . eight (4.0%) of the studied cases had detectable hbv-dna in the plasma samples ( table 1 ). the hbv genotyping was carried out for these samples by the inno-lipa hbv kit. all these patients were infected by hbv genotype d. 55 (27.8%) of the participants were hbcab positive, and a strong relationship was observed between the sex of the studied participants and anti-hbcab in plasma specimens (p < 0.001) ( table 1) . this survey demonstrated that none of the iranian hiv-infected individuals were hiv-rna/hcv-rna/hbv-dna positive simultaneously, 21.2% were hiv-rna/hcv-rna positive and 4.0% were hiv-rna/hbv-dna positive. no significant association was observed between the hiv viral load in coinfected patients and monoinfected patients (p = 0.054). nine (4.4%) of the studied hiv-infected patients were hbsag and anti-hcv ab positive. all the information about demographic and laboratory parameters of these patients are presented in table 4 . despite the existence of successful prevention and treatment methods, the simultaneous infection of hiv, hbv and hcv is still a worldwide health issue. with the use of antiretroviral medicines and longer life expectancy in 10 hiv-infected patients, the complications of this chronic disease and its intersection with other viral infections are more evident [30] . in iran, the prevalence of hiv and other blood-related viral infections, such as hcv is relatively low in the general population [31] . the present survey was conducted on 198 individuals who were infected with hiv to investigate the molecular epidemiology of hcv/hbv coinfection in these individuals. this study showed that none of the hiv-infected people were hiv-rna/hcv-rna/hbv-dna positive simultaneously, 4.0% were hiv-rna/hbv-dna positive and 21.2% were hiv-rna/hcv-rna positive. hcv infection is more common in people infected with hiv than in hiv-negative individuals [32] . the rate of hcv/hiv coinfection is different around the world and is heavily dependent on geographical location, socioeconomic conditions of that particular location and high-risk groups [32] . nearly 37 million people are infected with hiv so far and about 70 million people all over the world are infected with hcv [2, 13] . approximately 2,278,400 people have hiv/hcv coinfection in the world, and about 62% of them are people that have been infected through injecting drugs [32] . the present study showed that approximately 81 (40.9%) of the patients are those who have injected drugs and about 46 (23.2%) of them are those who had idu sexual partner (that includes only women) ( table 1 ). the epidemiologic parameters such as injection drug abuse, needle sharing, tattooing, history of imprisonment and history of having unprotected sex revealed higher prevalence of hcv-coinfection in these patients compared with monoinfected cases. in the coinfected patients, the level of liver enzymes alt/ast was significantly high. while the first way of hiv transmission in iran is from sharing injection needles among idus [33] , hiv transmission cases in idus has begun to decline from 2005, a trend that has continued so far, and today hiv transmission through unprotected sexual contact is increasing (36.8%) [34, 35] . in this study the number of idus was 81 (40.9%). since the transmission of hcv by sexual contact is a rare phenomenon, the number of people coinfected with hcv/hiv is expected to decrease in the future [3, 36] . in the current study, 75 (37.9%) of the hiv-positive patients had a history of unprotected sex and probably the hiv virus in these patients transferred through unprotected sex. perhaps this is the reason for the decrease in the number of patients with hcv/hiv coinfection (21.2%) compared with previous reports [37, 38] . of course, in recent years, the increase in the transmission of hcv in males that have sexual intercourse with other males has been seen due to high-risk sexual behaviors. in addition, there have been cases of hcv spontaneous clearance in people who inject drugs (pwid) after art [39, 40] . the hcv genotyping on the plasma specimens showed a prevalence for subtypes, 1a (40.5%), 1b (16.7%) and 3a (33.3%). in four samples, the divergence genotype detected a mixed infection of two subtypes (1a/3a-1ab/3a) ( [41] [42] [43] . according to various reports from around the world, it seems that more research is required in this field with a wider population. like hcv infection, all hiv-infected patients should be screened for hbv infection. cirrhosis, hcc and hepatotoxicity after arts are the effects of hbv/hiv coinfection [44] . hbv vaccination should be done in all hiv individuals with hbv-negative laboratory tests. similar to hcv, hcc occurs in hbv/hiv-coinfected patients without cirrhosis [45] . hbv viral load is higher in hiv/hbv-coinfected patients than hbv-monoinfected individuals [46] . the present study found that 55 (27.8%) of these patients have anti-hbcab and 25 (12.6%) of them have hbsag. in some people, antibodies of the hbv core can be detected without anti-hbsag and hbeag [47] . our study confirms previous reports that male subjects are at high risk of developing hbv infection. typical methods for the transmission of both hbv and hiv are the sexual pathway and injection drug abuse [48] , while transmission of hcv by sexual pathway is unusual and given that in recent years the pathway for hiv transmission has changed, the prevalence of hcv is changing, but a significant change in the prevalence of hbv is unlikely to occur [49] . in a study on blood donors with an nat test in tehran, the incidence and residual risk for hiv was lower than those in developed countries, whereas hbv and hcv was higher compared to developed countries. in the iranian population, hiv infection is lower than the other countries, and screening tests are effective for blood donors. in the case of hcv, an increased incidence of hcv infection has been observed in the iranian society and in blood donors in recent years; this may be due to the highest number of idus in iran compared to other middle eastern countries [50] . incidence and high residual risk in iran indicates the nature of the endemic hbv virus. due to the launch of hbv vaccination in iran in 1993, we have to wait for the effects of the vaccination among the iranian population. based on that study, blood donors should have a more accurate technique similar to the accurate nat-screening techniques [51] . in patients with coinfection of hiv with hcv and/or chronic hbv, progressive liver fibrosis, cirrhosis and hcc can occur and coinfection of hiv with hbv and/or hcv can affect the management of hiv infection and complicate it [44, 52] . therefore, it is best to identify infection of hepatitis as quickly as possible. the result of this study revealed that none of the participants were hiv-rna/hcv-rna/hbv-dna positive simultaneously. to the best of our knowledge, the current survey is the first research that has analyzed the presence of the molecular epidemiology of hcv/hbv coinfection in iranian hiv-infected individuals; therefore, the results of this study cannot be compared with the result of other iranian research. there have been reports of coinfection with hbv and hcv in hiv-positive people, for example, 0.5% in singapore [53] , 0.62% in germany [54] and 1.7% in serbia [55] . it seems that further research focusing on this issue is needed. although there are studies that indicate seroprevalence of hiv/hcv/hbv coinfection in iran. for example, bakhti et al. found that 8% of hiv-infected individuals are coinfected with hcv/hbv (hbsag and anti-hcv ab positive), and in a meta-analysis, bagheri amiri et al. reported that coinfection of hiv/hbv/hiv was close to zero in the general population, street children and healthcare workers, while it peaked to 1.25% in pwid [56] . this study revealed that in iranian hiv-infected individuals, 4.4% of the individuals are seropositive for hbv/hcv (hbsag and anti-hcv ab positive). according to a previous study, prevalence of cirrhosis in hiv/hbv/hcv triple-infected patients was higher than hiv/hbv-or hiv/hcv-coinfected individuals [57] . therefore, prevention programs for hiv/hbv/hcv coinfection are in need of development. this study reveals that there is a high prevalence of hcv infection (21.2%) in hiv-infected individuals (hiv-rna/hcv-rna positive), as well as 4% of these people infected with hbv (hiv-rna/hbv-dna positive). also, the result of this survey highlighted that none of the hiv-infected subjects were hcv-rna/hbv-dna positive simultaneously. therefore, it seems that in hiv-positive patients, in addition to routine diagnosis of all the authors of this article are very grateful to those who volunteered to participate in this research. the current research was funded by research deputy of iran university of medical sciences (iums), tehran, iran with grant number 8921215087. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. ethical approval for this research was obtained from the local ethics committee of iran university of medical sciences (iums), tehran, iran, that is accordance with helsinki declaration (ethical code: ir.iums.fmd.rec 1396. 8921215087). all of the volunteers participating in this study were informed about this research prior to their enrollment. in addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. hiv diagnosis and treatment through advanced technologies world health organization global health observatory current diagnostic methods for hiv interference of apoptosis by hepatitis b virus characterization of hepatitis b virus with complex structural variations hiv-hepatitis b virus coinfection: epidemiology, pathogenesis, and treatment global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity hepatitis b: screening, awareness, and the need to treat hepatitis b virus infection in the general population of iran: an updated systematic review and meta-analysis hepatitis c virus (hcv) genotyping by annealing reverse transcription-pcr products with genotype-specific capture probes identification of a novel hepatitis c virus genotype from punjab, india: expanding classification of hepatitis c virus into 8 genotypes molecular epidemiology of hepatitis c virus in cambodia during 2016-2017 hepatitis b, hepatitis c, and mortality among hiv-positive individuals seroprevalence of hepatitis c virus: the first population-based study from iran the impact of ifn-γ gene polymorphisms on spontaneous clearance of hcv infection in fars province, southern of iran the presence of autoantibodies to cytoplasmic rod and ring particles in the serum of patients with chronic hepatitis c virus infection hepatocellular carcinoma after sustained virological response with interferon-free regimens in hiv/hepatitis c virus-coinfected patients hiv-1, hepatitis b virus, and risk of liver-related mortality in the multicenter cohort study (macs) hepatitis b and long-term hiv outcomes in co-infected haart recipients survival in patients with hiv infection and viral hepatitis b or c: a cohort study management of hiv and hepatitis virus coinfection human immunodeficiency virus/hepatitis c virus (hcv) co-infected patients with cirrhosis are no longer at higher risk for hepatocellular carcinoma or end-stage liver disease as compared to hcv mono-infected patients liver fibrosis and hepatitis b coinfection among art naive hiv-infected patients at a tertiary level hospital in northwestern tanzania: a cross-sectional study detection of hepatitis b virus covalently closed circular dna in the plasma of iranian hbeag-negative patients with chronic hepatitis b detection of hbv genome in the plasma and peripheral blood mononuclear cells of iranian hbsag negative patients with hiv infection: occult hbv infection distribution of hepatitis b virus genotypes in azerbaijani patients with chronic hepatitis b infection prevalence of jc polyomavirus large t antigen sequences among iranian patients with central nervous system tumors hepatitis c genotypes in finland determined by rflp occult hepatitis c virus infection in iranian patients with cryptogenic liver disease trends in underlying causes of death in people with hiv from co-infection of human immunodeficiency virus, hepatitis c and hepatitis b virus among injection drug users in drop in centers prevalence and burden of hcv co-infection in people living with hiv: a global systematic review and meta-analysis trend of hiv/aids prevalence and related interventions administered in prisons of iran − 13years' experience antiretroviral drug resistance mutations in naïve islamic republic of iran aids progress report hiv-1 genetic diversity and transmitted drug resistance frequency among iranian treatment-naive, sexually infected individuals epidemiological distribution and genotype characterization of the hepatitis c virus among hiv patients in kashan high prevalence of hcv coinfection in hiv-infected individuals in shiraz, islamic republic of iran sexually transmitted hepatitis c infection: the new epidemic in msm? occasional spontaneous clearance of chronic hepatitis c virus in hiv-infected individuals hiv and hcv coinfection: prevalence, associated factors and genotype characterization in the midwest region of brazil prevalence of hepatitis c virus (hcv) coinfection in hiv infected individuals in south india and characterization of hcv genotypes detection and analysis of hepatitis c virus in hiv-infected patients in the guangxi province of china long-term liver diseases after initiation of antiretroviral therapy in hiv-infected patients with and without hbv or hcv coinfection risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus dna level hiv-hepatitis b virus co-infection: epidemiology, pathogenesis and treatment isolated antibody to hepatitis b core antigen in human immunodeficiency virus type-1− infected individuals hepatitis c, hepatitis b, and human immunodeficiency virus infections among non-intravenous drug-using patients attending clinics for sexually transmitted diseases epidemiology of hepatocellular carcinoma: new trends prevalence and trend of hepatitis c virus infection among blood donors in iran: a systematic review and meta-analysis incidence and residual risk of hiv, hbv and hcv infections among blood donors in tehran hepatic decompensation in antiretroviral-treated patients co-infected with hiv and hepatitis c virus compared with hepatitis c virus-monoinfected patients: a cohort study factors associated with hepatitis b and c co-infection among hiv-infected patients in singapore daclatasvir plus sofosbuvir, with or without ribavirin, in real-world patients with hiv-hcv coinfection and advanced liver disease comparison of demographic, epidemiological, immunological, and clinical characteristics of patients with hiv mono-infection versus patients co-infected with hcv or/and hbv: a serbian cohort study hbv and hcv coinfection prevalence in iran-a systematic review and meta-analysis hepatic decompensation in patients with hiv/hepatitis b virus (hbv)/hepatitis c virus (hcv) triple infection versus hiv/hcv coinfection and the effect of anti-hbv nucleos(t)ide therapy key: cord-308382-h8ldbzip authors: lin, serena y. c.; magalis, brittany rife; salemi, marco; liu, hsin‐fu title: origin and dissemination of hepatitis b virus genotype c in east asia revealed by phylodynamic analysis and historical correlates date: 2018-10-17 journal: j viral hepat doi: 10.1111/jvh.13006 sha: doc_id: 308382 cord_uid: h8ldbzip hepatitis b virus disease progression in east asia is most frequently associated with genotype c (hbv/c). the increasing availability of hbv/c genetic sequences and detailed annotations provides an opportunity to investigate the epidemiological factors underlying its evolutionary history. in this study, the bayesian phylogeography framework was used to investigate the origins and patterns in spatial dissemination of hbv/c by analyzing east asian sequences obtained from 1992 to 2010. the most recent common ancestor of hbv/c was traced back to the early 1900s in china, where it eventually diverged into two major lineages during the 1930s‐1960s that gave rise to distinct epidemic waves spreading exponentially to other east asian countries and the usa. demographic inference of viral effective population size over time indicated similar dynamics for both lineages, characterized by exponential growth since the early 1980s, followed by a significant bottleneck in 2003 and another increase after 2004. although additional factors cannot be ruled out, we provide evidence to suggest this bottleneck was the result of limited human movement from/to china during the sars outbreak in 2003. this is the first extensive evolutionary study of hbv/c in east asia as well as the first to assess more realistic spatial ecological influences between co‐circulating infectious diseases. hepatitis b virus disease progression in east asia is most frequently associated with genotype c (hbv/c). the increasing availability of hbv/c genetic sequences and detailed annotations provides an opportunity to investigate the epidemiological factors underlying its evolutionary history. in this study, the bayesian phylogeography framework was used to investigate the origins and patterns in spatial dissemination of hbv/c by analyzing east asian sequences obtained from 1992 to 2010. the most recent common ancestor of hbv/c was traced back to the early 1900s in china, where it eventually diverged into two major lineages during the 1930s-1960s that gave rise to distinct epidemic waves spreading exponentially to other east asian countries and the usa. demographic inference of viral effective population size over time indicated similar dynamics for both lineages, characterized by exponential growth since the early 1980s, followed by a significant bottleneck in 2003 and another increase after 2004. although additional factors cannot be ruled out, we provide evidence to suggest this bottleneck was the result of limited human movement from/to china during the sars outbreak in 2003. this is the first extensive evolutionary study of hbv/c in east asia as well as the first to assess more realistic spatial ecological influences between co-circulating infectious diseases. east asia, hbv genotype c, human mobility, phylogeography, population bottleneck, sars the different genotypes, except for genotypes d and g, which are scattered worldwide. 4 genotype a is prevalent in africa, north america and europe; genotypes b and c are the major genotypes circulating in asia 4 and, even in the usa, are the most common among asian patients 5 ; genotype e is prevalent in africa; f/h in central and south america; i in taiwan; and j in japan. 4, 6, 7 hbv genotype c (hbv/c), in particular, is the most prevalent genotype in almost every east asian country, and it also accounts for a large number of infections in the usa (prevalence of 41% and 23% along the western and eastern coasts, respectively 5 ). in korea, genotype c constitutes almost 100% of the infections, 8 approximately 50% in hong kong, 9 and 85% in japan. 10 in mainland china, genotype c is predominant in the northern part of china (beijing, xingjiang and gansu), while genotype b is prevalent in the central and eastern part of china (hunan and fujian), with an overall prevalence of 41% for genotype b and 53% for genotype c. 11 similarly in taiwan, where hbv genotype b is the most prevalent (68%), genotype c still accounts for almost one-third of the infections (32%). 12 and c4 in the aborigines from australia. 14 chronic infection with hbv/c has been associated with significantly higher risk than with other genotypes for progression to lc and hcc. 15, 16 in taiwan, in particular, chronic hepatitis and hcc are still the 9th ranking cause of death. 16 indeed, several studies have suggested that disease outcomes are related to specific genetic variants. [17] [18] [19] due to the disease activity and the risk of hcc development with which hbv/c is associated, it is important to investigate the molecular evolution and demographic history of this genotype in highly endemic countries notably the high prevalence of genotype c in usa and the prevalence (14.8%) of chronic hbv infections among asian immigrants who likely acquired infections in their country of origin. 20 we consider this as an asianrelated infection network, and therefore the hbv/c sequences from usa will be included in the study. previous studies based on bayesian coalescent analysis estimated the hbv evolutionary rate to be approximately 10 −4 -10 −5 nucleotide substitutions/site/ year 21, 22 and traced back the time of the most recent common ancestor (tmrca) of the currently circulating human genotypes to ~1500 years ago, which in turn separated from the avian hbv lineage ~6000 years ago. 21 however, specific genotypes, or lineages within genotypes responsible for current outbreaks, may have a more recent origin, and their successful spread could be the result of specific historical or geopolitical correlates during the past decades potentially related to an unprecedented increase in human mobility. therefore, our main objective was to infer the origin and epidemic history of hbv/c in east asia and investigate ecological factors affecting dissemination and epidemic outbreaks of the virus. we mined the genbank database (https://www.ncbi.nlm.nih.gov) to compile a comprehensive data set of all currently available hbv/c sequences. the gold standard method for hbv genotyping is wholegenome sequencing followed by phylogenetic analysis. 23 therefore, to infer a reliable demographic history of hbv/c, we focused on fullgenome sequences with known sampling time and country of origin sequences included in the final data set were required to satisfy the following criteria: nonrecombinant sequences with no uncertainty concerning genotype assignment; sequences isolated only from human serum or plasma (sequences from liver tumour were excluded to avoid the potential confounding factor of tissue-specific convergent evolution of sequences sampled from different patients); sequences not epidemiologically linked (ie, not linked through a direct transmission chain); and when multiple sequences from the same subjects were available, only one sequence was randomly selected. genotyping classification was confirmed by phylogenetic analysis using neighbor joining (nj) tree reconstruction, with the gtr nucleotide substitution model, gamma-distributed rate heterogeneity among sites (gtr + g), and 1000 bootstrap replicates, from an alignment including the full-genome sequences obtained from genbank and well-established genotype (a to j) reference strains (accession numbers shown in table s1 ). calculations were carried out within mega6 software. 24 data collection times of hbv sequences included in the full-genome data set (n = 429) spanned from 1992 to 2010. to ensure that a specific country was not falsely over-represented in the alignment, a sampling ratio was calculated using the proportion of genotype c in chronic hbv cases (china 53%, korea 98%, japan 85%, taiwan 32%, usa 41%) normalized by hbv prevalence in the general population of each country (china 10%, 25 korea 5.9%, 26 japan 4%, 27 taiwan 15%, 28 usa 15%, 20, 29 which resulted in a sampling ratio china:korea:japan:taiwan:usa of 1.6:1.7:1:1.4:1.8. sequences were, then, randomly selected from each country according to this ratio to generate an alignment including a final alignment of 120 strains representative of the virus prevalence in each country spanning from 1992 to 2010 (see table s2 ) to infer maximum likelihood (ml) trees, nj trees and bayesian coalescent inference. the best-fitting nucleotide substitution (gtr + g) model was selected using a hierarchical likelihood ratio test within paup* v4.0. 30 nj and ml trees were then inferred according to the best-fitting model using mega6 and phyml 3.0 (http://www.atgc-montpellier. to assess the molecular clock signal carried in the temporally sampled viral sequences, a cross-platform software, tempest (formerly known as path-o-gen; http://tree.bio.ed.ac.uk/software/tempest/), is used to explore the association between genetic divergence through time and the sampling dates. time-scaled phylogenetic trees, evolutionary rates and demographic histories of hbv/c strains were evaluated using the bayesian coalescent framework implemented in beast v1.8.2 (http://beast. community/index.html), which uses a markov chain monte carlo (mcmc) sampling method to obtain posterior distributions of tree topologies and parameter estimates. bifurcating nodes with posterior probability greater than 0.95 were considered statistically well supported. six different evolutionary models were tested: strict vs relaxed molecular clock, each one with a constant size, exponential growth or bayesian skygrid (nonparametric) demographic prior. 32 depending on the model, mcmcs were run for 500 million to 1.5 billion generations (sampling every 0.01% of the run) until the effective sampling size of each parameter estimate (after burn-in of 10%-25%, depending on the model) was >200 to ensure proper mixing of the markov chain. for each run, the marginal likelihood was estimated via path sampling (ps) and stepping stone (ss) methods 33 and the resulting bayes factors (bf) (ratio of marginal likelihoods) used to select the best-fitting clock/demographic model. 34 in practice, following the original work of kass and raftery, 35 in ml and nj trees, the putative location of each ancestral lineage (internal branch) was inferred by assigning a discrete character to each sequence corresponding to the country of origin and reconstructing ancestral states by maximum parsimony. a more in-depth phylogeographic analysis, incorporating both spatial and temporal information, was also performed with beast 36 using a discrete trait, symmetric substitution model with bayesian stochastic search variable selection (bssvs). the mcc tree was converted to a keyhole markup language file (kml file) using spread 37 software and projected onto a geographical map using google earth (available online: http://www.google.com/earth) to produce a graphical animation of the estimated spatiotemporal patterns of hbv/c evolution. longitude and latitude of each centre of the cities or countries were marked orderly in a text-delimited file for spread. the entry numbers at customs arrival point of eastern asian countries, as well as departure trends, were collected from a number of national databases (see below) to evaluate potential correlations between viral demographic history and human mobility during the past two to three decades (depending on available data for each country). human mobility data in taiwan the nj tree inferred from genotype c full-genome sequences was rooted with genotype b strains (see figure 1 ) in order to investigate the timeframe of hbv/c spatial dispersion patterns in east asia within the bayesian coalescent framework, we selected from all available full-genome sequences a random sub-sample according to the relative ratio of hbv/c prevalence in each country ( site/year), in agreement with previous estimates. 21 the overall topology of the bayesian maximum clade credibility (mcc) tree ( figure 3a ) inferred from the data set is almost the same as ml and nj phylogenies inferred from the full data set (figure 1 and figure s1 ). the majority of the available strains (99.5%) clustered within similar to the data set including all lineages, the relaxed molecular clock and nonparametric skygrid demographic prior were determined to be the best-fitting models when sequences of each of the three major lineages described in the previous section were analysed separately (table 1) the prevalence and dynamics of hbv/c infection are a major public health concern in east asia. our study resulted in two significant findings. first, we showed that during the past three decades since 1995, when successful vaccination programs started to curtail the number of "mother-to-infant" transmissions, overall hbv prevalence has been decreasing, especially in japan. 48 the authors declare no conflict of interest. hsin-fu liu http://orcid.org/0000-0003-0082-2269 world health organization. who guidance on development of influenza vaccine reference viruses by reverse genetics typing hepatitis b virus by homology in nucleotide sequence: comparison of surface antigen subtypes molecular epidemiological study of hepatitis b virus infection in two different ethnic populations from the solomon islands hepatitis b virus genotypes hepatitis b virus genotypes in the united states: results of a nationwide study a genetic variant of hepatitis b virus divergent from known human and ape genotypes isolated from a japanese patient and provisionally assigned to new genotype molecular characterization of hepatitis b virus and a 9-year clinical profile in a patient infected with genotype i distribution of hepatitis b virus genotypes in korea hepatitis b virus genotype c is associated with more severe liver fibrosis than genotype b geographic distribution of hepatitis b virus (hbv) genotype in patients with chronic hbv infection in japan geographic distribution, virologic and clinical characteristics of hepatitis b virus genotypes in china genotypes and clinical phenotypes of hepatitis b virus in patients with chronic hepatitis b virus infection genotype c of hepatitis b virus can be classified into at least two subgroups a novel variant genotype c of hepatitis b virus identified in isolates from australian aborigines: complete genome sequence and phylogenetic relatedness hepatitis b virus genotype c takes a more aggressive disease course than hepatitis b virus genotype b in hepatitis b e antigen-positive patients viral genotype and hepatitis b virus dna levels are correlated with histological liver damage in hbeag-negative chronic hepatitis b virus infection hepatitis b virus genotype a is more often associated with severe liver disease in northern india than is genotype d core promoter mutations and genotypes in relation to viral replication and liver damage in east asian hepatitis b virus carriers screening for chronic hepatitis b among asian/pacific islander populations bayesian estimates of the evolutionary rate and age of hepatitis b virus molecular evolution of hepatitis b virus over 25 years hepatitis b virus genotyping: current methods and clinical implications mega6: molecular evolutionary genetics analysis version 6.0 epidemiological serosurvey of hepatitis b in china-declining hbv prevalence due to hepatitis b vaccination hepatitis b vaccinations among koreans: results from 2005 korea national cancer screening survey epidemiology of hepatitis b virus in japan, especially in nagasaki epidemiology of hepatitis b virus infection in the asia-pacific region epidemiology of hepatitis b in the united states estimation of levels of gene flow from dna sequence data improving bayesian population dynamics inference: a coalescentbased model for multiple loci improving the accuracy of demographic and molecular clock model comparison while accommodating phylogenetic uncertainty make the most of your samples: bayes factor estimators for high-dimensional models of sequence evolution bayesian phylogenetics with beauti and the beast 1.7 spread: spatial phylogenetic reconstruction of evolutionary dynamics viral phylodynamics and the search for an 'effective number of infections' sars epidemic area severe acute respiratory syndrome coronavirus sequence characteristics and evolutionary rate estimate from maximum likelihood analysis cultural revolution memoirs written and read in english: image formation, reception and counternarrative: thesis(ph.d.)-university of minnesota life and death in shanghai world health organization changes hong kong, guangdong travel recommendations summary of probable sars cases with onset of illness from 1 sars in healthcare facilities control measures for severe acute respiratory syndrome (sars) in taiwan molecular evolution and phylodynamics of acute hepatitis b virus in japan characterising two-pathogen competition in spatially structured environments key: cord-000736-6f8vyziv authors: pripuzova, natalia; wang, richard; tsai, shien; li, bingjie; hung, guo-chiuan; ptak, roger g.; lo, shyh-ching title: development of real-time pcr array for simultaneous detection of eight human blood-borne viral pathogens date: 2012-08-17 journal: plos one doi: 10.1371/journal.pone.0043246 sha: doc_id: 736 cord_uid: 6f8vyziv background: real-time pcr array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors. findings: we developed a real-time pcr array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (hiv-1 and -2), hepatitis b virus (hbv), hepatitis c virus (hcv), human t-cell leukemia virus-1 and -2 (htlv-1 and -2), vaccinia virus (vacv) and west nile virus (wnv). one hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with sybr green chemistry. the specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. the array detected: 10 genome equivalents (geq)/ml of hiv-2 and hcv, 50 geq of hiv-1 (subtype b), hbv (genotype a) and wnv. it detected 100–1,000 geq/ml of plasma of hiv-1 subtypes (a – g), group n and crf (ae and ag) isolates. further evaluation with a panel consisting of 28 hiv-1 and hiv-2 clinical isolates revealed no cross-reactivity of hiv-1 or hiv-2 specific primers with another type of hiv. all 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. the pcr array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for hiv-1, hcv or hbv at as low as several geq per pcr reaction. conclusions: the viral array described here demonstrated adequate performance in the testing of donors’ clinical samples. further improvement in its sensitivity for the broad spectrum of hiv-1 subtypes is under development. rapid progress and improvement in molecular technologies have allowed researchers to switch from the traditional approaches of virus detection in clinical samples to multiplexing for simultaneous detection of multiple pathogens in a single assay. a number of different pcr based assays for detection and discovery of multiple pathogens have been developed [1] [2] [3] [4] . detection microarrays are proven to be useful in the identification and discovery of viruses homologous to known species. they have been used to guide the selection of samples for further analysis by sequencing [1] [2] [3] 5] . however, microarrays based on nucleic acid hybridization are too complex in design and performance for the routine donors testing, and exhibit a comparatively low sensitivity of detection, usually around 10021,000 genome copies of target virus per analyzed sample [1] [2] . several pcr based assays coupled with oligonucleotide microarray technology (so called resequencing arrays) have been designed to allow simultaneous detection or genotyping of a target group of viruses, such as some critical blood-borne pathogens (3 viruses) [6] , respiratory viruses (16-21 viruses) [7] [8] , and respiratory adenoviruses (6 different serotypes) [9] . such pcr based approach allows increasing the sensitivity of detection down to 10-100 copies of the target rna or dna in a sample. pcr multiplexing should be highly useful when both the volume of the samples and the time of testing are critical, as in the donor eligibility (de) testing for tissue or organ transplantation [10] . current regulation requires that de testing be performed using assays approved and licensed by the u.s. food and drug administration (fda). however, the automated assay systems that are designed to screen large numbers of samples, without the strict limitation of sample volumes, may not be completely suitable or ideal for the needs of de testing for tissue or organ transplantation. the main goal of the study presented here was to evaluate the feasibility of developing a sensitive and specific assay for rapid detection and identification of a group of target viral pathogens. the following viral pathogens were included in our array: human immunodeficiency virus types 1 and 2 (hiv-1 and hiv-2), human t-cell leukemia virus-1 and -2 (htlv-1 and htlv-2), hepatitis c virus (hcv) and west nile virus (wnv), all with singlestranded rna genome; vaccinia virus (vacv) and hepatitis b virus (hbv), both with double-stranded dna genome, hbv also has single-stranded rna stage. some of the listed viruses are included to the required de testing for tissue transplantation. besides, historically, some of the targeted viruses have been found to be allograft-transmitted to recipients [11] [12] . in the present study, a real-time pcr array with sybr-green chemistry targeting these eight viral pathogens listed above was developed and evaluated with analytical and clinical panels. the array demonstrated acceptable performance in the testing with both analytical panels and donors' clinical samples. the research study conducted at fda using previously frozen blood samples was reviewed by department of health and human services, food and drug administration, research involving human subjects committee (rishc protocol #10-008b entitled ''detection of infectious agents in previously frozen blood samples from patients with various illnesses and healthy blood donors''). the 17 clinical plasma samples positive for hbv, hcv or hiv-1 used in this study were existing clinical diagnostic samples kept in nih blood bank. information of these left over samples had been recorded in such a manner that subjects can not be identified, directly or through identifiers. the written informed consent from the participants was waived under 45 cfr 46.101 (b) (4) . the six plasma samples positive for hiv-1 were estimated to contain 50 to ,90,000 genome copy numbers per ml; six plasma samples positive for hcv were estimated to contain 780 to ,123,000 genome copy number/ml and five plasma samples positive for hbv were estimated to contain ,150 to 16,000 copy number/ml. the copy numbers of viruses in these samples were provided by the nih blood bank. no information about the subtypes or genotypes of these viruses was available. the amount of each clinical sample was sufficient to be tested only once by the pcr array in the study. all positive pcr products obtained in the testing using the pcr array were confirmed for validity by sequencing in the facility for biotechnology resources of fda/ cber. we used the ''insignia'' program (http://insignia.cbcb.umd. edu/query.php), a bioinformatics on line tool developed in the center for bioinformatics and computational biology, university of maryland [13] to choose a specific dna or rna ''signature'' (a sequence, with customized length and g/c content) for targeted viruses. comparative sequence analysis of the complete genomes was performed using mvista (http://genome.lbl.gov/vista/ mvista/submit.shtml). multiple nucleotide sequence alignments (nsas) were then created to visualize the most conserved genome areas using mega4 (http://www.megasoftware.net). specific criteria for the primers and amplicon selection for the sybr green based pcr array were: 1) the same range of annealing temperature (t) -57-60uc -for all primers, 2) high g/c content for primers, allowing higher specificity of annealing, and 3) an amplicon size in the range of 100-200 b.p. in order to have a high pcr amplification efficiency and to sufficiently distinguish the products from primer dimers based on melting t peak (tm). all primers were checked for potential dimer formation using ''primer express'' software (version 3.0, applied biosystems). after design, all primers were again checked using the national center for biotechnology information (ncbi) basic local alignment search tool (blast) (http://blast.ncbi.nlm.nih.gov/blast.cgi) to avoid any cross-reactivity with other species. newly designed and previously published primer sets adapted for the final version of the real-time pcr array are listed in table 1 . in addition, primers specific for the human beta-globin gene were included in the array as an internal control for the quality of dna/rna preparation as well as for estimation of viral copy number per host cell if needed (the last row of table 1 ). total cellular dna of the chronically infected cell cultures (for hiv-1, hiv-2; htlv-1, htlv-2 and vacv), viral genome cdna copy spiked into human dna (for hcv and wnv), and dna isolated from human plasma of the infected individual (for hbv) were used as the positive templates in the initial testing and are listed in the last column of table s1 . dna or rna panels were created by cloning of specific synthetic templates for each virus into the pgem-t-easy vector (promega) by ta cloning, following by in vitro transcription to obtain rna standards for hcv and wnv. all created plasmids are listed in table s1 . nucleotide numbers in table s1 refer to the location of the partial viral genome cloned into pgem-t-easy vector according to the following complete genome sequences available in genbank: htlv-1 -l03562.2, htlv-2 -m10060.1, hiv-1 -k02083.1, hiv-2 -j03654.1, hbv -af462041.1, hcv -af271632.1, vacv -ay243312.1, wnv -hq596519.1. to establish the real-time pcr standard curve the copy number was calculated for each plasmid carrying one copy of the specific viral gene. the size of each plasmid (x b.p.) was used to determine the molecular weight in daltons (g/mol): w (g/mol) = x b.p. (330 da 6 2). the copy number of the target viral gene (molecules/ml) was determined from the plasmid concentration (c dna ) and the molecular weight of each plasmid molecule: copy number = c dna (ng/ml) 6 6.02 6 10 23 (avogadro's number)/w. knowing the number of plasmid molecules with the target viral gene in a ml, a series of dilutions was made to generate a pcr standard curve. the developed analytical standards were used to calculate the intra and inter-assay reproducibility of quantification for each virus-specific primer set. mean c(t) values, standard deviation (sd) and coefficient of variation (cv) were calculated from the data obtained in three replicates of each standard dilution for the intraassay reproducibility, and in three real-time pcr assays consisted of three replicates each (nine total) for the inter-assay reproducibility. cv was calculated as sd/mean c (t) * 100%. preparation of viral rna/dna for pcr array analysis 0.5-1 ml of human plasma was used for the total viral rna/ dna extraction using ''qiaamp viral rna mini-kit'' (qiagen) and trna (sigma) was used as a carrier rna during the preparation. after the final elution step rna/dna in 160 ml of buffer ave was precipitated with 100% ethanol and 3 mm nacl at 220uc overnight. an rna/dna pellet was washed with 70% ethanol, dissolved in 10 ml of depc-treated water and then immediately used for cdna synthesis with superscript ii rt (invitrogen) and random hexamers (invitrogen) in a total reaction volume of 20 ml. the volume of cdna/dna sample was then adjusted up to 30 ml with depc-treated water and the whole pcr was performed using ''bio-rad cfx96 real-time system'' with ''power sybr green pcr master mix'' (applied biosystems). one reaction (25 ml total) contained: 12.5 ml of pcr master mix, 0.5 mm of each primer and 1.25 ml of dna/cdna template. in the single virus testing (sections 3 and 4 of the ''results'') we used 2.5 ml of dna/cdna template from 20 ml of sample after cdna synthesis. ''universal'' pcr conditions for all primer sets included to the array were: 95uc for 8 min (one cycle), then 50 cycles of: denaturation at 95uc for 15 s, annealing and extension at 60uc for 1 min, followed by melting curve read from 65uc to 95uc with increment 0.2uc for 5 s. real-time pcr data were downloaded in 96-well plate format from ''bio-rad cfx manager 2.1'' to ms excel and analyzed manually. two types of samples served as the background control for determination of the c(t) cut off. the 1 st type of negative control was 50 ng of human cellular dna. the 2 nd type of negative control was negative donors' plasma. data were collected in separate experiments from 8 human cellular dna controls and 3 negative donors' plasma. to standardized the c(t) cut off for all primers the threshold was set at the pcr machine default setting. based on a false positive rate of less than 5% the following method [14] was used to estimate the c(t) cut off from the range of c(t) obtained with negative samples for all 24 primer sets in the array: 1. calculate the margin of error of the confidence interval (ci), w = t* 6 sd/!n, where: n -number of obtained c(t) values, sd-standard deviation, df = n21, and t* (for 95% confidence) is a ''critical value of the t distribution'' [14] . 2. one side ci covers this range: m-w, where m is sample mean. the c(t) cut off calculated from the range (n = 50) of the 1 st type of negative control data was c(t)#41.03. the c(t) cut off calculated from the range (n = 50) of the 2 nd type of negative control data was c(t)#42.7. even some overlap between the c(t) measurements of truly positive and truly negative samples was detected in pcr array data, the tm parameter was used to define if the obtained pcr product is specific by comparison to the expected tm peak range. the analytical sensitivity of each primer set was determined in the single virus testing using fda/cber panels (kindly provided by dr. stephen kerby, fda/cber) consisting of various amounts of the viruses (0-1,000 genome copies/ml) spiked into the ''normal'' human plasma. panels for the following viruses were used in testing: hiv-1 (three different panels), hiv-2, hbv (based on genotype a), hcv (genotype 1b), and wnv (based on strain hu2002). these panels have been used for testing of commercially licensed nat assays and their development was described previously [15] . three separate hiv-1 rna panels were used for testing. the first consisted of various amounts of an hiv-1 group m subtype b isolate: 0, 5, 10, 25, 50, 100, and 500 copies/ml. the second consisted of 25 samples representing various concentrations of hiv-1 groups o and n, and group m subtypes a, c, d, e, f and g: 10, 100 and 1,000 copies/ml for each virus. the third panel consisted of hiv-1 circulating recombinant forms (crfs) ae and ag: 100, 1,000, and 10,000 copies/ml for each crf. np24 caacttcatccacgtttcacc a -to simplify the process of evaluation we used our primer names with sequential numbers, however some of the primers have been designed previously with their original names in the articles listed in the last column of this [25] [26] . hiv-1 groups and subtypes are based on designations reported by the nih arrrp. low passage virus stocks were prepared and median tissue culture infective dose (tcid 50 )/ml of virus containing supernatant determined in fresh human pbmcs isolated as previously described [27] . infectious unit (iu) of the virus stocks in tcid 50 s were titrated for each isolate and the number of iu used for viral rna isolation and pcr was calculated based on the dilution factor (1:100) and ranged from ,10 to 2,500 or 1 to 3.4 log 10 tcid 50 per pcr reaction. pcr approach based on sybr green chemistry, allowing simultaneous detection of multiple targets, was chosen to be applied for the array performance. virus-specific primer sets targeting at least three different genomic sites for each viral pathogen were designed for the real-time pcr array. we used the ''insignia'' program, a bioinformatics tool that helps to choose a specific dna or rna ''signature'' for different bacteria and viral pathogens that are included in the pre-built ''insignia'' database [13] . sequences of the highly conserved regions, such as coding viral polymerase or structural proteins were selected to design the candidate primers. in addition, we performed nucleotide sequence alignments (nsa) of the complete viral genomes and of the most conserved genome areas for different subtypes/genotypes or different isolates of all targeted viruses. some previously published primers designed for pcr detection of the target viruses were also adapted and evaluated. overall, a total of 120 primers were initially designed using specific criteria for the current real-time pcr array (see materials and methods) to cover the eight targeted viruses: hiv-1, hiv-2, hbv, hcv, htlv-1, htlv -2, wnv, and vacv. the primers were designed and tested for their effectiveness and specificity of amplifying the respective target under uniform pcr conditions (using the same annealing temperature for all primers). each candidate primer pair was first tested for its specificity and sensitivity of pcr amplification. this was assessed in a real-time pcr cross-testing against human pbmc dna (50 ng/pcr reaction) from a healthy donor, dna from human cell cultures infected by various target viruses, or human dna spiked with a known amount of genome copies of various viruses. it was important to ensure that the selected primers targeting a specific virus would not non-specifically amplify any dna in a sample. the melting temperature (tm) peak of the product amplified from the target virus should be clearly differentiable from the tm peak of primer dimers or any non-specific products produced in pcr. dna or rna panels were created from the cloned synthetic templates (listed in table s1 ) by spiking of 2-10 4 genome copies of each virus into 50 ng of human background dna. the sensitivity of each candidate primer set for each target virus was assessed using these panels. example of the experimental testing of hiv-1 specific primer set targeting gag gene (np3/4) for its sensitivity with dna analytical standards is shown in figure s1 . serial dilutions of a plasmid dna corresponding to 5-10 4 hiv-1 genome copies spiked into 50 ng of normal human dna, hiv-1 infected cells (h9/iiib) (''positive'' control dna) and uninfected human pbmc (''negative'' control dna) are used in the experiment. only a single melting peak tm = 72.5uc corresponding to hiv-1 specific product was observed and no unspecific amplification was registered. figure s1b revealed a standard curve showing the correlation between copy number of the target gene and ct values with a slope = 23.77. the limit of sensitivity was determined in this assay to be 10 viral genome copies/pcr. after evaluation, a total of 24 primer pairs targeting the eight different viruses were chosen for the real-time pcr array based on their specificity and sensitivity (table 1) . among them, five of the primer sets were previously published and the other 19 primer sets were originally designed in the current study. table 1 shows the sequences of the previously published primer sets selected for the real-time pcr array with the reference to the original source. analytical sensitivity expressed in genome copy/pcr for each primer set (table 1 ) was estimated using dna/rna analytical panels, as described above. coverage of variants (i.e., different subtypes or genotypes) for each virus (table 1 ) was estimated using the nsas. degenerative nucleotides were introduced into some of the primer sets based on the nsas performed in-house to obtain a broader coverage. the tm peak range of the product stated in table 1 for each primer set was established during further testing of selected primers with analytical and clinical panels. in addition, the intra and inter-assay reproducibility of quantification for all primer sets was evaluated using three replicates of each standard dilution (of dna or rna analytical standards) in each of three real-time pcr assay runs. the coefficient of variation (cv) for the c (t) values was #3.3% and #6.7% for intra-and inter-assay, respectively. all the data depicting mean c (t), standard deviation (sd), and cv for each primer set selected for the real-time pcr array with each standard concentration are shown in table s2 . to further evaluate the sensitivity of the selected primers, we used fda/cber panels (kindly provided by dr. stephen kerby, fda), consisting of various amounts of viruses spiked into ''normal'' human plasma, that are specifically developed and used for the evaluation of commercially licensed nat assays. table 2 summarizes the testing results for the selected primers against the target viruses. one out of four primer sets targeting hiv-1, np3/4, could detect the subtype b hiv-1 rna at the concentration of 50 copies/ml of human plasma. two other hiv-1 specific primer sets (np51/52 and np170/171) detected the hiv-1 rna at 100 copies/ml of plasma. the 4th primer set, np175/174, (targeting the conserved pol region and containing degenerative nucleotides to support broader variant coverage) could detect the virus only at 500 viral genome copies/ml of plasma. hiv-2 rna was detected with primer set np86/87 at the concentration of 10 copies/ml and with the primer set np76/77 at the concentration of 50 copies/ml. another primer set (np84/ 85) detected hiv-2 at 100 copies/ml. hbv (genotype a) dna was detected with the primer set np11/97 at 50 copies/ml and with np94/100 at 100 copies/ml. the third hbv specific primer set (np11/97-mod) detected hbv at 500 copies/ml of plasma. both hcv specific primer sets targeting the viral 59ntr detected hcv rna at the concentration of 10 copies/ml of plasma. two of the wnv-specific primer sets (np21/22, targeting e protein gene, and np176/177, targeting ns5) detected wnv at 50 copies/ml and the third primer set (np178/179, also targeting ns5) gave a positive signal at 100 copies/ml of plasma. the four hiv-1 specific primer sets were further evaluated by testing them against another fda/cber analytical panel containing a broad spectrum of hiv-1 subtypes. as summarized in table 3 , the hiv-1 subtype f (group m) and group o isolates in table 2 . the results of sensitivity testing of the real-time pcr array primer sets specific for hiv-1, hiv-2, hbv, hcv, and wnv the with fda/cber analytical plasma panels. table 3 . sensitivity of four hiv-1 specific primer sets selected for the real-time pcr array in testing with fda/cber analytical hiv-1 broad spectrum panel. detection limit (copy number/ml of plasma) was evaluated using fda/cber analytical panel, containing pre-set copy number of hiv-1 spiked into 1 ml of ''normal'' human plasma. rna from 1 ml of plasma was converted to cdna and divided into eight pcr reactions; two pcr repeats were performed for each primer set. the estimated copy number per pcr reaction is shown in parentheses. the panel could not be successfully amplified by any of the four selected primer sets (detection limit is .1,000 rna copy/ml of plasma). all other subtypes and crfs of group m, and the group n isolate could be amplified with at least one out of the four primer sets at 50 to 1,000 rna copies/ml of plasma (table 3) . the tm peaks of the pcr products amplified from different subtypes of hiv-1 varied within 1.5uc ( table 3 ). all of the four selected hiv-1 specific primer sets amplified hiv-1 subtype b (group m) with the highest sensitivity (50-500 copies/ml of plasma). to examine the specificity and the ability of the array to detect different isolates within subtypes of hiv-1 and different isolates of hiv-2 by the array, we additionally tested our primers with another panel, developed by the southern research institute. this panel contained three different isolates from each subtype (a to g) of hiv-1 group m, three isolates from hiv-1 group o, one isolate from hiv-1 group n and three different isolates of hiv-2. the infectious dose of each hiv isolate in the panel was determined by tcid 50 (median tissue culture infective dose) titration and the dose of virus used in each pcr reaction was calculated. the results of testing of hiv-1 specific primers are shown in figure 1 . in this experiment we evaluated the coverage of hiv-1 variants and estimated the relative sensitivity of the four hiv-1 specific primer sets based on cycle threshold (c(t)) values obtained with each isolate tested. two primer sets targeting the conserved pol regions (np170/171 and np175/174) were able to detect most of the hiv-1 subtypes with a high sensitivity (c(t) = 15-25). only one primer set (np175/174) detected both the group n isolate and all three isolates of group o with ct = 20-35. we did not detect crossreactivity of hiv-1 or hiv-2 specific primers with the other type of hiv. all three hiv-2 isolates studied (1-3 log 10 tcid 50 /pcr input) were detected with all hiv-2 specific primer sets with a low ct value: 12-20 (data not shown). after completion of sensitivity testing of primer sets with analytical panels we finalized the expected tm peaks range for each amplicon and arranged a working array in 96-well plate format ( figure s2 ). to evaluate the specificity and possible crossreactivity of primers in the array, 20-100 genome copies of each virus were spiked into 50 ng of human dna to be used as positive templates and the same amount of human dna was used as a negative control in each experiment. the array was tested with all targeted viruses using dna standards (listed in table s1 ). each positive template was tested in duplicate; one human dna negative control and one no-template control (ntc) were included in every testing. in these experiments the definition of ''positive'' signal was set up as the threshold cycle cut-off of c(t)#41 (see materials and methods) and all tm peaks are within the expected range ( figure s2) . no cross-reactivity was detected for any primer sets in the array using dna standards with this setting (data not shown). seventeen (17) clinical plasma samples (obtained from nih blood bank) from donors who tested positive for hiv-1, hbv, or hcv were used to evaluate the array sensitivity and specificity. the genome copy of each virus in virus-positive plasma samples was previously determined by the nih blood bank using commercial assays approved for donor screening. representative results from the testing of three hbv-positive plasma samples (pt.#13-pt.#15) using our array are shown on figure 2 . the threshold cycle cut off of the assay performed with plasma samples was set up as c(t)#43 (see materials and methods). the wells with c(t) lower than the cut-off and the obtained tm peaks within the expected tm range indicate positive amplification of hbv target genes and are highlighted with blue circles (figure 2 , green color code for hbv). for samples #13 and #14 tm peaks of the products, obtained with only two hbv specific primer sets (wells g2, g3-pt.#13 and wells g5, g6-pt.#14), were within the expected range, with c(t) values of 36.7-42.9 and 36.5-36.6 respectively. for sample #15, all three hbv specific primer sets gave the tm peaks within the expected range (wells g7, g8 and g9), and the c(t) values range was 33.8-34.2. the genome copy number of hbv in plasma samples #13-15 was 151-518 copies/ ml, corresponding to 6-21 copies/pcr. the wells (white color code, red circles) with the human beta-globin gene specific primer set, serving as the internal control, produced positive signals from all three plasma samples tested, with c(t) values of 25.5-26.3 (wells h3, h6 and h9 figure 2 ), which shows that the quality of the rna/dna sample preparation was equally good for the plasma of these three patients. the final tm range for each primer set was adjusted after testing of this clinical panel. based on the results from the testing of clinical samples and according to nsa performed during the design, two primer sets targeting the s gene region of hbv (np11/97 and np11/97-mod) have two different tm ranges depending on the hbv genotype ( figure 2 -wells g1 and g3). the lower tm range (74.5-75.5uc) was detected for genotype a and the higher tm range (76.8-77.4uc) -for genotypes b, c, and d. the difference in tm ranges occurred due to single nucleotide polymorphism (snp) in the amplicon sequence leading to a difference in the g/c nucleotide content of the products. table 4 summarizes the results from testing the 17 virus-positive clinical samples using the developed array. at least two different specific primer sets could detect a target virus in all cases, with one exception: one hiv-1 positive sample (#4) with ,4 viral genome copies per pcr reaction was amplified with only one primer set (np175/174). hiv-1 positive clinical samples (#1-#3) with 51-80 viral genome copies/ml of plasma or 2-3 copies/pcr reaction were detected with at least two primer sets. all six hcv-positive samples (#7 to #12) with 16-2,570 viral genome copies/ml of plasma tested positive with both hcv-specific primer sets. three hbv-positive samples (#15, #16, #17) at 21-66 copies/pcr tested positive by the three hbv-specific primer sets and two hbv-positive samples (#13, #14) with 6-10 copies/pcr tested positive with two out of three hbv-specific primer sets. it is important to note that none of the primer sets showed any crossreactivity with other viruses in the panel of clinical samples tested. in the study presented here we applied a real-time pcr array approach in a 96-well plate platform for detection of a group of target viral pathogens. in contrast to taqman pcr, which is commonly used for viral diagnostics, this platform based on pcr with sybr green chemistry supports simultaneous detection and identification of 24 different targets corresponding to eight different viruses. pcr based on sybr green staining of the double stranded dna is economically affordable and allows for the detection of mutants with snps within the amplicon sequence [34] . the strategy of primer selection for the array included the sequential use of different bioinformatics programs to identify highly-specific primer sets with a maximal variants' coverage of the targeted viruses, while working under ''universal'' pcr conditions. experimental selection process using a panel of dna or rna analytical standards allowed choosing two to four most sensitive and specific primer sets for each targeted virus. the sensitivity (in copy number per pcr reaction) and the intra and inter-assay reproducibility of quantification were characterized for each primer set selected for the array. fda/cber analytical plasma panels were used to assess the detection sensitivity of the primer sets selected for the working array. the hiv-2 and hcv rna was detected at as low as 10 genome copies/ml of human plasma by one out of three and two out two primer sets, respectively. similarly, wnv rna and hbv dna were detected at 50 genome copies/ml of plasma by two out of three and one out of three selected primer sets respectively. all four of the selected hiv-1 specific primer sets detected group m, subtype b, the most common subtype of hiv-1 in the americas and western europe [35] , at 50-00 genome copies/ml of plasma. however, the array was able to amplify all other subtypes, excluding subtype f, of hiv-1 group m with only one primer set (np170/171), targeting the conserved pol region, at 100-1,000 genome copies/ml of plasma. in addition, the array amplified the group n isolate at 1,000 copies/ml with another primer set (np175/174) targeting the conserved pol region. all the selected hiv-1 specific primer sets of the array failed to amplify the group o isolate (detection limit is .1,000 copies/ml of plasma). in comparison, the limit of detection (lod) of recently improved commercial multiplex nat assays for the broad spectrum of hiv-1 group m isolates is 40 copies/ml, and the lod for group o is 200 copies/ml [36] [37] [38] . thus, specific primer sets targeting group o isolates of hiv-1, as well as subtype f (group m) will need to be included in the array to increase the coverage of all existing subtypes/groups of the hiv-1. no crossreactivity was shown for hiv-1 or hiv-2 specific primers with the other type of hiv in a testing with medically relevant levels of viruses in a diversity panel containing 25 different hiv-1 isolates and three natural hiv-2 isolates collected worldwide. there is presently no us food and drug administration (fda) approved pcr-based nat testing for blood donors' screening for htlv-1 and htlv-2. there is no official fda (or world health organization (who)) viral panel released for these two viruses. it is also difficult to obtain htlv-1 or htlv-2 positive blood donors' samples in the united states. in the absence of the analytical panels and clinical samples we tested htlv-1, htlv-2 and vacv specific primers only with cell culture derived dna and with dna standards. the minimum detection limit of htlv-1 and htlv-2 specific primer sets estimated with analytical dna standards was 5-10 genome copies/pcr reaction, which is in the same range as for other primers included to the array. the coverage of the viral variants for the primers targeting these viruses was estimated in silico using multiple sequence alignments performed with complete genome sequences available in gen bank. the working array arranged in 96-well plate format was subsequently tested for specificity and potential cross-reactivity with human dna and with each of the targeted viruses. none of the primer sets selected for a particular target virus in the array produced non-specific cross-reaction toward the other viruses. comparative performance of the array was also evaluated through the testing of 17 clinical specimens from the united states patient population. all 17 samples were correctly identified in our pcr array with a high sensitivity to contain hiv-1, hcv, or hbv. we found that a combination of several primer sets targeting each virus in the array allows for the detection of different variants of the virus; however, it makes the absolute quantification with uniform dna/rna standards a challenge. quantification by the assay is not always possible when the genetic group of the viral isolate being tested is different than the assay standards. this is one of the reasons why in certain cases the commercial assays underestimate viral loads by up to 1-10 log 10 copies per ml [37] [38] [39] . nevertheless, relative quantification can be done using all primer sets selected for the array, and the absolute quantification can be performed with dna/rna standards using the primers targeting the most conserved genome areas. there are several commercial qualitative multiplex nat assays now available on the market simultaneously targeting three most important blood-borne viral pathogens (hiv-1, hcv and hbv) [40] . one of them was recently approved by u.s. fda for screening of blood and organ donors (http://www.fda.gov/biologicsblood vaccines/bloodbloodproducts/approvedproducts/licensedproduc tsblas/blooddonorscreening/infectiousdisease/ucm306073.htm). we compared the lod of these multiplex nat assays [40] with the results of our working pcr array in sensitivity of testing against these important viruses. the lod for hbv are 38.1-195 geq/ml by ''procleix ultrio tigris'' and 9.2-37. other pcr-coupled techniques have been developed previously for highly-sensitive pathogens' detection that could reach the sensitivity of the assay up to 1 genome copy per pcr reaction. for example the bioactive amplification with probing (bap), utilizing a nested pcr and magnetic bead-based hybridization with the specific probe, has been developed for the detection of bovine and avian viruses [41] [42] [43] . in spite of the exceeding sensitivity of such assays targeting a single virus, it may be difficult to adapt the approach or method to meet the main objective of simultaneous detection of multiple target viral pathogens by an array using universal pcr conditions. it is important to note that this array was developed to be adapted by any laboratory. comparison of experimentally obtained tm peaks to the range of expected specific tm peaks allows rapid identification or exclusion of the viral pathogen in a sample. this is an initial study to examine the suitability of using pcr arrays for the detection of a group of target viruses. the current array was developed utilizing five previously published and table 4 . tm and c(t) values obtained with primer sets specific for hiv-1, hcv, or hbv in testing of 17 human clinical samples in the format of pcr array targeting eight different viruses. 19 originally designed primers sets. however, it can be expanded to a larger number of targets for the same virus. targeting of several genome areas increases the detection sensitivity of the target virus and provides an intra-assay confirmation of positive signals. additionally, any new virus of interest can be added to the list of targeted pathogens. efforts are underway to test the utility of this real-time pcr array using samples with more diverse biological origin and pathogen content. panmicrobial oligonucleotide array for diagnosis of infectious diseases testing and validation of high density resequencing microarray for broad range biothreat agents' detection crossspecies transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony identification of pathogenic vibrio species by multilocus pcr-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of georgia pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity an oligonucleotide microarray for multiplex real-time pcr identification of hiv-1, hbv, and hcv a low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens application of taqman low density arrays for simultaneous detection of multiple respiratory pathogens use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses eligibility determination for donors of human cells, tissues, and cellular and tissue-based products; final rule and notice. 21 cfr part 1271 infections and human tissue transplants: review of fda medwatch reports reported infections after human tissue transplantation before and after new food and drug administration (fda) regulations, united states insignia: a dna signature search web server for diagnostic assay development intuitive biostatistics development and evaluation of hiv-1 subtype rna panels for the standardization of hiv-1 nat assays genetic variation of hiv type 1 in four world health organization-sponsored vaccine evaluation sites: generation of functional envelope (glycoprotein 160) clones representative of sequence subtypes a, b, c, and e. who network for hiv isolation and characterization human immunodeficiency virus type 1 t-cell tropism is determined by events prior to provirus formation dual infection of the central nervous system by aids viruses with distinct cellular tropisms biologic and genetic characterization of a panel of 60 human immunodeficiency virus type 1 isolates, representing clades a, b, c, d, crf01_ae, and crf02_ag, for the development and assessment of candidate vaccines subgroup g hiv type 1 isolates from nigeria variability of human immunodeficiency virus type 1 group o strains isolated from cameroonian patients living in france identification of a new human immunodeficiency virus type 1 distinct from group m and group o biological and molecular variability of human immunodeficiency virus type 2 isolates from the gambia genetically divergent strains of human immunodeficiency virus type 2 use multiple coreceptors for viral entry aids verursacht durch hiv-2 preliminary characterization of an hiv-2 isolate derived from a german virus carrier development of hexadecyloxypropyl tenofovir (cmx157) for treatment of infection caused by wild-type and nucleoside/nucleotide-resistant hiv dna amplification for direct detection of hiv-1 in dna of peripheral blood mononuclear cells quantitation of hepatitis b virus dna in plasma using a sensitive cost-effective ''in-house'' realtime pcr assay a genotypeindependent real-time pcr assay for quantification of hepatitis b virus dna rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a taqman reverse transcriptase-pcr assay hpv dna detection and typing in cervical scrapes complete coding sequences of the rabbit pox virus genome sybr green-based real-time quantitative pcr assay for detection of west nile virus circumvents falsenegative results due to strain variability human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications evaluation of the roche cobas taqman and abbott realtime extraction-quantification systems for hiv-1 subtypes comparative rna quantification of hiv-1 group m and non-m with the roche cobas ampli prep/cobas taqman hiv-1 v2.0 and abbott real-time hiv-1 pcr assays a new real-time quantitative pcr for the diagnosis and monitoring of hiv-1 group o infection performance characteristics and comparison of abbott and artus real-time systems for hepatitis b virus dna quantification comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: procleix tigris and cobas s 201 development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing (bap) for detection of bovine ephemeral fever virus development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing for detection of avian reovirus development of a reliable assay protocol for identification of diseases (rapid)-bioactive amplification with probing (bap) for detection of newcastle disease virus key: cord-296222-w5m23ikh authors: hu, song; jiang, li-bin; zou, xiao-jing; yi, wei; tian, de-ying title: hepatitis b virus upregulates host expression of α-1,2-mannosidases via the pparα pathway date: 2016-11-21 journal: world j gastroenterol doi: 10.3748/wjg.v22.i43.9534 sha: doc_id: 296222 cord_uid: w5m23ikh aim: to assess the effects of hepatitis b virus (hbv) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms. methods: we measured the expression levels of man1a1, man1a2, man1b1, and man1c1 in cell lines hepg2.2.15, hepn10, hepad38 and hepg2 by western blot. viral antigens (hbsag and hbeag) in the culture medium were measured using the chemiluminescence method. hbv dna quantification assays were performed using a commercial real-time pcr kit. protein levels of human liver tissue α-1,2-mannosidases were also evaluated by western blot. plasmids containing seven individual viral genes of hbv (ptt22-hbx, ptt22-hbs, ptt22-pres2, ptt22-pres1, ptt22-hbc, ptt22-hbe, and ptt22-hbp) or control plasmids (ptt22-vector) were transfected into hepg2 cells. mk886 (pparα) and gw9662 (pparγ) inhibitors were used to explore the effects of hbv on α-1,2-mannosidase expression after the pparα and pparγ pathways were blocked. results: we showed that the expression of α-1,2-mannosidases was higher in stably transfected hbv cells than in controls. the expression levels of α-1,2-mannosidase were higher in ad38 cells than those in nd10 cells, which were in turn greater than those in g2.2.15 cells, and positively correlated with the expression of hbsag in all the cell lines. levels of α-1,2-mannosidase in non-tumorous liver tissues of hbv-related hcc patients were also higher than in the tissues from non-hbv-related hcc patients. moreover, transfecting hepg2 cells with a component of the hbv viral envelope also increased the expression of α-1,2-mannosidases. however, this envelope protein component could not induce man1c1 expression in the presence of a pparα inhibitor, mk886. we also found that mk886 did not affect the expression of man1c1 in ad38 cells without tetracycline in the culture medium. this phenomenon was not observed in the case of gw9662. conclusion: our results indicate that hbv increases the expression of α-mannosidases both in vitro and in vivo via activation of the pparα pathway by its envelope protein. hepatitis b virus (hbv) infection is the most common chronic viral infection in the world. an estimated 2 billion people are infected, and more than 350 million are chronic carriers of the virus [1] . due to an insufficient immune response, some individuals with hbv infection can develop chronic hepatitis, which can eventually result in liver cirrhosis and hepatocellular carcinoma (hcc). while the underlying mechanisms for hbv-induced chronic hepatitis remain unclear, several studies indicate that dendritic cell (dc) function is impaired in patients with chronic hepatitis b [2, 3] . dcs are potent antigen-presenting cells (apcs) that can present antigen to t cells and activate naive t cells. multiple receptor molecules on the surface of dcs, including toll-like receptors (tlrs) and c-type lectin receptors (clrs), participate in the recognition and uptake of pathogens, and can regulate the expression of co-stimulatory molecules [4] . in particular, dcspecific icam-3 grabbing non-integrin (dc-sign) is an important clr that is mainly expressed on the surface of mature and immature dcs [5] . dc-sign plays an important role in the recognition of pathogen-associated molecular patterns (pamps) [5] . moreover, previous studies have shown that dc-sign is involved in the immune escape of multiple pathogenic microorganisms, including hiv-1, ebola virus, hepatitis c virus (hcv), dengue fever virus, cytomegalovirus (cmv), sars-coronavirus, mycobacterium tuberculosis, helicobacter pylori, fungus, and some parasites [6, 7] . unfortunately, the mechanisms behind immune escape mediated by dc-sign remain unclear. it may be that dc-sign directly recognizes mannose oligosaccharides found on the viral envelope of hbv. through modifications to these mannose oligosaccharides on its viral envelope, hbv could potential evade the host immune system. indeed, it has been shown that dc-sign does not recognize wild-type hbv but does recognize a high-mannose type hbv produced by the application of a class ⅰ α-mannosidase inhibitor, kifunensine [8, 9] . moreover, in our previous studies, we found that the high-mannose type hbv could promote the maturation of dcs, increase il-12 secretion, and effectively stimulate the proliferation of allogeneic lymphocytes, and that these effects were blocked by specific anti-dc-sign antibodies [10] . therefore, demannosylation appears to be beneficial to hbv to escape dc-sign recognition and avoid the consequent immune elimination. demannosylation of glycoproteins occurs through a family of mannosidases that trim mannan in the n-oligosaccharide. human class ⅰ α-mannosidases include the er α-1, 2-mannosidase i (man1b1), and three golgi α-1,2-mannosidases, namely α-mannosidase ia (man1a1), α-mannosidase ib (man1a2), and α-mannosidase ic (man1c1). generally, man1b1 firstly trims α-1,2-mannose from man9glcnac2-asn, and then man1a1, man1a2, and man1c1 trim the residual α-1,2-mannose, to generate man5glcnac2-asn. these processes are dependent on the varying levels of the mannosidase subtypes in different cell types [11] . there are glycosylation sites on the surface of hbv envelope proteins, which could be glycosylated by the host [12] . therefore, it is not surprising that hbv infection has previously been shown to upregulate the expression of er degradation-enhancing α-mannosidases (edems) in order to increase demannosylation [13] . however, few studies have investigated the effects of hbv on the expression of class ⅰ α-mannosidases. peroxisome proliferator activated-receptors (ppars), a group of ligand-activated nuclear transcription factors, may be involved in α-mannosidase expression during hbv infection. indeed, tumor necrosis factor-α (tnf-α) has been shown to downregulate the expression of man1c1 to increase the expression of high-mannose type proteoglycans, and this can be reversed by the activation of the ppar signaling pathway [14] . based on the results of these previous studies, we hypothesized that hbv could promote the demannosylation of the viral protein by increasing the expression of α-mannosidase i in the host cells. in turn, dc-sign can no longer recognize the hbv glycoprotein coat, allowing the virus to escape from immune attack. in the present study, we investigated the effects of hbv infection on the expression of α-mannosidase i, and also determined the mechanisms driving the altered expression. we show for the first time that hbv infection increases the expression of α-mannosidase i via the pparα signaling pathway. human hepatocellular carcinoma cells (hepg2, hepg2.2.15, ad38, and n10) were cultured in dmem at 37 ℃ in a 5% co2 incubator. the medium was supplemented with 10% fbs, 100 iu/ml penicillin, and 100 iu/ml streptomycin. cells were changed into fresh medium every third day, and split by trypsinization at a confluence of about 90% [15] . ad38 cells, which are a variant of hepg2 cells, express the hbv genome under the control of a tetracycline (tet)-off promoter. therefore, the ad38 cell culture medium also contained tetracycline (1 μg/ml) when not requiring the expression of hbv genes [16] . hepg2.2.15 and n10 cells are secretory hbv cell lines derived from g2 [17] . viral antigens (hbsag and hbeag) in the culture medium were measured using the chemiluminescence method with commercial assay kits (wantai, beijing, china). hbv dna quantification assays were performed using a commercial real-time pcr kit (kehua, shanghai, china). plasmids containing seven individual viral genes of hbv (i.e., ptt22-hbx, ptt22-hbs, ptt22-pres2, ptt22-pres1, ptt22-hbc, ptt22-hbe, and ptt22-hbp) and the control plasmid (ptt22-vector) were a kind gift from dr. quan yuan and tianying zhang (national institute of diagnostics and vaccine development in infectious diseases, xiamen university, china). transient transfections with plasmids were performed using lipofectamine 2000 reagent (invitrogen, united states) according to the manufacturer's protocol. hepg2 cell lines only express pparα and pparγ but not pparβ [18] . therefore, pparα (mk886) and pparγ (gw9662) inhibitors were used at a concentration of 10 μmol/l, unless otherwise noted. mk886 and gw9662 (sigma-aldrich) were added to the cells 6 h after transfection. human liver tissues were obtained from patients who were treated at tongji hospital, tongji medical college, huazhong university of science and technology between july 2014 and december 2014, and included 12 non-tumorous samples from hbv-related hcc patients and 12 samples from non-hbv-related hcc patients. all tissue samples were collected and stored at -80 ℃ before investigation. cells were lysed 72 h after transfection. cell and tissue samples were lysed in rira (sigma-aldrich) buffer with pmsf (sigma-aldrich). lysates were centrifuged for 10 min at 12000 × g, and protein in the supernatant was quantified using the bca method (pierce). sds-page and western blot analysis were performed according to standard procedures. mouse anti-hbs, anti-hbx, anti-hbp, and anti-hbc antibodies were kindly provided by dr. quan yuan from xiamen university. goat anti-man1a1 (santa cruz; 1:500 dilution), rabbit anti-man1a2 (proteintech; 1:200 dilution), rabbit anti-man1b1 (genetex; 1:500 dilution), mouse anti-man1c1 (abcam; 1:500 dilution) antibodies were used as the primary antibodies. mouse anti-β-actin (proteintech; 1:1000 dilution) was used as a reference for protein quantification. goat anti-mouse igg (proteintech; 1:10000 dilution), goat anti-rabbit igg (proteintech; 1:10000 dilution), and donkey antigoat igg (santa cruz; 1:10000 dilution) were used as the secondary antibodies followed by enhanced chemiluminescence (ecl; thermofisher scientific) detection. the ethics committee of tongji hospital, tongji medical college, huazhong university of science and technology authorized the study protocol. written informed consent was obtained from all participants regarding the aims and objectives of the study. irb id:tj-c20140411. the valid date was from 05/2014 to 05/2015. data are mean ± sd of at least three replicates. the expression of the α-1,2-mannosidase levels were compared between groups using the student t test. statistical analysis was performed using spss software. differences were considered statistically significant at a value of p < 0.05. in order to investigate whether hbv could upregulate man1c1 protein levels increased in cells transfected with hbs, pres2, and pres1; however, the other viral proteins (hbc, hbx, hbe, and hbp) had no significant effects on α-1,2-mannosidase expression ( figure 3f and g). hbv has the genes for three envelope proteins: shb, mhb (pres2 + s) and lhb (pres1 + pres2 + s) [19] . because of the similarity in the shb motifs for ptt22-hbs (shb), ptt22-pres2 (mhb), and ptt22-pres1 (lhb), we focused on ptt22-hbs for further study. as the downregulation of man1c1 expression can be reversed by activation of the ppar signaling pathway [14] , we investigated whether the hbvinduced increase in α-1,2-mannosidase expression is associated with the ppar signaling pathway. as hepg2 cells only express pparα and pparγ [18] , mk886 (pparα) and gw9662 (pparγ) inhibitors were used to explore the effects of hbv on α-1,2-mannosidase expression after the pparα and pparγ pathways were blocked. the results showed that after the application of the pparα inhibitor mk886, the effects of hbv on α-1,2mannosidase expression were neutralized; however, no such effects were found when a pparγ inhibitor was applied ( figure 4a ). in order to further confirm whether the ppar signaling pathway plays a role in the hbv-mediated increase in α-1,2-mannosidase levels, we tested the effect of gw9662 and mk886 on the expression of α-1,2-mannosidase in ad38 cells. we found that mk886 did not affect the expression of man1c1 in ad38 cells without tet, indicating that mk886 inhibited the increase in α-1,2-mannosidase levels caused by the tet withdrawal. however, this phenomenon was not observed in the case of gw9662 ( figure 4c ). most enveloped viruses, including hbv, contain envelope protein polysaccharides, which are extensively glycosylated [20] . protein glycosylation has multiple functions, and sometimes assists in evading immune surveillance [21] . viruses use the host cell glycosylation system to synthesize and modify their envelope proteoglycans, and thus, the structures of viral envelope glycoproteins can be affected by the glycosylation mechanisms in the host [22] . indeed, previous studies have shown that applying low-doses of glucosidase inhibitors to host cells changes the phenotype of the viral envelope proteins [23] . this, in turn, can reduce viral infection and affect the virus assembly and/or particle release [23] . for example, the α-glucosidase inhibitor nb-dnj was previously shown to prevent the hbv from correct folding and the release of viral envelope molecules, and thus, could dose-dependently reduce the virus level [9] . on the other hand, while the α-mannosidase i inhibitor the expression of α-1, 2-mannosidases, we measured the expression of man1a1, man1a2, man1b1, and man1c1 in hepatoma cells with or without hbv transfection. man1a1, mana2, man1b1, and man1c1 protein levels in the hepg2.2.15 and n10 cell lines with stable hbv-transfection were higher than in hepg2 cells (figure 1 ). to confirm whether hbv infection was the cause of the upregulation, ad38 cells, which express the hbv genome under the control of a tetracycline (tet)-off promoter, were further investigated. the expression of hbv genes in the ad38 cell line was restricted in the presence of tet. when tet was absent, these cells produced 3.5 kb hbv pregenomic rna and secreted virus-like particles into the supernatant. we found that α-1,2-mannosidase expression in the ad38 cells without tet was higher than in cells with tet ( figure 1a and b) . the expression levels of mannosidases were higher in ad38 cells than those in nd10 cells, which were in turn greater than those in g2.2.15 cells. to investigate the association between the expression of α-1,2mannosidase and virus secretion, we assessed virus secretion in the cell culture supernatants of various hbv cell lines. among the three cell lines, ad38 without tet showed the highest secretion of hbv-dna (10 7.8 ± 0.1 iu/ml), hbeag (21.9 ± 0.8 ncu/ml), and hbsag (87.5 ± 2.4 iu/ml) ( figure 1c-e) . further, the expression of α-1,2-mannosidase in all the cell lines was positively correlated with the expression of hbsag ( figure 1d ). next, we investigated whether hbv could upregulate the expression of α-1, 2-mannosidases (man1a1, man1a2, man1b1, and man1c1) in human liver samples. levels of α-1,2-mannosidase in non-tumorous liver tissues of the hbv-related hcc patients were higher than in the tissues from non-hbv-related hcc patients ( figure 2 ). these findings suggest that hbv increases the expression of α-1,2-mannosidases both in vivo and in vitro. since previous studies have shown that man1c1 plays a vital role in glycosylation [14] , subsequent experiments focused on the role of this protein, as a representative of the α-1,2-mannosidases. to further confirm the effect of hbv on α-1,2mannosidase expression, plasmids containing seven individual viral genes of hbv (ptt22-hbx, ptt22-hbs, ptt22-pres2, ptt22-pres1, ptt22-hbc, ptt22-hbe, and ptt22-hbp) or control plasmids (ptt22-vector) were transfected into hepg2 cells. western blot was used to determine the expression of hbx ( figure 3a ), hbp ( figure 3b ), hbc ( figure 3c) , hbs, pres2, and pres1 ( figure 3d ); whereas elisa was used to estimate the expression of hbe ( figure 3e ). the kifunensine did not affect the production or secretion of hbv virus particles, it did increase its recognition by dc-sign, resulting in activation of the immune response [8, 9] . moreover, in our previous studies, we found that demannosylation is beneficial to hbv to escape dc-sign recognition [10] . therefore, the upregulation of host α-mannosidases by hbv, as observed in this study, appears to contribute to viral escape [10] . hbv contains three envelope proteins made up of the pres1, pres2, and s domains: the large (l, pres1/pres2/s), middle (m, pres2/s), and small (s) envelope proteins [24] . the l and s proteins can only be singly glycosylated (p39 and gp42 for the l protein, and p24 and gp27 for s protein). there is also a n-glycosylation site (nxs/t) at n146 of the s region. on the other hand, the m protein can be bi-glycosylated (at the gp33 and gp36 sites) [25] . we used plasmids encoding different hbv proteins, including the l, m, and s proteins, to transfect hepg2 cells. surprisingly, we found that only the l, m, and s proteins could increase the expression of man1c1; however, such an effect was not found for the other hbv proteins. as all the l, m, and s proteins include the s domain, we hypothesized that it was this region that induced the increase of man1c1. therefore, the s protein was used to represent the envelope protein in the consequent experiments. as it was unclear whether this envelope protein increased man1c1 expression via a direct or indirect effect, we further investigated the exact mechanisms involved in hbv-induced man1c1 expression. we examined whether the hbv envelope protein activated the pparγ pathway to increase the expression of man1c1 protein. however, we found that inhibition of the pparγ pathway with gw9662 did not affect the hbv envelope protein-induced upregulation of man1c1 expression. on the other hand, when the pparα pathway was inhibited with mk886, the envelope protein could not upregulate man1c1 expression. our results indicate that man1c1 expression is associated with the pparα pathway, whereas previous studies have shown that man1c1 expression in endothelial cells is associated with the pparγ pathway [14] . we speculate that the expression of ppar subtypes could be altered in different cell types, which could be at least partially responsible for the difference between our findings and the previous studies. we also tested the effect of mk886 on the expression of α-1,2mannosidases in ad38 cells. increases in the levels of man1c1 were inhibited by mk886 in ad38 cells. therefore, we speculate that the effects of the hbv envelope protein in upregulating the expression of class ⅰ α-mannosidases are closely associated with the pparα pathway. we only focused on man1c1 in this study. furthermore, the measurement of hbv glycosylation status together with the effect on hbv glycosylation upon knock-down of the different mannosidase members in hbv expressing or not cells might better explain the correlation between hbv production, mannosidase level and hbv glycosylation status. we will consider all the mannosidase members and explain the correlation between hbv production, mannosidase level and hbv glycosylation status in future experiments. in conclusion, the findings of the present study showed that hbv could increase the expression of class ⅰ α-mannosidases both in vitro and in vivo. moreover, the hbv envelope protein increases the figure 5 ). these findings suggest that the class ⅰ α-mannosidases could be used as drug targets to inhibit the demannosylation of hbv, thereby improving the binding of the virus to dc-sign and disrupting the immune tolerance to prevent and treat viral infection. we would like to thank professor xiao-ping chen from the tongji hospital, tongji medical college, huazhong university of science and technology for providing the liver samples, and mr. quan yuan and tian-ying zhang from the xiamen university for providing the cells and western blot showed that after the addition of mk886, the expression of man1c1 did not alter in hepad38 cells without tet as compared to that in the hepad38 cells with tet. moreover, there was no effect of gw9662 on the expression of man1c1 in ad38 cells with or without tet treatment. d: quantification of the α-1,2-mannosidases expression from the band intensities normalized by actin. the data shown represent the mean ± sd of three independent experiments, a p < 0.05. hepatitis b virus (hbv) infection is the most common chronic viral infection in the world. an estimated 2 billion people are infected, and more than 350 million are chronic carriers of the virus. few studies have investigated the effects of hbv on the expression of class ⅰ α-mannosidases. tumor necrosis factor-α has been shown to downregulate the expression of man1c1 to increase the expression of high-mannose type proteoglycans, and this can be reversed by the activation of the ppar signaling pathway. based on the results of these previous studies, the authors hypothesized that hbv could promote the demannosylation of the viral protein by increasing the expression of α-mannosidase i in the host cells. class ⅰ α-mannosidases could be used as drug targets to inhibit the demannosylation of hbv and prevent immune tolerance of the virus. the authors will consider all the mannosidase members and explain the correlation between hbv production, mannosidase level and hbv glycosylation status in future experiments. hbv infection is the most common chronic viral infection worldwide. because of insufficient immune response, some hbv-infected patients will develop chronic hepatitis and possibly liver cirrhosis and hepatocellular carcinoma. in the present study, the authors aimed to explore one of the mechanisms able to impair dendritic cell function in patients suffering from chronic hepatitis b; they hypothesized that hbv could promote the demannosylation of hbv glycoprotein coat by increasing the expression of α-mannosidase 1 allowing hbv to escape from host immunity. hepatitis b virus infection hepatitis b virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis b virus mitochondria: sensors and mediators of innate immune receptor signaling dc-sign, a dendritic cell-specific hiv-1-binding protein that enhances trans-infection of t cells a multivalent inhibitor of the dc-sign dependent uptake of hiv-1 and dengue virus identification of pathogen receptors on dendritic cells to understand their function and to identify new drug targets branched oligosaccharide structures on hbv prevent interaction with both dc-sign and l-sign drastic reduction in the production of subviral particles does not impair hepatitis b virus virion secretion role of dendritic cell-specific icam-3-grabbing nonintegrin on dendritic cells in the recognition of hepatitis b virus class 1 [man1a1 (golgi alpha-mannnosidase ia), man1a2 (golgi alpha-mannosidase ib), man1b1(er alpha-mannosidase i), man1c1 (golgi alpha-mannosidase ic) hepatitis b virus large and middle glycoproteins are degraded by a proteasome pathway in glucosidase-inhibited cells but not in cells with functional glucosidase enzyme activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production endothelial surface n-glycans mediate monocyte adhesion and are targets for antiinflammatory effects of peroxisome proliferator-activated receptor γ ligands up-regulation of il-12 expression in patients with chronic hepatitis b is mediated by the pi3k/akt pathway inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication production of hepatitis b virus particles in hep g2 cells transfected with cloned hepatitis b virus dna 85-kda cpla(2) plays a critical role in ppar-mediated gene transcription in human hepatoma cells characterization of early hepatitis b virus surface protein oligomers pathogen recognition by dc-sign shapes adaptive immunity vertebrate protein glycosylation: diversity, synthesis and function virus entry into animal cells treatment of hepatitis b virus-infected cells with alpha-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity occult hbv infection hepatitis b virus (hbv) envelope glycoproteins vary drastically in their sensitivity to glycan processing: evidence that alteration of a single n-linked glycosylation site can regulate hbv secretion key: cord-264713-38dlh3wg authors: vernet, guy title: molecular diagnostics in virology date: 2004-08-20 journal: j clin virol doi: 10.1016/j.jcv.2004.06.003 sha: doc_id: 264713 cord_uid: 38dlh3wg molecular biology has significantly improved diagnosis in the field of clinical virology. virus discovery and rapid implementation of diagnostic tests for newly discovered viruses has strongly beneficiated from the development of molecular techniques. viral load and antiviral resistance or subtyping assays are now part of the biological monitoring of patients chronically infected by human immunodeficiency virus (hiv), hepatitis b virus (hbv), hepatitis c virus (hcv) and cmv. it will be important to add to this panel assays for other viruses of the herpesviridae family. qualitative assays for the detection of blood-borne viruses have increased safety of blood donation and organ transplantation. screening of other blood-borne viruses (parvovirus b19, hav), multiplexing of detection and test automation to improve practicability and reduce costs will be the next steps. a major evolution in the near future will be the generalization of nat for the diagnosis of viral etiology in patients, mostly with respiratory, cns or gastro-intestinal diseases. major technical improvements have been made to avoid obstacles that still limit this generalization, i.e. genetic variability of viruses, multiplex detection, contamination risk. commercial offers already exist but menus must be extended to limit the validation and documentation work associated with home-brew assays. real-time amplification has allowed the development of new nat platforms but automation and integration of all steps of the reaction are still required to reduce hands-on-time, time-to-result and costs, and to increase throughput. molecular biology has revolutionized all domains of viruses diagnosis including the rapid identification of emerging or re-emerging viruses, viral safety of blood products or organ transplants and viral disease management. one of the major driving forces for the introduction of molecular techniques in virology has been the absence of easy and performing multiplication techniques similar to those developed for bacteriology. the most striking illustration of the power of molecular techniques concerns blood transmitted viruses-human immunodeficiency virus (hiv), hepatitis b virus (hbv) and hepatitis c virus (hcv) for which spectacular progresses in the detection and treatment of viral diseases have been made following the introduction of qualitative and quantitative nucleic acid tests (nat). the recent discovery of a new human coronavirus responsible for severe acute respiratory syndrome (sars) epidemic is another example. it is obvious that nat will encourage the development of antiviral drugs which, compared to antibiotics, has been delayed partly because performing efficiency assessment techniques were lacking. during the last 15 years, many new human pathogens have been discovered among which eight were viruses with various pathogenicity. molecular techniques have played a central role in their discovery (table 1) . the construction of cdna libraries by cloning techniques has been used to identify hcv and hepatitis e virus. representational difference analysis (rda) was successful in identifying human herpes virus 8 (hhv-8), and the hepatic viruses gbv-c and ttv. rda allows the detection of viral sequences by comparing whole nucleic acids sequences in cells from humans or animals before and after infection. reverse-trancription polymerase chain reaction (rt-pcr) with random or degenerate primers has been used to identify human metapneumovirus (hmpv), the virus sen and the coronavirus associated to sars (sars-cov). molecular techniques alone have allowed the characterization of very important human pathogens like hhv-8, responsible of kaposi sarcoma or hcv which induce acute or chronic hepatitis, cirrhosis and hepatocarcinoma. however, other viruses detected in a similar way are still waiting for the demonstration of their clinical importance. this illustrates the need to verify koch's postulate and the importance of keeping laboratory competencies for classical virology-tissue culture, electron microscopy and animal experiments-which plays a major role in virus discovery, together with epidemiological studies. large epidemiological studies are also required to assess the clinical interest of new pathogens. molecular diagnostics have been very rapidly implemented in clinical virology laboratories following the discovery of hmpv (van den hoogen et al., 2001; peiris et al., 2003; boivin et al., 2003) and sars-cov (ksiazek et al., 2003; drosten et al., 2003; anderson, 2003) . a prospective study on the prevalence of hmpv could be initiated as early as during the 2000-2001 winter, although the virus has been discovered in 2000 only. there has been only a few weeks lag between sequencing sars-cov and availability of the first commercial nat. nat are more and more used to exclude blood donations from patients infected by viruses (allain, 2003) . hcv and hiv testing has been implemented as part of routine screening in blood banks in 14 and 7 european countries respectively. in france, two commercial offers -procleix (gen-probe, inc. usa) and nuclisens extractor (biomerieux, france) associated with cobas ampliscreen (roche diagnostics, switzerland)-are used to screen donations for hiv and hcv infections. this allowed the reduction of residual risk from 1 in 400,000 to 1 in 2.5 millions for hiv and 1 in 760,000 to 1 in 5 millions for hcv (assal et al., 2003) . however, there is room from improvement as few contaminated blood units are still missed due to the lack of sensitivity induced by pooling strategies. cost constraints must also be considered if further improvements are to be considered. cost effectiveness of hiv and hcv nat addition to serology testing is already very low: in usa it has been calculated that the cost of each saved life is 4.7-11.2 millions us$ per year (jackson et al., 2003) . hepatitis b virus screening using molecular biology should also be included as even the most recent antigen assays (hbsag) assays miss infected blood units. as an example, single-sample hbv testing would allow the detection of 35-50 additional contaminated units among 10 7 units tested (biswas et al., 2003) . monovalent or trivalent assays (hiv, hbv, hcv) are proposed by roche diagnostics (ampli nat) and gen-probe (procleix ultrio) but blood units should not be pooled to provide sufficient sensitivity. procleix ultrio detects single seroconversions 20 days earlier than abbott prism-hbsag but only 13 days in pools of 8 and 11.5 days in pools of 24 (cambié, 2002) . alternatively, an ultracentrifugation step could be introduced following pooling to obtain a higher sensitivity compared to current antigen assays (roth et al., 2002) . screening for west-nile virus contamination has been implemented in us blood banks. from late june to mid-september 2003, approximately 2.5 million donations were screened. twelve hundred eighty-five (0.05%) were initially reactive for wnv by using nucleic acid-amplification tests and 601 (0.02% of the total donations) are considered presumptive viremic donations (i.e. a donation that is repeatedly reactive by the primary and/or alternate nat assay or a primary nat assay with a very high signal) (cdc, 2003) .other viruses (parvovirus b19, hepatitis a virus) are also transmitted by blood donation and may be part of the screening in a near future. however, nat may not always be the best diagnosis approach and antigen tests or antibody tests may be efficient and less expensive alternatives. automation and integration of nat is necessary to reduce costs and quarantine delays for blood units supply. in this respect, the recent approval by us food and drug administration (fda) of the tigris molecular diagnostic system (gen-probe, san diego, usa) is a major breakthrough as it has been designed to process 500 samples in 8 h. integration and time-to-result are also very important parameters to be considered to insure viral safety of transplant organs, especially lung, heart and liver. it is very important to determine the status of transplants regarding infection by hiv, hbv, hcv and viruses from the herpesviridae family. nat have significantly improved identification of viruses as etiologic agents of human diseases affecting various organs, especially respiratory and gastro-enteric tracts and central nervous system (cns). as a consequence, rapid antiviral treatments can be initiated and considerably reduce morbidity and mortality as, for example, in the case of herpes encephalitis. the increasing number of available antiviral drugs will even accentuate the need for positive viral identification. similarly, unnecessary antibiotic treatments can be avoided or reduced and hospitalisation durations shorten. treatments of chronic viral diseases are very efficiently monitored by viral load assays. however there are still obstacles that prevent a wider dissemination of molecular assays. the extreme genetic variability of some viruses, especially rna viruses (which rna-polymerases have no proofreading activity), often makes their diagnosis difficult. the most striking examples are found in the norovirus family which contains viruses responsible for the vast majority of gastro-intestinal epidemics in adults. hiv diagnosis is also quite difficult to achieve due to its high genetic variability. gardner et al. (2003) have deduced from sequence alignments that a real-time taqman assay should contain not less than nine primer and probe sets to detect with the same sensitivity all hiv strains in a geographically representative panel. to reduce the impact of variability on amplification and detection efficiency, one can use primers and probes with 2 o-methyl bases, degenerate bases or "universal" bases, such as inosine or nebularine. touchdown pcr protocols, in which the annealing temperature slightly decreases during the successive amplification cycles to bracket the melting temperature tm of the reaction, provides sensitivity even when primers have mismatches with target sequences of divergent species in a viral family. finally, degenerate primers or probes, with mixtures of the bases found in sequence databases among various species, may be useful to detect all species of a viral family. it is often desirable to provide the capability for panel detection, i.e. to detect several viruses that can be responsible for a disease. for example, coyle et al. have described at the winter meeting of european society for clinical virology (copenhagen, january 2004) a molecular viral respiratory strip for the detection of 12 common respiratory viruses. whenever possible, consensus primers able to detect all viruses from a family or genus must be used. there are several examples of such consensus primers for enterovirus (kammerer et al., 1994) , flavivirus (scaramozzino et al., 2001) or herpesviridae (tenorio et al., 1993) . however, their ability to amplify all viruses with the same efficiency must be carefully evaluated. if such an approach is not possible, two different possibilities exist for panel detection: multiplex detection in single tubes or parallel detection in individual tubes. mixing primers in a single amplification tube to achieve multiplex detection of viruses usually results in decreased sensitivity of assays compared to single tests. for example, we have observed, using a dna-microarray assay (see below), that the analytical sensitivity of multiplex rt-pcr detection of six viruses, i.e. influenza a, influenza b, rsv a/b, parainfluenza 1, 2 and 3 is reduced by a factor of <1-2 logs compared to single detections, depending on the virus. nevertheless, this multiplex assay was able to identify correctly 21/22 infections in respiratory specimen (one rsv b infection was misidentified as rsv a; unpublished data). the formation of primer dimers is generally considered as the major cause of sensitivity loss but careful optimisation of all parameters of amplification including primer, enzymes, nucleotides and salts concentrations as well as protocol conditions are required to obtain expected performances. realtime assays (see below) that monitor signal apparition during the amplification step are also limited in their capacity to realize multiplex detection by the number of available wavelengths in existing equipments which currently allows the detection of three viruses only. instead of mixing several pairs of primers in a single tube, nucleic acids purified from the original clinical specimen can be distributed into several tubes for independent amplification and detection. major drawbacks of this approach are the reduction of sensitivity because of lower amounts of nucleic acid available for each individual amplification, higher hands-on times required to manipulate all the different tubes, difficulty to automate the distribution of small volumes of purified nucleic acids without introducing cross-contaminations and higher costs due to the need for enzymes in each tube. internal controls (ic) are important components to monitor each step of the assay from extraction of nucleic acids to detection. because inhibitors of the amplification reaction, which are frequent in some specimen types, will also impact its amplification, the presence of an ic is a strong validation in case of negative result. niesters (2002) has described an original approach for internal control of nat: the use of viral universal controls that can be added to each specimen and be amplified with specific primers, preferably in a multiplex format with primers for the virus to detect. seal herpes virus 1 and phocine distemper virus can be used to control nat for dna and rna viruses respectively. of course, animal viruses which can not infect humans are required. other strategies for ic involve synthetic materials, i.e. plasmids or transcripts which contain sequences able to bind the test primers and a specific probe. clinical virology laboratories have high expectations in term of automation and integration of molecular assays. a major bottleneck in the workflow of these laboratories is at the level of sample preparation. table 2 shows automated systems for nucleic acid purification that are currently commercialised. most of them use the nucleic acid binding properties of silica (boom et al., 1990) . as many as 96 samples can be handled in a single run on the biorobot 9604 (qiagen gmbh, germany). time-to-result and, more important, hands-on-time tend to decrease, with the most recent equipments requiring no longer than 20 min of technician time for more than 24 samples. new labelling technologies that do not need solid-phase separation have allowed the development of real-time molecular assays in which the detection of amplicons is done as soon as they appear during amplification. the most simple real-time detection chemistry uses the sybr green dye which specifically binds during double-stranded dna generated during pcr. several probe technologies (fig. 1 ) have also been designed for real-time assays like taqman ( fig. 1a ) or molecular beacons (fig. 1b) in which a quencher molecule is removed from the vicinity of the fluorescent marker upon binding to rna or dna generated during amplification cycles. in the fret technology (fig. 1c) , two probes, one with a fluorescence donor and one with a fluorescence acceptor molecule are designed to bind adjacent sequences of the amplified material to generate signal (mackay et al., 2002) . real-time techniques have been designed for pcr or nasba amplification and have several advantages which facilitate automation and reduce time-toresult (30-120 min) and hands-on-times. they are performed in closed devices which do not need to be opened to transfer amplified material for end-point detection, thus reducing the risk of laboratory or samples cross-contamination. the use of real-time platforms makes the general organisation of molecular biology laboratories easier by reducing the constraints on activities segregation in different rooms to control contaminations. table 3 shows existing real-time automates. the next generation of nat platforms will integrate sample preparation, amplification and detection in a single test device genexpert (cepheid, usa) is the first fully integrated system which allows the detection of bacillus anthracis or group b streptococcus in approximately 45 min directly from clinical specimen. several viral assays based on real-time nasba on this platform will soon be available from biomerieux. several ivd companies offer commercial nat for the diagnosis of viral diseases. besides technical difficulties, another obstacle to the development of molecular assays is the importance of resources needed to optimise, produce and validate home-brew assays and to build up documentation required for qualification of techniques and laboratories. new european community regulations will even increase this need. it is the role of in vitro diagnostic (ivd) companies to provide reagents which are the results of careful optimisation and are produced according to high quality manufacturing procedures. they have clinical and regulatory affairs departments that conduct validation studies and assemble documentation required to get approval of the reagents. however, even for ivd companies, the conception and validation process is time-consuming and timeto-market may be long. this is especially a problem when a diagnostic tool is urgently needed in case of emergence of a new virus. one possibility to reduce time-to-market is to release "research use only" (ruo) assays or assays that have the "ce analytical" approval in europe or the status of "analyte specific reagent" (asr) in the usa. in this case, commercial products that have excellent analytical sensitivity and are manufactured according to quality standards of the ivd industry can be used by clinical virology laboratories, which have the responsibility to validate their use as diagnostic tools and obtain authorization to use them. infections by hiv and hepatitis b and c viruses are usually well diagnosed using serology. only diagnosis of early primo-infections may benefit from nat. platforms like amplicor amplicor from roche diagnostics or easyq from biomerieux that are usually used for viral load measurement during therapy (see below) are also suitable for the early detection of hiv or hcv infections. many other infections and especially acute infections for which igm appear only several days after onset of symptoms cannot be efficiently diagnosed using serology assays. forty to sixty percent of community acquired pneumonia that require hospitalisation 2 have no known aetiology despite intensive investigation and this percentage is even higher when less severe lower respiratory tract infections (lrti) are considered (l. kaiser, personal communication). in a recent study, henrickson et al. (2004) , have shown, using multiplex rt-pcr that 40% of children hospitalised for lrti are infected with the seven most common respiratory viruses. similarly, a recent survey of encephalitis leading to hospitalisation in the usa from 1988 to 1997 has revealed that nearly 60% had no aetiology (khetsuriani et al., 2002) . absence of specific antiviral treatments for most viruses, which limits prescription of biological tests and weak performances of diagnostic tests based on viral culture or serology are major explanations of this situation. nat, which have been implemented by many large european hospitals, significantly improve viral diagnosis. however, there are several obstacles to the generalization of molecular diagnostics in smaller, decentralized laboratories. a major obstacle is the fact that, except for hiv, hbv, hcv and cmv, virology nat are most often home-brew assays which sometimes suffer from bad performances and poor batch to batch consistency. however, quality of nat is the percentage of false-positive results which reflects laboratory or cross-contaminations and used to be high, dropped to 5.5% (wallace et al., 2003) . table 5 shows some products currently commercialised by major companies for viruses other than hiv, hbv and hcv, although the list may not be exhaustive. most of them are asr or ruo kits although some are ec marked. the majority of these reagents have been designed to run on realtime platforms. results shown by liolios et al. in 2001 illustrate the interest of multiplex detection of respiratory viruses by nat. one hundred forty-three clinical specimen were tested using the hexaplex assay from prodesse inc. (usa) which detects six viruses in a single tube using pcr and detection with microplate capture and peroxidase-labelled probes. 9 samples were detected with the prodesse assay only and not with immunofluorescence or viral culture (table 6) . table 6 multiplex nat for the detection of respiratory viruses (hexaplex, prodesse inc.) dna-microarrays or dna-chips are very powerful detection tools that can be combined with amplification techniques to detect viruses or virus variants (reviewed in clewley, 2004) . wang et al. (2002) have described a microarray spotted with 70-mer oligonucleotides which represent the five most conserved sequences (more than 20/70 bases are conserved among all representative sequences in a virus sequence alignment) of each virus of interest. the chip contains 1600 different probes. following pcr amplification of genetic material in the clinical specimen using random primers, hybridisation onto the microarray was able to identify respiratory viruses in the enterovirus, rhinovirus, paramyxovirus, adenovirus and herpesvirus families. however, this technique has not yet been extensively validated for routine diagnosis in a clinical virology laboratory. we have developed assays combining rt-pcr and dna-microarrays for the detection of viruses. the arrays are manufactured using the photolithography in situ synthesis technology (affymetrix, usa) and contain 20-mer oligonucleotides. sequence signatures are identified using extensive sequence databases because they are conserved in all viruses of a genus or family or in all subtypes or isolates of a virus and are not found in other viruses. for each base of the signature, probes perfectly matching the target and probes with a mismatch at the interrogated position are present on the array. if polymorphisms are present in or near the signature, variant probes may also be present. consensus primers have been designed for enterovirus, flavivirus, herpesviridae, parainfluenza and influenza virus, rsv and adenovirus. they can be combined in multiplex detection pcr or rt-pcr assays for the diagnosis of viral respiratory or cns infections. following amplification, dna is labelled using a newly developed diazomethyl chemistry (laayoun et al., 2003) . a complete line of instruments (sample preparation, thermocycler, hybridisation station and laser scanner) is available to perform the assay. as described above, a respiratory assay designed to detect six major viruses (parainfluenza 1, 2 and 3, rsv a/b, influenza a and b) in a single specimen has demonstrated high clinical sensitivity in preliminary evaluation. fig. 2 illustrates the discriminatory capacity of this technology for cns viruses. assays for the identification of human papilloma virus (hpv) are commercialised by several companies. the detection of a highly pathogenic hpv type has a very high predictive value for cervical carcinoma. however, the number of hpv types that are more or less closely associated to cervical cancer is high. dna-microarrays may thus be appropriate for the multiplex detection of all these types. such an assay has been described by and is distributed by biomedlab co. (south-korea). it is based on a consensus amplification and on 30-mer probes that are able to detect and discriminate 22 hpv types and has shown an association of 92.5-97.5% between hpv positivity and lesions of different severity or carcinoma whereas hpv infection was only found in 35.1% of cases when cytology was normal. viral load platforms are available from several ivd companies (versant from bayer diagnostics; cobas ampliprep/ amplicor from roche diagnostics, minimag/nuclisens easyq from biomerieux). significant progress have been made in the ability of most hiv assays to detect all subtypes of hiv-1 but no commercial assay exist for hiv-2. the analytical sensitivity of hbv viral load assays should be increased to reach the same performances as those of hiv assays, especially in the case of infections by variants with low replication competencies. for efficient treatment monitoring of immunosuppressed patients, for example in the case of organ transplantation, viral load assays should also be developed for epstein-barr virus, varicella-zooster virus and hhv6. genotyping tests are now commercially available and are part of the biological follow-up of treated patients although home-brew assays are still used in most laboratories (korn et al., 2003) . hiv and hbv resistance assays as well as hcv subtyping assays are generally based on the sequencing technology (trugene hiv and hbv from bayer healthcare; vi-roseq hiv-1 from celera diagnostics; 5 genotyping hcv kit from bayer healthcare). hybridisation techniques, such as lipa assays (innogenetics, belgium) are also used. however, the number of probes that can be spotted on nitrocellulose strips is limited and only few polymorphisms can be detected which is convenient for hcv subtyping but does not for resistance tests which are based on the detection of a high number of mutations. in addition, hiv and hbv have highly variable genomes and naturally occurring polymorphisms that are present in the vicinity of resistance mutations may affect the binding efficiency of probes. dna-microarrays are an alternative for resistance or typing reagents for viruses or bacteria . we have developed an assay based on rt-pcr and detection with fig. 2 . biomerieux dna-microarray for the detection of neurotropic viruses. following nucleic acid purification, amplification is performed by pcr using a single touchdown protocol in three tubes, one for herpesviridae (one primer pair for hhv 1, 2, 3, 5 and 6), one for enteroviruses (one primer pair for all serotypes) and one for flaviviruses (one primer pair for all viruses). amplification products are combined and labelled using diazomethyl chemistry and hybridised on a dna-microaaray which contains 20-mer oligonucleotides. two or four probes are used for the detection of each base of sequence signatures determined for each virus. a total of 20,000 probes are synthesised on this dna-microarray which has been designed for the simultaneous detection of viruses from the herpesviridae family and from the flavivirus, enterovirus, paramyxovirus, poliomavirus, bunyavirus and orthopoxvirus genus. a: amplicons generated resolved using the bioanalyzer (agilent technologies). b: image of the dna-microarray obtained with a confocal laser reader. c: resolution capability of the array. closely related viruses hybridise with very different efficiency on heterologous probes. high density probe arrays, designed to detect 204 antiretroviral resistance mutations simultaneously in gag cleavage sites, protease, reverse transcriptase, integrase and gp41. this assay has been tested on a panel of 99 hiv-1 patients on a total of 4465 relevant codons in comparison with the classic sequence-based method. key resistance mutations were correctly identified in 95 and 92% of codons in protease and reverse transcriptase, respectively (gonzalez et al., 2004) . we have also developed a similar assay for the detection of polymorphisms in the complete hbv genome: 78 antiviral resistance mutations in pol gene, 146 vaccine, diagnostic or immunotherapy mutation in s gene and 209 mutations in basic core promoter, pre-core, core, x, pre-s1 and pre-s2 regions that may have an impact on disease evolution or treatment efficiency. this assay is currently under evaluation in the frame of hepbvar a european collaborative group for the study of emerging variants of hepatitis b virus. in the coming years, more and more laboratories will offer to clinicians viral diagnosis based on nucleic acid tests. many biological and instrumentation problems that have slowed the generalization of molecular assays have been resolved but several others remain and need to be addressed. major im-provements are expected in the integration and automation of nat diagnostic platforms to reduce hands-on-time, time-toresult and costs and to increase throughput. ivd companies have engaged in development programs to provide clinical virologists with equipments and application menus adapted to their diagnosis needs. technical constraints and recent improvements of molecular assays transfusion risks of yesterday and of today correlation of cervical carcinoma and precancerous lesions with human papillomavirus (hpv) genotypes detected with the hpv dna chip microarray method a novel coronavirus associated with severe acute respiratory syndrome application de la biologie moléculaireà la sécurité virale transfusionnelle: le dépistage génomique viral comparative sensitivity of hbv nats and hbsag assays for detection of acute hbv infection human metapneumovirus infections in hospitalized children rapid and simple method for purification of nucleic acids update: detection of west nile virus in blood donations-united states a role for arrays in clinical virology: fact or fiction identification of a novel coronavirus in patients with severe acute respiratory syndrome limitations of taq-man pcr for detecting divergent viral pathogens illustrated by hepatitis a, b, c, and e viruses and human immunodeficiency virus detection of hiv-1 antiretroviral resistance mutations with highdensity dna probe arrays national disease burden of respiratory viruses detected in children by polymerase chain reaction the cost-effectiveness of nat for hiv, hcv, and hbv in whole-blood donations nested pcr for specific detection and rapid identification of human picornaviruses burden of encephalitisassociated hospitalizations in the united states quality control trial for human immunodeficiency virus type 1 drug resistance testing using clinical samples reveals problems with detecting minority species and interpretation of test results characterization of a novel coronavirus associated with severe acute respiratory syndrome aryldiazomethanes for universal labeling of nucleic acids and analysis on dna chips comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens real-time pcr in virology clinical virology in real-time children with respiratory disease associated with metapneumovirus in hong kong nat for hbv and anti-hbc testing increase blood safety comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns5 gene sequences a newly discovered human pneumovirus isolated from young children with respiratory tract disease species differentiation and antibiotic susceptibility testing with dna microarrays linkage between the journal and quality control molecular diagnostics (qcmd) microarray-based detection and genotyping of viral pathogens key: cord-001515-x11t9pbv authors: kosinska, anna d.; liu, jia; lu, mengji; roggendorf, michael title: therapeutic vaccination and immunomodulation in the treatment of chronic hepatitis b: preclinical studies in the woodchuck date: 2014-12-23 journal: med microbiol immunol doi: 10.1007/s00430-014-0379-5 sha: doc_id: 1515 cord_uid: x11t9pbv infection with hepatitis b virus (hbv) may lead to subclinical, acute or chronic hepatitis. in the prevaccination era, hbv infections were endemic due to frequent mother to child transmission in large regions of the world. however, there are still estimated 240 million chronic hbv carriers today and ca. 620,000 patients die per year due to hbv-related liver diseases. recommended treatment of chronic hepatitis b with interferon-α and/or nucleos(t)ide analogues does not lead to satisfactory results. induction of hbv-specific t cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. vaccination with commercially available hbv vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of hbv infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. the woodchuck (marmota monax) and its hbv-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with prime-boost vaccination using dna vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. in this review, we summarize these encouraging results obtained with these therapeutic vaccines. in addition, we present potential innovations in immunostimulatory strategies by blocking the interaction of the inhibitory programmed death receptor 1 with its ligand in this animal model. more than 240 million people worldwide are persistently infected with hepatitis b virus (hbv) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma (hcc) [1] . an effective and affordable therapy to achieve sustained suppression of hbv replication and remission of liver disease is urgently needed. pegylated interferon-alpha 2a (ifn-a) is recommended for the treatment of chronic hepatitis b (chb) in the current consensus guidelines of many countries. compared with conventional recombinant ifn-a, however, pegylated ifn-a alone or in combination with nucleoside analogues does not significantly increase the rate of sustained response [2, 3] . nucleos(t)ide analogues, such as, entecavir and tenofovir, suppress hbv replication and result in the improvement of liver architecture. however, these agents cannot eradicate hbv genomes from the liver and may further limited by the development increasingly select drug-resistant mutants with prolonged use [4, 5] . therapy with additional antiviral drugs targeting other steps in the viral life cycle, in combination with immunomodulatory options, might be more beneficial and effective. more than 90 % of acutely infected adults resolve clinical symptoms and maintain lifelong protective immunity by mounting a vigorous, multi-specific immune response abstract infection with hepatitis b virus (hbv) may lead to subclinical, acute or chronic hepatitis. in the prevaccination era, hbv infections were endemic due to frequent mother to child transmission in large regions of the world. however, there are still estimated 240 million chronic hbv carriers today and ca. 620,000 patients die per year due to hbv-related liver diseases. recommended treatment of chronic hepatitis b with interferon-α and/ or nucleos(t)ide analogues does not lead to satisfactory results. induction of hbv-specific t cells by therapeutic vaccination or immunomodulation may be an innovative strategy to overcome virus persistence. vaccination with commercially available hbv vaccines in patients with or without therapeutic reduction of viral load did not result in effective immune control of hbv infection, suggesting that combination of antiviral treatment with new formulations of therapeutic vaccines is needed. the woodchuck (marmota monax) and its hbv-like woodchuck hepatitis virus are a useful preclinical animal model for developing new therapeutic approaches in chronic hepadnaviral infections. several innovative approaches combining antiviral treatments using nucleos(t)ide analogues, with primeboost vaccination using dna vaccines, new hepadnaviral antigens or recombinant adenoviral vectors were tested in the woodchuck model. in this review, we summarize these encouraging results obtained with these therapeutic vaccines. in addition, we present potential innovations this article is part of the special issue "therapeutic vaccination in chronic hepatitis b-approaches, problems and new perspectives". to hbv proteins. by contrast, patients with chronic hepatitis b tend to have delayed, transient or narrowly focused t cell responses [6] [7] [8] . patients who spontaneously recover from hbv infection might experience reactivation of hbv under immunosuppressive treatments. thus, the specific immune responses to hbv remain crucial for the long-term control of hbv infection even after resolution of the acute infection. for chronically infected patients, immunostimulatory and immunomodulatory strategies to boost or to broaden the weak virus-specific t cell response have been proposed to reach an effective control of viral infection. since more than 20 years, numerous clinical trials exploited the conventional prophylactic vaccine based on the hepatitis b surface antigen (hbsag) for therapeutic vaccination (table 1) . these studies demonstrated reductions in viremia, seroconversion of the hepatitis b "e" antigen (hbeag) to anti-hbe and hbv-specific t cell responses in some patients after vaccination. however, the antiviral effect was only transient and did not lead to an effective control of the hbv [9] [10] [11] [12] [13] [14] [15] [16] [17] . a more sophisticated therapeutic vaccination based on hbsag complexed with human anti-hbs was proposed by the group of wen et al. [18] . immunogenic complexes (ic) stimulate robust t cell responses by increasing uptake of hbsag through fc receptors on antigen-presenting cells (apc) and, therefore, enhance hbsag processing and presentation. it was demonstrated that this vaccine administered to hbeag-positive patients led to decrease of hbv dna in serum and hbeag seroconversion in some subjects [19] . in a phase ii b clinical trial, hbeag seroconversion was observed in about 21.6 % of treated patients. moreover, a moderate decrease in serum hbv dna and hbsag levels was observed after treatment [20, 21] . very recently, a large phase iii clinical trial with 12 injections of ic complex failed to show any therapeutic efficacy when compared to the placebo control injected only with alum [22] . overstimulation with ic-based vaccine did not increase but decreased efficacy of the therapeutic vaccination. these results underline that an appropriate immunization protocol is crucial for the efficacy of therapeutic vaccination. dna vaccines using plasmids expressing viral proteins have gained popularity given their ability to induce strong cellular and humoral immune responses. several phase i clinical studies investigated the therapeutic efficacy of plasmid dna vaccines expressing hbsag in chronic hbv carriers. these studies showed evidence for the safety of hbv dna vaccination (for details see below), but t cell responses were restored or activated at only a low level. furthermore, dna vaccines expressing only hbsag did not result in significant suppression of viremia in chronic carriers of hbv [23, 24] . from results of these studies, it can be concluded that the therapeutic vaccination alone is not sufficient to achieve pre-s1/pre-s2/s, hbcag, polymerase yoon et al. [27] the control over hbv. high load of virus particles and large amounts of hbsag in the liver and peripheral blood may be responsible for the immune tolerant status in the patients. therefore, pretreatment with nucleos(t)ide analogues has been proposed to achieve better cd8 t cell response and subsequent therapeutic efficacy after administration of dna vaccines. recently, the results of the trial of this combination therapy have been published. in a large double-blind study, 70 patients were treated effectively with nucleos(t)ide analogues for a median of 3 years resulting in undetectable levels of hbv dna and thereafter randomized into two groups: one received five intramuscular injections of dna vaccine expressing hbsag and one received placebo. nucleos(t)ide analogues were stopped. although this combination therapy was fairly well tolerated, the hbv dna vaccine did not decrease the risk of relapse in hbv-treated patients and did not restore the anti-hbv immune response despite effective viral suppression by analogues [25, 26] . during a study in korea, 27 patients randomly received either adefovir (adv) alone or adv in combination with hbsag-expressing dna vaccine. therapeutic vaccination was safe and tolerable in chb patients. vaccine-induced hbv-specific t cell responses and hbeag seroconversion were weaker in korean patients than in caucasian patients [27] . asian patients, who are generally infected via vertical transmission, may have a higher level of immune tolerance than caucasians who are usually infected later in life. improved vaccines for breaking immune tolerance may be needed to develop effective therapeutic hbv dna vaccines. the aim of a study in vietnam was to evaluate viral suppression following combined treatment with a new vaccine containing all three envelope proteins of hbv (pre-s1/pre-s2/s) and lamivudine in chb patients. the enhanced suppression of viremia in the combination group was reversed after the discontinuation of vaccine treatment, suggesting that booster doses are required for a sustained viral response. anti-hbs was detected in 55/120 vaccine recipients, but only three patients demonstrated hbsag loss, indicating that the vaccine-induced anti-hbs was unable to completely neutralize hbsag in the serum [28]. the eastern woodchuck (marmota monax) is naturally infected by woodchuck hepatitis virus (whv) which was discovered in 1978 [29] . whv was found to be closely related to hepatitis b virus (hbv) [30] and classified as the second member of the genus ortho-hepadnavirus, family hepadnaviridae. in contrast to hbv-associated hcc in patients without a preferred integration site of hbv dna, a frequent integration of the whv genome close to the n-myc and c-myc gene has been observed in woodchucks developing hcc [31] . infections of woodchucks with whv have been shown to be endemic in the mid-atlantic states of the usa, whereas in the state of new york and new england woodchucks are rarely infected with whv. recently, a chinese marmot marmota himalayana was found to be susceptible to whv infection [32] (fig. 1 this review is focusing on the characterization of woodchuck genes related to innate and adaptive response, the recent development of new tools to determine virusspecific t cell response, therapeutic vaccines, and finally immunostimulatory and immunomodulatory approaches to treat chronic whv infection. these new findings in this preclinical model will help the development of new strategies to treat chronic hbv infection in patients. in recent years, many efforts have been devoted to cloning and characterization of components of the woodchuck immune system. a number of immune function-related genes including cytokines and their receptors, immune cell surface markers and other immune function-related proteins have been cloned and characterized. so far, important woodchuck cytokines and their receptors such as tnf-α, ifn-α, ifn-γ, il-12, il-15, gmcsf, lymphotoxin (lt)-α and il-10r have been cloned and tested for their biological activities [54] [55] [56] [57] [58] [59] [60] [61] . in patients, ifn has been used in the treatment of chb for many years. therefore, the ifn system has also been characterized in woodchucks. woodchuck ifn-α was shown to reduce whv surface antigen expression in a dose-dependent fashion in whv-infected woodchuck hepatocytes [62] . the woodchuck ifn-α/β system and their expression in peripheral blood lymphocytes (pbls) from naïve and whv-infected woodchucks have also been studied [63] . the woodchuck ifn-α genes could be classified into ten subtypes and three pseudotypes. poly(i:c) stimulation on naïve woodchuck pbls could induce ifn-α subtypes one, four and five production, indicating a selective expression of woodchuck ifn-α subtypes. moreover, pbls from chronically whv-infected woodchucks showed a reduced ability to produce woodchuck ifn when stimulated with poly(i:c). the complete or partial sequences of the type i ifn receptors (ifnars) of woodchucks were also obtained and analysed by fan et al. [64] . ifn-α or ifn-γ stimulation significantly upregulated ifnar2 expression in primary woodchuck hepatocytes. a decreased ifnar1 and ifnar2 expression was observed in woodchucks chronically infected with whv. these data are essential for studying type i ifn-related innate immunity and therapy in hepadnaviral infection in the woodchuck model. il-10 is a pleiotropic cytokine acting on a variety of immune cells through its cell surface receptor (il-10r). it has been suggested to resuscitate antiviral immunity by interfering with il-10/il-10r pathway. an increased production of il-10 was observed in patients with chb [65] , which hints that blockade of il-10r might become a feasible therapeutic approach for chb. very recently, jiang et al. [54] successfully cloned woodchuck il-10r and generated antibodies against this molecule. the blockade of woodchuck il-10r enhanced the proliferation and degranulation of specific t cells from chronically whv-infected woodchucks in vitro. this work provides a basis for potential therapeutic approaches in chronic hbv infection. important woodchuck immune cell surface molecules which have been cloned so far can be divided into two categories based on their function: molecules involved in innate immunity and molecules involved in adaptive immunity. toll-like receptors (tlrs) are a class of molecules that play a key role in the innate immune system. recent progress in this field revealed that there are significant interactions between the tlr system and pathogens in chronic viral infections [66] . so far, tlr2, tlr3, tlr4, tlr7, tlr8 and tlr9 have been successfully cloned in woodchucks [67] . in a recent study, zhang et al. [66] showed that tlr2 ligands induced the activation of nf-κb, pi3k/akt and different arms of mapk signalling pathways and the production of pro-inflammatory cytokines in woodchuck hepatocytes. tlr2-mediated innate immune responses reduced replication and gene expression of hbv in hepg2.2.15 cells and whv in primary woodchuck hepatocytes (see also article from zhang and lu, in this issue). in chronic whv carriers woodchuck model, relatively low levels of tlr2 expression were found in pbmcs and in liver tissues. tlr2 expression in pbmcs was inversely correlated with whv dna titres in acute whv infection and in entecavir-treated chronic whv carriers. an effective immune response against viral infections depends on the activation of cd8 t cells that can clear infection by killing virus-infected cells. therefore, sequence information of woodchuck cd3, cd4 and cd8 has been used to determine the kinetic of the influx of t cells into the liver during incubation period and acute or chronic whv infection. in week two post-infection, an influx of cd3+ lymphocytes could be observed and reached higher levels prior and during the recovery phase. the peak level of cd4+ and cd8+ t cells coincided with recovery. during transient infection, t cells can accumulate in the liver and reach up to two-thirds of the total number of liver cells [35] . in the adaptive immune response, cd28 and ctla-4 are known to play important roles for the regulation of t cell activation by delivering costimulatory signals. the complete coding regions of woodchuck cd28 and cytotoxic t-lymphocyte-associated antigen 4 (ctla-4) have been cloned and sequenced [68] . woodchuck cd28 showed a similarity of 76 and 70 % to its human and mouse homologues, respectively, according to the deduced amino acid sequences. woodchuck ctla-4 has a higher similarity of 86 and 75 % to the corresponding human and mouse ctla-4 molecules, respectively. the strict conservation of critical amino acid residues like cysteine and asparagine residues in woodchuck cd28 and ctla-4 suggests that both molecules may structurally resemble their human or mouse homologues. a hexapeptide motif mypppy which has been supposed to be essential for the interaction with cd80 is present in both woodchuck cd28 and ctla-4 [68] . the advances in sequencing technology provide new tools to characterize genes of the woodchuck immune system in large scale. fletcher et al. [69] performed the sequencing, assembly and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. by using this new platform, they characterized the transcriptional response to persistent whv infection and whv-induced hcc. liu et al. have also performed de novo woodchuck transcriptome assembly by using deep sequencing technology (unpublished data). with the help of this advanced technology, sequence information of important immune genes such as apobec3 of woodchucks has been revealed. it has been shown that upregulation of apobec3 led to specific and non-hepatotoxic degradation of nuclear hbv cccdna [70] . therefore, future cloning and characterizing of apobec3 in the woodchuck model will evaluate the therapeutic potential for chb. in summary, these efforts on establishing the translational value of the woodchuck model can provide new insight into characterizing immune pathways which may play a role in the persistence of hbv infection. studies in patients underline the important role of hbvspecific t cell response as a leading factor of viral clearance. for many years, the lack of appropriate methods to evaluate antigen-specific t cell responses was the serious limitation of this model. the establishment of the assays for monitoring of cellular immune response in woodchucks is of great importance for a reliable evaluation of therapeutic and immunomodulatory strategies for treatment of chb in the woodchuck model. development of the 2[ 3 h]-adenine-based proliferation assay enabled to detect the t-helper lymphocyte responses after stimulation of woodchuck pbmcs [39, 41] . in addition, several t-helper epitopes within whcag [39, 41] were identified in pbmcs from acutely whv-infected animals. significant progress in studying the t cell response of woodchucks was achieved by introduction of the flow cytometric cd107a degranulation assay that enables the detection of whv-specific cytotoxic t cells (ctls) in woodchuck pbmcs and splenocytes [38] . several studies demonstrated that detection of cd107a, as a degranulation marker, is a suitable method for determination of antigenspecific cytotoxic t lymphocytes [71, 72] . introduction of the immunological tools for studying of the t cell response in woodchucks revealed a significant similarity between the pathogenesis of whv infection in woodchucks and hbv in humans. it was demonstrated that acute self-limiting and resolved whv infections correlate with robust multifunctional t-helper and cytotoxic t cell responses, while whv chronic carriers demonstrate weak or no virus-specific t cell responses against the viral proteins (table 2) [38, 39, 41]. moreover, these studies confirmed that the efficient cellular immune response to viral antigens results in liver injury and is necessary for viral clearance. recently described advancements in the characterization and monitoring of the woodchuck immune system during the whv infection made this animal model particularly useful for development of the immunomodulatory approaches in chb. the pioneer investigations with therapeutic vaccines based on whv core [73] or surface [76, 77] reporting that the t cell response to hbv was successfully restored in patients treated with lamivudine. in addition, the quantity of antigen particularly the whv surface antigen (whsag) to which the immune system is exposed can induce different degrees of functional impairment of antiviral t cells, up to physical t cell deletion [78, 79] . combination therapy using lamivudine and serumderived whsag vaccination showed no effect on induction of anti-whs antibodies or reduction of whv dna [80] . our group evaluated the efficacy of the combination therapy in the woodchuck model by combining lamivudine treatment, dna vaccination (three plasmids expressing whsag, whcag and woodchuck ifn-γ) and whsag/ anti-whs immunogenic complexes vaccination [81] . the triple combination led to a decrease in whv viral load up to 2.9 log, in serum whsag up to 92 % and in development of anti-whs antibodies. nevertheless, these effects were not sustained and all parameters reached the baseline levels shortly after withdrawal of lamivudine treatment. in addition, the vaccination did not induce whv-specific t cell responses in the majority of woodchucks, even in animals that exhibited virological responses. later, we modified this protocol by using the more potent antiviral drug entecavir (etv) and increasing the number of the immunizations (with plasmids expressing whsag and whcag from three to six) (lu et al., unpublished results). a significant delay of the rebound of viremia was observed in woodchucks which received additional vaccination, compared to controls treated only with etv. in another study, chronic whv carriers received a treatment of the potent antiviral drug clevudine in combination with an alumadsorbed whsag vaccine. combination treatment resulted in significant and sustained reduction of whv dna loads and whsag concentrations in most treated animals. compared to vaccination alone, combination treatment induced a more robust anti-whs response [82, 83] . the results of these studies clearly showed that combination of antiviral treatment and vaccination is more effective in inducing virus-specific t cell responses than therapeutic vaccination alone. nevertheless, the efficacy of these approaches was still too limited when applied for treatment of chb. the vaccination strategies used in some of these studies were even not able to boost a functional antiviral t cell response. a significantly better induction of whcagspecific t cells using more potent vaccines, such as recombinant viral vectors, may be required to achieve sustained antiviral response and viral clearance. recombinant adenoviral vectors (adv) proved to elicit a vigorous and sustained humoral and t cell responses to the transduced antigen [84, 85] . adenoviral vectors also act as a natural adjuvants causing dc maturation, enhanced antigen presentation and secretion of antiviral cytokines, such as ifn-α, tnf-α and il-6 [86] . however, even single immunization with recombinant adenoviruses may induce immunity, predominantly neutralizing antibodies, against the vector itself. this negative effect of the adenovirusinduced immunity against the vaccine may be overcome by heterologous prime-boost regimen. in particular, subsequent priming immunizations with plasmid dna vaccine followed by a booster vaccination with adv seem to be a very promising strategy. dna prime-adenovirus boost regimen proved to induce more robust and potent immune response in comparison with plasmid dna alone and provided protection against the pathogen challenge in several animal models of infectious diseases [87] [88] [89] (see also article from e. barnes in this issue). recently, our group has investigated whether the heterologous prime-boost immunization strategy using plasmid dna and recombinant adenoviral vectors may improve the efficacy of the therapeutic vaccination in chb in the woodchuck model. a new dna plasmid (pcgwhc) and an adenoviral serotype 5 vector (ad5whc) and a chimeric ad5 displaying ad35 fibre (ad35whc) showing high expression levels of whcag were constructed [90] . the increased antigen expression was achieved by insertion of an intron sequence in the expression cassette of the vaccines. preliminary results showed that the new vaccines are able to induce strong and sustained whcag-specific t cell response in mice and naïve woodchucks. interestingly, immunization with advs led to rapid and massive production of anti-whs antibodies and as a result resolution of infection after the whv challenge [90] . the dna prime-adv boost immunization strategy was further used as a therapeutic vaccine against chronic whv infection in combination with antiviral treatment with etv. six chronically whv-infected woodchucks were treated for 23 weeks with etv. starting from week eight, four of the six etv-treated animals received subsequently nine intramuscular immunizations with: dna plasmids expressing whcag (pcgwhc) and whsag (pwhsim), ad5whc and ad35whc. whsag-and whcag-specific t-helper and cytotoxic t cell responses were detected in all chronic carriers that received immunizations, but not in etv only treated animals. in addition, woodchucks receiving the combination therapy showed a prolonged suppression of whv replication and lower whsag levels compared to controls. excitingly, two of four immunized carriers remained whv dna negative after the end of etv treatment and developed anti-whs antibodies [91] . these data are encouraging and demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks. persistent hbv infection is associated with functional exhaustion of virus-specific cd8 t cells [92] . this defect in virus-specific t cells is one of the primary reasons for the inability of the host to eliminate the persisting pathogen. although it has been shown that nucleoside analogues treatment can induce the recovery of hbv-specific ctl activity in patients [76] , this effect is only transient [77] . those findings are consistent with our data obtained from the woodchuck model, in which etv treatment alone only induced either only transient ctl responses [91] or no responses at all [93] . therefore, additional strategies that can potently enhance t cell response need to be enroled for the treatment of chb infection. recent studies in chronic virus infection models indicate that the interaction between the inhibitory receptor programmed death-1 (pd-1) and its ligands plays a critical role in t cell exhaustion [94] [95] [96] [97] . in chronic hbv infections, upregulation of pd-1 on virus-specific t cells was observed, and restoration of the t cell function has been achieved by blocking the pd-1/pd-ligand 1 (pd-l1) interaction in vitro [98] . recently, the therapeutic effect of pd-1/pd-l1 blockade has also been investigated for chronic hcv infection in chimpanzees [99] and in patients [100] . however, limited effect on restoring t cell function was observed in these studies which used only pd-1/pd-l1 blockade. it has been recently clarified that the proportion of cd8 t cells expressing pd-1 and the levels of pd-1 on virus-specific t cells are strongly correlated with viral load in the plasma [101] [102] [103] . antiretroviral treatment resulted in the dramatic decline of plasma viral load, coincident with a decrease in the pd-1 expression level on virus-specific cd8 t cells [101, 103] . in line with this, a better restoration of t cell functions upon in vitro anti-pd-l1 treatment was observed in chronic hbv patients with lower viremia [104] . therefore, a combination therapy that includes direct antiviral drug and pd-l1 blockade is a reasonable strategy for the treatment of chronic hbv infection. in line with these findings, zhang et al. [105] and liu et al. [93] successfully cloned and characterized the woodchuck pd-1/pd-l system in the whv infection woodchuck model. a significant positive correlation between the viral load and the pd-1 expression on total cd8 t cells in chronic whv infection was observed. both the proportion of pd-1+ cd8 t cells and the levels of pd-1 expression on cd8 t cells were significantly higher in the woodchucks with chronic whv infection compared to naïve animals and resolvers. more importantly, during etv treatment of those chronic carriers, a reduction of serum viral load was correlated with a dramatic decrease in the level of pd-1 expression on cd8 t cells [93] . in vitro blockade of woodchuck pd-1/pd-l1 pathway by using a rabbit polyclonal pd-l1 blocking antibody could partially restore the t cell function in whv-infected woodchucks [105] . moreover, in vivo blockade of the pd-1/pd-l1 pathway on cd8 t cells, in combination with nucleoside analogue treatment and dna vaccination, synergistically enhanced the function of virus-specific t cells. the combination therapy potently suppressed whv replication, leading to sustained immunological control of viral infection, anti-whs antibody development and complete viral clearance in some woodchucks [93] . although similar approaches have been tried in other viruses in the past, such as lcmv, the data presented here may be an advance for the hbv field to new approaches for eliminating the virus itself rather than only suppressing its replication. the woodchuck is a valuable preclinical model for developing new therapeutic approaches in chronic hepadnaviral infections. even though several innovative approaches combining antiviral treatment with nucleoside analogues, dna vaccines and protein vaccines were tested in chronically infected woodchucks, the effectiveness of those strategies was very limited. strategies investigated so far were often hampered by weak t cell responses observed after immunization, suggesting a strong need for alternative strategies to enhance t cell functions during chronic hbv infection. recently, our group published two independent proof-of-concept studies, showing that using a very potent t cell vaccine and blockade of negative signalling in t cells may lead to the resolution of chronic hepatitis b in some woodchucks (table 3) . these data are encouraging and implicate the feasibility and usefulness of the immunotherapeutic strategies for the treatment of chronically hbv-infected patients. nevertheless, which factors influence the effect of therapeutic vaccination remains to be further investigated. it has been noticed that satisfactory therapeutic effects could not be documented in the studies using hbsagbased prophylactic vaccines. in the mean time, evidence has supported that hbcag-specific immunity is endowed with antiviral and liver-protecting capacities in chb patients and animal models. with the increasing number of available vaccine formulation, a more crucial question raised recently: what is the optimal combination of these vaccines. obviously, it is necessary to test the mutual influences of different types of vaccines to maximize their effects and avoid the negative interference between the vaccines. also, the question how hbv infection leads to defective immune responses to hbv proteins remains to be investigated. this issue is the key to a more rational design of new therapeutic approaches. figure 2 summarizes the ideas of a potential combination treatment for patients with chronic hepatitis b. the presence of viral components may be a main reason for t cell tolerance in chronic hbv infection. antiviral treatment with nucleoside analogues efficiently reduce hbv replication and release of new virions and may partly restore hbv-specific cd8 and cd4 t cell responses, thereby allowing successful therapeutic vaccination. however, hbv proteins are still produced as the transcription of mrnas for the s protein and the core protein on hbv cccdna is not affected by antiviral treatment. even when hbv dna disappears during antiviral treatment, hbsag and hbcag/hbeag are present in the liver or in blood at high levels. it is proposed to reduce hbv protein load by small interfering rnas (sirnas), which lead to the sequence-specific degradation of homologous mrna. using this rna interference (rnai) with chemically synthesized or vector-expressed sirnas, many clinically relevant viruses including the human immunodeficiency virus, hbv and hcv could be inhibited. in in vitro experiments showed that whv transcripts could be degraded by sirnas [106] . at the same time, the degradation of viral rnas resulted in the activation of multiple pathways of host innate immune responses [107] . however, future in vivo studies are required to demonstrate the usefulness of this technology. combining gene-silencing approach with nucleoside analogues may further facilitate the stimulation of the immune system by therapeutic vaccines. the epigenetic regulation provides an alternative to interfere with hbv gene expression. hbv minichromosome in hepatocytes is under the complex control of epigenetic mechanisms, and its transcriptional activity could be influenced by methylation, histone acetylation and other mechanisms [108] . therefore, exploring epigenetic drugs to modify, these regulatory processes may achieve an effective suppression of hbv gene expression and thereby replace antiviral treatment with nucleoside analogues. the stimulation of innate immune responses may contribute to the control of hbv infection. in this special issue, zhang and lu provided a review dedicating to the role of tlr system. interferons and interferon-stimulated genes (isgs) represent still an important part for anti-hbv treatment. a recent review about this aspect described the current progress (pei et al., in press) . recently, the antiviral functions of isgs are under studies. for example, interferon-induced protein with tetratricopeptide repeats 1 and 2 is a cellular factor that was shown to limit hepatitis b virus replication in hepatoma cells [109] . another recent report by lucifora et al. [70] about the role of apobecs in the degradation of cccdna was highly interesting, but remained to be controversial [110, 111] . future investigation is required to elucidate the functions of isgs and their relative contribution for control of hbv infection, before exploring these genes for antiviral treatment. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity pegylated interferon alfa-2b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial peginterferon alfa-2a, lamivudine, and the combination for hbeag-positive chronic hepatitis b cellular and virological mechanisms of hbv drug resistance hepatitis b virus resistance to nucleos(t)ide analogues long-lasting memory t cell responses following selflimited acute hepatitis b the hepatitis b virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic t-lymphocyte response cytotoxic t lymphocyte responsiveness after resolution of chronic hepatitis b virus infection specific vaccine therapy in chronic hepatitis b: induction of expression and purification of woodchuck tumour necrosis factor alpha molecular cloning of the woodchuck cytokines: tnf-alpha, ifn-gamma, and il-6 woodchuck gamma interferon upregulates major histocompatibility complex class i transcription but is unable to deplete woodchuck hepatitis virus replication intermediates and rnas in persistently infected woodchuck primary hepatocytes molecular characterization of woodchuck interleukin 15 (wil-15) and detection of its expression in liver samples of woodchucks infected with woodchuck hepatitis virus (whv) molecular cloning and expression of woodchuck granulocyte-macrophage colony stimulating factor expression of a new woodchuck ifn-alpha gene by a helper-dependent adenoviral vector in woodchuck hepatitis virus-infected primary hepatocytes molecular characterization of woodchuck type i interferons and their expression by woodchuck peripheral blood lymphocytes molecular characterization of the type i ifn receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (whv) hepatitis b virus-specific t-cell proliferation and cytokine secretion in chronic hepatitis b e antibody-positive patients treated with ribavirin and interferon alpha role of tolllike receptor 2 in the immune response against hepadnaviral infection lipopolysaccharide-induced innate immune responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon-independent pathways molecular characterization of cd28 and cytotoxic t-lymphocyte-associated antigen 4 (ctla-4) of woodchuck (marmota monax) transcriptomic analysis of the woodchuck model of chronic hepatitis b specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna sensitive and viable identification of antigen-specific cd8+ t cells by a flow cytometric assay for degranulation ex vivo identification, isolation and analysis of tumor-cytolytic t cells the woodchuck: an animal model for hepatitis b virus infection in man therapeutic vaccination of woodchucks against chronic woodchuck hepatitis virus infection induction of antibodies to the pres region of surface antigens of woodchuck hepatitis virus (whv) in chronic carrier woodchucks by immunizations with whv surface antigens lamivudine treatment can overcome cytotoxic t-cell hyporesponsiveness in chronic hepatitis b: new perspectives for immune therapy transient restoration of anti-viral t cell responses induced by lamivudine therapy in chronic hepatitis b molecular signature of cd8+ t cell exhaustion during chronic viral infection reinvigorating exhausted hiv-specific t cells via pd-1-pd-1 ligand blockade t-helper cell response to woodchuck hepatitis virus antigens after therapeutic vaccination of chronically-infected animals treated with lamivudine combination of an antiviral drug and immunomodulation against hepadnaviral infection in the woodchuck model immunization with surface antigen vaccine alone and after treatment with 1-(2-fluoro-5-methyl-beta-larabinofuranosyl)-uracil (l-fmau) breaks humoral and cellmediated immune tolerance in chronic woodchuck hepatitis virus infection immunogenic effects of woodchuck hepatitis virus surface antigen vaccine in combination with antiviral therapy: breaking of humoral and cellular immune tolerance in chronic woodchuck hepatitis virus infection replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines recombinant adenovirus induces maturation of dendritic cells via an nf-kappab-dependent pathway development of a preventive vaccine for ebola virus infection in primates attenuation of simian immunodeficiency virus siv-mac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag prime-boost vaccination with plasmid dna and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against hiv dna prime-adenovirus boost immunization induces a vigorous and multifunctional t-cell response against hepadnaviral proteins in the mouse and woodchuck model combination of dna prime-adenovirus boost immunization with entecavir elicits sustained control of chronic hepatitis b in the woodchuck model t cells and viral persistence: lessons from diverse infections enhancing virus-specific immunity in vivo by combining therapeutic vaccination and pd-l1 blockade in chronic hepadnaviral infection restoring function in exhausted cd8 t cells during chronic viral infection pd-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination pd-1:pd-l1 interactions contribute to the functional suppression of virus-specific cd8+ t lymphocytes in the liver enhancing siv-specific immunity in vivo by pd-1 blockade characterization of hepatitis b virus (hbv)-specific t-cell dysfunction in chronic hbv infection immunotherapy of chronic hepatitis c virus infection with antibodies against programmed cell death a randomized, double-blind, placebo-controlled assessment of bms-936558, a fully human monoclonal antibody to programmed death-1 (pd-1), in patients with chronic hepatitis c virus infection pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression dysfunction and functional restoration of hcv-specific cd8 responses in chronic hepatitis c virus infection pd-1 is a regulator of virus-specific cd8+ t cell survival in hiv infection antiviral intrahepatic t-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis b the expression of pd-1 ligands and their involvement in regulation of t cell functions in acute and chronic woodchuck hepatitis virus infection inhibition of woodchuck hepatitis virus gene expression in primary hepatocytes by sirna enhances the cellular gene expression rnai induces innate immunity through multiple cellular signaling pathways regulation of hepatitis b virus replication by epigenetic mechanisms and micrornas interferon-induced proteins with tetratricopeptide repeats 1 and 2 are cellular factors that limit hepatitis b virus replication specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna virology. response to comment on "specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna a pilot study of the cy-1899 t-cell vaccine in subjects chronically infected with hepatitis b virus. the cy1899 t cell vaccine study group clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis b in vivo immunization by vaccine therapy following virus suppression by lamivudine: a novel approach for treating patients with chronic hepatitis b therapeutic vaccination of chronic hepatitis b patients with virus suppression by antiviral therapy: a randomized, controlled study of co-administration of hbsag/as02 candidate vaccine and lamivudine the authors thank dr. wolfram gerlich for his critical comments on this manuscript. a number of studies cited in this review were funded by deutsche forschungsgemeinschaft (gk 1045 and sfb/trr60). open access this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord-255697-trig04hd authors: cheng, vincent chi-chung; chan, jasper fuk-woo; hung, ivan fan-ngai; yuen, kwok-yung title: viral infections, an overview with a focus on prevention of transmission date: 2016-10-24 journal: international encyclopedia of public health doi: 10.1016/b978-0-12-803678-5.00514-2 sha: doc_id: 255697 cord_uid: trig04hd viruses are obligatory intracellular pathogens with a simple structure consisting of assembled proteins enclosing the nucleic acid genome with or without a lipid envelope. despite increasing availability of rapid nucleic acid amplification assays for laboratory diagnosis, effective antivirals, and safe vaccines, the control of most viral infections depends on time-honored surveillance and infection control measures. moreover most viruses can be readily destroyed by common disinfectants. this article is focused on the epidemiology, diagnosis, and control of common and emerging viral diseases. traditionally, the epidemiological control of most viral infections depends on the isolation of cases, quarantine of contacts, personal protection by infection control measures and mass vaccination, because specific antiviral treatment is generally not available for most viral infections ( table 1) . this scenario is rapidly changing with the increasing availability of rapid diagnostic tests which use nucleic acid amplification, and the development of an increasing number of effective antiviral agents. common acute viral diseases such as respiratory, diarrheal, exanthematous, or neurological infections can overlap with each other and appear as seasonal epidemics, which peak in incidence every few years and coincide with the accumulation of sufficient number of nonimmune hosts in the young population. arboviral disease activity often coincides with arthropod vector activity such as mosquito breeding in hot rainy seasons which are associated with increased incidence of hemorrhagic fever or neurological diseases such as dengue hemorrhagic fever, west nile virus, or japanese encephalitis in southeast asia. many chronic blood-borne viral illnesses such as human immunodeficiency virus (hiv), hepatitis b virus (hbv), and hepatitis c virus (hcv) are still taking a major toll in certain geographical regions due to specific human behaviors or vertical transmission. some of these chronic viral infections such as hbv, hcv, hiv, polyomaviruses, and papillomaviruses are also linked to the genesis of cancers. over 70% of emerging viral infections such as the ebola virus, severe acute respiratory syndrome (sars) coronavirus, and middle east respiratory syndrome coronavirus (mers-cov) are associated with acute explosive outbreaks after the virus jumped the species barrier from bats or other animals into humans ( table 2) . this article will focus on the prevention and control of viral infections, while other articles in this encyclopedia will cover information on specific viruses. unlike bacteria, fungi, and parasites, viruses are too small to be visible under light microscopy. moreover, viruses are obligate intracellular pathogens and do not grow in artificial culture medium. collecting the correct clinical specimens during the peak of viral shedding in appropriate viral transport medium is crucial for accurate diagnosis. electron microscopy is not sensitive and has a limited role in the examination of feces from viral gastroenteritis and vesicular fluid from skin lesions caused by herpesviruses and poxviruses. virus isolation in cell lines or chick embryo is the gold standard for virological diagnosis but seldom alters clinical management due to its long turnaround time. viral antigen detection by immunofluorescence, enzyme immunoassay, and point-of-care rapid lateral flow immunochromatographic assays has significant impact on therapeutic and infection control strategies. the most important rapid virological tests are nucleic acid amplification tests such as real-time or multiplex reverse transcription-polymerase chain reaction (rt-pcr) assays that are useful for accurate diagnosis and subsequent viral load monitoring during antiviral treatment. genotyping by nucleic acid amplification and sequencing for detection of mutations associated with antiviral resistance directly from clinical specimens are now available for many antiviral agents and are routine for antiretroviral drugs used to treat hiv infection. though nucleic acid amplification tests still have practical limitations in the field settings of developing areas, such tests are now routine in most hospitals in developed countries. antibody testing by enzyme immunoassay for igm in acute infection, igg for immune status of exposed individuals, and retrospective diagnosis by the presence of rising antibody titers in paired acute and convalescent sera of symptomatic patients is useful for making clinical and epidemiological decisions. antibody screening is especially important in antenatal visits of expectant mothers, blood donors, and organ donors and recipients before transplantation. next-generation sequencing performed directly on clinical specimens may revolutionize virological diagnosis in the coming decade. the timely and accurate diagnosis of viral infections has important implications for effective epidemiological control in the community and infection control for hospital outbreaks. transmission of blood-borne viruses can result from sexual intercourse and maternal-fetal transmission in the community setting, needle stick injury, and other exposure-prone procedures in the health-care setting. in a study from the usa, the annual death rate of health-care workers from occupational events was estimated to be 17-57 per 1 million workers, and most of these deaths resulted from infection-related complications of blood-borne viruses (sepkowitz and eisenberg, 2005) . the overall risk of transmission of blood-borne viruses by hollow needle stick injury is 33%, 3%, and 0.3% if the source is a hepatitis carrier with positive hbe antigen or high viral load, hepatitis c carrier with viremia, and hiv, respectively. compliance with standard precautions including wearing gloves when handling blood during patient care practice, disposing sharp needles into puncture-resistant box, and avoidance of recapping needles remain the most important ways to prevent nosocomial acquisition of blood-borne viruses (garner, 1996) . active immunization for hbv can protect health-care workers from hbv infection with an efficacy of 80-95% (dienstag et al., 1984; sabido et al., 2007; shim et al., 2011) . postimmunization anti-hbs antibody level should be measured at 4-8 weeks after completion of the 3-dose immunization regimen given at baseline, 1 month, and 6 months. a good responder is defined as a person whose anti-hbs antibody level is greater than 100 iu/l. if the hepatitis b antibody level is between 10 and 100 iu/l, a booster dose of vaccine should be given. for nonresponders whose anti-hbs antibody is less than 10 iu/l, another course of hbv vaccine should be given. the response rate is about 61% in repeated hbv vaccination by the same route as the initial vaccination (struve et al., 1994) . alternatively, immunization with high-dose intradermal recombinant hbv vaccine, given in up to four doses, can achieve immunity in 88% of health-care workers who failed to respond to intramuscular vaccination and boosters (levitz et al., 1995) . the anti-hbs titer was persistently higher than the protective level for at least 10 years after primary hbv vaccination (floreani et al., 2004) . when a health-care worker sustains a needle stick injury, he/ she should be advised to rinse the wound with tap water and allow natural bleeding. the source patient's blood is collected to check for the presence of hiv, hbv, and hcv. if the status of blood-borne viruses of the source patient is positive or unknown, postexposure prophylaxis (pep) should be offered according to current guidelines (kuhar et al., 2013 ). the exposed health-care worker will be closely followed up for counseling, baseline and follow-up hiv testing, and monitoring for drug toxicity. if a newer fourth-generation combination hiv p24 antigen-hiv antibody test is utilized for follow-up hiv testing, it may be concluded 4 months after exposure. otherwise, follow-up hiv testing is performed 6 months after the exposure (kuhar et al., 2013) . for hbv, pep with hepatitis b immune globulin (hbig) and/or hbv vaccination should be considered for occupational exposures after evaluation of the hbsag status of the source, and the vaccination and vaccine-response status of the exposed person (2001). for hcv, pep is not currently recommended. however, an open-label pilot trial was conducted to determine the safety, tolerability, and acceptance of peginterferon alfa-2b as pep. among 213 health-care workers exposed to an hcv antibody-positive source, 51 hcws enrolled in the study and 44 (86%) elected to undergo peginterferon alfa-2b as the study group. seven subjects who elected not to undergo pep were treated as the control group. in this pilot study, peginterferon alfa-2b was proven to be safe without serious adverse rural residents with contact with bats (organ transplantation) dietzschold and koprowski (2004) and kusne and smilack (2005) effects. however, the lack of hcv transmission in both the study and control groups did not support the routine use of pep in health-care workers after hcv exposure (corey et al., 2009) . it is likely that the new polymerase and protease inhibitors used in the treatment of hcv infection will result in new strategies for pep of hcv exposures. blood-borne viruses can also be transmitted from healthcare workers to patients, especially during exposure-prone procedures in dental and cardiothoracic operations. the most well-known example involved an hiv-positive dentist working in florida, usa, who infected five of his patients after performing invasive dental procedures on them . sequencing of the hiv proviral envelope gene showed that the viruses infecting the dentist and the five patients were closely related . however, the overall risk for transmission of hiv from a health-care worker to a patient is very small. in a study conducted by the centers for disease control and prevention (cdc) of 22 171 patients being cared by 51 hiv-positive health-care workers, 113 (0.5%) patients became hiv positive. epidemiologic investigation did not implicate health-care workers as the source of infection in any of these patients (robert et al., 1995) . in contrast, transmission of hbv and hcv from health-care workers to patients was more frequently documented. between august 1991 and july 1992, 19 of 144 (13%) patients who were operated on by a thoracic surgeon with acute hbv infection became hbvinfected. sequencing of 160 bases in the core region of hbv showed an indistinguishable pattern among the strains of the surgeon and nine infected patients (harpaz et al., 1996) . subsequently, numerous health-care worker-to-patient transmissions of hbv and hcv were reported. among all these reported cases, the viral loads of the index health-care workers were more than 10 6 genome copies per ml (buster et al., 2003; gunson et al., 2003) . in this connection, the society for healthcare epidemiology of america (shea) issued a guideline for the management of health-care workers who are infected with hiv, hbv, and hcv to impose restriction on different categories of exposure-prone procedures according to the viral load (henderson et al., 2010) . epidemiologically important respiratory viruses such as influenza a virus are predominantly transmitted by the droplet route. by definition, the virus can spread within 1 m from the index case. however, individuals infected with influenza a virus may produce as many as 40 000 droplets of 0.5-12 mm in size and expel them at a velocity of 100 m s à1 upon sneezing (tang et al., 2006) . droplet nuclei of less than 3 mm may suspend in air and do not settle onto the ground (knight, 1980) . therefore, an explosive outbreak with high clinical attack rate as a result of aerosol transmission may occur under special conditions. in a jet airliner with nonfunctioning ventilation system, 72% of 54 passengers developed influenza-like illness within 72 h after being kept on ground for 3 h due to delay in flight time (moser et al., 1979) . as the virus may survive on inanimate surfaces for 12-48 h, and on the surface of hands for 10-15 min (kampf and kramer, 2004; kramer et al., 2006) , influenza virus can be transmitted indirectly by contact with hands from the contaminated environment to the pharyngeal mucosa. symptomatic influenza may develop after intranasal inoculation of 1 tcid 50 of influenza a virus (tellier, 2006) . hand hygiene is always the core component of infection control measures in both community and hospitals to prevent the transmission of influenza a virus. wearing face masks by either the index case as source control or the health-care workers as contacts has shown to be equally effective in the control of nosocomial transmission of pandemic influenza a h1n1 (cheng et al., 2010) . hand hygiene and face masks have been shown to prevent household transmission of influenza virus when implemented within 36 h of the index patient's symptom onset . oseltamivir pep halted an outbreak of pandemic influenza a h1n1 in a secondary school (asiedu-bekoe et al., 2012), but not in nursing homes (van der sande et al., 2014) . prevention of nosocomial transmission of influenza a virus requires multiple actions. early identification of symptomatic cases by direct antigen detection from nasopharyngeal specimens and initiation of droplet precautions by wearing surgical masks, along with staff education, could achieve reductions in nosocomial pandemic influenza to near zero (cheng et al., 2010) , while a similar protocol was also effective in minimizing the risk of nosocomial transmission of avian influenza a/h7n9 virus (cheng et al., 2015) . to ensure hand hygiene compliance, directly observed hand hygiene was adopted to control the spread of respiratory viruses in hospitals (cheng et al., 2010 (cheng et al., , 2007b . alcohol-based hand rub is delivered to every health-care worker and conscious patient once every 2-3 h in the clinical areas, which may further reduce the spread of respiratory viruses. varicella zoster virus (vzv) and measles are predominantly transmitted by aerosols and deposited in distal airways (roy and milton, 2004) . the exact mechanism of airborne transmission remains to be elucidated. however, an early study demonstrated that nosocomial outbreak of vzv occurred even when the index case was strictly isolated in a single room (gustafson et al., 1982) . there was a lack of nosocomial spread of vzv when all index cases were placed in negative pressure airborne infection isolation rooms (anderson et al., 1985) . measles virus can survive in the air for at least 1 h, as shown in an outbreak where three susceptible children who contracted measles were never in the same room with the source patient and one of the three arrived at the office 1 h after the source patient had left (bloch et al., 1985) . a massive community outbreak of measles occurred in a modern suburban elementary school in new york in spring, 1974, when the index case produced 28 secondary cases in 14 different classrooms. the epidemic subsided after two subsequent generations when 60 children had been infected. from estimates of major physical and biologic factors, it was possible to calculate that the index case produced approximately 93 units of airborne infection (quanta) per minute, which was higher than that of patients with laryngeal tuberculosis (riley et al., 1978 (riley et al., , 1962 . early recognition and placement in airborne infection isolation room of index case of vzv and measles may reduce the risk of nosocomial outbreaks. standard and transmission-based precautions are important to prevent the spread of respiratory and gastrointestinal viral infection ( table 3) . some of the respiratory viruses such as respiratory syncytial virus (rsv), parainfluenza virus, and the gastrointestinal viruses, norovirus, and rotavirus are predominantly spread by direct contact. as an illustrative example, rsv is the most frequent cause of nosocomial infection in pediatric wards and causes lower respiratory tract disease in 40% of young children. prolonged shedding of rsv for 3-11 days has been observed in immunocompetent children (hall, 2000) , and the virus can survive on inanimate surfaces for 6 h (kramer et al., 2006) . all these factors contribute to fomite-mediated transmission of rsv in the hospital. the risk of nosocomial rsv transmission was not related to age or underlying disease, but to length of hospitalization (hall et al., 1975) . contact precautions with cohort nursing and wearing gloves and gowns during patient care resulted in a significant reduction in nosocomial transmission of rsv in three consecutive winters (madge et al., 1992) . in another study, the incidence of nosocomial acquisition of rsv was significantly decreased after implementation of wearing gloves and gowns and isolation of cases even though the duration of rsv shedding remained unchanged before and after the intervention (leclair et al., 1987) . for the gastrointestinal viruses, norovirus is the most famous agent to cause outbreaks in the community and hospital. transmission is predominantly by the fecal-oral route. numerous community outbreaks of norovirus have been reported in restaurants, resorts, cruise ships, schools, and nursing homes (arvelo et al., 2012; britton et al., 2014; kuo et al., 2009; lai et al., 2013; wikswo et al., 2011) . the emergence of a new variant of norovirus, genogroup ii, type 4 (gii.4), in australia, europe, and north america associated with increased acute gastroenteritis activity has been reported since 2006 (bruggink and marshall, 2010; hasing et al., 2013; kanerva et al., 2009; yen et al., 2011) . norovirus is a nonenveloped rna virus which is relevantly resistant to common disinfectants. as norovirus is unculturable, feline calicivirus has been used as a surrogate for in vitro and in vivo testing for different preparations of disinfectants (gehrke et al., 2004; lages et al., 2008) . in the who formulation ahr, formula i preparation contains ethanol (80% v/v) which, based on the above studies, may possess reasonable virucidal activity for norovirus when the contact time is prolonged for up to 30 s. successful control of nosocomial outbreaks of norovirus by directly observed hand hygiene has been reported, especially during high-risk nursing care practices such as changing napkins and feeding . a proactive infection control approach with the provision of 'added test' was implemented to prevent the occurrence of nosocomial outbreak when the new variant of norovirus, table 3 infection control measures for transmission-based precautions in resource-poor areas (cheng et al., 2011) . rt-pcr for norovirus was performed as an 'added test' by the microbiology laboratory for all fecal specimens that were requested for bacterial culture, clostridium difficile culture or cytotoxin, and rotavirus antigen detection without a request for norovirus detection. during the study period, almost 50% of newly diagnosed norovirus infections were detected by the added test. timely implementation of infection control measures by single room isolation of index case with strict contact precautions significantly reduced the incidence of hospital-acquired norovirus infection from 131 (baseline) to 16 cases per 1000 potentially infectious patient-days (p < 0.001) (cheng et al., 2011) . disease outbreak such as sars, pandemic influenza, and ebola the outbreak of sars in 2003 was the first emergence of an important human pathogen in the twenty-first century. sars emerged as an outbreak of atypical acute community-acquired pneumonia in late 2002. the epidemic may have started when a bat sars coronavirus jumped into caged palm civets in a wildlife market and became adapted and amplified to jump from civet to human. infected chefs and animal handlers transmitted the adapted virus to health-care workers and then the epidemic became amplified into the community. the epidemic was rapidly and globally disseminated when a 'super-spreader' of sars, who was a medical professor from a teaching hospital in guangzhou, went to hong kong on 21 february 2003. during his stay in hotel m, he transmitted sars-cov to other residents, and the secondary cases spread the disease to hospitalized patients in hong kong, and to other countries including vietnam, singapore, and canada. eventually, a total of 8096 patients were infected in over 30 countries among five continents and 774 (9.5%) of them died (cheng et al., 2007a) . nosocomial outbreaks were reported in many parts of the world including toronto, hong kong, guangzhou, kaohsiung, singapore, and vietnam during the sars epidemic. there were a total of 716 secondary and tertiary cases of sars as a result of the admission of infected index patients. health-care workers constituted 410 (52.3%) of the secondary and tertiary cases (cheng et al., 2013) . as there were no known effective antiviral agents or vaccine for the treatment and prevention of sars, infection control measures and extensive tracing to quarantine the contact person became the most important interventions for sars control. the longitudinal follow-up of sars patients revealed that the viral load gradually increased on day 5 after symptom onset and peaked at day 10. early isolation of source patients can prevent ongoing transmission of sars in the community. in hospitals, temporary suspension of clinical services in both inpatient and outpatient settings was adopted (gopalakrishna et al., 2004; liu et al., 2006; nishiura et al., 2005; reynolds et al., 2006) , while home quarantine of health-care workers who had contact with sars patients was also mandated in some centers (dwosh et al., 2003) . provision of personal protective equipment (ppe) such as n95 respirators, gloves, gowns, and goggles, and placement of suspected or confirmed cases of sars in airborne infection isolation rooms were enforced when resources were available. the appropriate use of ppe was also important for staff protection. many health-care workers apparently lacked a clear understanding of how best to remove ppe without contaminating themselves. little information about the appropriate sequence of removing ppe was available at that time (puro and nicastri, 2004) . infection control measures are particularly important for emerging viral infections without effective antiviral therapy and vaccine. recently, the largest outbreak of ebola virus disease (evd) in west africa (guinea, sierra leone, liberia, nigeria, and équateur province of democratic republic of the congo) resulted in a total of 21 724 cases and 8641 deaths as of 21 january 2015. ebola virus is transmitted via contact with contaminated body fluid or the contaminated environment, and therefore the practice of contact precautions with appropriate ppe is of utmost importance when handling suspected or confirmed evd cases. health-care workers should preferably work in pairs so as to mutually observed against lapses in infection control measures. they are required to put on the ppe in the following sequence: n95 respirator, water-repellent cap or hood, fulllength shoe cover or boot, water-resistant gown, face shield, and finally long nitrile gloves. if the patient has hemorrhagic symptoms, double nitrile gloves should be worn. in view of the high virulence and mortality, patients suspected to have evd should be isolated in airborne isolation rooms, although the who allows cohorted nursing in designated areas with dedicated instruments, where access should be restricted in developing countries with limited isolation facilities. degowning remains the most critical procedure for healthcare workers. the most contaminated ppe should be removed first, starting with the long nitrite gloves, water-resistant gown, full-length shoe cover or boot, face shield, water-repellent cap or hood, and finally n95 respirator. hand hygiene with alcohol-based hand rub should be performed in each step of degowning. when the hand is visibly soiled, it should be washed with soap and water. health-care workers must be well trained and audited for the proper procedure of gowning and degowning. when the suspected or confirmed case of evd dies, the health-care and mortuary workers are required to wear ppe as described above. the dead body is placed in double bags with leak-proof characteristic of no less than 150 mm thick. absorbent material should be put under the body and placed in the first bag. the surface of each body bag is wiped with 10 000 ppm sodium hypochlorite solution. the bags are sealed and labeled with the indication of highly infectious material (category 3) and moved to the mortuary immediately. viewing in funeral parlor, embalming and hygienic preparation are not allowed. the dead body should not be removed from the body bag and should be sent to cremation as soon as possible. in august 2014, who declared the evd outbreak in west africa a public health emergency of international concern. preparedness and response plans were made available by health authorities in nearly all countries worldwide. the aim was to detect the first imported case for early isolation in order to prevent local transmission in the community and healthcare settings. therefore, risk assessment at ports, emergency rooms, and outpatient clinics for any patient fulfilling both clinical and epidemiological criteria for evd is important. for the clinical definition, patient suffering from elevated body temperature or subjective fever or symptoms including severe headache, fatigue, muscle pain, vomiting, diarrhea, abdominal pain, or unexplained hemorrhage should be alerted, while the epidemiological definition includes close contact with a confirmed or probable case of evd or residence in or history of travel to an affected area or countries (guinea, liberia, sierra leone) within 21 days before symptom onset. for health-care workers working in volunteer medical services or nongovernment organizations, who have had direct contact with patients in the affected areas or countries, should also perform medical surveillance or be placed in quarantine for at least 21 days after leaving the affected areas or countries. medical evaluation should be sought promptly if there are any symptoms of fever, diarrhea, vomiting, or bleeding during quarantine or medical surveillance. with reference to the experience in the community spread of pandemic influenza a virus infection, nonpharmacological interventions with social distancing, such as school closures, have been evaluated in previous modeling and epidemiological studies (bell et al., 2009; bootsma and ferguson, 2007; ferguson et al., 2006; markel et al., 2007) . during the influenza pandemic in 2009, school closures were practiced in the usa and australia (borse et al., 2011; effler et al., 2010) , because school closures were associated with a 65% reduction in the mean total number of contacts for each student as reported in a retrospective questionnaire survey in the united kingdom (jackson et al., 2011) . in hong kong, kindergartens and primary schools were closed when local transmission of influenza a virus was identified in 2009, followed shortly afterward by secondary school closures for summer vacations. influenza a virus transmission was estimated to be reduced by 25% (wu et al., 2010) . home quarantine was also shown to reduce the incidence of pandemic influenza a in the workplace (miyaki et al., 2011) . in fact, home quarantine has been used to control the community spread of sars in beijing, taiwan, singapore, and toronto (centers for disease control and prevention (cdc), 2003; cava et al., 2005; hsu et al., 2006) . home quarantine can be considered for the control of the spread of ebola virus in affected countries although in resource-limited settings effectively implementing these strategies can be challenging. the local government and health authorities have already implemented home quarantine for 3 days as an urgent infection control measure. however, if it is technically and politically feasible, home quarantine may be extended for up to 21 days (one incubation period) for ebola virus disease. however, public health staff is expected to face unprecedented challenges in implementing an extensive quarantine policy, as they have a dual role of monitoring compliance and providing support to people in quarantine. countries in close proximity to the affected areas require implementing border control measures to screen for any suspected case of ebola virus or even considering closing the border for 21 days. although these measures may adversely affect international travel and local economies, it may be worthwhile to implement such strict measures to control this reemerging infectious disease with high mortality and psychological fear in a timely manner. currently available antivirals against influenza a include the adamantanes (amantadine and rimantadine), neuraminidase inhibitors (oseltamivir, zanamivir, and peramivir) and a pyrazinecarboxamide derivative (favipiravir). only the neuraminidase inhibitors and pyrazinecarboxamide derivatives are active against currently circulating influenza a viruses. oseltamivir and favipiravir are available orally. zanamivir is available either as a dry powder that is delivered by oral inhalation or recently, intravenous formulation is available. peramivir is only available in the intravenous formulation. randomized controlled trials in patients with seasonal influenza suggested that the use of neuraminidase inhibitor can shorten the duration of illness by approximately 1 day. a recent meta-analysis had demonstrated that early neuraminidase inhibitor treatment (within 2 days of symptom onset) was associated with a reduction in mortality (muthuri et al., 2014) . two prospective clinical trials have demonstrated that treatment with convalescent plasma or hyperimmune intravenous immunoglobulin for patients with severe influenza infection was associated with lower viral load, cytokine level, and reduced mortality . clinical trials on various antiviral treatments against evd are underway. these agents include bcx4430 (a novel nucleoside analog) (julander et al., 2014) , brincidofovir, favipiravir, tkm-ebola, and zmapp (a chimeric monoclonal antibody) in guinea, sierra leone, and liberia (bishop, 2015) . when there is no highly effective antiviral for the treatment of a severe viral illness, especially in patients at the extremes of age or with medical comorbidities, and infection control measures are difficult to implement or comply with, vaccination is the final option to prevent massive outbreaks. influenza vaccine is the most widely used annual vaccine in the community and health-care setting to protect at risk or any person to develop influenza-related complications and prevent institutional outbreaks. seasonal influenza-related excess hospitalization and death were estimated to be 10 000 and 1100 per year, respectively, in hong kong, a subtropical city (chiu et al., 2002; wong et al., 2004 wong et al., , 2006 . in a meta-analysis assessing influenza vaccine efficacy and effectiveness in elderly patients, the inactivated influenza vaccine could reduce the risk of hospitalization as a result of pneumonia by 21-38%, and cardiovascular disease by 18-30%, and all cause of mortality by 39-56% (nichol, 2008) . control of virus disease outbreak by vaccination is particularly valuable for exposed individuals, when the viral diseases have a long enough incubation period so that the exposed individuals have sufficient time to develop protective immune responses before symptomatic disease set in. measles (incubation period of 7-18 days), mumps (incubation period of 12-25 days), rubella (incubation period of 14-23 days), and varicella (incubation period of 10-21 days) are relevant examples. reactive vaccination for measles outbreak has been shown to be an effective measure to reduce the scale of outbreaks. in the democratic republic of congo, weekly reported cases reduced respectively by 89.3% and 68.9% in the 3 weeks following mass vaccination campaigns (alberti et al., 2010) . similarly, nationwide mass vaccination interrupted the transmission of paralytic poliomyelitis in albania. in 1996, a total of 138 paralytic cases occurred with an attack rate of 10 per 100 000 population among adults aged 19-25 years. the epidemic was controlled by two rounds of mass vaccination with trivalent oral poliovirus vaccine targeted to persons aged 0-50 years (prevots et al., 1998) . while routine laboratory diagnostic tests and specific antimicrobial agents are generally available for the treatment of bacterial, fungal, and parasitic infections, we are just entering the stage when rapid nucleic acid tests and a greater array of antiviral agents are available for tackling viral infections. the broad array of viruses worldwide causes substantial morbidity and mortality, ranging from respiratory viruses, arthropod-related viruses, to the most deadly blood-borne viruses. novel emerging or reemerging viruses are causing major epidemics from time to time especially in densely populated areas where human populations have close contact with wild animals (wildlife markets) and food animals (wet markets and abattoirs). such epidemics such as the ebola virus disease can be explosive in countries with failed governance and poor health infrastructures. currently, there is a lack of antiviral treatment for most of these infections. therefore, prevention by implementing effective infection control and vaccination is of utmost importance to contain these viruses. see also: arboviruses; hiv safety guidelines; hepatitis, viral; influenza; measles; mumps; rabies; respiratory syncytial virus; rubella. reactive vaccination as an effective tool for measles outbreak control in measles mortality reduction settings lack of nosocomial spread of varicella in a pediatric hospital with negative pressure ventilated patient rooms norovirus outbreak of probable waterborne transmission with high attack rate in a guatemalan resort mass oseltamivir prophylaxis halts pandemic influenza a h1n1 2009 outbreak in a secondary school in ashanti region pandemic influenza as 21st century urban public health crisis potential and emerging treatment options for ebola virus disease measles outbreak in a pediatric practice: airborne transmission in an office setting the effect of public health measures on the 1918 influenza pandemic in u.s. cities closing schools in response to the 2009 pandemic influenza a h1n1 virus in new york city: economic impact on households norovirus outbreak at a wildland fire base camp ignites investigation of restaurant inspection policies doctor to patient transmission of hepatitis b virus: implications of hbv dna levels and potential new solutions the experience of quarantine for individuals affected by sars in toronto clinical management and infection control of sars: lessons learned severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection outbreak of human metapneumovirus infection in psychiatric inpatients: implications for directly observed use of alcohol hand rub in prevention of nosocomial outbreaks successful control of norovirus outbreak in an infirmary with the use of alcohol-based hand rub prevention of nosocomial transmission of swine-origin pandemic influenza virus a/h1n1 by infection control bundle infection control preparedness for human infection of influenza a h7n9 in hong kong prevention of nosocomial transmission of norovirus by strategic infection control measures influenza-related hospitalizations among children in hong kong transmission of human immunodeficiency virus in a dental practice pilot study of postexposure prophylaxis for hepatitis c virus in healthcare workers facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial efficiency of quarantine during an epidemic of severe acute respiratory syndrome-beijing, china hepatitis b vaccine in health care personnel: safety, immunogenicity, and indicators of efficacy rabies transmission from organ transplants in the usa identification and containment of an outbreak of sars in a community hospital household responses to pandemic (h1n1) 2009-related school closures ebola virus: from discovery to vaccine strategies for mitigating an influenza pandemic long-term persistence of anti-hbs after vaccination against hbv: an 18 year experience in health care workers guideline for isolation precautions in hospitals. part i. evolution of isolation practices, hospital infection control practices advisory committee inactivation of feline calicivirus, a surrogate of norovirus (formerly norwalk-like viruses), by different types of alcohol in vitro and in vivo clinical features of nipah virus encephalitis among pig farmers in malaysia isolation and characterization of viruses related to the sars coronavirus from animals in southern china hepatitis b virus (hbv) and hepatitis c virus (hcv) infections in health care workers (hcws): guidelines for prevention of transmission of hbv and hcv from hcw to patients an outbreak of airborne nosocomial varicella nosocomial respiratory syncytial virus infections: the "cold war" has not ended nosocomial respiratory syncytial virus infections isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus transmission of hepatitis b virus to multiple patients from a surgeon without evidence of inadequate infection control emergence of a new norovirus gii.4 variant and changes in the historical biennial pattern of norovirus outbreak activity in alberta shea guideline for management of healthcare workers who are infected with hepatitis b virus, hepatitis c virus, and/or human immunodeficiency virus identification and molecular characterization of hendra virus in a horse in queensland confidence in controlling a sars outbreak: experiences of public health nurses in managing home quarantine measures in taiwan convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe school closures and student contact patterns bcx4430, a novel nucleoside analog, effectively treats yellow fever in a hamster model epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs prolonged norovirus outbreak in a finnish tertiary care hospital caused by gii.4-2006b subvariants viruses as agents of airborne contagion how long do nosocomial pathogens persist on inanimate surfaces? a systematic review updated us public health service guidelines for the management of occupational exposures to human immunodeficiency virus and recommendations for postexposure prophylaxis a non-foodborne norovirus outbreak among school children during a skiing holiday transmission of rabies virus from an organ donor to four transplant recipients in-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus a norovirus outbreak in a nursing home: norovirus shedding time associated with age severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats prevention of nosocomial respiratory syncytial virus infections through compliance with glove and gown isolation precautions immunization with high-dose intradermal recombinant hepatitis b vaccine in healthcare workers who failed to respond to intramuscular vaccination epidemiologic study and containment of a nosocomial outbreak of severe acute respiratory prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic an effective quarantine measure reduced the total incidence of influenza a h1n1 in the workplace: another way to control the h1n1 flu pandemic an outbreak of influenza aboard a commercial airliner efficacy and effectiveness of influenza vaccination rapid awareness and transmission of severe acute respiratory syndrome in hanoi french hospital, vietnam. am molecular epidemiology of hiv transmission in a dental practice outbreak of paralytic poliomyelitis in albania, 1996: high attack rate among adults and apparent interruption of transmission following nationwide mass vaccination sars and the removal of personal protective equipment factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi airborne spread of measles in a suburban elementary school infectiousness of air from a tuberculosis ward. ultraviolet irradiation of infected air: comparative infectiousness of different patients investigations of patients of health care workers infected with hiv. the centers for disease control and prevention database airborne transmission of communicable infection-the elusive pathway timing of hepatitis b vaccination: its effect on vaccine response in health care workers occupational deaths among healthcare workers anti-hepatitis b core antibody is not required for prevaccination screening in healthcare workers seroconversion after additional vaccine doses to non-responders to three doses of intradermally or intramuscularly administered recombinant hepatitis b vaccine. scand effectiveness of postexposition prophylaxis with oseltamivir in nursing homes: a randomised controlled trial over four seasons factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises review of aerosol transmission of influenza a virus updated u.s. public health service guidelines for the management of occupational exposures to hbv, hcv, and hiv and recommendations for postexposure prophylaxis disease transmission and passenger behaviors during a high morbidity norovirus outbreak on a cruise ship influenza-associated mortality in hong kong influenza-associated hospitalization in a subtropical city school closure and mitigation of pandemic (h1n1) impact of an emergent norovirus variant in 2009 on norovirus outbreak activity in the united states key: cord-009813-o8ai730r authors: wang, wei; xiong, liang; wang, pengyun; wang, fubing; ma, qingfeng title: major vault protein plays important roles in viral infection date: 2019-11-26 journal: iubmb life doi: 10.1002/iub.2200 sha: doc_id: 9813 cord_uid: o8ai730r viral replication and related protein expression inside the host cells, and host antiviral immune responses can lead to the occurrence of diverse diseases. with the outbreak of viral infection, a large number of newly diagnosed and died patients infected with various viruses are still reported every year. viral infection has already been one of the major global public health issues and lead to huge economic and social burdens. studying of viral pathogenesis is a very important way to find methods for prevention, diagnosis, and cure of viral infection; more evidence has confirmed that major vault protein (mvp) is closely associated with viral infection and pathogenesis, and this review is intended to provide a broad relationship between viruses and mvp to stimulate the interest of related researchers. viruses are acellular that cannot naturally reproduce outside of the living host cells and only assemble themselves depending on the host cellular metabolism. 1 virion, known as the complete viral particle, consists of nucleic acid surrounded by capsid, which is enveloped with lipids in some viruses. virion is less than 300 nm in diameter, and its self-assembly is very fast, viral replication inside of the host cells may manipulate and damage the host cells, and the antiviral immune response of the host can damage tissue simultaneously. under the effort of viral toxicity and host immunity, the host is prone to get many kinds of diseases. hepatitis b virus (hbv) and hepatitis c virus (hcv) can cause chronic infection, which can lead to liver cirrhosis and subsequently develop hepatocarcinoma, the patients with viral hepatitis serve as reservoirs of infectious virus. 2 some viruses, including hepatitis a virus (hav), human enterovirus, ebola virus, sars virus, and avian influenza, can cause an outbreak of epidemic infection. [3] [4] [5] [6] the typical antibiotics are not effective of antiviral infection, antigenic drift of viruses can make effective treatments ineffective, 7 and treatment of viral infection is still one of challenges for humanity. abbreviations: aids, acquired immunodeficiency syndrome; atf, activating transcription factors; c/ebpβ, ccaat-enhancer-binding protein β; egf, endothelial growth factor; eif4a, eukaryotic initiation factor 4a; erk, extracellular signal-related kinase; irf7, interferon regulatory factor 7; mapk, mitogen-activated protein kinase; mda5, melanoma differentiation-associated protein 5; mdm, monocytederived macrophages; mvp, major vault protein; myd88, myeloid differentiation primary response 88; nf-kb, nuclear factor kappa-lightchain-enhancer of activated b cells; pbmc, peripheral blood mononuclear cells; pkm2, pyruvate kinase isozyme m2; prrs, pattern recognition receptors; pten, phosphatase and tensin homolog deleted on chromosome 10; srsfs, serine/arginine-rich splicing factors; stat-1, signal transducer and activator of transcription-1 . recent studies have shown that many host-encoded proteins are associated with viruses: heat shock protein 70 is incorporated into the virions of human immunodeficiency virus type 1 (hiv-1) 8 ; serine/arginine-rich splicing factors (srsfs) are related to viral replication, srsf2 promotes anogenital tumorigenesis by maintaining the stability of e6e7 mrnas of human papillomavirus 16 (hpv16), which is the pathogen of anogenital cancer; hiv-1 replication is increased by srsf1, srsf4, and srsf10 within the host cells 9 ; 36 host-encoded proteins are presented in influenza virions 10 ; mvp is involved in antiviral immune response 11 ; and the study of hostencoded proteins in relation to viruses contributes to finding novel targets for antiviral drugs. vaults, the large ribonucleoprotein particles, are composed with mvp, poly (adp-ribose) polymerase, telomerase-associated protein-1 (tep1), and one or more noncoding rna. 12, 13 the human mvp, encoded by mvp gene that is located in chromosome 16p11.2, 14 is highly conserved during evolution 15, 16 and predominant component of vaults. [17] [18] [19] [20] the expression of mvp is very strong and widespread, 21 the mvp is mainly located in the cytoplasm and associated with the cytoskeleton, and a small amount is localized at or around the nuclear membrane and the nuclear pore complex. 22, 23 current studies have confirmed that mvps are associated with multidrug resistance in treatment of non-small lung cancer, 24 human colon cancer, 25 and mesial temporal lobe epilepsy with hippocampal sclerosis. 26 mvp/vaults play important roles in several signal transduction pathways, suppress c-jun-mediated ap-1 transactivation by associating with cop1, 27 participate the phosphoinositide 3-kinase pathway by interacting with endogenous phosphatase and tensin homolog deleted on chromosome 10 (pten) with the help of ca2+ modulation, 28 act as a signaling scaffold protein of extracellular signal-related kinase (erk)/mitogen-activated protein kinase (mapk) pathway by interacting with src in response to endothelial growth factor (egf), 29 and affect the jak-stat signaling pathway by responding and interfering the interferon (ifn)-gamma-mediated stat1 signals. 30 growing evidences also confirmed that mvp is closely associated with other multiple cellular processes, such as nuclear-cytoplasmic transport, 31 malignant transformation, 32 senescence/ aging, 33 autophagy, 34 and innate immunity. 35 interestingly, mvp has been linked to several types of viral infectious diseases as well as to virus-mediated immune responses. 29, 36 here, we focus on the roles of mvp in the intracellular viral replication and host immune responses. the innate immune response, including the production of ifn-1, is the first barrier of eliminating invaded pathogens early. 37 in host cells, tlrs, rig-1 (rig-i-like receptor dsrna helicase enzyme), and mda5 (melanoma differentiation-associated protein 5) act as pattern recognition receptors (prrs), ifn-stimulated proteins, and sensors for viral infection. [38] [39] [40] the interferon regulatory factor 7 (irf7) plays the master transcriptional role in viral infection-induced ifn production and immune responses, [41] [42] [43] activates ifn-β production mediated by myd88 (myeloid differentiation primary response 88)independent rig-1/mda5 pathway, also activates ifn-α production mediated by the myd88-dependent tlrs pathway. [44] [45] [46] the ifn-1 inhibits viral replication (including hcv, influenza a virus [iav], and hiv) by the production of ifn-stimulated effective proteins. 11, 47, 48 after host cells or tissues are infected by hcv, prrs of host cells recognize stimulation signals of products of hcv processing, the interaction between prrs and stimulation signals activates ikba kinase to phosphorylate ikbα, 49 which is associated with nf-kb protein complex in the cytoplasm, phosphorylated ikbα is released from nf-kb complex and degraded by ubiquitin-proteasome pathway, 50 free nf-kb complex translocates to the nucleus, and subsequently activates mvp transcription under coactivators including hcv protein ns5a. 11 hcv infection also induces mvp expression through the sp1 signal pathways, and the infection of vesicular stomatitis virus (vsv), iav, and enterovirus 71 (ev71) has the same effect with hcv infection. 11 inducible mvp is helpful for the nuclear translocation of irf7 and nf-kb, and performs antiviral activity by promoting endogenous ifn-1 production and expression of the ifn-stimulated genes. the production of ifn is the critical step in an innate immune response, and mvp plays strong antiviral activity in an ifn-1-dependent manner. with the advent of effectively prophylactic vaccines and antiviral drugs, hbv infection remains a global public health problem, 51,52 an estimated 240 million people with chronic hbv infection are hbv carriers, 53 deadly complications of hbv chronic infection (including cirrhosis and hepatocellular carcinoma) result in approximately 600,000 deaths per year, 54 and hbv infection brings heavy economic pressure for individuals and heavy social burden for the world. as a type of pathogen, hbv causes host cells to produce ifns to increase protective defense of host immune system, 55 ifns play important roles of antivirus by regulating the host immune system, and have been used to treat some cancers 56 and hbv infection. 57, 58 hbv virus infection leads to the production of type 1 ifns by two main pathways. toll-like receptors 3/4 (tlr 3/4) recognize viral nucleotides and glycolipids and recruit the adaptor protein trif (tir-domaincontaining adapter-inducing ifn-β), trif interacts with traf6 (tumor necrosis factor [tnf] receptor-associated factor 6) to activate nf-kb (nuclear factor kappa-lightchain enhancer of activated b cells), and activated nf-kb provokes ifnb production. 59, 60 another pathway is triggered by tlr7/8 and tlr9, tlrs recognized viral nucleotides in the endosome recruit myd88, 61 in turn recruit irak1/4 (interleukin-1 receptor-associated kinase 1/4) 62 to the complex and interact with traf6 (tnf receptorassociated factor 6), 63, 64 and then activate irf5/7 (ifn regulatory factor 5/7) to induce ifnα expression. mvp is a virus-induced protein, and the level of mvp in peripheral blood mononuclear cells (pbmcs), sera, and liver tissue derived from patients with chronic hepatitis b (chb) is higher than healthy individuals; mvp expression is also increased in hbv stable expression cell lines (hepg2.2.15 and huh7.37) and hbv-infected hepatocarcinoma cell lines (hepg2 and huh7). 11 during hbv infection, tlrs recruit and activate myd88, which interacts with irak1/4, irf5/7, and traf6 to form a complex, 62 the middle domain (aa 310-620) of mvp can interact with myd88, high expressed mvp joins the myd88-mediated complex by interacting with myd88 to promote ifn-1 production through translocation of irf7 and nf-kb from the cytoplasm to the nucleus. 11, 65 however, hbsag and hbeag competitively bind the myd88-binding region of mvp and suppress the ifn-1 production by disrupting mvp/myd88 interaction; the ifn-1 increment effect induced by mvp is counterattacked through hbeag and hbsag binding to mvp 11 (figure 1 ). evidence suggests that hbv has other strategies to suppress the host immune response. hbv polymerase (pol) may inhibit ifn-ɑ-induced myd88 induction, 66 hbeag suppresses tlr-induced ifn-β, 67 hbsag can block the irf-7 mediated ifn-ɑ production pathway, 68 and multiple mechanisms lead to hbv immune escape. when the host is attacked by harmful pathogens including viral infection, one of protective immune response is inflammation to eliminate damage. 69 ifn to interfere viral replication, 55 interleukin 6 (il-6) acted as a proinflammatory cytokine, 70 and interleukin 8 (il-8) served as a chemokine for neutrophils and monocytes 71 are important mediators of immune response, and activation of il-6 and il-8 gene expression is regulated by transcription factors. 72, 73 activator protein 1 (ap-1), composed of proteins belonging to c-fos, c-jun, activating transcription factors (atf) and maf families, 74 is a heterodimeric complex and acts as a transcription factor. 75 the function of ap-1 complex is heavily dependent on the c-fos and c-jun subunits, 76 ap-1 complex binds dna at ap-1 specific sites at the promoter and enhancer regions of target genes and increases target gene expression, 77, 78 and researchers had confirmed that the ap-1 complex is involved in il-6 and il-8 regulation. 79, 80 ccaatenhancer-binding protein β (c/ebpβ) is a member of the c/ebp transcription factor family, the gene of c/ebpβ can be translated into three polypeptides: the 38 kda and 34 kda liver-enriched transcriptional activating proteins (laps), and the 20-kda liver-enriched transcriptional inhibitory protein (lip). 81 c/ebp proteins interact with certain gene promoters containing ccaat box motif, then recruit co-activators to promote gene expression. 81 the promoters of il-6 and il-8 consists of the ccaat box motif region, wherein c/ebpβ can bind and affect il-6 and il-8 expression. 82 f i g u r e 1 hbsag and hbeag weaken the effect of mvp on promoting ifn production in order to restrict the spread of infected virus, some activated transcription factors contribute to the production of inflammatory-related cytokines and chemokines. iav, as a kind of negative single-stranded rna viruses (ssrna), produces replicative intermediate double-stranded rna (dsrna) in the infected cells, 83 dsrna and the synthetic dsrna analog polyinosinic-polycytidylic acid (poly[i:c]) are recognized by tlr3, 29, 84 then activate the tlr3-ifn production pathway to robustly express type i ifns. 59, 60 mvp, as a regulator in the proinflammatory response and an effector in ifn signaling pathway, increases to against viral replication during viral infection. 65 mvp has been proven to be a nuclear-cytoplasmic transport protein 30 and interacts with c-fos of the ap-1 complex components and c/ebpβ-laps. 48 the interaction promotes the ap-1 complex and c/ebpβ-laps translocation from the cytoplasm to nucleus and follows to activate the il-6 and il-8 expression by the ap-1 complex and c/ebpβ-laps binding to the il6 and il8 promoters, and mvp plays a synergistic role in the expression of il-6 and il-8. 48 the expression of mvp, il-6, and il-8 increases simultaneously in iavinfected a549 or dsrna-stimulated pbmcs, and the expression of il-6 and il-8 is impaired in mvp knockdown cells and knockout mice 48 ; mvp plays a pivotal role in virus-triggered proinflammatory response by mediating the ap-1 and c/ebpβ signaling pathways. the model of mvp functions for proinflammatory response is summarized in figure 2 . hepatitis e virus (hev), belonged to the genus hepevirus, is classified as a positive-strand rna virus ([+] ssrna virus), 85 and hev infection is an important public health problem. hev is mostly transmitted via the fecal-oral route in developing countries under poor sanitary conditions, 86, 87 and often spread in many countries by food borne, 88 blood transfusion, 89, 90 and zoonotic origin. 91 hev can cause chronic infection in immunosuppressed patients, pegylated ifn-alpha-2b is used in the treatments for chronic hepatitis e (che) virus infection in liver transplant patients, 92 pegylated ifn-alpha-2a is used in the treatments for che virus infection in a hemodialysis patient, 93 and ribavirin as monotherapy may be effective in the treatment for che virus infection in solid-organ transplant patients. 94 silvestrol is a natural cyclopenta(b)benzofuran and acts as an inhibitor of the eukaryotic initiation factor 4a (eif4a) via hindering translation initiation from the 5 0capped and 5 0 -utr of mrnas. 95 the hev is a (+) ssrna virus containing 5 0 -cap and 5 0 -utr structure, 96 the released hev particles from persistently hev-infected a549 cells treated with silvestrol are robustly reduced, which are caused by the decrease of the intracellular hev capsid protein. 97 silvestrol also affect the expression and localization of antiviral host factor mvp, the mvp amount of the cytoplasm is reduced after treating with silvestrol in hev-infected cells, and the mvp transfers from the cytoplasm to the perinuclear area 97 that affects mvp-mediated ifn production. 98 the translation of mvp is highly activated to play an antiviral role by hev infection; however, the change of translation and cytoplasmic localization affected by the silvestrol treatment counteracts part of antiviral effect, mvp plays a complex interplay between the anti-hev replication and the effect of treating with silvestrol for hev infection. the infection of hiv is the pathogenesis of acquired immunodeficiency syndrome (aids) and one of major global public health issues. 99 hiv infected immune cells, including monocytes, lymphocytes, and macrophages, act as stable rival reservoirs, 100 and are main barrier to f i g u r e 2 mvp plays a pivotal role in the proinflammatory response caused by (−) ssrna viral infection eradicate virus by antiviral therapy. 101 the level of cystatin b, a cysteine protease inhibitor, is higher in blood monocyte-derived macrophages (mdm) than in placental macrophages, which are more resistant to hiv-1 infection than mdm. 102, 103 the expression of cystatin b is upregulated in hiv-1-infected mdm, 104 and cystatin b promotes hiv-1 replication by interacting with pyruvate kinase isozyme m2 (pkm2), 105 which is associated with the cocaine enhancement of hiv-1 replication. 106 in hiv-infected mdm, upregulated cystatin b interacts with mvp 105 and signal transducer and activator of transcription-1 (stat-1). 103 mvp, as an ifn-responsive protein, directly inhibits tyrosine phosphorylation of stat-1 to weaken ifn-induced antiviral response by interfering the jak/stat signal pathway, 29 then promote hiv replication. cystatin b directly interacts and decreases tyrosine phosphorylation of stat-1, and inhibits ifn-β response and stat-1 translocation from the cytoplasm to nucleus to reduce jak/stat signal pathway activity, and ultimately promote hiv replication. 105 under the cooperation of the cystatin b and mvp, hiv replication is activated by the damage of jak/stat signal pathway activity mediated by the low tyrosine phosphorylation of stat-1. mvp is involved in the diversely cellular processes, including multiresistant cancers, 24-26 signal transmission pathways, [27] [28] [29] [30] and immune response associated with viral infection and treatment. 11, 48, 65, 97, 105 viruses with divergent virulence and spreadways can cause diverse human diseases with different types and degrees of damage, as a response of viral infection, studies have confirmed that mvp is enhanced in diverse viral infection, including hbv, hcv, hiv, iav and vsv, and so on. the infection of (−) ssrna viruses (including hcv, vsv, iav, and ev71) or dsrna stimulation activates proinflammatory response by inducing the expression of mvp, il-6, and il-8, enhanced mvp further increase the expression of il-6 and il-8 by translocating transregulatory elements (ap-1 protein complex and c/ebpβ-laps) to the nucleus, 65 and lipopolysaccharide synthesized during viral replication also activates the tlr4 signaling pathway to induce cytokines, chemokines, and ifn-1 against iav replication 107 ; however, the value of mvp in the diagnosis, treatment, and prognosis of viral infection remains unclear and additional studies are still required. hbsag and hbeag compete to bind with mvp, facilitate hbv replication and survival by attenuating the effect of mvp-induced ifn, 48 and ifn and nucleotide analogs (nas) are used for the treatment of patients infected with hbv, the stage of liver diseases is important in guiding antiviral therapy 108 ; however, the effect of mvp on the severity of liver disease and the efficacy of different treatments is unclear. silvestrol, as a potent antiviral compound, inhibits hev assembly by interfering hev capsid protein translation, but deactivates the antiviral effect of mvp by translocating mvp to the perinuclear membrane 97 ; cystatin b, as a cysteine protease inhibitor, increases hiv replication by interacting with mvp and pkm2 to inhibit ifn response and tyrosine phosphorylation of stat-1. 105 mvp plays an opposite role in hiv infection by comparing with iva and hbv infection, weakens the antiviral efficacy of silvestrol in the treatment of hev infection, and additional studies are necessary to clarify the role of mvp more clearly in viral infection. i would like to thank my collaborators for their kind help to organize the thoughts and concepts. synthetic viruses: a new opportunity to understand and prevent viral disease significance of hbv dna by pcr over serological markers of hbv in acute and chronic patients. indian outbreak of infection with hepatitis a virus (hav) associated with a foodhandler and confirmed by sequence analysis reveals a new hav genotype ib variant survey of enterovirus infections from hand, foot and mouth disease outbreak in china who ebola response team. ebola virus disease in west africa -the first 9 months of the epidemic and forward projections cross-species virus transmission and the emergence of new epidemic diseases the influenza viruses specific incorporation of heat shock protein 70 family members into primate lentiviral virions physiological and pathological function of serine/arginine-rich splicing factor 4 and related diseases cellular proteins in influenza virus particles human hepatitis b virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small rna the vault complex the lrp gene encoding a major vault protein associated with drug resistance maps proximal to mrp on chromosome 16: evidence that chromosome breakage plays a key role in mrp or lrp gene amplification vaults. ii. ribonucleoprotein structures are highly conserved among higher and lower eukaryotes cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the mvp n termini and vparp the drug resistance-related protein lrp is the human major vault protein the 193-kd vault protein, vparp, is a novel poly(adp-ribose) polymerase characterization of mvp and vparp assembly into vault ribonucleoprotein complexes vaults and telomerase share a common subunit, tep1 vaults are upregulated in multidrugresistant cancer cell lines evidence that vault ribonucleoprotein particles localize to the nuclear pore complex characterization of the sea urchin major vault protein: a possible role for vault ribonucleoprotein particles in nucleocytoplasmic transport mechanisms underlying lung resistance-related protein (lrp)-mediated doxorubicin resistance of non-small cell lung cancer cells molecular basis for the expression of major vault protein induced by hyperosmotic stress in sw620 human colon cancer cells major vault protein (mvp) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis major vault protein, in concert with constitutively photomorphogenic 1, negatively regulates c-jun-mediated activator protein 1 transcription in mammalian cells pten associates with the vault particles in hela cells crosstalk between src and major vault protein in epidermal growth factordependent cell signalling the major vault protein is responsive to and interferes with interferon-gamma-mediated stat1 signals phosphatase and tensin homologue deleted on chromosome 10 (pten) has nuclear localization signal-like sequences for nuclear import mediated by major vault protein lung resistance-related protein as a predictor of clinical outcome in advanced testicular germcell tumours on the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis listeria and autophagy escape: involvement of inlk, an internalin-like protein host resistance to lung infection mediated by major vault protein in epithelial cells evaluation of mdr1, lrp, mrp, and topoisomerase iialpha gene mrna transcripts before and after interferonalpha, and correlation with the mrna expression level of the telomerase subunits htertand tep1 in five unselected human melanoma cell lines innate immunity to virus infection pathogen recognition and innate immunity rig-i-mediated antiviral responses to single-stranded rna bearing 5 0 -phosphates length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 irf-7 is the master regulator of type-i interferon-dependent immune responses irf family of transcription factors as regulators of host defense convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response toll-like receptors in innate immunity the immunobiology of the tlr9 subfamily pattern recognition receptors and control of adaptive immunity a diverse range of gene products are effectors of the type i interferon antiviral response inducible major vault protein plays a pivotal role in double-stranded rna-or virus-induced proinflammatory response nf-kappab regulation in the immune system ikk-1 and ikk-2: cytokine-activated ikappab kinases essential for nf-kappab activation global epidemiology of hepatitis b virus infection: new estimates of agespecific hbsag seroprevalence and endemicity time trends of chronic hbv infection over prior decades-a global analysis estimations of worldwide prevalence of chronic hepatitis b virus infection: a systematic review of data published between hepatitis b virus infection an overview of the immune system the role of interferon in cancer therapy: a current perspective the role of interferon therapy in hepatitis b combination therapy (interferon alfa and ribavirin) in the treatment of chronic hepatitis c: a rapid and systematic review distinct poly(i-c) and virus-activated signaling pathways leading to interferonbeta production in hepatocytes the host type i interferon response to viral and bacterial infections the family of five: tir-domaincontaining adaptors in toll-like receptor signalling the ikappab kinase complex regulates the stability of cytokineencoding mrna induced by tlr-il-1r by controlling degradation of regnase-1 sequential control of toll-like receptor-dependent responses by irak1 and irak2 traf6 is a signal transducer for interleukin-1 major vault protein: a virusinduced host factor against viral replication through the induction of type-i interferon hepatitis b virus polymerase impairs interferon-alpha-induced stat activation through inhibition of importin-alpha5 and protein kinase c-delta hepatitis b virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells hbsag inhibits tlr9-mediated activation and ifn-alpha production in plasmacytoid dendritic cells chronic inflammation: importance of nod2 and nalp3 in interleukin-1beta generation the pro-and anti-inflammatory properties of the cytokine interleukin-6 tumor-produced interleukin-8 attracts human myeloid-derived suppressor cells and elicits extrusion of neutrophil extracellular traps (nets) activation of il-8 gene expression by helicobacter pylori is regulated by transcription factor nuclear factor-kappa b in gastric epithelial cells transcription factors nf-il6 and nf-kappa b synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8 fos/ap-1 proteins in bone and the immune system ap-1 subunits: quarrel and harmony among siblings the role of jun, fos and the ap-1 complex in cell-proliferation and transformation the c-fos protein interacts with c-jun/ap-1 to stimulate transcription of ap-1 responsive genes translational regulation mechanisms of ap-1 proteins effects of il-10 and il-4 on lps-induced transcription factors (ap-1, nf-il6 and nf-kappa b) which are involved in il-6 regulation. leukemia human cytomegalovirus induces interleukin-8 production by a human monocytic cell line, thp-1, through acting concurrently on ap-1 and nf-kappab-binding sites of the interleukin-8 gene ccaat/enhancer binding proteins are critical components of the transcriptional regulation of hematopoiesis the c/ebpb isoform 34-kda lap is responsible for nf-il-6-mediated gene induction in activated macrophages, but is not essential for intracellular bacteria killing cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome hepatitis e virus hepatitis e: discovery, global impact, control and cure public health risks associated with hepatitis e virus (hev) as a food-borne pathogen seroprevalence of hepatitis e virus (hev) and detection of hev rna with a transcriptionmediated amplification assay in blood donors from catalonia hepatitis e virus in blood components: a prevalence and transmission study in southeast england chronic hepatitis e with cirrhosis in a kidney-transplant recipient treatment of chronic hepatitis e in liver transplant recipients with pegylated interferon alpha-2b three-month pegylated interferon-alpha-2a therapy for chronic hepatitis e virus infection in a haemodialysis patient ribavirin for chronic hepatitis e virus infection in transplant recipients antitumor activity and mechanism of action of the cyclopentabbenzofuran, silvestrol hepatitis e virus (hev) protease: a chymotrypsinlike enzyme that processes both non-structural (porf1) and capsid (porf2) protein inhibition of hepatitis e virus spread by the natural compound silvestrol nuclear localization of the major vault protein in u373 cells emerging concepts in the immunopathogenesis of aids quantification of latent tissue reservoirs and total body viral load in hiv-1 infection the latent reservoir for hiv-1: how immunologic memory and clonal expansion contribute to hiv-1 persistence proteomic analyses associate cystatin b with restricted hiv-1-1 replication in placental macrophages cystatin b associates with signal transducer and activator of transcription 1 in monocytederived and placental macrophages restricted hiv-1 replication in placental macrophages is caused by inefficient viral transcription inhibition of interferon response by cystatin b: implication in hiv replication of macrophage reservoirs enhancement of hiv-1 replication by opiates and cocaine: the cytokine connection the tlr4-trif pathway protects against h5n1 influenza virus infection update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld 2018 hepatitis b guidance the authors declare no potential conflict of interest.orcid pengyun wang https://orcid.org/0000-0001-6801-0705 fubing wang https://orcid.org/0000-0002-5971-2622 qingfeng ma https://orcid.org/0000-0002-1676-3235 key: cord-003993-3bozjfv7 authors: cagliani, rachele; forni, diego; sironi, manuela title: mode and tempo of human hepatitis virus evolution date: 2019-10-25 journal: comput struct biotechnol j doi: 10.1016/j.csbj.2019.09.007 sha: doc_id: 3993 cord_uid: 3bozjfv7 human viral hepatitis, a major cause of morbidity and mortality worldwide, is caused by highly diverse viruses with different genetic, ecological, and pathogenetic features. technological advances that allow throughput sequencing of viral genomes, as well as the development of computational tools to analyze such genome data, have largely expanded our knowledge on the host range and evolutionary history of human hepatitis viruses. thus, with the exclusion of hepatitis d virus, close or distant relatives of these human pathogens were identified in a number of domestic and wild mammals. also, sequences of human viral strains isolated from different geographic locations and over different time-spans have allowed the application of phylogeographic and molecular dating approaches to large viral phylogenies. in this review, we summarize the most recent insights into our understanding of the evolutionary events and ecological contexts that determined the origin and spread of human hepatitis viruses. human hepatitis viruses are extremely diverse and consequently belong to different viral families (table 1 ). in recent years, the availability of high-throughput technologies has revealed that relatives of human hepatitis viruses can be found in a wide variety of animals. this finding, as well as the increasing availability of the genome sequences of human-infecting viruses sampled across different geographic areas, has largely expanded our knowledge about the genetic diversity and evolutionary origin of these human pathogens. in this review, we thus focus on the latest insights into the possible events and ecological contexts that determined the origin and spread of human hepatitis viruses. a short presentation of the most widely used approaches to estimate the ages of viral lineages is also provided to contextualize recent research efforts on these viruses. molecular dating analyses using virus genetic data can be particularly informative due to the rapid rate of evolution of many viral species. by converting genetic differences among sequences into time units, molecular dating provides information on the timing of viral spread or emergence. most molecular dating approaches are based on maximum-likelihood or bayesian phylogenetic frameworks [5, 6] , and they usually exploit two strategies: molecular clock calibration using the sampling dates of the viral sequences (tip dating) and/or calibration using information on some internal nodes of the phylogeny. tip dating is well-suited to study relatively recent events (e.g., epidemics or intra-host evolution), but requires that a temporal signal is present in the dataset (i.e., that the sequences have accumulated a measurable amount of change between sampling times) [6] . calibration using internal nodes can in principle allow to dig deeper into the past, but requires some a priori knowledge about the virus evolutionary history (e.g., host-virus co-evolution, paleontological information). the widespread use of molecular dating has however revealed that the relationship between genetic divergence and time is complex, as evolutionary rates tend to vary with the time frame of measurement. in particular, high evolutionary rates are observed in the short term, whereas low rates are inferred in long time span studies [7] [8] [9] . this pattern was observed for many viral lineages and is sometimes referred to as the time-dependent rate phenomenon (tdrp) [10] . failure to account for the tdrp can potentially lead to erroneous molecular dating results [10, 11] . the tdrp reflects mutation rate in very short timescales and substitution rate in very long timescales, during which transient deleterious mutations are removed by the action of natural selection, leading to lower rate estimates [12] . another factor most likely contributing to the tdrp is the saturation of nucleotide substitutions, which is extremely rapid in viral genomes, especially when the polymerase is error-prone [12] . thus, recent analyses indicated that, for all baltimore classification groups, viral evolutionary rates tend to decrease continuously with the timescale of measurement [10, 11] . because the rate of decay is consistent with a power law relationship between substitution rate and sampling timescale, a model using a simple regression was at first proposed to estimate the tdrp effect on viral phylogenies [10] . very recently, this approach was implemented in a bayesian statistical framework, in which evolutionary rates can vary among different time epochs [13] . before the introduction of such an approach, effective attempts to correct for the tdrp were performed by the use of nucleotide substitution models that allow site-and branch-specific variation in selective pressure (selectioninformed models). these models, which were applied to analyze the ancient evolution of some viral lineages, at least partially correct for the effects of both purifying selection and substitution saturation in branch length estimation [14] [15] [16] . hav is mainly transmitted via the faecal-oral route through exposure to contaminated food or water, or through direct contact with infected people. hav is a single-strand, positive rna virus with a genome of approximately 7.5 kb in length ( table 1 ). the hav genome contains a single orf flanked by a relatively long 5 0 utr and a 3 0 utr. the 5 0 utr harbors an internal ribosome entry site that directs the cap-independent translation of hav proteins. the orf encodes a polyprotein processed in 11 mature proteins: 5 structural proteins involved in capsid formation (vp4, vp2, vp3, vp1, and px, deriving from p1 segment) and 6 nonstructural proteins with a role in rna genome amplification (2b, 2c, 3a, 3b, 3cpro, and 3dpol, deriving from p2 and p3 segments) [4, 17] . based on genomic structure, hav belongs to the family picornaviridae within the genus hepatovirus. nevertheless, many characteristics distinguish hav (and hepatoviruses in general) from other picornaviridae family members. some peculiar features include the primary tropism for hepatocytes, the ability to shed as nonenveloped virus in feces and as enveloped particles in blood, as well as some genomic features such as low g/c ratio, low cpg levels, and strong codon bias [18, 19] . hav was identified as the etiologic agent of hepatitis a by feinstone and colleagues [20] in 1973. unlike hbv and hcv, which establish chronic infections in humans, hav infection is usually acute and generates lifelong immunity. this condition is able to determine the disappearance of the virus in small and isolated populations [21, 22] and did not probably favor its maintenance in early human communities. it is thus legitimate to wonder how hav survived and evolved during early human history, a question that remains presently unanswered. for a long time, it was thought that hepatoviruses were restricted to humans and non-human primates (nhps), with genetically distinct variants classified as six main different genotypes [23] : three isolated from humans (hav, genotype i-iii) and subclassified in 6 subgenotypes (ia, ib, iia, iib, iiia, iiib) and three of simian origin (shav, genotype iv-vi). however, despite genetic heterogeneity, hav viruses belong to a single common serotype. in recent years, the advent of new sequencing approaches has led to an exponential increase in the identification of new viral speacute cies, including highly diverse non-primate hepatoviruses. several hav-related viruses were identified in different mammalian orders. in particular, a number of hav-like viruses were recovered in placental mammals, mainly in bats and rodents, but also in tree shrews, hedgehogs, seals and chinese woodchucks [24] [25] [26] . recently, de oliveira carneiro and colleagues [27] identified a novel hav-related virus in didelphis aurita, a brazilian common opossum, further extending the host range of mammal-infecting hepatoviruses. moreover, viruses related to mammalian hepatoviruses were detected in reptiles, amphibians, and fish [28] . these advances allowed new insights into the evolutionary history of hav. the phylogenetic relationships among hepatovirus that infect small mammals only partially reflects those among their hosts, suggesting multiple, non-recent cross-species and cross-order host switches during hepatovirus evolution [24] . this observation is supported by recombination events observed in hepatoviruses that have been identified in genetically and geographically distant hosts [29] . these cross-species transmission events also involved the opossum hepatovirus, which most likely originated from an ancestral host switch from rodents into marsupials [27] . conversely, hepatovirus phylogenies suggest no host switch involving a primate donor. this evidence, the absence of recombination events between havs and non-primate hepatoviruses, as well as the observation that primate hepatoviruses form, regardless of the genomic region considered, a monophyletic group in the topology of hepatovirus phylogenies, support the hypothesis that humans and nhp have acquired hepatoviruses from other animal reservoirs relatively recently [24, 29] . however, if and when this hypothetical host-jump occurred into primates remains to be clarified. phylogenetic and ancestral state reconstruction suggested a likely cricetid rodent origin for primate havs and marsupial hepatoviruses, whereas a laurasiatherian host origin was proposed for all mammalian hepatoviruses [24, 27] (fig. 1) . in this scenario, the evolutionary history of hepatoviruses is evocative of that of hantaviruses, as the origin of mammalian hantaviruses is traced back to bats and insectivores [30] . thus, the supposed origin of hepatoviruses in insectivorous laurasiatherian mammals, as well as the preservation of some structural and functional characteristics similar to present-day insect picorna-like viruses (dicistroviridae) [31] led drexler and colleagues to hypothesize a more ancient evolutionary origin of havs, with an ancestry in a primordial insectborne virus [24] . in conclusion, hav emergence in humans likely represents a relatively recent evolutionary event, probably of zoonotic origin. nonetheless, the ancestor of human hepatoviruses has yet to be identified. the characterization of other hepatoviruses in primates, and mammals in general, will be instrumental to the identification of the hav ancestors and will clarify the evolutionary history of hepatoviruses. hbv was the first human hepatitis virus to be isolated and identified in 1970 [32] . hbv transmission varies depending on the prevalence of infection. in areas with a low prevalence (<2%), the most common mode of transmission is through infected blood or high-risk behaviors (e.g. unprotected sex or injecting drug use). in high-and intermediate-prevalence areas, hbv is commonly spread through perinatal and horizontal (especially among children) routes [33] . hbv belongs to the hepadnaviridae family, which comprises two genera: orthohepadnavirus (mammal-infecting viruses) and avihepadnavirus (bird-infecting viruses) ( fig. 2a) . its genome, a partially double-stranded circular dna of about 3.2 kb (table 1) , is composed of four overlapping frameshifted open reading frames (orfs) [34] . viral replication is carried out by a reverse transcriptase with no proofreading ability and considerable variability exists among strains. thus, at least nine genotypes (a-i) plus a tentative one (j), with a heterogeneous global distribution, have been described to date [35] [36] [37] [38] [39] (fig. 2b) . despite its worldwide diffusion and the accumulation of detailed knowledge on the associated pathologies, the origin and evolutionary history of hbv are still debated [34, 40] . hepadnaviruses were detected in several mammals, including nhps, rodents and bats, birds, fish, and reptiles [41, 42] (fig. 2a) . recently, lauber et al. discovered a family of fish viruses with genomic features similar to those of hbv, dating the origin of the hepdnaviridae family to at least 300 million years ago [43] ; this finding, together with the discovery of endogenous hepadnavirus elements integrated in the genome of birds and reptiles [44] [45] [46] [47] , suggests a long and complex relationship between this viral family and its hosts. concerning hbv, different theories were proposed to explain its origin, but all of them have some sort of limitation. hbv was initially thought to have emerged quite recently in the new world from genotypes f/h infecting amerindians [48, 49] ( fig. 2a and b). however, the discovery of an ancient strain in a 16th century asian mummy, as well as the worldwide diffusion of hepadnaviruses in nhps, questioned this hypothesis [50] (fig. 2a ). an alternative theory suggests that hbv followed the out-of-africa migration of modern humans, which occurred approximately 60,000 years ago [51] [52] [53] . in particular, paraskevis et al. found a good correspondence between the demographic histories of hbv and those of human populations [53] . these authors also showed that the substantial divergence of the f and h genotypes ( fig. 2a) , a major evidence in favor of the new world origin hypothesis [49] , was probably due to positive selection acting on those branches [54] . nonetheless, the extensive application of ancient dna sequencing revealed a more complex scenario. in fact, two european neolithic hbv genomes did not cluster with any extant human strain in the phylogenetic tree, but did cluster with nhp viruses [55] (fig. 2a ). other authors [9] , who sequenced 12 ancient hbv genomes of different ages (800-4500 years old), obtained a similar result, with the ancient strains clustering with known modern genotypes or forming new clades ( fig. 2a) . this implies that some hbv lineages of the past went extinct ( fig. 2a) . moreover, muhlemann and coworkers showed that the geographic distribution of ancient samples does not match the modern genotype distribution [9] . they thus suggested that early evolutionary scenarios can be concealed and overwritten by more recent migratory events [9] . a third hypothesis for the origin of hbv posits that hepadnaviruses co-speciated with their primate hosts in the new world and in the old world. thus, multiple zoonotic transmissions would have originated hbv genotypes found in humans [56] . this scenario is supported by the diffusion of hepadnaviruses in diverse primate species and by the inferred divergence time of the orthohepadnavirus and avihepadnavirus genera, that is very similar to that of their host classes [56] . however, the recent identification of a novel hepadnavirus in capuchin monkeys confirmed that new world monkeys are infected by viruses that are very distantly related to hbv ( fig. 2a) , indicating that they do not represent the direct ancestors of genotypes h and f [41] . instead, evolutionary analyses with human and nhp viral strains placed the origin of hbv ancestors in hominoid old world primates, preceding the formation of the human lineage [41] . in summary, although considerable progress was achieved in recent years, a high level of uncertainty concerning the ultimate origin of hbv still exists. the particular organization of the viral genome (i.e. overlapping reading frames in a short genome) limits the variability of most of nucleotide positions (i.e. to avoid the introduction of nonsynonymous mutations) and results in a relatively slow mutation rate. this characteristic, along with the action of natural selection on particular genotypes [54] , as well as the adaptation to different human populations [34] , contributes to hbv variability and complicates inferences about its origin. finally, different studies [9, 50, 57] have shown that, as for other viruses (see section 2), hbv substitution rates are affected by viral sampling time frames. indeed, the evolutionary rates generated using information from ancient hbv genomes were shown to fit well with the tdrp regression line calculated for baltimore group vi and vii viruses (i.e., reverse-transcribing viruses) [11] . crucially, these results indicate that, whereas tip calibration approaches have demonstrated to be useful in the reconstruction of recent epidemiological events [58] [59] [60] , limiting analysis to extant strains for the reconstruction of ancient hbv evolution can be misleading [9] and that approaches that correct for the tdrp should be envisaged. hcv is an enveloped virus belonging to the flaviviridae family (genus hepacivirus). in analogy to other members of the family, hcv has a 9.6 kb positive-stranded linear rna genome. the virus encodes a single polyprotein that is processed by cellular and viral proteases to yield at least 10 mature products. hcv was identified in 1989 by houghton and colleagues as a cause of non-a and non-b hepatitis [61] . if left untreated, hcv can persist lifelong in humans, often resulting in cirrhosis and hepatocellular carcinoma. presently, the hcv worldwide seroprevalence is estimated to be $1% [1] , with about 71 million persons living with chronic infection [1] . the hcv epidemic apparently started recently, in the 1930s-1940s, as a consequence of practices that determined parenteral or percutaneous exposure (e.g., blood transfusion, vaccination campaigns, and intravenous drug injection) [62] [63] [64] . for instance, one of the most affected countries is egypt, where the virus was most likely disseminated through nationwide vaccination programs or contaminated blood-derived products [65] . in fact, sexual or vertical transmission of hcv are relatively rare, and the overwhelming majority of infections occur via the parenteral/percutaneous route. thus, due to historical reasons and to the transmission pattern, a small number of so-called ''epidemic" hcv subtypes (1a, 1b, 3a, and 2a) account for most infections worldwide [64, 66] . hcv is, however, genetically heterogeneous and the epidemic subtypes represent a minor fraction of viral diversity. eight major hcv genotypes (hcv-1 to -8) have been described, and these are further divided into at least 90 subtypes (https://talk.ictvonline. org/ictv_wikis/flaviviridae/w/sg_flavi/56/hcv-classification) (fig. 3a) . several of these genotypes and subtypes were identified and classified only recently [67, 68] , suggesting that a considerable proportion of hcv diversity remains undescribed. moreover, a number of natural inter-genotype recombinants were reported [24, 27] was generated using phyml [136] . hepatovirus host silhouettes are colored according to taxonomic order. the human hav subgenotypes are also reported. (https://talk.ictvonline.org/ictv_wikis/flaviviridae/w/sg_flavi/38/ table-4-recombinant-rf-hcv-genomes). hcv genotypes display antigenic variability and viral genetic diversity is geographically structured: in sub-saharan africa and south-east asia highly divergent subtypes of the same genotype dominate transmissions across contiguous areas (fig. 3a) . these subtypes are referred to as ''endemic" [62, 64] and their presence is consistent with a long-standing association of hcv with populations living in these regions. because parenteral exposure became common only in the relatively recent past, several hypotheses were formulated to account for the maintenance of endemic hcv transmission. some authors proposed that traditional practices such as circumcision, tattooing, piercing or acupuncture facilitated and maintained hcv infection among human populations [64, 69] . others indicated that sexually transmitted infections (stis) that disrupt mucosal integrity are responsible for increased sexual hcv transmission [70] . this was indeed shown to be the case in modern high-risk populations [71] and stis have probably been common throughout human history [72] . an alternative scenario is that the bite of arthropods, especially those taking large blood [138] ). graphical representation of genotype (gt) diversity. the number of distinct recognized subtypes is represented on the y axis. circle size is proportional to the average pairwise distance between subtypes. genotype 7 is marked with an asterisk as only two subtypes are known. (b) phylogenetic tree of the rdrp domain of known hepaciviruses. the tree was obtained using raxml with 1000 bootstrap replicates [139] . nodes with support equal to or higher than 0.7 are marked with a black dot. (c) hypothetical viral phylogenies that illustrate the effect of viral lineage extinction on the evolutionary inference about the origin of hcv and ehv/chv. in the left panel, a horse-to human transmission event is hypothesized, with the following extinction of the transmitted ehv lineage. in the right panel, a reverse zoonosis introduces a hcv-like virus in horse populations; the following extinction of several ehv lineages accounts for the low genetic diversity of extant strains. meals, can mechanically transmit hcv, possibly from other animal reservoirs such as horses [73, 74] . hcv infection is in fact restricted to our species but, thanks to extensive field work, a number of hepaciviruses have been described in domestic and wild mammals, as well as in reptiles and fish [28, [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] (fig. 3b ). at present, the largest diversity of hepacivirus species seems to be hosted by rodents and bats (fig. 3b) . instead, as previously noted [75] , the lowest genetic diversity is observed for hepaciviruses that infect cattle and horses (fig. 3b) , suggesting that husbandry practices may have resulted in the artificial selection of specific viral strains or facilitated recent viral transmission from some other animal source (e.g., commensal rodents). non-human primates also host hepaciviruses, but these are highly divergent from hcv. overall, the phylogenetic relationships among hepaciviruses poorly mirror those among their hosts (fig. 3b ), suggesting several crossspecies and cross-order host switches during viral evolution. up to now, the closest relatives of hcv were identified in horses/donkeys (equine hepacivirus, ehv) and dogs (canine hepacivirus, chv) (fig. 3b) . because chv is less genetically diverse than ehv, the canine virus possibility originated as a recent cross-species transmission from horses [85] , hinting at the ability of hepacivirus to shift among genetically distant hosts. despite these advances, the events that led to the origin of hcv are still unknown. taking as a fact the relatedness of the human virus to ehv/chv, possible scenarios include that: i) hcv originated from a cross-species transmission of ehv, ii) that ehv was transmitted to horses by humans infected with hcv, which leaves the question on hcv origin open; iii) that hcv and ehv originated from the cross-species transmissions of the same (or similar) virus, with subsequent host adaptation and divergence. if this were the case, multiple crossspecies transmission events may have originated distinct hcv genotypes [85] . teasing apart these possibilities clearly requires understanding of the timing and circumstances of hcv (and ehv) evolution. up to date, no archaeological sample carrying traces of hcv or ehv has been described and the oldest hcv sequence dates to 1953 [86] (1979 for ehv [82] ). thus, molecular dating efforts have relied on extant sequences, with the difficulties associated with the tdrp. studies that did not account for the tdrp provided estimates of the time to the most recent common ancestor (tmrca) of hcv genotypes in a range between $200 and 1000 years ago [63, 64, 76, 87, 88] ; the origin of the horse virus was dated around 1800 ce [85] . a study that separately accounted for the rate of synonymous and nonsynonymous substitutions estimated hcv to have originated at least 2000 years ago [89] . recently, a method based on an a selection-informed model was used to estimate the divergence time of hcv genotypes and the origin of extant ehv/chv strains [70] . this approach, provided estimates of $3000 years ago for the tmrca of extant hcv genotypes (with a low-bound estimate of $5000 years before present) and of $800 years ago for ehv/chv [70] . if these dates are taken to provide at least an indication of the real evolutionary scenario, the possibility that hcv was transmitted to humans by horses infected with ehv can be excluded, an observation in line with the low diversity of ehv/chv [82] . the origin of ehv/chv as reverse zoonosis (i.e., the transmission of a human virus to animals) seems also unlikely, as in this case horse viruses should cluster within hcv diversity, unless the hcv lineage that originated ehv went extinct in the last 800 years. indeed, as the hbv story exemplifies, viral lineage extinction can occur and was previously documented for other human pathogens such as parvovirus b19 [90] , and variola virus [91] . as anticipated above, breeding practices may facilitate this process in the case of animal viruses. we know, for instance, that a minimum of two horse lineages went extinct during the domestication process and that horse genetic diversity has largely been shaped by events that occurred in the last few centuries [92] . it is thus possible that human-mediated selection on the host also resulted in the artificial selection of viral lineages. this would explain the relatively recent origin of extant ehv strains and their low diversity. if viral lineage extinction did occur, the time frames of ehv and hcv evolution would be underestimated and the scenario of hcv originating as a zoonosis from horses (or ehv as a reverse zoonosis) may still hold (fig. 3c) . of course, the alternative possibility that ehv and hcv were transmitted independently to their present-day hosts by a third unknown reservoir is also in line with data on extant diversity and, if the transmission to horses occurred recently, does not require to postulate viral lineage extinction. thus, a number of open questions remain on the origin of hcv. hopefully, technological advances that allow sequencing of trace genetic material from ancient samples will provide information on viral strains hosted by humans and horses back in the past. at the same time, the extensive application of metagenomic approaches to different animal hosts across diverse geographic regions will expand our knowledge on hepacivirus diversity and eventually uncover the direct ancestor of hcv (if it still exists). indeed, the possibility that the different hcv genotypes derived from independent cross-species transmission events [85] would imply that viruses related to hcv are relatively common in the wild, thus giving good chances to be recovered in large field surveys. hdv is a defective virus incapable of autonomous propagation [93] . its genome, a self complementary circular rna of $1700 nucleotides, encodes a single protein (the hdv-encoded delta antigen) ( table 1) . hdv requires hbv surface proteins, that are complexed with the delta antigen, to form transmissible virions [94] . thus, hdv is usually considered a satellite of hbv, although recent data have shown that other enveloped viruses can promote hdv propagation, at least in vitro (e.g. hcv, dengue virus, vesicular stomatitis virus) [95] . genetic heterogeneity among strains is quite high for hdv, which is thus classified in eight different genotypes (from 1 to 8), although a three major genogroup classification was recently proposed (group 1 for previous genotype 1, group 2 for genotypes 2 and genotypes from 4 to 8, and group 3 for previous genotype 3) [96] hdv genetic diversity is highest in africa, suggesting that the defective virus emerged in and spread from this continent [40, 97] . the evolutionary origin of hdv is nonetheless unknown. hdv-like circular rnas were only recently described in birds, reptiles, amphibians, fish and insects [98] [99] [100] . however, these hdvlike elements were not found to be associated with hepadnavirus infection, reinforcing the idea the hdv is not necessarily only transmitted in conjunction with hbv-related viruses, at least in these animals [100] . current evidence suggests that hdv evolved from the human cellular transcriptome [101] . the first indications that a virus was responsible for waterborne, epidemic hepatitis came from studies of asian outbreaks in the 1950-70s and, in analogy to hcv, the agent was referred to as ''epidemic non-a, non-b hepatitis" [102] [103] [104] . hepatitis e virus was eventually isolated and sequenced in the early 1990s [105, 106] . since then, a number of hev strains responsible for human infection were identified. hev is a positive-strand rna virus belonging to the hepeviridae family (table 1) . in common with all other members of this viral family, the hev genome comprises three partially overlapping open reading frames (orfs): orf1 and orf2 encode a non-structural [136] . the piscihepevirus branch is in red, orthohepevirus branches are in blue. the enlargement shows phylogenetic relationships for viruses belonging to the orthohepevirus a species, with representative hosts. (b) geographic distribution of anthropotropic (hev-1 and hev-2) and enzootic (hev-3-hev-4) hev strains. genotypes were assigned to countries irrespective of their prevalence. thus, even if a single case was reported in a given country, the genotype was recorded as present. cases that could be clearly ascribed to migration/travels were excluded. data derive from forni et al. [121] , with updates from [110, [140] [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] . (c) time-scaled phylogenetic tree of a non-recombing orf1 region [121] . branch lengths represent evolutionary time. the time-frames of historical events mentioned in the text are reported. the rabbit and camel silhouettes mark the split of the rabbit-infecting and camel/dromedary-infecting genotypes. the pig silhouette marks the human-restricted/enzootic genotype split. polyprotein and the viral capsid, respectively, whereas orf3 codes for a small phosphoprotein. a fourth orf (orf4), overlapping the helicase domain in orf1, was recently described but seems to be specific for some hev genotypes (hev genotype 1, hev-1) [107] . members of the hepeviridae family are currently classified into two genera, orthohepevirus and piscihepevirus [108] (https://talk. ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rnaviruses/w/hepeviridae). the piscihepevirus genus includes only one species (piscihepevirus a) with one member (cutthroat trout virus), whereas the orthohepevirus genus is divided into four species of viruses infecting mammals and birds (orthohepevirus a-d) [108] (fig. 4a) . this classification is, however, likely to change in the near future following the identification of novel hepeviruses in fish other than trout and in amphibians [28] (fig. 4a) . human-infecting hev strains are genetically heterogeneous and display distinct epidemiologic patterns, but all belong the orthohepevirus a species. orthohepeviruses a account for a minority of the overall diversity of hepeviruses that infect vertebrates and their closest relative is a presently unclassified virus detected in a swedish moose (fig. 4a ) [109] . field surveys revealed a high prevalence of hev in moose populations from sweden and other baltic regions [110, 111] . in general, ungulates represent major orthohepeviruses a reservoirs. at present, eight orthohepevirus a genotypes are recognized (hev-1 to -8) (fig. 4a ). hev-1 and hev-2 infect only humans and cause waterborne outbreaks mainly in tropical and subtropical regions (fig. 4b) . conversely, genotypes 3 and 4 account for the majority of hepatitis e human cases in industrialized countries. hev-3 and hev-4 also infect several other domestic (mainly pigs) and wild (e.g., ungulates and small carnivores) animals, their transmission to humans being usually zoonotic [2] (fig. 4a) . phylogenetic analyses showed that hev-3 and hev-4 sequences derived from human cases are interspersed within those isolated from swine, indicating that pig-infecting hev-3 and hev-4 can easily cross the species barrier and infect humans [112] . notably, though, evolutionary rates are higher for genotypes 3 and 4 than for the human-specific genotypes, suggesting cyclical adaptation to different mammalian hosts [113] . a distinct hev-3 clade, mainly detected in rabbits (hev-3ra), can also cause human hepatitis e [114] [115] [116] [117] (fig. 4a) . the remaining genotypes hev-5/hev-6 and hev-7/hev-8 have been detected in boars and camels, respectively [2] (fig. 4a) . however, they are also thought to have zoonotic potential, as hev-5 and hev-7 can infect cynomolgus monkeys [118, 119] and hev-7 was detected in a patient who consumed camel meat and milk [120] . thus, viruses belonging to all hev genotypes seem to be transmissible to humans. conversely, experimental infection with hev-1 and hev-2 indicated that these viruses have a host range restricted to primates [2] . hev genotypes are therefore usually referred to as enzootic (hev-3 and ã�4) or human-restricted/anthropotropic (hev-1 and -2). although several human hepatitis e cases have a zoonotic origin and orthohepeviruses a are found in diverse mammals, recent data indicated that one or more reverse zoonoses led to the emergence and radiation of hev genotypes [121] . in fact, character state reconstruction on a large phylogeny revealed that humans were the most likely hosts of the ancestor of extant orthohepeviruses a [121] . this notion is in line with the observation that most, if not all, hev genotypes can infect our species, whereas other animals are differentially susceptible to distinct hev genotypes. moreover, increasing evidence suggests that reverse zoonotic events (also known as zooanthroponoses) are all but rare, and examples include other rna viruses such as rotaviruses, enteroviruses, and human influenza viruses [122] [123] [124] [125] . for both swine influenza a viruses and swine vesicular disease virus onward transmission in pigs is well documented [123, 125] and is facilitated by intensive husbandry practices. molecular dating using a selection-informed method inferred that the ancestor of extant orthohepeviruses a existed $6800 to $3200 years ago, most likely in east asia [121] . these inferences well correlate with historical circumstances that may have favored hev emergence and host range expansion. in this period, sedentary agriculture promoted the appearance of large human settlements in several asian regions and pig husbandry practices started to intensify in east asia [126] [127] [128] [129] [130] (fig. 4c) . crowded living conditions and poor sanitation possibly favored the emergence and spread of the waterborne, human-specific hev strains. the close contact between humans and pigs most likely promoted hev zooanthroponotic transmission and emergence of the enzootic strains (fig. 4c) . additional reverse zoonotic transmissions may have also originated the camel-infecting and rabbit-infecting strains. in fact, the estimated timing of hev-7/8 emergence (4055 bce to 1192 bce) [121] encompasses the time of domestication of bactrian and dromedary camels [131] [132] [133] (fig. 4c) . as for hev-3ra, it was estimated to have diverged from hev-3 around 600 ce, in europe [121] . this time frame corresponds to the middle ages, when historical evidence indicates that rabbits were kept in warrens and bred for meat [134] (fig. 4c ). of course, these estimates do not necessarily imply that camels and rabbits acquired hev from humans, as the domestication process may have exposed these animals to various viral sources, including other domestic (e.g., pigs) and peridomestic mammals. these scenarios provide a credible framework for orthohepevirus a origin, as well as for the diversification of hev genotypes, and selection-informed methods should at least partially correct for the tdrp, as they explicitly model purifying selection [14] [15] [16] . nonetheless, the above-mentioned data on hbv [9] suggest caution in the inference of time and location of ancestral events based on extant viral diversity. also, pig infection with hev-3 and hev-4 is generally asymptomatic [135] , possibly indicating a long-standing host-virus association that might even predate swine husbandry development. it should also be noted that the ultimate origin of orthohepevirus a remains unknown. humans may have acquired hev through cross-species transmission from other animals. however, known orthohepeviruses that infect mammals and birds are distantly related to orthohepevirus a, indicating that none of them represents the source of human-infecting hev. likewise, the origin and evolutionary relationship between the moose virus and human-infecting orthohepeviruses remain unclear. by allowing the large-scale identification of novel viral species, as well as the sequencing of viral genomes from archaeological samples, technological advances have largely expanded our knowledge on the evolution and origin of human hepatitis viruses. these insights have been paralleled by the development of computational tools and theoretical frameworks to analyze and mine viral sequence data (e.g., the above-mentioned recognition of the tdrp and the development of approaches to correct for it). the overall picture emerging from these studies clearly indicates that, with the possible exception of hdv, viruses related to human hepatitis viruses infect several other mammalian and non mammalian vertebrates. the specific events that originated these human pathogens remain, however, largely unknown. for hcv and hev, the evolutionary history of the human viruses is likely intertwined with that of related viruses that infect domestic animals. conversely, in the case of hav and hbv, the closest relatives of the human viruses are found in nhps. in general, these observations indicate that a deeper understanding of the evolutionary dynamics of human viruses has a relevance not only to improve our ability to treat and prevent present infections, but also to gain insight into the ecological contexts that may fuel the emergence of novel human pathogens, with particular reference to zoonotic ones. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. world health organization (who) zoonotic hepatitis e virus: classification, animal reservoirs and transmission routes hepatitis a: old and new type a viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention bayesian molecular dating: opening up the black box inferences from tip-calibrated phylogenies: a review and a practical guide bayesian estimates of the evolutionary rate and age of hepatitis b virus enigmatic origin of hepatitis b virus: an ancient travelling companion or a recent encounter ancient hepatitis b viruses from the bronze age to the medieval period time-dependent rate phenomenon in viruses prisoners of war -host adaptation and its constraints on virus evolution time-dependent estimates of molecular evolutionary rates: evidence and causes bayesian inference of evolutionary histories under time-dependent substitution rates a case for the ancient origin of coronaviruses purifying selection can obscure the ancient age of viral lineages evolutionary origins of human herpes simplex viruses 1 and 2 hepatitis a virus genome organization and replication strategy a pathogenic picornavirus acquires an envelope by hijacking cellular membranes fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis a virus capsid hepatitis a: detection by immune electron microscopy of a viruslike antigen associated with acute illness hepatitis ain greenland: importance of specific antibody testing in epidemiologic surveillance prevalence of antibody to hepatitis a and hepatitis b viruses in selected populations of the south pacific genetic variability and molecular evolution of hepatitis a virus evolutionary origins of hepatitis a virus in small mammals discovery of a novel hepatovirus (phopivirus of seals) related to human hepatitis a virus a novel hepatovirus identified in wild woodchuck marmota himalayana a novel marsupial hepatitis a virus corroborates complex evolutionary patterns shaping the genus hepatovirus the evolutionary history of vertebrate rna viruses evolutionary origins of enteric hepatitis viruses phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents hepatitis a virus and the origins of picornaviruses virus-like particles in serum of patients with australia-antigen-associated hepatitis hepatitis b virus epidemiology hepatitis b virus: the challenge of an ancient virus with multiple faces and a remarkable replication strategy genotypes and genetic variability of hepatitis b virus heterogeneous recombination among hepatitis b virus genotypes a genetic variant of hepatitis b virus divergent from known human and ape genotypes isolated from a japanese patient and provisionally assigned to new genotype possible origins and evolution of the hepatitis b virus (hbv) the global hepatitis b virus genotype distribution approximated from available genotyping data origins and evolution of hepatitis b virus and hepatitis d virus a novel hepatitis b virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses bat hepadnaviruses and the origins of primate hepatitis b viruses deciphering the origin and evolution of hepatitis b viruses by means of a family of nonenveloped fish viruses endogenous hepadnaviruses, bornaviruses and circoviruses in snakes early mesozoic coexistence of amniotes and hepadnaviridae endogenous hepadnaviruses in the genome of the budgerigar (melopsittacus undulatus) and the evolution of avian hepadnaviruses the first full-length endogenous hepadnaviruses: identification and analysis hepatitis b virus has a recent new world evolutionary origin reconstructing the origins of human hepatitis viruses tracing hepatitis b virus to the 16th century in a korean mummy complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis b virus, four of which represent two new genotypes subtypes, genotypes and molecular epidemiology of the hepatitis b virus as reflected by sequence variability of the s-gene dating the origin and dispersal of hepatitis b virus infection in humans and primates dating the origin of hepatitis b virus reveals higher substitution rate and adaptation on the branch leading to f/h genotypes neolithic and medieval virus genomes reveal complex evolution of hepatitis detection of hepatitis b virus infection in wild-born chimpanzees phylogenetic relationships with human and other primate genotypes the paradox of hbv evolution as revealed from a 16th century mummy epidemic history and evolutionary dynamics of hepatitis b virus infection in two remote communities in rural nigeria molecular epidemiology of hepatitis b virus in misiones molecular diversity in irregular or refugee immigrant patients with hbvgenotype-e infection living in the metropolitan area of naples isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome phylogeny and molecular evolution of the hepatitis c virus the epidemic behavior of the hepatitis c virus the origin of hepatitis c virus genetic epidemiology of hepatitis c virus throughout egypt global distribution and prevalence of hepatitis c virus genotypes identification of a novel hepatitis c virus genotype from punjab, india: expanding classification of hepatitis c virus into 8 genotypes identification of 19 novel hepatitis c virus subtypes-further expanding hcv classification global epidemiology of hepatitis c virus infection evolutionary analysis provides insight into the origin and adaptation of hcv sexually acquired hepatitis c virus infection: a review infectious diseased and human population history investigating the endemic transmission of the hepatitis c virus exploring the possibility of arthropod transmission of hcv highly divergent hepaciviruses from african cattle characterization of a canine homolog of hepatitis c virus identification of rodent homologs of hepatitis c virus and pegiviruses surveying the global virome: identification and characterization of hcv-related animal hepaciviruses evidence for novel hepaciviruses in rodents bats are a major natural reservoir for hepaciviruses and pegiviruses serology-enabled discovery of genetically diverse hepaciviruses in a new host differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys identification of a novel hepacivirus in domestic cattle from germany detection of non-primate hepaciviruses in uk dogs hepacivirus cross-species transmission and the origins of the hepatitis c virus evolutionary analysis of hepatitis c virus gene sequences from 1953 origin of hepatitis c virus genotype 3 in africa as estimated through an evolutionary analysis of the fulllength genomes of nine subtypes, including the newly sequenced 3d and 3e full-length genomes of 16 hepatitis c virus genotype 1 isolates representing subtypes 1c, 1d, 1e, 1g, 1h, 1i, 1j and 1k, and two new subtypes 1m and 1n, and four unclassified variants reveal ancestral relationships among subtypes the origin of hepatitis c virus genotypes ancient human parvovirus b19 in eurasia reveals its long-term association with humans 17(th) century variola virus reveals the recent history of smallpox tracking five millennia of horse management with extensive ancient genome time series transmission of the hepatitis b virus-associated delta antigen to chimpanzees hepatitis delta virus enveloped viruses distinct from hbv induce dissemination of hepatitis d virus in vivo a comprehensive bioinformatic analysis of hepatitis d virus full-length genomes genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains a divergent hepatitis d-like agent in birds identification of a novel deltavirus in boa constrictors novel hepatitis d-like agents in vertebrates and invertebrates hepatitis delta virus: a fascinating and neglected pathogen study of an epidemic of non-a, non-b hepatitis. possibility of another human hepatitis virus distinct from post-transfusion non-a, non-b type infectious hepatitis in delhi (1955-56): a critical studyepidemiology. 1957 epidemic and endemic hepatitis in india: evidence for a non-a, non-b hepatitis virus aetiology isolation of a cdna from the virus responsible for enterically transmitted non-a, non-b hepatitis hepatitis e virus (hev): molecular cloning and sequencing of the full-length viral genome endoplasmic reticulum stress induced synthesis of a novel viral factor mediates efficient replication of genotype-1 hepatitis e virus international committee on taxonomy of viruses hepeviridae study group novel hepatitis e like virus found in swedish moose prevalence and phylogenetic analysis of hepatitis e virus in pigs, wild boars, roe deer, red deer and moose in lithuania high prevalence of hepatitis e virus in swedish moose -a phylogenetic characterization and comparison of the virus from different regions close similarity between sequences of hepatitis e virus recovered from humans and swine genotypespecific evolution of hepatitis e virus rabbit hepatitis e virus infections in humans rabbit hev in immunosuppressed patients with hepatitis e acquired in switzerland transmission of hepatitis e virus from rabbits to cynomolgus macaques hepatitis e virus strains in rabbits and evidence of a closely related strain in humans genotype 5 hepatitis e virus produced by a reverse genetics system has the potential for zoonotic infection production of infectious dromedary camel hepatitis e virus by a reverse genetic system: potential for zoonotic infection chronic infection with camelid hepatitis e virus in a liver transplant recipient who regularly consumes camel meat and milk origin and dispersal of hepatitis e virus reverse zoonotic disease transmission (zooanthroponosis): a systematic review of seldom-documented human biological threats to animals reverse zoonosis of influenza to swine: new perspectives on the human-animal interface a systematic review of evidence that enteroviruses may be zoonotic evolutionary histories of coxsackievirus b5 and swine vesicular disease virus reconstructed by phylodynamic and sequence variation analyses bar-yosef of the neolithic demographic transition and its consequences rapid, global demographic expansions after the origins of agriculture changes in regional settlement patterns and the development of complex societies in southeastern shandong social complexification and pig (sus scrofa) husbandry in ancient china: a combined geometric morphometric and isotopic approach agricultural origins and the isotopic identity of domestication in northern china the prehistory of the silk road the history of the camel bone dating project the appearance of the domestic camel in southeast arabia rabbits and the specious origins of domestication recent knowledge on hepatitis e virus in suidae reservoirs and transmission routes to human estimating maximum likelihood phylogenies with phyml distribution of hbv genotypes in latin america global prevalence and genotype distribution of hepatitis c virus infection in 2015: a modelling study raxml version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies complete genome sequence of a hepatitis e virus genotype 1e strain from an outbreak in nigeria hepatitis e in italy: 5 years of national epidemiological, virological and environmental surveillance hepatitis e virus genotypes and subgenotypes causing acute hepatitis quantification and genetic diversity of hepatitis e virus in wild boar (sus scrofa) hunted for domestic consumption in central italy dakovic rode o. genetic diversity of hepatitis e virus (hev) strains derived from humans, swine and wild boars in croatia from a case of incidental infection of hepatitis e virus (hev) genotype 1 in a domestic pig hepatitis e virus genotype 1 and hepatitis a virus dual infection in pediatric patients with a low socioeconomic status from mexico epidemiology and genotype 3 subtype dynamics of hepatitis e virus in belgium first detection of hepatitis e virus genotype 3 as a common infectious agent in patients with chronic liver damage in mexico a new hepatitis e virus genotype 2 strain identified from an outbreak in nigeria hepatitis e virus seroprevalence and correlates of anti-hev igg antibodies in the rakai district hepatitis e virus infection in different groups of estonian patients and people who inject drugs the worldwide burden of viral hepatitis in terms of death and disability is enormous. in 2015, viral hepatitis caused 1.34 million deaths, the majority of which imputable to the effects of chronic hbv (hepatitis b virus) and hcv (hepatitis c virus) infection [1] . an estimated 5% of hbv-infected persons are also co-infected with hdv (hepatitis delta virus), which worsens the clinical outcome compared to hbv monoinfection [1] . less than 5% of overall hepatitis mortality is caused by hav (hepatitis a virus) and hev (hepatitis e virus), that are usually associated with acute, self-limiting disease [1] . however, the case fatality rate of hev is particularly high in specific groups, notably pregnant women and elderly individuals [2] . although rare, infection with hav can also cause acute liver failure and death, and the risk increases with age [3] . despite the existence of a safe and effective vaccine, hav remains a common cause of acute viral hepatitis in many regions of the world [4] . key: cord-001247-pxzbirqd authors: sun, lu; zhang, yu; zhao, bao; deng, mengmeng; liu, jun; li, xin; hou, junwei; gui, mingming; zhang, shuijun; li, xiaodong; gao, george f.; meng, songdong title: a new unconventional hla-a2-restricted epitope from hbv core protein elicits antiviral cytotoxic t lymphocytes date: 2014-03-22 journal: protein cell doi: 10.1007/s13238-014-0041-4 sha: doc_id: 1247 cord_uid: pxzbirqd cytotoxic t cells (ctls) play a key role in the control of hepatitis b virus (hbv) infection and viral clearance. however, most of identified ctl epitopes are derived from hbv of genotypes a and d, and few have been defined in virus of genotypes b and c which are more prevalent in asia. as hbv core protein (hbc) is the most conservative and immunogenic component, in this study we used an overlapping 9-mer peptide pool covering hbc to screen and identify specific ctl epitopes. an unconventional hla-a2-restricted epitope hbc141–149 was discovered and structurally characterized by crystallization analysis. the immunogenicity and anti-hbv activity were further determined in hbv and hla-a2 transgenic mice. finally, we show that mutations in hbc141–149 epitope are associated with viral parameters and disease progression in hbv infected patients. our data therefore provide insights into the structure characteristics of this unconventional epitope binding to mhc-i molecules, as well as epitope specific ctl activity that orchestrate t cell response and immune evasion in hbv infected patients. around 350 million people worldwide are chronically infected with hepatitis b virus (hbv). chronic hbv infection is a major cause of cirrhosis, liver failure, and hepatocellular carcinoma (hcc) (lavanchy, 2005; neuveut et al., 2010) . hbv-specific cd8 + t lymphocytes (ctl)-mediated immune response is multi-specific, polyclonal, and vigorous during acute hepatitis b (ahb), which plays a vital role in viral control and viral clearance, as well as disease pathogenesis (yukihiro, 2012; westover and hughes, 2007; tan et al., 2008) . whereas hbv-specific ctl response is minimal or undetectable in chronic hepatitis b (chb) with viral persistence and immune tolerance, indicating the key role of hbvspecific t-cell response in determination of disease progression and outcome (bertoletti and gehring, 2006; . the hbv genome of ∼3.2 kb in length efficiently encodes several overlapping viral proteins, including the polymerase, core, hbe, envelope (pre-s1, s2, s), and x proteins. analysis of ctls specific for viral epitopes within core (sendi et al., 2009; liu et al., 2012; ) , envelope (liu et al., 2008) , polymerase (rehermann et al., 1995) , and x (hwang et al., 2002) proteins showed that the highly conserved hbv core protein (hbc) elicits the strongest ctl responses than other viral proteins, suggesting that hbc-specific t cell response may play a leading role in viral control and clearance. to identify immune-dominant hbv-specific ctl epitopes, especially epitopes from hbc protein, is therefore necessary for monitoring t cell responses during disease progression, as well as for developing epitope-based therapeutic vaccines against chb (inchauspe and michel, 2007; gordon et al., 2013; liu et al., 2013a, b) . so far 60 hbv-specific human leukocyte antigen (hla) class i restricted and 32 hbv-specific hla class ii restricted epitopes have been identified in all 8 hbv genotypes (desmond et al., 2008; liu et al., 2008; guo et al., 2011; chen et al., 2013; tan et al., 2013) . however, most of these identified epitopes are derived from hbv of genotypes a and d, few hla class i restricted epitopes have been defined in virus of genotypes b and c, which are more prevalent in asia. meanwhile, currently the mostly used methods to predict and identify t cell epitopes are either by computer algorithms based on the mode of peptide binding to mhc molecules, or measurement of t cell responses of pbmcs simulated with panels of overlapping peptides (liu et al., 2011) . these epitope identification strategies may ignore unconventional t cell epitopes as computational analysis is largely based on the characteristic of anchor residues. in this study, an overlapping 9-mer peptide pool was used to screen and identify hbv genotypes b-and c-derived t cell epitopes. a new unconventional cd8 + t cell epitope hbc141-149 derived from viral core protein, which shares partial sequence identity with previously reported hbc141-151 (bertoni et al., 1997) and hbc139-148 (lee et al., 1997) , was discovered. its immunological function and clinical relevance were further assessed. identification of a new hla-a*0201-restricted epitope from hbv core protein hbv core protein is the most conservative and immunogenic component of hbv proteins. we used an overlapping 9-mer peptide pool covering the whole length of core protein (aa 1-150) and its variants (totally 191 peptides) for t2 binding assay to screen genotype b-and c virus-derived hbcag-specific t cell epitopes , as shown in fig. 1a . several peptides were found to have high affinity for binding to hla-a*0201 molecules, as evidenced by the fi (0.72 for hbc183-191, 2.41 for hbc141-149, 2.64 for hbc60-68 (v60), 2.47 for hbc18-27, and 0.04 for hbc82-90), as shown in fig. 1b . hbc141-149 spanning from hbc aa 141 to 149 was chosen as the focus of this study due to its high binding affinity among the top hits from screening. to determine the minimal sequence length of the epitope, panels of n-or c-terminally truncated or extended peptides of hbc141-149 were synthesized. the fis of truncated peptide hbc141-148 (stlpettv) and hbc142-149 (tlpettvv) were only 0.124 and 0.151, respectively (fig. 1c) . the fis of decapeptides hbc140-149 (lstlpettvv) and hbc141-150 (stlpettvvr) were only 0.033 and 0.005, respectively (fig. 1d ). all hbc141-149 truncated and extended peptides displayed little binding to hla-a*0201, which indicates that the 9-mer peptide hbc141-149 is the optimized epitope in length. in addition to t2 cell binding assay, the capability of hbc141-149 to bind to hla-a*0201 was observed in the refolding assay (fig. 1e ). the structure of hla-a*0201/hbc141-149 complex next, the complex of hla-a2 and hbc141-149 was prepared for crystallization to characterize the binding features of hbc141-149. the crystal structure of the hla complex was determined to 2.3 å resolution (table 1 ). the structure of hla-a*0201/hbc141-149 shows that hbc141-149 possesses a typical conformation of an hla-a2-restricted 9-mer epitope ( fig. 2a and 2b ). the unambiguous electron densities of hbc141-149 clearly show that position 2 (t2) and position 9 (v9) are buried in pockets b and f, respectively (fig. 2c ). compared to the typical hla-a2-restricted epitopes which have an anchor residue leu or met at position 2, hbc141-149 has thr at position 2. the side chain oh of thr does not disrupt its inserting into the hydrophobic pocket b properly (fig. 2d) . instead, the side chain oh of thr can form a strong hydrogen bond interaction with h atom of glu on the α1 domain of the heavy chain, which helps hbc141-149 binding to the hla-a2 heavy chain and stabilizes the entire complex. the side chains of amino acids p4, e5, and v8 protrude out from the hla-a2 surface and may be involved in t cell receptor (tcr) attachment and recognition. to the best of our knowledge, this is the first structure showing the binding features of hla-a2 to hbc141-149 with an unconventional p2 anchor thr. hbc141-149 peptide generates specific ctl response in hla-a2.1/kb transgenic mice then, the immunogenicity of hbc141-149 epitope was determined in vivo. female hla-a2.1/kb transgenic mice were immunized with hbc dna-prime/hbc141-149 peptide boost regimen three times, using heat shock protein gp96 as adjuvant (li et al., 2011) . hbc141-149-specific ctl was detected by elispot assay 1 week after the last immunization. as can be seen in fig. 3a , similar to the hbc18-27 peptide-immunized mice (positive control), a strong ctl response was observed in splenocytes from hbc141-149 peptide-immunized mice (sfc, 145.4 ± 58.6) . no peptidespecific cd8 + t cell response was detected from hbc82-90 peptide-immunized mice (negative control). similar results were observed in the killing assay using hbv plasmidtransfected 293t cells (fig. 3b ) or hbc141-149 peptidepulsed t2 cells (fig. 3c ) as target cells. to further determine the epitope-specific ctls, fresh pbmcs from hla-a2 + ahb patients were stimulated with hbc141-149 peptide and detected by ex vivo ifn-γ elispot assays. as shown in fig. 3d , a much higher peptide-specific ctl response was observed in pbmcs stimulated with hbc141-149 or the immunodominant peptide hbc18-27 as the positive control than negative control peptide hbc82-90 (119.68 ± 76.66 for hbc141-149 or 164.58 ± 112.41 for hbc18-27 vs. 20.94 ± 17.57 for hbc82-90, both p < 0.01). the high standard deviations observed in elispot assay may reflect the random between-patient variation. taken together, these results indicate that hbc141-149 is an hla-a2-restricted cd8 + tcell epitope and is naturally processed in patients with hbv infection. hbc141-149 epitope elicits antiviral t cell immunity in hla-a2.1/hbv transgenic mice next, we examined whether hbc141-149 epitope was able to induce anti-hbv t cell response using f1 hybrids of hbv transgenic balb/c mice and hla-a2.1/kb transgenic mice as the experimental model, which are hbv immunotolerant. hla-a2.1/hbv transgenic mice were immunized with an hbc dna prime/ hbc141-149 peptide boost formulation. as shown in fig. 4a , compared to mice immunized with negative control peptide, the number of sfcs increased by around 6-fold in hbc141-149 peptide-immunized mice. similar result was obtained in cytotoxicity assay using hbv plasmid-transfected 293t cells as target cells. we then evaluated hbc141-149 peptide-induced tcell response could lead to inhibition of hbv. immunization with hbc141-149 led to a 35.5% decrease in serum hbsag levels (p < 0.05) (fig. 4c) , and a significant reduction of viral dna levels (p < 0.05) (fig. 4d ) at week 8 compared to the negative control. meanwhile, significant lower levels of serum hbsag and viral dna were observed after immunization (at week 8) than those before immunization (at week 0) in hbc141-149-treated mice but not in control peptidetreated mice. and these results indicate that the epitope-specific ctl response induced by the hbc141-149 could significantly inhibit hbv replication in the transgenic mice. in vitro refolding of hbc141-149 peptide with hla-a*0201 heavy chain and β2m. gel filtration chromatography was used to analyze the refolded complexes on a superdex200 16/60 column. the hla complex with the expected molecular mass of 45 kda eluted at the volume of 15.9 ml. the hla complex (peak 2) was analyzed by sds-page electrophoresis and coomassie blue staining (line 2). finally, to address the clinical relevance of the hbc141-149 epitope in chb, mutations within this epitope were analyzed in 197 chb and 64 aclf patients (table 2 ). in chb, compared to patients infected with wild-type isolates (hbc141-149, stlpettvv), patients infected with hbc141-149 mutants had much higher alt levels (247.7 ± 18.62 vs. 545.2 ± 137.7, p < 0.05) (fig. 5a ) and aspartate aminotransferase (ast) levels (187.6 ± 13.78 vs. 1668 ± 700.5, p < 0.05) (fig. 5b) . notably, compared to chb patients infected with hbc141-149 wild-type viruses, patients with hbc141-149 mutants had much higher (approximately 3.3-fold) hbv dna loads (p < 0.05) (fig. 5c ). there were no statistical differences in sex and age between patients infected with the wild-type and mutant isolates. the prevalence of the hbc141-149 mutations increased with the disease progression in hbv-infected patients (fig. 5d ). to investigate the impact of these sequence variations within hbc141-149 epitope on its immunogenicity, three 9-mer peptides containing main epitope variations of hbcv149i, hbct147a, and hbct147c in chb patients were synthesized, respectively, for hla-a2.1/kb transgenic mice immunization. as seen in fig. 5e , compared to the wild-type hbc141-149 peptide-immunized mice (sfc, 96.7 ± 6.02), ctl responses by elispot assay were significantly decreased in hbcv149i mutant peptide-(sfc, 66.3 ± 5.69) or hbct147a mutant peptide-immunized mice (sfc, 73 ± 7.81). taken together, these results suggest that the hbc141-149 mutations associated with necroinflammation and higher hbv levels, and it may also be associated with poor prognosis, which may be due to viral immune evasion. in this study, we identified a new hla-a2-restricted cd8 + t cell epitope hbc141-149 by screening an overlapping 9-mer peptide pool covering hbv core protein. this unconventional hla-a2 restricted epitope was further determined and structurally characterized by hla-a2 transgenic mouse model and crystallographic analysis. moreover, we demonstrated that the hbc141-149 epitope exhibits antiviral capability in hla-a2.1/hbv transgenic mice. finally, our results show that mutations in hbc141-149 epitope correlate with clinically relevant parameters in chb. our work may therefore provide a comprehensive evaluation of the impact of the newly defined epitope on viral specific t cell response and suggests a possible immune evasion for maintenance of viral persistence in patients with hbv infection. the identification of hbv-specific t cell epitopes is mostly based on the binding mode of the peptides to mhc molecules in silico prediction or assessing t cell responses with panels of overlapping peptides in hbv-infected patients. these methods are effective and rapid to purposefully identify epitopes, however, a considerable number of atypical or unconventional epitopes may be ignored or missed by these methods due to the limitation of anchor residues analysis-based computation (liu et al., 2011) . in this study, an overlapping 9-mer peptide pool was used to screen b and c genotype-derived hbc-specific ctl epitopes, and hbc141-149 was identified as a new 9-mer hla-a2restricted t cell epitope. importantly, similar numbers of hbc141-149-specific and immunodominant epitope hbc18-27-specific cd8 + t cells were observed in ahb patients (fig. 3c) , indicating that hbc141-149 is an immunodominant epitope in hbv-infected patients. however, the newly defined epitope hbc141-149 (stlpettvv) in this study possesses thr at position 2. different t cell epitope prediction programs, including syfpeithi (http://www.syfpeithi. de/scripts/mhcserver.dll/epitope-prediction.htm), net-mhc (http://www.cbs.dtu.dk/services/netmhc/), and bimas (http: //www-bimas.cit.nih.gov) show the binding score of the epitope hbc141-149 to hla-a2 is only 0, 1.465, and 0.414, respectively. as hbc141-149 does not get a high score, it could be omitted in conventional screening. interestingly, as for hbc141-149 (stlpettvv), the side chain oh of thr2 can form a stronger hydrogen bond interaction with glu on α1 domain of the heavy chain, which helps its inserting into lu sun et al. pocket b (fig. 2d ) of hla-a*0201 and stabilize the entire complex. therefore, our data indicated that thr may also act as a dominant role as a p2 anchor for hla-a2-binding peptides, which may be taken into consideration in the future designation of these online prediction programs. cd8 + t cells are the main effector cells responsible for viral clearance as well as disease pathogenesis during hbv infection (thimme et al., 2003; harari et al., 2006; ouyang et al., 2013) . as a consequence of long-term interaction between hbv and infected patients, the virus evolves mutations to reduce epitopes for evading immune detection and clearance, especially escape from t cell recognition (maman et al., 2011; westover and hughes, 2007) . indeed, nonsynonymous mutations in hbv epitope have been found to be associated with disease progression in chb (kim et al., 2011; frelin et al., 2009) . consistent to these studies, we found that mutations in the newly defined epitope are positively related to viral parameters and pathogenesis of liver disease. the effect of mutations within hbc141-149 on viral replication capability, as well as the potential impact of the epitope-specific ctls on the interplay of hbv and chronically infected patients remains to be addressed. in summary, this study indicates that hbc141-149 is an immunodominant hla-a2-restricted ctl epitope with atypical binding characteristics to hla-a2 molecules, has potent anti-hbv immune activity and the clinical significance in patients with hbv infection. we further demonstrated that mutations within this epitope may affect disease progression in chb. given the key role of cd8 + t cells in viral clearance, our work provides valuable insight for the functional implications of hbc141-149 epitope-specific t cell response in hbv infection. understanding the epitope-specific ctl function in the complex regulation networks that orchestrate t cell response, viral persistence and immunoevasion in chb will allow to predict disease progression and develop immunotherapeutic approaches against hbv infection. all the enrolled patients' clinical characteristics are described in table 2 . a total of 29 ahb patients were enrolled for blood collection, which were divided into two groups: hla-a2-positive (n = 19) and hla-a2-negative (n = 10). all patients were negative for hcv, hdv, and hiv-1 infection. 10 ml of blood samples were collected from each patient. all patients were hospitalized in beijing 302 hospital from september 2010 to september 2011. all study participants have written informed consent and the study was approved by the ethics committee of beijing 302 hospital. hbv transgenic balb/c mice were purchased from transgenic engineering lab, infectious disease center (guangzhou, china), which were generated with a viral dna construct, phbv1.3, containing 1.3 copies of the hbv genome. serum hbv s antigen (hbsag) and viral dna, as well as hbc expression in hepatocytes in mice's liver, were tested positive for all transgenic mice. the hla-a2.1/kb transgenic mice (zhang et al., 2007) , and t2 cells labeled with cfse were loaded with 20 μg/ml peptide at 37°c for 1 h as target cells (c). the target cells were then mixed with hbc141-149-stimulated splenocytes at different ratios: 1:1, 1:10, and 1:20, or hbc18-27, hbc82-90-stimulated splenocytes served as positive or negative controls. after 4 h, the mixed samples were stained with pi, and the killing of target cells were analyzed by facs. the data shown are the mean ± sd of five mice. (d) ahb patients (n = 29) were divided into hla-a2-positive (n = 19) and hla-a2-negative (n = 10) groups. pbmcs (2 × 10 5 /well) from these patients were stimulated with the indicated peptides for detection of peptide-specific ctls by elispot assay. hbc82-90 peptide served as negative control for background evaluation in elispot assay. pbmcs from hla-a2-patients were used for specificity evaluation of ctls. paired samples t-tests were used for elispot assay in ahb patients. *p < 0.05 and **p < 0.01 by t-test. data are representative of two independent experiments. by professor huang wl (imcas, beijing, china). the hla-a2.1/kb mice and hbv transgenic mice were crossed to gain the f1 hybrids of hla-a2.1/hbv transgenic mice. all f1 hybrids were screened for serum hbsag by elisa, viral dna by real-time pcr, and hla-a2 by pcr-ssp (protrans, deutschland) before experimental manipulations. the wild-type hbc gene was cloned into pcdna3.1 (invitrogen) and the recombinant plasmid was designated pcdna3.1-hbc. phbv1.3 containing 1.3 copies of the full-length hbv genomic sequence was maintained in the lab. the hbc sequences of genotypes b and c were attained using the protein database from ncbi and a total of 171 hbc sequences of genotype b and 159 hbc sequences of genotype c included. the sequences were compared and served as a basis on peptide synthesis. if the variation rate of a certain amino acid (aa) was more than 10%, a series of peptides associated with this variation would be synthesized. we adopted the overlapping method (8-aa overlap) to synthesize a total of 191 nonapeptides (9-mers) covering hbc1-150 aa . all of these peptides were synthesized at jier biological (shanghai, china), and their purity was determined as >95%. the hbc18-27 (flpsdffpsv) and hbc82-90 (rel-vvsyvn) peptides were used as positive and negative controls, respectively. the 293t fig. 3 . fresh splenocytes (5 × 10 5 ) isolated from immunized mice were stimulated with wild-type or mutant peptide, respectively. peptide-specific ctls were detected by ifn-γ elispot assay. by the manufacturer. cells and supernatants were harvested at 24 h, 48 h, and 72 h after transfection, respectively. t2 cells were used to perform mhc stabilization assays as previously described (zhou et al., 2006) . the binding activity of each peptide was calculated as the fluorescent index (fi), and the fi was determined by: (mean fitc fluorescence with the given peptide − mean fitc fluorescence without peptide) (mean fitc fluorescence without peptide). peptides regarded as epitopes with high affinity should meet the following criteria: fi ≥1. refolding, protein crystallography, and structure determination recombinant proteins of hla-a*0201 heavy chain and β2m were expressed in escherichia coli (garboczi et al., 1992.) and the gradual dilution method was performed during refolding process (liu et al., 2012) . then, superdex 200 10/300 gl gel filtration chromatography followed by resource-q anion-exchanged chromatography (ge healthcare) was used for the concentration and purification of the soluble portion of the refolded complex. the hanging-drop vapor diffusion method was performed at 18°c to crystallize the purified complexes. at a final concentration of 10 mg/ml in 0.1 mol/l bis-tris (ph 6.5) and 25% (w/v) polyethylene glycol 3350, hla-a0201/hbc141-149 crystals were obtained. equipped with an r-axis v||++ image-plate detector, rigaku micromax007 rotating-anode x-ray generator was operated at 40 kv and 20 ma (cu κα; λ = 1.5418 å) to collect crystallographic data at 100 k in house. with protein data bank (pdb) entry 1jf1 as the search model, the structure of hla-a*0201/hbc141-149 was determined by molecular replacement with the program molrep. mice (6-8 weeks old) were immunized i.m. with 50 μg of plasmid pcdna3.1-hbc or pcdna3.1 (control) at week 1 and subcutaneously with 50 μg of hbc141-149 peptide bound to 30 μg of heat shock protein gp96 as adjuvant (li et al., 2011) at weeks 3 and 4, respectively. mice were sacrificed 1 week after the last immunization. splenocytes were isolated as previously described (liu et al., 2009) . each group contained five to seven mice. to detect epitope-specific t cells, enzyme-linked immunosorbent spot (elispot) assay was performed according to the manufacturer's instruction. briefly, ninety-six well pvdf plates (bd-pharmigen, san diego, ca) were precoated overnight at 4°c with the coating ab and blocked for 1 h at 37°c. patient pbmcs (2 × 10 5 ) or murine splenocytes (10 6 ) were added to each well together with 50 μg/ml of peptide and incubated at 37°c for 24-48 h with phytohemagglutinin (pha)-stimulated t cells as positive controls. each test was performed at least in triplicate. the spots were counted and analyzed using an elispot reader (cellular technology ltd, usa). 293t cells were labeled with 2 μmol/l cfse as target cells after transfection with phbv1.3 and seeded into a 96-well plate. then ctls were added at different ratios: 1:1, 10:1, and 20:1. plates were incubated for 4-6 h at 37°c, and cfse positive target cells were stained with propidium iodide (pi) using a vybrant apoptosis assay kit (invitrogen, usa). in addition, t2 cells were loaded with 20 μg/ml hbc141-149 peptide at 37°c for 1 h as target cells, and seeded into a 96-well plate. the cytotoxicity assay was performed as described above. each assay was performed in triplicate. detection of hbsag and hbeag by elisa, and hbv dna by realtime pcr elisas and real-time pcr for detection of serum hbsag, hbeag and viral dna copies were performed as described (fan et al., 2013) . differences between groups were determined using student's t-test. pearson's χ test was used to detect the correlation between variation rate and disease progress in hbv infected patients. clinical statistical analyses were performed with spss version 16.0 software (spss inc., chicago, illinois). p values <0.05 were considered significant. the immune response during hepatitis b virus infection human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic t lymphocyte responses in patients with acute hepatitis an immunodominant hla-a*1101-restricted cd8 + t cell response targeting hepatitis b surface antigen in chronic hepatitis b patients a systematic review of t-cell epitopes in hepatitis b virus: identification, genotypic variation and relevance to antiviral therapeutics increased expression of gp96 by hbx-induced nf-κb activation feedback enhances hepatitis b virus production a mechanism to explain the selection of the hepatitis e antigen-negative mutant during chronic hepatitis b virus infection hla-a2-peptide complexes: refolding and crystallization of molecules expressed in escherichia coli and complexed with single antigenic peptides antiviral therapy for chronic hbv infection and development of hepatocellular carcinoma in a u.s. population identification and functional studies of hla-a0201 restricted ctl epitopes in the x protein of hepatitis b virus functional signatures of protective antiviral t-cell immunity in human virus infections hla-a2 1 restricted peptides from the hbx antigen induce specific ctl responses in vitro and in vivo vaccines and immunotherapies against hepatitis b and hepatitis c viruses number of mutations within ctl-defined epitopes of the hepatitis b virus (hbv) core region is associated with hbv disease progression worldwide epidemiology of hbv infection, disease burden, and vaccine prevention peptide-specific ctl induction in hbv-seropositive pbmc by stimulation with peptides in vitro: novel epitopes identified from chronic carriers hansenula polymorpha expressed heat shock protein gp96 exerts potent t cell activation activity as an adjuvant a mutant hbs antigen (hbsag)183-191 epitope elicits specific cytotoxic t lymphocytes in acute hepatitis b patients treg suppress ctl responses upon immunization with hsp gp96 revival of the identification of cytotoxic t-lymphocyte epitopes for immunological diagnosis identification of hla-a*0201-restricted cd8 + t-cell epitope c64-72 from hepatitis b virus core protein conserved epitopes dominate cross-cd8+ t-cell responses against influenza a h1n1 virus among asian populations cross-allele cytotoxic t lymphocyte responses against 2009 pandemic h1n1 influenza a virus among hla-a24 and hla-a3 supertype-positive individuals immune-induced evolutionary selection focused on a single reading frame in overlapping hepatitis b virus proteins mechanisms of hbv-related hepatocarcinogenesis cd8(low) t-cell subpopulation is increased in patients with chronic hepatitis b virus infection the cytotoxic t lymphocyte response to multiple hepatitis b virus polymerase epitopes during and after acute viral hepatitis ctl escape mutations of core protein are more frequent in strains of hbeag negative patients with low levels of hbv dna host ethnicity and virus genotype shape the hepatitis b virus-specific t-cell repertoire immunoprevalence and immunodominance of hla-cw0801 restricted t cell response targeting the hbv envelope transmembrane region abbreviations aclf, acute on chronic liver failure; ahb, acute hepatitis b; ctl, cytotoxic t lymphocyte; elispot, enzyme-linked immunosorbent spot; fi, fluorescent index; hbc, hbv core protein; hbeag, hbv e antigen; hbsag, hbv s antigen; hbv, hepatitis b virus; sfc, spotforming cells. lu sun, yu zhang, bao zhao, mengmeng deng, jun liu, xin li, junwei hou, mingming gui, shuijun zhang, xiaodong li, george f. gao, and songdong meng declare that they have no conflict of interest. for studies with human subjects in this article: all patients were hospitalized in beijing 302 hospital from september 2010 to september 2011. written informed consent was provided by all study participants. the study was approved by the ethics committee of beijing 302 hospital.for studies with animals in this article: animals received humane care, and the study of mice was in strict accordance with "the this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. key: cord-334150-t6n95laz authors: peng, liang; liu, jing; li, yang-mei; huang, zhan-lian; wang, pei-pei; gu, yu-rong; zheng, yu-bao; gao, zhi-liang title: serum proteomics analysis and comparisons using itraq in the progression of hepatitis b date: 2013-09-18 journal: exp ther med doi: 10.3892/etm.2013.1310 sha: doc_id: 334150 cord_uid: t6n95laz the aim of this study was to analyze the changes in serum protein levels in the progression of hepatitis b using isobaric tags for relative and absolute quantitation (itraq) analysis, in addition to comparing the serum protein levels of patients with chronic hepatitis b (chb), patients with hepatitis b virus-induced acute-on-chronic liver failure (hbv-induced aclf) and normal individuals. protein analysis was performed on 15 serum samples using itraq. the study population included healthy controls (n=5), patients with chb (n=5) and patients with hbv-induced aclf (n=5). western blotting was used to verify the results in an additional nine serum samples from healthy controls, patients with chb and patients with hbv-induced aclf (n=3, respectively). using itraq analysis, 16 different serum proteins with ≥1.5-fold differences in expression levels were identified in the patients with chb and aclf compared with the healthy controls. five of those proteins, c-reactive protein precursor, hemoglobin β chain variant hb s-wake, apolipoprotein j precursor, platelet factor 4 precursor and vitronectin, which demonstrated the greatest differences in their expression levels and the most significant correlation with liver diseases, were subsequently verified using western blotting. the western blotting results were consistent with the results from the itraq. two of the five proteins are not classified by biological process, and the biological functions of all the proteins in hbv-induced aclf remain unclear. this preliminary study demonstrated that a correlation between the expression of various serum proteins and the different pathogenetic conditions induced by hbv may exist. the analysis of a larger number of samples is required to identify potential protein biomarkers that may be involved in the pathogenesis and progression of hepatitis b. in china, the hepatitis b surface antigen (hbsag) seropositive rate for the general population (between 1 and 59 years of age) is 7.18% (1) . globally, there are ~93 million individuals with hepatitis b virus (hbv) infections, 20 million of which are chronic (2) . hepatitis b is the most common risk factor for liver cirrhosis and hepatocellular carcinoma (hcc), and the mortality of hbv-induced acute-on-chronic liver failure (aclf) may exceed 60%. hbv infections are not easy to cure, and the mechanisms of the virus are unclear, particularly with regard to protein expression and regulation function in the pathogenic process. proteomics analysis is a powerful technology used in a myriad of studies, including those focused on liver diseases (3) (4) (5) (6) (7) . isobaric tags for relative and absolute quantitation (itraq), as a quantitative method, is a common tool in proteomics and has been suggested to be as sensitive as (or more sensitive than) the differential in gel electrophoresis (dige) technique (8) . specifically, the itraq method has been used to study a variety of diseases and has been shown to be effective and accurate (4, 5, 9) . the purpose of this study was to analyze serum protein levels using itraq in normal controls, as well as patients with chronic hepatitis b (chb) and hbv-induced aclf, and to verify those results using western blotting. the ultimate aim was to identify the differences in serum protein levels that were closely associated with the progression of hepatitis b and hbv-induced aclf. the results of this study may provide crucial information regarding viral mechanisms and the pathogenic process. patients and specimens. serum samples from healthy individuals and patients with chb and hbv-induced aclf were obtained from the department of infectious diseases and the department of traditional chinese medicine of the third affiliated hospital of sun yat-sen university (guangzhou, china) (tables i and ii) . hbv-induced aclf was diagnosed using the previously described criteria (10, 11 sample preparation and protein extraction. plasma was fractionated with proteinminer protein enrichment small-capacity kit, cat# 163-3006, (proteinminer ; bio-rad laboratories, hercules, ca, usa) according to the manufacturer's instructions. protein solutions were reduced for 1 h at 56˚c with 10 mm dithiothreitol (dtt) and were cysteine-blocked with 55 mm iodoacetamide (iaa) at room temperature for 10 min. each sample was precipitated with four-times the volume of cold acetone. the total protein concentration was determined using the bradford assay. protein digestion and peptide tagging. protein solutions [100 µg; 5 mg/ml in 0.5 m triethyl ammonium bicarbonate (teab) containing 0.1% sodium dodecyl sulfate, ph 8.5]were digested for 24 h with 10 µg l-1-(4-tosylamido)-2-phenylethyl tosylphenylalanyl chloromethyl ketone (tpck)-treated trypsin. each peptide solution was labeled for 3 h at room temperature using an itraq reagent that had been reconstituted in 70 liters of ethanol, in accordance with the itraq reagents multiplex kit instructions (applied biosystems, foster city, ca, usa). the reaction was terminated by adding milliq water, and the samples were labeled with 114, 115, 116 and 117 mass-tagged itraq reagents. strong cation exchange chromatography (scx). scx was performed to remove excess itraq reagents and interfering substances for the mass analysis. the labeled peptides were then dried in a vacuum centrifuge and resuspended in 200 ml buffer a, prior to being loaded on a phenomenex luna 55 µm scx 100a column [250x4.60 mm (length x internal diameter), 5 µm; phenomenex, inc., torrance, ca, usa] on an agilent 1100 hplc unit (agilent technologies, santa clara, ca, usa). buffer a consisted of 10 mm kh 2 po 4 and 25% acetonitrile at ph 3.0, while buffer b consisted of 10 mm kh 2 po 4 , 25% acetonitrile and 2 m kcl at ph 3.0. the 60 min gradient comprised 100% buffer a for 30 min, 0-5% buffer b for 1 min, 5-30% buffer b for 15 min, 30-50% buffer b for 5 min, 50% buffer b for 4 min and 50-100% buffer a for table ii . clinical characteristics of the nine serum samples for western blotting verification. 5 min. thirteen fractions were collected using a foxy jr fraction collector (dionex corp., sunnyvale, ca, usa). fractions 2 and 3 were pooled according to the chromatogram profile, based on the peak intensity. all fractions were then dried in a vacuum concentrator and stored at -20˚c, prior to further analysis using mass spectrometry. peptides from the scx fractions were dissolved in 0.3 µl 100% formic acid and diluted to 5 µl in 0.05% trifluoroacetic acid (tfa). the peptides were loaded onto a microtof-q ii-nano lc system (bruker daltonics, inc., billerica, ma, usa) and mass spectra were acquired in the 250-1,600 m/z range every second for 60 min. database screening. data were processed using biotools software (bruker daltonics, inc.). the files were subsequently submitted to an in-house mascot server (matrix science ltd., london, uk) for database screening. the data were screened against the human sequence database [national center for biotechnology information non-redundant (ncbinr)]. the search was performed using trypsin as a specific enzyme. a maximum of one missed cleavage was permitted and oxidation (m), itraq 4 plex (k) and itraq 4 plex (n-term) were selected as variable modifications. the data obtained on the microtof-q were searched with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.8 da. protein ratios were normalized using the overall median ratio for all the peptides in the sample for each separate ratio in every individual experiment. the ratio for a given protein was calculated by taking the average of all the peptide ratios that briefly, the protein lysates were separated by polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride (pvdf) membrane and subjected to immunoblotting with the following antibodies: anti-apolipoprotein j precursor antibody (ab16077; dilution, 1:2,000; abcam, cambridge, uk), anti-hemoglobin β (sc-21757; dilution, 1:500; santa cruz biotechnology, inc., santa cruz, ca, usa), anti-crp precursor (ab32412; dilution, 1:1,000; abcam), anti-platelet factor 4 precursor (ab1488p; dilution, 1:5,000; millipore, billerica, ma, usa) and anti-vn (ab11591; dilution, 1:500; abcam) at 4˚c overnight. after washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence system (ecl; forevergen, guangzhou, china). tables i and ii summarize the clinical characteristics of the patients from whom the samples were collected. itraq identified 16 different proteins that had ≥1.5-fold differences in expression level between the patients with hbv-induced aclf and chb, respectively, and the healthy controls (table iii) . protein quantification software, based on the relative content of the isotopic reporter group and using m/z 114 as a reference, showed significant results (p≤0.05). some identified protein was lost in the corresponding reporter group, giving no quantification information. the specific information and re-identification will be published in a future study. the 16 proteins that were identified using itraq were classified into six categories, based on protein function (fig. 1) . the five proteins that demonstrated the greatest differences in their expression and the most significant correlation with liver diseases were verified using western blotting: crp precursor, hemoglobin β chain variant hb s-wake, apolipoprotein j precursor, platelet factor 4 precursor and vn ( fig. 2 and table iv) . two of the five proteins were not classified according to biological processes (apolipoprotein j precursor and platelet factor 4 precursor). proteomics research is a potentially useful and effective tool for studying pathogenesis, establishing prognosis and determining treatment outcomes in a variety of diseases. in the field of chb, proteomics is not widely performed, presumably due to the mechanism of chb being so complicated. the aim of this study was to describe the changes in serum protein levels in patients with chb and hbv-induced aclf, respectively, compared with healthy controls using itraq and western blotting. the authors hypothesized that this approach had the potential to ultimately be beneficial for the identification of proteins that were important in the progression of hbv infections. in the present study, 16 unique proteins were identified in patients with chb and those with hbv-induced aclf that had ≥1.5-fold differences in expression compared with those in the healthy controls. those proteins belonged to six different categories based on protein function, while two out of the five proteins that were analyzed using western blotting were not classified according to biological processes (apolipoprotein j precursor and platelet factor 4 precursor). the biological functions of the remaining proteins in hbv infection and progression that were analyzed have not yet been elucidated; however, a number of theories are discussed in the following section. crp, synthesized in the liver, is an acute phase protein that is rapidly identified in the plasma in cases of infection or tissue damage. crp is able to activate complement and enhance phagocytosis to facilitate the removal of pathogenic microorganisms and injured, necrotic and apoptotic cells. therefore, crp is important in natural immunity (12, 13) . originally, crp was considered to be a nonspecific biomarker of inflammation; however, following ~10 years of investigation, it has been demonstrated that crp participates in inflammation and atherosclerosis directly and has been indicated to be a prognostic factor and a risk factor (14) . in this study, it was revealed that levels of the precursor of crp were decreased in the patients with chb and markedly increased in the patients with aclf. nearly identical changes were observed for the hemoglobin β chain variant hb s-wake, which has been implicated in in orphan diseases (15) , although not in liver diseases. the apolipoprotein j precursor is important in cell aging and tumorigenesis with apolipoprotein j, lipid transfer inhibitor protein, complement c3d, corticosteroid-binding globulin and table iv . five serum proteins with the greatest differences in their expression levels and the most significant correlation with liver diseases, as verified using western blotting. apolipoprotein l1 (16, 17) . these latter five markers of fibrosis are secreted in the blood and show consistent changes with the increasing stage of fibrosis when compared with the markers used in the fibrotest and enhanced liver fibrosis (elf), hepascore and fibrospect tests. in the present study, the levels of apolipoprotein j precursor decreased in the progression of hbv from chb to aclf. therefore, further studies, with a larger number of samples, are required in order to better establish the role of apolipoprotein j precursor as a potential biomarker for hbv progression. platelet factor 4 is chemotactic for numerous cell types. it also functions as an inhibitor of hematopoiesis, angiogenesis and t-cell function, which may be used as a biomarker for establishing a prognosis in severe acute respiratory syndrome (18) . changes in platelet morphology, with accompanying increases in megathrombocyte fraction, may occur in chronic liver diseases, and thrombocytes are activated in chronic liver diseases and liver cirrhosis. platelet sensitivity to stimuli in patients with liver cirrhosis has been demonstrated to be higher than in healthy controls (19) and it was indicated that platelet factor 4 was activated and upregulated in chronic hepatitis and liver cirrhosis. in the present study, levels of platelet factor 4 precursor decreased in the progression of hepatitis b to liver failure. this result suggests that further studies are required to investigate the potential of platelet factor 4 precursor as a biomarker in the progression of chb. vn is another protein that commonly features in studies on liver disease. it is a multifunctional plasma glycoprotein produced by hepatocytes that is detectable in plasma and the extracellular matrix. glycosylation of vn modulates multimerization and collagen binding in liver regeneration (20) and alterations in vn glycosylation modulate substrate adhesion to rat hepatic stellate cells (hscs), which is responsible for matrix restructuring (21) . immunoblotting data identified increases in vn levels in cirrhotic livers, and vn immunoreactivity has been shown to be markedly increased in the cirrhotic liver matrix, irrespective of the apparent reduction in plasma vn. vn immunoreactivity in the liver may be considered to be a marker of fibrosis, particularly for chronic/mature fibrosis, paralleling previous observations concerning the enhanced orcein staining of cirrhotic septa (22) . these results suggest that vn may be important in the progression of liver disease and/or in hepatic fibrosis through its collagen-binding domain i, and that vn levels are likely to be higher in patients with chronic hepatitis, liver cirrhosis and liver cancer than in controls (20) (21) (22) (23) . in the present study, vn levels decreased in the progression of hepatitis b (from chb to hbv-induced aclf). the observation of decreased vn levels was consistent with two other studies (24, 25) . in one of these studies, it was demonstrated that, in chronic liver disease, the concentration of plasma vn was significantly lower than in healthy controls and was associated with the severity of liver disease. notably, levels of vn in the liver tissue were significantly increased in patients with chronic liver disease compared with those in normal controls. these results suggested that vn was deposited in injured tissue during the repair process and functioned as an adhesive protein (24) . in the other study, it was demonstrated that there were lower levels of plasma vn in chronic liver disease, which was possibly due to a decreased synthesis, deposition in injured tissues or a combination of the two (25) . the same group of authors also revealed that decreased levels of plasma vn and fibronectin (fn) and increased levels of serum laminin (lm) p1 in patients with chronic liver diseases were associated with hepatic dysfunction, and that changes in the levels of the glycoproteins involved in cell attachment were significant in the development of hepatic fibrosis in patients with chronic liver diseases (25) . based on the accumulating information on vn and liver fibrosis, it appears that vn requires further study. in the present study, the authors aimed to describe changes in serum protein levels in patients with liver dysfunction induced by hepatitis b and patients with severe liver damage (aclf). the purpose of this was to specifically identify one or more proteins that are able serve as biomarkers for predicting a pathogenic condition and for prognostic purposes, similar to α-fetoprotein (afp) for hepatocellular carcinoma (hcc). it was not possible in this study to obtain a series of serum samples from individual patients as they progressed through the various stages of hepatitis b infection to severe liver damage (aclf). however, using itraq and western blotting, several candidate proteins were identified (namely apolipoprotein j precursor, platelet factor 4 precursor and vn) that warrant further study using a larger number of samples. chinese society of hepatology and chinese society of infectious diseases, chinese medical association: the guideline of prevention and treatment for chronic hepatitis b management of hepatitis b in china comparative serum proteomic analysis of patients with acute-on-chronic liver failure: alpha-1-acid glycoprotein maybe a candidate marker for prognosis of hepatitis b virus infection novel biomarker candidates to predict hepatic fibrosis in hepatitis c identified by serum proteomics itraq-2dlc-esi-ms/ms based identification of a new set of immunohistochemical biomarkers for classification of dysplastic nodules and small hepatocellular carcinoma simple method for quantitative analysis of n-linked glycoproteins in hepatocellular carcinoma specimens network-based pipeline for analyzing ms data: an application toward liver cancer comparative study of three proteomic quantitative methods, dige, cicat, and itraq, using 2d gel-or lc-maldi tof/tof quantitative proteomics analysis of maternal plasma in down syndrome pregnancies using isobaric tagging reagent (itraq) chronic hepatitis b: update acute-on-chronic liver failure: consensus recommendations of the asian pacific association for the study of the liver (apasl) role of c-reactive protein velocity in the diagnosis of early bacterial infections in children after cardiac surgery variation of serum c-reactive protein (crp) over time in pediatric cancer patients with febrile illness and its relevance to identified pathogen relationship between coronary arterial remodeling, fibrous cap thickness and high-sensitivity c-reactive protein levels in patients with acute coronary syndrome a new sickling variant 'hb s-wake β [(glu6val-asn139 ser)]' found in a compound heterozygote with hb s β (glu6val) coinherited with homozygous α-thalassemia-2: phenotype and molecular characteristics clusterin/apolipoprotein j in human aging and cancer discovery of novel biomarker candidates for liver fibrosis in hepatitis c patients: a preliminary study proteomic analysis reveals platelet factor 4 and beta-thromboglobulin as prognostic markers in severe acute respiratory syndrome activation of blood platelets in chronic hepatitis and liver cirrhosis p-selectin expression on blood platelets and secretory activity of beta-thromboglobulin and platelet factor-4 changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration survival signals of hepatic stellate cells in liver regeneration are regulated by glycosylation changes in rat vitronectin, especially decreased sialylation vitronectin in the cirrhotic liver: an immunomarker of mature fibrosis collagen-binding activity of plasma vitronectin in chronic liver disease distribution of vitronectin in plasma and liver tissue: relationship to chronic liver disease changes in plasma vitronectin, fibronectin, and serum laminin p1 levels and immunohistochemical study of vitronectin in the liver of patients with chronic liver diseases the authors would like to thank miss. qin zhang and mr. bing-quan lai for their commitment to this study. this study was supported by the national natural science key: cord-009636-5kddituy authors: shirbaghaee, zeinab; bolhassani, azam title: different applications of virus‐like particles in biology and medicine: vaccination and delivery systems date: 2015-12-22 journal: biopolymers doi: 10.1002/bip.22759 sha: doc_id: 9636 cord_uid: 5kddituy virus‐like particles (vlps) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. vlps can elicit efficient protective immunity as direct immunogens compared to soluble antigens co‐administered with adjuvants in several booster injections. up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and e. coli were used to express recombinant proteins, especially for vlp production. recent studies are also generating vlps in plants using different transient expression vectors for edible vaccines. vlps and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. herein, we describe vlp production in different systems as well as its applications in biology and medicine. © 2015 wiley periodicals, inc. biopolymers 105: 113–132, 2016. v irus-like particles (vlps) known as viral "empty shells" maintain the same structural properties of virions, without genome. these constructs are considered very efficient as vaccine platforms and therapeutic delivery systems. 1 many antigens can readily be displayed on the surface of vlps. these antigens can be genetically or chemically fused to the vlp. 2 regarding to the reports, the immune stimulation by vlps contains: (a) stimulation of innate immunity through tlrs and pattern recognition receptors (prrs) due to the expression of multivalent structures; (b) induction of strong humoral response and also igm in t-cell independent way; and (c) enhancement of the uptake, processing and presentation by apcs through mhc i and mhc ii cross-presentation pathway due to the particulate nature of vlps. 3 vlps can be subcutaneously or intramuscularly injected. their small diameter facilitates entry into lymphatic vessels and direct drainage into local lymph nodes. once in the lymph node, vlps are taken up by lymph node resident dendritic cells (dcs). this uptake is enhanced by the size and form of vlps. vlps stimulate cd4 t cells via the mhc ii pathway, as well as highly efficient cross-presentation on the mhc class i pathway. 3 generally, viral-like particles, are considered as vaccine candidates because their natural properties such as multimeric antigens and their specific structures are suitable for the stimulation of efficient humoral and cellular immunity. currently, the development of recombinant subunit vaccines (suvs) has been significantly increased using heterologous expression systems. antigens derived from many bacterial, viral, fungal and parasitic pathogens were used for safe and effective vaccination. five vlp-based vaccines have been already approved including three for hbv and two for hpv, while in the veterinary field; a vlp-based vaccine against porcine circovirus type 2 (pcv2) has been approved. some vlp-based vaccines targeting human and animal diseases are recently in late stages of clinical trials. vlps have a positive value as academic, industrial, and commercial systems especially in gene therapy and design of nanomaterials. however, the study of the vlp-based applications (vaccination, gene and drug delivery, and imaging) must be followed to show the reliability, and cost efficiency of this technology. furthermore, the expression systems would be improved to achieve the best strategy for vlp production from different viral genes. this review will focus on vlp characteristics and its applications especially as vaccines or delivery systems for dna, sirna and drugs. it should be noted that in the vaccination field whenever a viral-like particle carries genetic material is called "vectored vaccines 4 " and in gene therapy, they are called viral vectors. however, for simplicity in this review, we called all particles entitled as viral-like particles (vlps). viral-like particles (vlps) have been generated for over thirty various infectious viruses in animals and humans. 5 vlps are composed of one or more structural (/capsid) proteins possessing natural properties for self-assembly, and are morphologically similar to authentic viruses. 5, 6 comparing to live viruses, vlps are non-replicating and non-infective due to the lack of infectious genetic material. 5 virus-like particles have the potential to be used as safe vaccine candidates without the need for any adjuvant. 7, 8 different viruses present different structures for generation of viral-like particles such as: a. simple viral capsids with one or two major proteins (e.g., parvoviruses, papillomaviruses, circovirses, calciviruses, hepatitis e virus (hev) and polyomaviruses). b. complex viral capsids with various protein layers, encoded by many distinct mrnas, or generated from a single polyprotein (e.g., picornaviruses). c. viral capsids with lipid envelopes including a lipid bilayer obtained from the host cell, as well as viral glycoprotein spikes (e.g., influenza, hiv and hepatitis c). 5, 9 figure 1 shows the general model of vlp along with its applications. the selection of expression vector is one of the major factors in vlp generation. the reports showed the successful production of 174 vlps indicating that bacterial systems, yeast and insect systems are used in 28%, 20%, and 28% of the cases. in addition, mammalian cells (15%) and plants (9%) were usually applied to produce vlps with special properties. 8 bacterial systems are often included the commercial e.coli strains and expression vectors, to produce non-enveloped vlps in high levels compared to other systems (table i) . 5 in addition, bacterial cells have been applied for generation of vlps which need several types of structural proteins, such as the avibirnavirus ibdv vp2, vp4, and vp3-polyproteins. 54 the reports indicated that the expression of the hepatitis b virus (hbv) capsid protein in e. coli leads to the formation of structures similar to the hbv core (hbc) particle. 55 bacterial figure 1 general model of vlp along with its applications: the picture shows the recombinant hpv16 l1 pentamers assembled in vitro into capsid-like structures. self-assembly of recombinant viral coat proteins into empty capsids is a promising strategy for production of virus-like particles (vlps) in vaccine design. the resulting vlps can induce a protective immune response by mimicking the authentic epitopes of virions. systems are not always a desired plan for vlp production due to several factors, such as (a) lack of ability to generate recombinant proteins with mammalian-like post-translational modifications (ptm), (b) failure to produce the correct disulfide bonds, (c) drawbacks of protein solubility, and d) the existence of lipopolysaccharides (lps)/or endotoxins in production of recombinant proteins (rp). 5 viral coat proteins (cps) can be efficiently produced as insoluble inclusion bodies, purified under denaturing conditions, refolded, and selfassembled, as indicated in the parvovirus b19 and the ccmv and cmv plant viruses. 56 a simple change in the cultivation conditions such as low-temperature can solve the problem of inclusion bodies and induce the formation of soluble vlps, as performed for two viral systems, the densovirus ihhnv, 57 and the potyvirus pvy. 58 some factors including the resistance markers of the expression plasmids and the composition of the cultivation medium can also change the vlp assembly (e.g., bacteriophage qb). 59 another strategy applied to increase expression levels and solubility involves the use of different fusion protein systems, e.g., glutathione-s-transferase (gst) fusion proteins such as the papillomavirus l1, the polyomavirus mupyv, and the picornavirus fmdv. [60] [61] [62] [63] other prokaryotic hosts have been recently used to generate vlp, e.g., lactobacillus. 8 the intracellular assembly of hpv16 l1 vlp was reported in lactobacillus casei, a lactose-inducible expression strain. 5 furthermore, the production of l1 vlps using lactobacillus developed new live mucosal prophylactic vaccines (table i) . 29, 30 a pseudomonas fluorescens (p. fluorescens) expression system is an efficient choice against e. coli, because of simple manipulation, high yields of active and soluble proteins, and largescale cultivation. some differences between p. fluorescens and e. coli including the various sizes of genome, and diverse metabolic approaches can influence the generation of recombinant proteins. 64 the capsid protein of a plant bromovirus, the cowpea chlorotic mottle virus (ccmv), has been recently expressed as a soluble form in p. fluorescens, and assembled into vlps in vivo. this construct was structurally similar to the natural viral particles provided from plants. 64 eukaryotic expression systems are a striking alternative to bacteria, especially for solving the problem of bacterial endotoxins in vaccine development. some structural genes of mammalian viruses expressed in yeast are able to form the vlp. this expression host has been efficiently applied to generate the first licensed hbv vaccine. 65 hbsag is one of the antigens commonly utilized for production of vlp-based hbv vaccine. hbsag has been expressed in pichia pastoris (p. pastoris), sac-charomyces cerevisiae (s. cerevisiae) and hansenula polymorpha (h. polymorpha) (table i) . 5, 16 it is critical to consider that the viral-like particles are not always formed during the cultivation procedure of the yeast cells. these studies showed that the selfassembly of the vlps in pichia system should be completed during the protein purification. 16, [66] [67] [68] the expression and selfassembly of recombinant bacteriophage q coat protein (q-cp) was indicated in saccharomyces cerevisiae and pichia pastoris. the yeast-derived q-vlps were greatly immunogenic in mouse similar to that in e.coli-derived q-vlps. 69 ms2 vlps produced in saccharomyces cerevisiae could package functional heterologous mrnas. for example, the linkage of the ms2 packaging sequence to the human growth hormone mrna allowed the packaging of the mrna in ms2 vlps. indeed, the high stability of ms2 vlps suggests them as an efficient delivery system for rna-based vaccines. 70 the p. pastoris system was also utilized as a potent alternative for expression of ccmv coat protein vlps due to easy manipulation and high expression levels. 71 in addition, this system has been utilized to express efficiently the premembrane and envelope glycoproteins of dengue virus type 2 (denv-2), 72 hbsag, 73, 74 hccag 75 resulting in the generation of vlps. 72 the major advantage of yeast systems is the ptm including phosphorylation or glycosylation, as indicated in hbv vlps. 8 the studies indicated that hbc phosphorylation plays a major role in viral replication and capsid formation. such yeast-derived hbc vlps are valuable for vaccination and diagnostics. 76 furthermore, the potent multigene expression systems have been constructed in yeasts. for example, the expression of three rotavirus structural genes from a single plasmid vector led to the generation of triple layered vlps in saccharomyces cerevisiae. 8, 77 however, the multimerization of protein into vlps is not supported for the enveloped viruses (e.g., gag vlps of hiv-2), suggesting that yeast does not have the essential factors of host. 8 thus, the generation of enveloped hiv-1 pr55gag vlps has been performed using s. cerevisiae spheroplasts, morphologically similar to immature viral particles. 5, 78 in general, the construction of yeast expression systems, especially hansenula and pichia strains, are more difficult than bacterial vectors. in addition, the yield of vlp production is less than that in e.coli. 8 other limitation of yeast system is its dissimilarity with mammalian cells in the ptm of proteins, especially glycosylation. 79,80 therefore, this system is more suitable for the generation of non-enveloped viral-like particles. another attractive system utilized broadly for production of vlp is the baculovirus-insect cell expression system, due to 118 shirbaghaee and bolhassani some advantages, such as the rapid growth ratios, the culture preparation in large-scale, and the ptm of the target proteins similar to mammalian cells. [81] [82] [83] the results showed that both yeast and insect cells were previously used for the vp1 expression of several polyomaviruses, and its assembly into viral-like particle. 84 in addition, insect cells were used to provide vlpbased vaccines, e.g., the approved hpv vaccine, cervarix. indeed, insect cells are able to generate both vlp types (i.e., enveloped and non-enveloped). there are enveloped vlps in clinical trials. 9 the main limitation of insect cell system is protein contamination with the enveloped baculovirus particles, suggesting the development of efficient plans for purification of vlps. 85 recently, co-expression of four genes of human influenza h3n2 virus (i.e., ha, na, m1, and m2) in insect cells led to generate influenza vlps which protected mice against h3n2 virus challenge. 86 these data suggested that viral-like particles are a hopeful vaccine candidate for h9n2 influenza and probably other subtypes of virulent avian influenza viruses. 87 the non-infectious viral-like particles of the alphavirus sav was also generated using the recombinant baculoviruses expressing sav capsid protein and two major immunodominant viral glycoproteins (e1 and e2) in insect cells. 88 moreover, baculovirus expression system was utilized to generate vlps from cowpea mosaic virus (cpmv), tomato bushy stunt virus, and entorovirus271 (ev71). 8, 89, 90 recently, non-replicative baculovirus have been developed to cope with the problem of baculovirus contamination. 91 stable systems using insect cells have been also tested. 92 moreover, silkworm expression systems were efficiently applied to generate vlps and the surface of vlps could be changed by some strategies, irrespective if their constructs are enveloped or not. silkworms show a high capability for production of recombinant proteins, in comparison with insect cells, and also easy and inexpensive protein preparation similar to e.coli expression system. 81 for over two decades, different mammalian cell lines have been developed as a source of commercial therapeutic proteins for clinical applications, 93 because of their ability for proper protein folding, assembly and ptm (e.g., the correct glycosylation pattern). 8, 93 however, high costs of production and potential safety concerns remained a challenge for these systems. the mammalian cells were progressively utilized to produce vlpbased vaccines 5,94 , e.g., for influenza viruses. for instance, the generation of a stable mammalian cell line (e.g., vero cells) expressing four influenza structural proteins (ha, na, m1, and matrix 2 (m2)) led to form hybrid vlps containing matrix proteins, and surface glycoproteins of h3n2 and h5n1 influenza types, respectively. 8, 95 another examples are the produc97 and hiv-1 vlp in cos-7/vero cells, 98 and hbv vlp in cho cells. [99] [100] [101] plant systems plants were successfully used to express specific gene products. the feasibility of recombinant plants for generation of vaccine antigens were shown in tobacco plants, potato tubers, and others. 102 this approach develops vaccine strategies which can stimulate mucosal as well as systemic immune responses. in addition, it can be delivered orally as part of a normal biologic function in human. 102 the antigen expressed in plant systems shows extensive disulphide crosslinking and oligomerization for formation of virus-like particles. for example, the hepatitis b major surface antigen has been expressed in several plant systems. 103 plants are able to express and assemble both types of vlps (i.e., enveloped and non-enveloped) as multimeric and chimeric proteins. the high expression of vlps in plant is easy and rapid (e.g., 1-2 weeks) using a tobacco mosaic virus (tmv) rna replicon system and/or a bean yellow dwarf virus (beydv) dna replicon system. 104 another advantage of plants is the use of plant virus particles as a delivery system to present foreign epitopes. furthermore, the problem of plant-specific glycans has been partially solved using the development of transgenic plants with "humanized" glycosylation pathways. 104 plantderived vlps can be used for oral delivery of vaccines. virallike particles are more resistant to digestive enzymes than soluble proteins in body, because of their highly ordered and packed structures. for example, the gastrointestinal virusesderived vlps including noroviruses and rotaviruses were utilized orally as potent candidates for mucosal immunization. 105 plant-derived vlps showed the same structures with vlps generated in other expression systems accompanied by a comparable or higher immunogenicity. some plant-derived vlps could induce protective humoral and cellular immunity and also safety in clinics. 105 the studies showed that the level of protein expressed in the recombinant plants is variable and often low. therefore, further increase in expression will be necessary for practical and efficient products. 102 recent progress in the glyco-engineering of plants allows human-like glycol-modification and optimization of desired glycan structures for increasing safety and functionality of recombinant pharmaceutical glycoproteins. 1 some plantbased systems can stabilize antigen and thus reduce storage and distribution costs. 103 different applications of virus-like particles 119 toxoplasma gondii (t. gondii) is an obligate intracellular parasite infecting the nucleated cells of warm-blood vertebrates. this parasite is able to stimulate strong humoral, cellular and mucosal immunity, and thus it can be used as an efficient delivery system for heterologous antigens. t. gondii was applied as a vector for live vaccination against infectious pathogens. [104] [105] [106] [107] [108] [109] recently, a non-pathogenic kinetoplastida, leishmania tarentolae, was utilized to express heterologous proteins. the studies showed that expression of mammalian glycoproteins in this parasite leads to their modification with mammalian-like oligosaccharides. [110] [111] [112] [113] recently, our group has focused on its use as a live vector or killed vaccine, [114] [115] [116] and also generation of viral coat proteins and their assembly as vlp in this system. 117 virus-like particles show an efficient strategy to deliver antigens to the immune system, inducing both arms of the adaptive immunity. 118 indeed, vlps present antigenic epitopes in the proper conformation, leading to induce humoral responses. 5 for example, preclinical trials with influenza vlps indicated their capacity to induce both humoral and cellular immune responses at low antigen doses. several authors have reported antibody response to parenterally or orally administered plant-derived antigens. 119, 120 as exogenous antigens, vlps are taken up by professional antigen presenting cells (apcs), especially dcs, followed by antigen processing and presentation via mhc class ii molecules, dc activation and maturation through up-regulation of co-stimulatory molecules and cytokine production, and stimulation of cd41 t helper cells. all these events can efficiently induce both humoral and cellular immunity. 5 in addition, the exogenous vlps can enter the cytosol of dcs, be processed and presented by mhc class i molecules to cytotoxic t lymphocytes (ctls) using cross-presentation. 5, 121, 122 furthermore, the b-cell activation using vlps is robust enough to induce t cell-independent igm antibodies. 7, 8 dcs loaded with yeast-derived hiv vlps can alter gag-specific memory cd81 t cells into effector cells through cross-presentation in chronically hiv-infected individuals, although some gag-specific t cells in these patients did not show any response. 123 the reports showed that the expression system used for generation of vlp might significantly affect direction, type and outcome of immune responses. 121 for example, potent and specific immunomodulatory effects were assigned to yeast-derived hiv vlps in comparison with other expression systems. 123 on the other hand, plant-or insectderived vlps, consisting of the l1 capsid protein of hpv, were both immuno genic to an equal degree. half of mice fed trans-genic potatoes expressing hpv vlps developed l1-specific antibodies. 124 the studies indicated that the vlps are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. vlp-derived peptides are loaded onto mhc i that have been recycled from the cell surface. 125 a study showed that uptake and activation of dc by vlp involves proteoglycan receptors, tlr4 and nf-kb, and can be inhibited by heparin. 126 several data suggest different routes of vlp uptake by dc and langerhans cells (lc). 127 for example, lcs and dcs internalize similar amounts of hpv-vlps in vaccine design, albeit through different uptake mechanisms. 128, 129 vlp uptake by dcs results in activation and cross-presentation of mhc i-restricted peptides with co-stimulation to t-cells. on the other hand, vlp uptake by lc leads to cross-presentation in the absence of costimulation. efficient vlp cross-presentation by lcs with costimulation can be achieved by addition of cd40 ligand. 128 the lack of a protective immune response after viral contact with lcs may explain why some women fail to induce an immune response against the virus. lcs endocytose hpv vlps via a non-clathrin, non-caveolae, actin-independent pathway, whereas dcs take up hpv vlps both by a clathrin-mediated mechanism and via macropinocytosis in an actin-dependent manner. this difference in endocytosis resulted in processing and presenting hpv vlp peptides by lcs similar to that by dcs on their surface, but in the absence of co-stimulation. with the addition of cd40l, lcs incubated with hpv vlps generated the efficient amounts of the pro-inflammatory cytokine (il-12) and could stimulate a hpv-specific immune response after incubation with t cells. 128 despite these differences, vlps taken up by dc and lc were able to prime naive cd81 t cells and induce cytolytic effector t cells in vitro. 127 furthermore, hiv-1 pr55 gag virus-like particles could stimulate strong humoral and cellular immune responses. vlp expressed by recombinant baculoviruses activated human pbmc to release pro-inflammatory (il-6, tnf-a), antiinflammatory (il-10) and th1-polarizing (ifn-c) cytokines as well as gm-csf and mip-1a in a dose-and time-dependent manner. furthermore, vlp-induced monocyte activation was shown by up-regulation of molecules involved in antigen presentation (mhc ii, cd80, and cd86) and cell adhesion (cd54). exposure of vlp to serum inactivated its capacity to stimulate cytokine production. 130 the linking of vlps to adjuvant molecules was also shown to improve the immunogenicity of the nano-bioparticles. 131 adjuvanted vlps [e.g., cpg odn1826 or poly (i: c) adjuvants] elicited a higher titer of total specific igg compared to vlps alone. furthermore, while vlps alone induced a balanced th2 pattern, vlps formulated with adjuvant elicited a th1-biased igg subclasses (igg2a and igg3), with poly (i: c) more potent than cpg odn1826 in 120 shirbaghaee and bolhassani animal model. 118 in addition, mice immunization with chimeric simian immunodeficiency virus (siv) vlps containing gm-csf significantly induced siv env-specific antibodies as well as neutralizing activity at higher levels than those elicited by standard siv vlps, siv vlps containing cd40l, or standard vlps mixed with soluble gm-csf. on the other hand, the incorporation of immunostimulatory molecules showed significantly increased cd41 and cd81 t-cell responses to siv env, compared to standard siv vlps. 132 formulation of vlps with rough lps (r-lps) adjuvant as well as dna primed-vlp boosted regimen were led to increase specific immune responses as compared to vlps alone, but among them the vlp/r-lps highly enhanced immune response. 133 recent studies demonstrated the potential of the hbc vlps as an oral immunogen. intraperitoneal immunization with the hbc vlp induced a strong, mixed th1/th2 response. in contrast, oral administration of the hbc vlp generated a high humoral response with mainly igg2a antibodies, directing toward a th1 response which is essential in the control of intracellular pathogens. 134 in addition, the intranasal monovalent adjuvanted norwalk vlp vaccine was well tolerated and highly immunogenic. 135 the studies showed that chimeric hpv-vlps are able to elicit potent ctl responses in mice against hpv16transformed tumors; however, the mechanism of t cell priming has remained obscure. hpv vlp could bind to human mhc class ii-positive apcs through interaction with fccriii, and immature dcs were activated after incubation with hpv vlp. 136 it was shown that binding and uptake of vlp by dc from fccrii, fccriii, and fccrii/iii deficient mice are reduced by up to 50% compared with wild-type mice. in addition, maturation of murine dc from fccrii/iii-deficient mice by vlp is also reduced, indicating that dc maturation, and thus ag presentation, is diminished in the absence of expression of fccr. 136 poor immunogenicity of mucosally administered proteins has been a major barrier for development of efficient oral vaccines. one way to overcome this obstacle is the use of appropriate adjuvants. also, delivery of antigen to mucosal surfaces as vlp provides an efficient way of inducing mucosal immunity. after oral or intranasal immunization with norwalk vlp, or rotavirus vlp without adjuvant, intestinal iga was detected in immunized mice, which were protected from virus challenge. 137 in addition, the plasma cell precursors that migrate to the genital tract are derived primarily from mucosal lymphoid tissues and often secrete iga. 138 the studies indicated that immune responses generated by mucosal administration of vlp were generally weaker than systemic administration. vlp specific iga was higher in intestine washes following intrarectally (i.r.) than intravaginally (i.va.) immunization, and higher in vaginal washes following intramuscularly (i.m.) than i.r. or i.va immunization. some studies suggested that the immunogenicity of virus particles at mucosal surfaces is probably a property of particulate antigens assembled as multimers of subunits. indeed, vlp might be actively taken up by mucosal apc through the integrin receptors. 137 lipoparticles are stable, highly purified, homogeneous, and specialized vlps containing high concentrations of an integral membrane protein. integral membrane proteins are involved in different biological functions and are targeted by 50% of existing therapeutic drugs. however, because of their hydrophobic domains, membrane proteins are difficult to manipulate outside of living cells. lipoparticles can incorporate a wide variety of the membrane proteins, including g proteincoupled receptors, ion channels, and viral envelopes. lipoparticles provide a platform for different applications such as antibody screening, production of immunogens, and ligand binding assays. [139] [140] [141] during the assembly of enveloped viruses, lipid ordered domains of the host cell plasma membrane, known as lipid rafts, frequently function as a natural target for viral proteins. the role of lipid rafts in the organization of complex combinations of immune receptors during antigen presentation and t cell signaling is extensively recognized. 142 on the other hand, in order to improve the immunogenicity of hiv-1 envelope glycoproteins, the fusion of gp120 was performed to a carrier protein, hepatitis b surface antigen (hbsag) which is capable of spontaneous assembly into viruslike particles. the hbsag-gp120 hybrid proteins assembled efficiently into 20-30 nm particles. the particles resembled native hbsag particles in size and density, consistent with a lipid composition of about 25% and a gp120 content of about 100 per particle. particulate gp120 folded in its native conformation and was biologically active, as shown by high affinity binding of cd4. because the particles are lipoprotein micelles, an array of gp120 on their surface closely mimics gp120 on the surface of hiv-1 virions. these gp120-rich particles can enhance the quality, and also quantity of antibodies elicited by a gp120 vaccine. 143 virus-like particles show an expanding spectrum of applications such as gene therapy, nanotechnology, vaccination, and diagnostics. 55, 77 recently, the studies showed a pattern of direct conjugation of some ligands, including nucleic acids and proteins attached to vlp surface. 144, 145 in addition, because of the superior accessibility of cysteine and lysine residues on vlps, bio-conjugation has been performed by commercial homo-or hetero-bifunctional linkers. [146] [147] [148] [149] for example, three foreign proteins were chemically conjugated to the vlp surface of cpmv by proper bifunctional cross-linkers. 147 on the other hand, the researchers could produce an alphavirus vlp surrounding a functional gold nanoparticle. 150 vlps have been also used to stimulate immune responses and generate antitumor responses, e.g., alphavirus-based virus-like replicon particles (vrp) expressing various melanoma antigens. [151] [152] [153] it is interesting that the first viral-associated cancer vaccines were founded on hbv vlp and hpv vlp to prevent hbvassociated hepatocellular carcinoma (hcc) and hpvassociated cervical carcinoma, respectively. 153, 154 it should be noted that these vlp formulations are viral vaccines that prevent a viral infection that may progress to carcinoma after a long time. we indicate some applications of vlps against viral diseases as following: in several studies, specific vaccine antigens were generated by various expression systems to induce protective immune responses and apply in licensed recombinant viral vaccines. 100, 155 some examples of preventive vlp-based vaccines are recently commercialized worldwide including glaxos-mithkline's engerixv r (hbv) and cervarixv r (hpv), and merck and co., inc.'s recombivax hbv r (hbv) and gardasilv r (hpv). other vlp-based vaccines undergo preclinical evaluation or clinical trials, including parvovirus-, influenza-, norwalk-derived vlps and also different chimeric vlps. 156 for generation of immunogenic vlps, eukary otic expression hosts including yeast (s. cerevisiae, p. pastoris and h. polymorpha) and mammalian cells (chinese hamster ovary cell line [cho]) were used. the studies indicated that the recombinant hbsag generated in cho and h. polymorpha have significant differences in size, molecular weight (mw), and monomer number. furthermore, the cho-derived viral-like particles include a combination of glycosylated and non-glycosylated hbsag proteins, similar to those in patients' sera, while yeastderived antigens were reported to be non-glycosylated. chobased vaccines were provided by pasteur-m erieux aventis in france (genhevac bv r ) and scigen in israel (sci-b-vac tm ). both vaccines contained not only the hbsag s pro tein but also the m protein (genhevac b) or the m and l protein (sci-b-vac). 156 on the other hand, gardasil approved by the fda in 2006 is a quadri valent hpv types 6/11/16/18 l1 vlp vaccine produced in s. cerevisiae. cervarix is the other licensed hpv vaccine approved by the fda in 2009. 156 cervarix is a bivalent hpv types 16/18 l1 vlp vaccine expressed via a recombinant baculovirus vector. 25, 157 different experiments have been concentrated on hpv vlp vaccination in mouse and human models including: (a) activation of immature human dcs by chimeric hpv16 vlps, (b) determination of systemic cytokine pattern elicited by hpv l1 vlps, (c) identification of gene expression signatures in hpv16 l1 vlp-induced human pbmcs, (d) generation of potent and prolonged neutralizing l1 antibodies using a single intramuscular (im) mice injection with recombinant adenoassociated virus encoding hpv16 l1 protein (raav-16l1), (e) augmentation of immunogenicity of hpv l1 dna vaccines using genetic linkage to a chemokine and secretory signal peptide sequences, (f) potent stimulation of systemic and mucosal immune responses to vlp vaccines using the encapsulation of a genetic cytokine adjuvant (e.g., il-2), (g) improvement of hpv16 vlp immunogenicity by linkage to the modified adjuvant, and m) nasal immunization of mice with hpv16 vlps. 158 hpv16 l1-e7 chimeric virus-like particles (cvlp) could induce e7-and l1-specific ctls. the therapeutic potential of the cvlp also indicated a considerable safety in high grade cervical intraepithelial neoplasia patients (cin 2/ 3). 159 several improvements in vaccine design by vlp are still in preclinical trials. some main examples are referred as following: a. co-injection of the hpv16 l1 vlp with e. coli heatlabile enterotoxin (lt) as an adjuvant significantly increased the levels of serum igg and vaginal iga after nasal or bronchial mice immunization. 160 antigens (hiv-1 p17/p24: ty vlp) was also immunogenic and well-tolerated in phase i clinical trials. 24,169 g. several groups have focused on improving bacteriophagebased vlp vaccines, e.g., rna bacteriophage qb. these chimeric vlp vaccines were targeted against noninfectious diorders including hypertension, allergy, neurodegenerative and autoimmune diseases (e.g., diabetes mellitus type ii and alzheimer), cancer (e.g., melanoma). the vaccine candidate against alzheimer (cad-106) was constructed to display chemically coupled amyloid beta (ab1-6) peptide derived from the n-terminal b cell epitope of ab protein, on the surface of qb-cp vlps. this vaccine could stimulate ab-specific igg and decrease amyloid accumulation in animal models expressing ab precursor protein, without eliciting t-cells or inflammatory reactions in brain tissue. 170, 171 in addition, the angiotensin ii vaccine was synthesized by covalently conjugation of a peptide derived from angiotensin ii to the rna bacteriophage qb vlp capsid. this modified vlp could decrease blood pressure in spontaneously hypertensive rats. 172 table i shows preclinical and clinical studies of vlps in vaccine development. generally, a major application of vlps is the stimulation of immunity against foreign protein epitopes by genetically fusing or chemically conjugating them to vlps entitled as chimeric vlp (cvlps). 173 antigens can be fused to vlps through either covalent or non-covalent bonds. the most common covalent bond is generated by the heterobifunctional chemical cross-linkers with amine and sulfhydryl-reactive arms. 104 for instance, cysteine-containing antigens can be conjugated to lysine residues of vlps surface at a high density (e.g., three peptides per coat protein molecules). the non-covalent conjugation strategy contains the use of streptavidin as linkers to attach biotinylated antigens and vlps through their efficient and specific interactions. 104 sv40 vlps can also encapsulate various materials such as dna (5 kb) and proteins as antigens. insertion of a special exogenous peptide into the surface loops of vp1 produced sv40 vlps with the ability of cell targeting. moreover, sv40 vlps stimulated innate immunity as a natural adjuvant. indeed, sv40 vlps may be a promising vaccine candidate to deliver heterologous antigens followed by the induction of ctls without synthetic adjuvants. 174 several chimeric vlp vaccines have entered clinical trials, such as the anti-influenza a m2-hbcag vlp vaccine (hbcag vlps displaying m2 epitope of influenza a), the anti-hiv p17/p24: ty vlp, two anti-malaria vaccines (hbcag vlps displaying malaria epitopes), the nicotine-qb vlp and the anti-ang ii qb vlp. 175 genetic linkage contains a stable bond between vlp and antigen. the studies showed that only peptides shorter than 30 amino acids (small peptides) can be presented without interfering with the correct assembly of vlps. other limitations contain the improper folding of displayed antigens and the formation of cvlps with heterogeneous size. to prevent these issues (e.g., assembly problems), structural studies have identified domains for different vlps such as hbcag, hbsag and hiv gag that were not necessary for vlp assembly as well as allowed insertion of foreign antigens. 104 the simplest way for generation of single component cvlps, is the insertion of peptides at the n-or c-terminal regions of chimeric vlps. multiple fusion positions should be identified to produce multicomponent cvlps inducing broad immune responses. 104 the direction and intensity of the immune responses are significantly influenced by the vlp type, the foreign antigen density, and its accessibility on or within the vlp. furthermore, preexisting immunity against the epitopes of the vlp as a delivery system may importantly change the response against the heterogenous antigen. for example, hbcag was also utilized to display a neutralizing epitope of hpv16 l2 protein. the nasal delivery of hbcag-hpv16 l2 epitope cvlps expressed in tobacco induced antigen-specific antibody responses in mouse model. on the other hand, an hpv16 l1-based chimeric vlp was generated in transgenic tomato to present several t-cell epitopes from hpv16 e7 and e6 proteins. the hpv l1-e7/e6 vlps elicited a neutralizing antibody response similar to that from an equal amount of the commercial vaccine (gardasil) in preclinical study. moreover, the chimeric vlps induced ctl responses against the e7 and e6 epitopes. chimeric hpv l1 vlps were also designed using genetic fusion to display epitopes of influenza m2 protein. 104 to overcome the problems associated with genetic fusion including the antigen size, conformation and vlp assembly, different applications of virus-like particles chemical conjugation approaches were applied to construct cvlps. in this strategy, target antigens and native vlps were generated individually and coupled together by attachment of the antigen to the surface of the pre-assembled vlps. two main advantages of this strategy include: (a) various sizes and types of antigens can be exposed, and (b) the antigen-vlp binding site can be manipulated for further presentation of the conjugated antigen. for example, vlps were used to display full-length and correctly folded proteins, such as interleukin-17 (il-17). 104 generally, vlps were used for delivery of protein/peptide, dna, sirna and drugs as a brief description in following: viral-like particles were used as a peptide/protein carrier, in vitro and in vivo. there are several examples for delivery of protein/peptide using vlps as following: a. chimeric vlp vaccines have been improved based on rna bacteriophage ap205, presenting peptides of selfantigens or pathogens fused to either the n-or cterminal regions of ap205 coat protein. ap205-derived vlps were highly immunogenic in mice. furthermore, influenza m2 vlps stimulated an efficient m2-specific antibody response and full protection against lethal influenza virus challenge. 176 b. vlps containing flt3 ligand (fl-vlps), a dc growth factor, could effectively increase immunogenicity in mice. dcs exposed to vlps also produced high levels of il-6. 177 c. a plant vlp-based approach was used to develop respiratory syncytial virus (rsv) vaccine. a target peptide displaying amino acids 170-190 of the rsv g protein was delivered on the surface of recombinant alfalfa mosaic virus (almv) particles. this construct induced high pathogen-specific immune responses in immunized animals. 178,179 d. in a recent study, a peptide from an external loop of mouse ccr5 protein was inserted into a neutralizing epitope of hpv l1. the particles generated by this chimeric l1 could elicit high levels of ccr5 antibodies that specifically recognized the surface of ccr5transfected cells and blocked in vitro infection of an mtropic hiv strain in mice. 161 in addition, chimeric vlps containing the full length hpv16 e7 oncoprotein linked to l2, or the n-terminal region of e7 fused to l1, could induce antigen-specific protection of mice from lethal challenge with e7-expressing tumor cells. 180-182 e. a pre-s1 epitope of hbv was also inserted into the ef loop of hpv vlp recognized by hbv-specific antibody. 6 chimeric vlps produced in e.coli carried a virus-neutralizing hbv pre-s1 epitope in the major immunodominant region (mir) and a highly conserved n-terminal hcv core epitope (aa 1 to 60) at the c-terminal region of the truncated hbv core vlps (hbc). the presence of two different foreign epitopes within the hbc molecule did not interfere with its vlp-forming potential, with the hbv pre-s1 epitope exposed on the surface and the hcv core epitope buried within the vlps. mice vaccination showed a specific t cell activation by both foreign epitopes and a highlevel antibody response against the pre-s1 epitope, whereas an antibody response against the hbc carrier was inhibited. 183 f. the researchers have shown that the nanosized hbc-vlps bearing mycobacterial antigen cfp-10 (hbc-vlp: cfp-10 fusion protein) induced an increased immune response in balb/c mice compared to mixtures of native antigen. 184 g. chimeric papillomavirus vlps based on the bovine papillomavirus type 1 (bpv-1) l1 protein were designed by replacing the 23-carboxyl-terminal amino acids of the bpv1 major protein l1 with a synthetic "polytope" minigene, containing known ctl epitopes of human pv16 e7 protein, hiv iiib gp120 p18, nef, and reverse transcriptase (rt) proteins, and an hpv16 e7 linear b epitope. the chimeric l1 protein assembled into vlps in insect cells. polytope vlps could deliver multiple b and t epitopes as immunogens to the mhc class i and class ii pathways. this study has demonstrated that hybrid vlps can be used as an efficient antigen delivery system to transfer more than one ctl epitope through mhc class i pathways. 185 h. the chimeric hpv vlps were generated in which hpv16 l2 neutralization epitopes (l2 residues 69-81 or 108-120) are inserted within an immunodominant surface loop (between residues 133 and 134) of the l1 major capsid protein of bpv1. immunization of rabbits with assembled particles elicited high l2-specific serum antibody responses. 186 193 l. the studies showed that the c-terminal region of gag fused by t cell epitopes from human cytomegalovirus pp65 led to the formation of hybrid vlps activating antigen-specific cd81 memory t cells ex vivo. 161 regarding to previous studies, the gag polyprotein is the only retroviral protein required for vlp formation. [194] [195] [196] vlps, derived from an avian retrovirus, were applied to deliver proteins to cells, either as part of gag fusion proteins (intracellular delivery) or on the surface of vlps. the construct is an effective system because the vlps are completely made of the gag fusion protein, and a single vlp will deliver 2000-5000 copies of gag fusion protein into a transduced cell. 197 delivery of foreign genes to the digestive tract mucosa by oral administration of non-replicating gene transfer vectors would be a very useful method for vaccination and gene therapy. 198 the studies indicated that plasmid dna could be packaged in vitro into a vlp composed of open reading frame 2 (orf2) of hev, which is an orally transmissible virus. these vlps could deliver this foreign dna to the intestinal mucosa in vivo, eliciting high mucosal and systemic immunity in mice, without the use of adjuvants. an orally administered hiv dna vaccine encapsulated in hev-vlps could induce mucosal and systemic cellular and humoral immune responses. 198 moreover, the ability of hpv vlps was examined to mediate delivery and expression of dna plasmids in vitro and in vivo. 199 hpv pseudoviruses were provided by disrupting hpv-vlp, mixing them with dna plasmids and reassembling them into the pseudoviruses (vlps with plasmids inside). the pseudovirus induced more potent immune responses than dna vaccines. the pseudovirus could be used in gene therapy by transferring the therapeutic genes into lymphoid tissues in human. 5 in addition, the recombinant hpv16 l1 vlps, produced in insect cells, could efficiently encapsulate a plasmid harboring either a gene for the gfp or b-galactosidase during in vitro disassembly-reassembly of vlps. 200 vlp-mediated delivery of a gfp reporter construct in vitro showed to be highly dependent on the presence of full-length l2 protein within the vlps. similarly, expression of gfp and luciferase reporter plasmids in vivo was efficiently enhanced by co-administration of l1/l2 vlps. in addition, co-administration of vlps with a hpv16 e6-expressing plasmid increased significantly e6-specific cellular immune responses. 201 the reports indicated that the recombinant major structural protein of the bk polyomavirus (bkv vp1) was shown to self-assemble into vlps with a diameter of 45-50 nm. the potential of bkv vp1 vlps was investigated to transfer gene into cos-7 cells using three methods for the formation of pseudovirions: disassembly/reassembly, osmotic shock and direct interaction between vlps and plasmid dna. the most efficient method is the direct interaction between vlps and linearized plasmid dna. the findings generally demonstrated that bkv vlps have exogenous dnabinding activity, as a promising vehicle for gene transfer studies. 200 sirna delivery there is a major challenge to identify novel approaches for specific and effective delivery of new types of drugs like sirnas and peptides. systemic delivery of small interfering rna (sirna) was restricted by its poor stability and low cellpenetrating properties. to overcome these limitations, an efficient sirna delivery system was designed using polyethyleneimine (pei)-coated vlps derived from adeno-associated virus type 2 (pei-aav2-vlps). generally, one of the strategies to integrate sirna into nanoparticles was to coat these particles with positively charged polymers, including pei, poly b-amino different applications of virus-like particles ester, or poly l-lysine. electrostatic coating could increase the efficiency of systemic sirna delivery due to its protective effects and improved cellular uptake. an insect/baculovirus expression system was used to generate aav2-vlps. pei-aav2-vlps could condense sirna, protect it from enzymatic degradation, transfer it with high efficiency and induce cell death in mcf-7 breast cancer cells, for breast cancer therapy. 201 furthermore, micrornas (mirnas) play an essential role in immunoregulation and may be involved in the pathogen esis of systemic lupus erythematosus (sle). among these sle-related mirnas, mir-146a, acts as a significant inhibitor of autoimmunity, myeloproliferation, and cancer. a novel mirna-delivery approach was described via bacteriophage ms2 vlps for evaluation of the therapeutic effects of mir-146a, in bxsb lupus-prone mice. treatment with ms2-mir-146a vlp increased the level of mature mir-146a, leading to a significant reduction in the expression of autoantibodies and total igg. furthermore, the levels of inflammatory cytokines, including ifn-a, il-1b and il-6 were decreased in mice. the stimulation of dysregulated mirnas by an ms2 vlp-based delivery system may be considered as a novel therapy. [202] [203] [204] the use of ms2 vlps was reported for selective delivery of nanoparticles, chemotherapeutic drugs, sirna cocktails, and protein toxins to human hcc. 205 in addition, the researchers used jc virus (jcv) vlps as a vector for delivering rnai in silencing the il-10 cytokine gene. jcv vlps were non-toxic, and showed the therapeutic use as a gene therapy approach for autoimmune diseases (aid) including sle. 206, 207 drug delivery a major challenge in pharmacology is to find methods that drugs (especially anti-cancer drugs) can be delivered specifically to target tissues. a potential strategy would be to package or encapsulate the drug molecules inside a particle which is bound to the cancerous tissue. such encapsulation would protect the drug from degradation in blood. for this purpose, it will be necessary to develop particles which can be modified on their outer surface to carry drug molecules into the target cells. novel nanocarriers such as dendrimers, liposomes, polymersomes, micelles, and vlps indicated high potency in improving drug delivery, and targeting strategies. all of these delivery systems make drugs more biocompatible, watersoluble, or colloidal, indicating low toxicity and high uptake in cells. 208 different virus-based materials were studied for drug delivery such as: the ccmv, the cpmv, the red clover necrotic mosaic virus (rcnmv), ms2 rna-containing bacteriophage, the bacteriophage qb, m13 bacteriophage, the tmv. 208 drug cargo can be loaded through covalent attachment of drugs or their analogs to particular reactive residues on the capsid pro-teins. 209 several cancer cell targeting ligands were attached to different types of vlps, including small molecules, antibodies, peptides and proteins, as well as dna aptamers. folic acid (fa) was broadly used in drug delivery targeted to cancer cells. uptake of fa into cells is mediated by the folate receptor (fr). 210 recently, lactobionic acid (la) was applied for the specific targeting of a rotavirus capsid vp6 to hepatocytes or hepatoma cells bearing asialoglycoprotein receptors (asgprs). 211 human holo-transferrin (tfn) is essential for iron homeostasis. tfn is especially recognized by the tfn receptor (tfnr), which is over-expressed on the surface of various tumor cells and efficiently taken up by cells in the clathrinmediated endocytosis. 212, 213 tfn has been conjugated to cpmv 214 and bacteriophage qb. 215 the cellular uptake of the qb-tfn particles was relative to the tfn density; while the internalization was prevented by comparable concentrations of free tfn. antibodies contain another group of targeting proteins that could be chemically linked to vlps. for instance, a single-chain (scfv) antibody that recognizes the carcinoembryonic antigen (cea) over-expressed in a variety of tumor cells, has been attached to cpmv. 216 an important strategy to improve cellular uptake of therapeutic molecules is the use of cell-penetrating peptides (cpps). 217 the hiv-1 tat peptide is one of the cpps that were extensively used in the delivery of vlps. 218, 219 in general, virus-like particles represents an attractive system for drug delivery in vitro. 220 the efficient delivery of hydrophobic drugs into target cells without the use of organic solvents or chemical linkage to delivery carriers is a critical issue in the biological field. recently, the intracellular delivery of hydrophobic dyes or drugs encapsulated in vlps through cyclodextrins (cds) showed high efficiency. as a model anticancer drug, paclitaxel (ptx)-cd complexes encapsulated inside vlps exhibited a dose-dependent cytotoxic effect with a 20-fold smaller ic50 than that of free ptx dissolved in dmso. 221 cell targeting is aimed to effective uptake of therapeutic and/ or diagnostic reagent in a special location such as a tumor. 222 targeting can also be achieved using proteins (mainly antibodies), peptides, nucleic acids (aptamers), small molecules, vitamins and carbohydrates. by attachment of targeting ligands, specificity for cell targeting was obtained by receptor-mediated endocytosis. for instance, bacteriophage ms2 vlps, were chemically conjugated to a targeting peptide (sp94) for the selective delivery of nanoparticles, chemotherapeutic drugs, sirna cocktails and protein toxins to human hcc. [223] [224] [225] recently, the chemical conjugation of human epidermal growth factor (egf) to simian virus 40 vlps allowed for cell 126 shirbaghaee and bolhassani selective targeting. 226 simian viruses 40 vlps have attracted a great attention in gene delivery due to their high stability and low toxicity in blood. 172 in design of polymeric nanoassemblies, chemical modification is necessary to conjugate the dye or probe for in vitro and in vivo imaging. however, in the case of nanobioassemblies, chemical or genetic modification can be applied for bioconjugation of fluorescent dyes or other probes. another advantage of nanobioassemblies such as vlps for bioimaging is their biological compatibility. quantum dots (qds) and gfp were used broadly for in vitro and in vivo imaging as alternatives to labelling. for example, fluorescent chimeric vlps of canine parvovirus were expressed in insect cells. 227 to create the fluorescent chimeric vlps of canine parvovirus, gfp was genetically engineered onto the n-terminal region of the viral protein vp2, as a visualization tool to understand mechanisms of viral infections. gfp was also used to design chimeric hiv vlps allowing protein to be followed during assembly and transmission using live-cell imaging. 228, 229 advantages of vlps include: (a) no need to propagate pathogenic organisms, (b) repetitive and ordered surface structures, (c) multivalent as well as particulate in nature, (d) safer than other vaccines because of non-infectious and non-replicating properties: the studies showed that there is no risk of disease progress in vaccinated groups with vlp-based vaccines as compared to attenuated viral vaccines, because they lack the genomic material needed for the replication and the spread of the viruses, (e) stable in extreme environmental conditions, depending on vlp structure (i.e., envelope or non-envelope), and (f) as carrier to express foreign antigen. 230 the potential of vlps to target dcs is a main advantage of vlp vaccines, for activating the innate and adaptive immune responses. they have a special benefit against other delivery systems in size, stability, and capacity to transfer biological molecules across cell barriers. particles in the 20-200 nm range can stimulate cd41, cd81 cells and especially generate th1 responses. in addition, despite a limited number of vlp vaccines approved for human use, they represent a promising platform for the development of novel mucosal vaccine strategies. indeed, vlps are sufficiently small, and the composition of their surface chemistry can be designed to minimize hydrophobic and electrostatic adhesive interactions with mucus. they can also be engineered for recombinant expression of multiple antigenic epitopes and for incorporation of co-stimulatory and immuno-regulatory proteins. however, vlp technology can be limited by difficulties of scale-up and the need for purification from the expression systems. 231 other limitation in chimeric vlp vaccine is to determine the compatibility of peptide with assembly of vlp and its immunogenicity property. under the host immune defence, pathogens undergo mutation which render the vlp vaccine ineffective and will be effective for only highly conserved b or t cell epitopes. 230 the major challenge is to develop novel production platforms that can deliver vlp vaccines while significantly reducing production times and costs. 104 viral-like particles (vlps) have shown high ability for the improvement of vaccines against infectious and non-infectious diseases. several recombinant expression systems were successfully applied for vlp production, with different efficiency. the use of vlps in vaccine development showed that they are considered safe. in addition, nano-sized vlps, can act as an adjuvant as well as antigen delivery system through increasing the antigen uptake by apcs. thus, it is not necessary for the use of adjuvants along with vlps to stimulate potent immune responses. vlps have shown a natural affinity to target host cells, and this property has been used for cell-targeting applications. regarding the advantages of vlps, it is necessary for further studies in various aspects especially easy and low-cost purification of vlps as well as their application as a delivery system in vivo. different applications of virus-like particles hum vaccine hepatitis b virus vaccine ip recombinant (genetically engineered): enivac hb hepavax-genev r . summary of product characteristics revac-b1tm. available at prescribing information. merck prescribing information. glaxosmithkline medicago to present additional positive clinical data at the 2011 eswi influenza conference medicago inc. news release exp rev vaccine hum vaccine immunother different applications of virus-like particles antimicrob agents chemother intranasal norwalk vaccine hum alves, p. m. exp rev vaccine exp rev vaccines different applications of virus-like particles curr top microbiol immunol the authors are grateful to elnaz agi and negar zohrei (dept. of hepatitis and aids, pasteur institute of iran) for technical assistance. key: cord-318143-s4q059g8 authors: cheng, zhikui; zhi, xiaoguang; sun, ge; guo, wei; huang, yayun; sun, weihua; tian, xiaohui; zhao, fei; hu, kanghong title: sodium selenite suppresses hepatitis b virus transcription and replication in human hepatoma cell lines date: 2015-10-16 journal: j med virol doi: 10.1002/jmv.24366 sha: doc_id: 318143 cord_uid: s4q059g8 hepatitis b virus (hbv) infection is one of the most serious and prevalent health problems worldwide. current anti‐hbv medications have a number of drawbacks, such as adverse effects and drug resistance; thus, novel potential anti‐hbv reagents are needed. selenium (se) has been shown to be involved in both human immunodeficiency virus and hepatitis c virus infections, but its role in hbv infection remains unclear. to address this, sodium selenite (na(2)seo(3)) was applied to three hbv cell models: hepg2.2.15 cells, and huh‐7 cells transfected with either 1.1 or 1.3× hbv plasmids. cytotoxicity of na(2)seo(3) was examined by cell counting kit‐8. levels of viral antigen expression, transcripts, and encapsidated viral dna were measured by enzyme‐linked immunosorbent assay, northern blot, and southern blot, respectively. there was no obvious cytotoxicity in either hepg2.2.15 or huh‐7 cells with <2.5 µm na(2)seo(3). below this concentration, na(2)seo(3) suppressed hbsag and hbeag production, hbv transcript level, and amount of genomic dna in all three tested models, and suppression level was enhanced in line with increases in na(2)seo(3) concentration or treatment time. moreover, the inhibitory effect of na(2)seo(3) on hbv replication can be further enhanced by combined treatment with lamivudine, entecavir, or adefovir. thus, the present study clearly proves that na(2)seo(3) suppresses hbv protein expression, transcription, and genome replication in hepatoma cell models in a dose‐ and time‐dependent manner. j. med. virol. 88:653–663, 2016. © 2015 wiley periodicals, inc. hepatitis b virus (hbv) infection is a major public health problem throughout the world, especially in east asia and africa. an estimated two billion people, roughly 30% of the world's population, have been infected with hbv, and over 350 million patients have chronic hepatitis b (chb) [liaw and chu, 2009] . each year, about 786,000 deaths are caused by hbv infection or its associated liver diseases [lozano et al., 2012] , including hepatic decompensation, cirrhosis, and hepatocellular carcinoma; hbv-infected individuals are at higher risk of all these hbv related diseases [lee et al., 2013] . although efficient prophylactic vaccines have been developed for prevention of hbv infection, there is still no cure for existing infection. the current clinical applied therapeutic agents for chb are mainly immunomodulators and nucleoside/nucleotide analogues, which can be used either separately or in combination [yuen and lai, 2001; papatheodoridis et al., 2008; scaglione and lok, 2012; trepo et al., 2014] . interferon alpha (ifn-a) is the best-known immunodulator, and controls hbv infection by stimulating the cellular antiviral cytokine expression to inhibit viral replication and by enhancing the host immune responses to eliminate the hbv-infected hepatocytes [yuen and lai, 2001] . however, its poor response against chb, high commercial cost, and various adverse effects (aes) limit the wide clinical application of this drug. multiple nucleoside (lamivudine, entecavir, and telbivudine) and nucleotide analogues (adefovir and tenofovir) have been approved for clinical application [liaw and chu, 2009 ]. these oral antiviral agents mainly target hbv polymerase and reverse transcriptase by rapidly and potently inhibiting their activities. although nucleoside/nucleotide analogues have fewer aes, they fail to eradicate hbv covalently closed circular dna (cccdna) in the nuclei of infected hepatocytes, which results in viral relapse after stopping the medication [yuen and lai, 2001; trepo et al., 2014] . in addition, the development of drug resistance during therapy also limits the effective application of nucleoside/nucleotide analogues. some of the most recent drugs, such as entecavir and tenofovir, have been observed to induce only a very low level of drug resistance, but long-term application may still induce expansion of hbv strains with mutations resisting these drugs [trepo et al., 2014] . thus, developing novel effective therapeutic approaches and agents is a necessary step against hbv infection. selenium (se) is an essential trace mineral for animals and humans. in addition to antioxidant activity, se has also shown anti-inflammatory and anticancer properties [rayman, 2000] . se functions in either its inorganic forms, such as sodium selenite (na 2 seo 3 ) and sodium selenate, or its organic forms, such as the amino acids selenocysteine (sec) and selenomethionine (semet), and multiple selenomolecles as intermediary metabolites [papp et al., 2007] . upon absorption, the inorganic salts in their oxidized forms [selenite (se 4þ ) or selenate (se 6þ )] can be reduced to selenide (se 2à ) by using reducing equivalents from reduced glutathione and reduced nicotinamide adenine dinucleotide phosphate (nadph) [zeng and combs, 2008] . as a storage mechanism, semet can replace normal methionine in protein synthesis, and then be released reversibly by the normal metabolic process as necessary [schrauzer, 2000] . sec is probably the most abundant biologically active form of selenium in vivo, and can be specially incorporated into selenoproteins as the 21st amino acid encoded by the uga codon [metanis and hilvert, 2014] . importantly, sec might play some important biological roles in vivo, as most of the 25 identified human selenoproteins have shown enzymatic redox function with catalytic or antioxidant activities conferred by sec [kryukov et al., 2003; papp et al., 2007] . besides the biological functions described above, se is also involved in the occurrence, virulence, and disease progression of some viral infections [rayman, 2000; moghadaszadeh and beggs, 2006 ]. for some rna viruses, including coxsackievirus b3 (cvb3/0) (cause of keshan disease), human immunodeficiency virus (hiv), influenza a virus, sars coronavirus, and ebola virus, the absence of se causes accumulation of mutations in their genome, leads to changes in the virulence-associated genetic structures [beck et al., 2003; harthill, 2011] . viral glutathione peroxidase (vgpx), a selenoprotein with antioxidant activity, has been found to be encoded by some rna virus such as hiv and hepatitis c virus [zhang et al., 1999] . in virus-infected cells, vgpx inhibits ros-induced apoptosis, a host response against infection, and further promotes viral replication [papp et al., 2007] . to date, only a few reports have described the possible relevance of se to hbv infection [yu et al., 1997; khan et al., 2012] , but direct evidence is still lacking, and the underlying mechanism remains to be elucidated. in the present study, hepatoma cell hepg2.2.15 and huh-7 transfected with two different hbv plasmids were utilized in order to investigate the effect of na 2 seo 3 on hbv. the data clearly clarify that application of na 2 seo 3 inhibits hbsag/ hbeag expression, hbv transcription, and genome replication in the examined models. this is the first report with direct evidence confirming the suppressive effect of se on hbv replication. the human hepatoma cell lines hepg2.2.15 and huh-7 were obtained from the china center for type culture collection (cctcc), and maintained in dulbecco's modified eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 mg/ml penicillin, and 100 mg/ml streptomycin (all from gibco life technologies) at 37˚c in a 5% co 2 incubator. hepg2.2.15 is derived from hepg2 cells by being stably transfected with a construct containing two head-to-tail dimers of the hbv genome (genbank accession: u95551.1), so 200 mg/ml g418 was also added to maintain the cell line. plasmid pch-9/3091 (1.1âhbv) is an hbv construct containing 1.1 copies of the hbv genome (subtype ayw) driven by the human cytomegalovirus (hcmv) immediate early i protein (ie1) promoter [nassal, 1992] (kind gift from dr michael nassal), and p1.3hbv (1.3âhbv) is another hbv construct generated by inserting 1.3 copies of the hbv genome (subtype adw), starting from the enhancer-i-x promoter region, into pgem-3z [doitsh and shaul, 2003 ] (kind gift from dr. yosef shaul). to study viral replication, plasmid pch-9/3091 and 1.3âhbv were transfected into 3 â 10 6 huh-7 cells in 6 cm plates using lipofectamine tm 2000 (invitrogen, waltham, ma) according to the manufacturer's instructions. after hepg2.2.15 was cultured for 24 hr or huh-7 was transfected for 24 hr, then the medium was replaced by fresh medium with na 2 seo 3 or 30 nm entecavir (etv) (both sigma aldrich, st. louius, mo), which had been dissolved in 1â sterile phosphate-buffered saline (pbs). the treatment starting day was defined as day 0, and samples were collected at the indicated time points. hepg2.2.15 and huh-7 cells (1â10 4 cells/well) were cultured in 96-well plates, and na 2 seo 3 at the indicated concentrations was added to the culture 24 h later. the cells were harvested at the indicated time points, and assayed by colorimetry (cell counting kit-8; dojindo laboratories). to quantify hbsag and hbeag production, the supernatants of the cells with the indicated treatment were collected, and tested for the presence of hbsag and hbeag using a commercial enzymelinked immunosorbent assay (elisa) kit (kehua bio-engineering). the results were normalized to the mock-treatment (pbs) control sample. cells were washed twice with pbs and homogenized in trizol (invitrogen). total rna was isolated according to the manufacturer's protocol. potential dna contamination in the extraction was eliminated with recombinant rnase-free dnase i (takara). samples were incubated at 65˚c for 5 min for denaturation, and then 20 mg rna was electrophoresed for 3 hr at 100 v in 1.2% (w/v) agarose gel in the presence of formaldehyde. as described previously [tian et al., 2013] , the resolved rna was then blotted onto a positively charged nylon membrane, and hybridized with a 32 p-labeled random-primed probe specific for the hbv genome. the visualized image was obtained using cyclone plus storage phosphor system (perkinelmer, waltham, ma), and the cell level in the northern blot assay were normalized to 28s/18s rna. intracellular viral core dna was isolated using the method described previously [feng et al., 2013] . briefly, cells were lysed in either 1 ml (hepg2.2.15, 10 7 cells per 10 cm plate,) or 0.6 ml (huh-7, 3â10 6 cells per 6 cm plate) lysis buffer (50 mm tris-hcl ph 7.5, 140 mm nacl, 0.5% np-40). after removing the cell debris by centrifugation, the supernatants were treated with 60 u dnase i (takara) and 5 ml rnase a (takara), and then incubated at 37˚c for 2 hr in the presence of 8 mm mgcl 2 to completely digest the non-encapsidated dna and rna. core particles were degraded with 200 mm proteinase k (tiangen), and encapsidated dna was extracted with phenol chloroform and precipitated by ethanol. following separation in a 1% agarose gel (w/v), the isolated encapsidated hbv dna was blotted onto a positively charged nylon membrane (millipore) and hybridized with the same probe used for the northern blot assay. finally, the signals were visualized using cyclone plus storage phosphor system (perkin elmer). the cell level in the southern blot assay was normalized to the b-actin protein level. reaction (qpcr) the quantitative real-time polymerase chain reaction was performed with the sybr premix ex taq ii (takara) using a lightcycler 96 real-time pcr system (roche, penzberg, germany). the hbv genome sequence (genebank accession no. dq219811) was amplified with primer 5 0 -acc aat cgc cag tca gga ag-3 0 and 5 0 -acc agc agg gaa ata cag gc-3 0 ; and b-actin (genebank accession no. nm_001101), serving as the internal reference, was amplified with primer 5 0 -cat gta cgt tgc tat cca ggc-3 0 and 5 0 -ctc ctt aat gtc acg cac gat-3 0 . the cycling program was run at 95˚c for 5 min, followed by 45 cycles at 95˚c for 10 sec, 60˚c for 10 sec, and 72˚c for 10 sec. all results for the toxicity assay and elisa are shown as mean ae standard error from at least three independent experiments. statistical differences were analyzed using unpaired two-tailed student's t tests. differences were considered to be statistically significant at p < 0.05. results of southern or northern blot assays are representative images selected from at least three independent experiments. to eliminate the possibility that any observed influences of na 2 seo 3 on hbv replication were caused by cytotoxicity, the influence of na 2 seo 3 on huh-7 and hepg2.2.15 proliferation was examined using cck-8 assay. no significant toxicity of na 2-seo 3 on huh-7 cells was observed at concentrations 2.5 mm, whereas na 2 seo 3 treatment beyond this concentration displayed inhibition of huh-7 growth in a dose-dependent manner (fig. 1a) . hepg2.2.15 cells showed greater tolerance against na 2 seo 3 treatment. treatment with 10 mm na 2 seo 3 for three days and 5 mm na 2 seo 3 for five days did not change the growth of hepg2.2.15 compared with the mocktreated control. beyond this concentration, the cell numbers of hepg2.2.15 decreased gradually, indicating cytotoxicity of na 2 seo 3 (fig. 1b) . for both cell types, no significant differences were observed during the na 2 seo 3 treatment period at or below the safe concentration of 2.5 mm for huh-7 and 5 mm for hepg2.2.15. however, the numbers of viable cells were obviously decreased during the treatment period beyond the safe concentration ( fig. 1a and b) . for huh-7, the decline in cell growth at one, three, and five days was relatively consistent (fig. 1a) , but hepg2.2.15 treated for five days showed a more dramatic decrease in cell growth compared with days one and three (fig. 1b) . hence, 2.5 mm, which showed no significant cytotoxicity for either cell type, was set as the maximum concentration of na 2 seo 3 treatment for subsequent experiments. to study the effect of na 2 seo 3 on hbv viral protein production, hepg2.2.15 cells or hbv plasmid-transfected huh-7 cells were treated with na 2 seo 3 . four concentrations, 0.5, 1.5, 2.0, and 2.5 mm, of na 2 seo 3 were investigated for cytotoxicity (fig. 1) . pbs and 30 nm etv were used as controls. at three days posttreatment (huh-7) or post-culture (hepg2.2.15), supernatants of the cultures were collected, and hbsag and hbeag expression were measured by elisa. for hepg2.2.15, 0.5 mm na 2 seo 3 was enough to cause a significant reduction in hbsag and hbeag expression compared with the mock-treated control, and na 2 seo 3 at the examined concentrations inhibited hbv antigen production in hepg2.2.15 in a dosedependent manner ( fig. 2a and b) . in huh-7 cells transfected with 1.1â hbv plasmid, 0.5 mm na 2 seo 3 failed to decrease hbsag and hbeag levels. a dose-dependent suppression was observed, starting from 1.5 mm na 2 seo 3 ( fig. 2c and d) . viral antigen in 1.3â hbv plasmid-transfected huh-7 cells showed even stronger resistance to na 2 seo 3 . hbsag and hbeag levels started to decline with 2 mm na 2-seo 3 , and the decline was further enhanced by 2.5 mm na 2 seo 3 treatment ( fig. 2e and f) . next, a fixed concentration (2.5 mm) of na 2 seo 3 was used to assess viral antigens expression over a time course. for hepg2.2.15 cells, 2.5 mm na 2 seo 3 showed significant inhibition of hbv antigen expression from day 2, and these repressive effects increased with time ( fig. 3a and b) . on the final assessment day (day 4), 2.5 mm na 2 seo 3 treatment had caused a reduction of 62.81 ae 2.59% and 54.69 ae 7.88% in production of hbsag and hbeag, respectively. a similar time-dependent suppression enhancement was also observed in hbv plasmidtransfected huh-7 cells, with the maximum decrease in viral antigen levels also occurring on day 4. compared with the mock-treated control, 2.5 mm na 2 seo 3 treatment in huh-7 cells transfected with 1.1 or 1.3â hbv plasmid produced a drop in hbsag level to 41.16 ae 10.55% and 42.14 ae 10.15% ( fig. 3c and e) , respectively, and a drop in hbeag level to 29.73 ae 8.33% and 32.32 ae 9.03%, respectively ( fig. 3d and f). there was no obvious decrease in 1.3â hbv plasmid-transfected huh-7 cells on day 1, but at this time point, hbeag was already significantly suppressed in 1.1â hbv plasmid-transfected huh-7 cells (fig. 3d) . notably, etv treatment did not affect viral antigen production in any of the models (figs. 2 and 3) . because hbsag and hbeag are products of pres/s mrnas and precore mrna, levels of various hbv mrnas were further analyzed in hepg2.2.15 and 1.1/1.3â hbv plasmid-transfected huh-7 cells in the presence of na 2 seo 3 . four concentrations, 0.5, 1.5, 2.0, and 2.5 mm, of na 2 seo 3 were used, and the concentration of 2.5 mm was further analyzed at different time points. the hbv rnas in all the examined models were decreased by na 2 seo 3 treatment, but with slight differences. in hepg2.2.15 cells, 0.5 mm na 2 seo 3 had no obvious effect on viral transcription, but 1.5 mm dramatically decreased hbv rna level, which was further decreased with 2.0 mm na 2 seo 3 treatment (fig. 4a) . when the concentration was fixed at dose-dependent suppression of hbsag/hbeag expression by na 2 seo 3 . hepg2.2.15 and 1.1 â or 1.3â hbv-transfected huh-7 cells were treated with na 2 seo 3 at the indicated concentrations or with 30 nm etv. on day 3, culture supernatants were collected and assayed for hbsag and hbeag concentration by elisa. all data were normalized to the mock-treated control. ã p < 0.05; ãã p < 0.01. fig. 3 . time-dependent suppression of hbsag/hbeag expression by na 2 seo 3 . hepg2.2.15 and 1.1âor 1.3â hbv-transfected huh-7 cells were treated with 2.5 mm na 2 seo 3 or 30 nm etv. supernatants of the na 2 seo 3 -treated cultures were collected daily for four days, and etvtreated supernatant was harvested on day 4. hbsag and hbeag concentrations were measured by elisa. all data were normalized to the mock-treated control. ã p < 0.05; ãã p < 0.01. 2.5 mm, progressive repression by na 2 seo 3 of hbv rna level in hepg2.2.15 cells occurred in line with the length of treatment, starting from day 1 (fig. 4a) . moreover, the viral transcription level in hepg2.2.15 was quantified using qpcr, and the host gene b-actin was chosen as the internal reference. the result of qpcr (fig. s1 ) was consistent with northern blot assay (fig. 4a ). viral mrna was inhibited by na 2 seo 3 treatment in dose-dependent and time-dependent manners, which was reduced to 49.28 ae 3.82% in the presence of 2.5 mm na 2 seo 3 for four days. in huh-7 cells transfected with 1.1âhbv plasmids, there was a dose-dependent suppression of viral mrna by na 2 seo 3 (fig. 4b ). in addition, perceptible repression of hbv transcription was observed from day 2 of 2.5 mm na 2 seo 3 treatment, which also occurred in a time-dependent manner (fig. 4b) . na 2 seo 3 -induced suppression of hbv transcripts also occurred in huh-7 cells transfected with 1.3âhbv plasmids. as shown in figure 4c , suppression was evident with as little as 0.5 mm na 2 seo 3 , and progressed in a dose-dependent manner. na 2-seo 3 treatment at 2.5 mm caused a dramatic suppression of hbv rnas from day 1; however, the timedependent suppression effect was not as clear as in the other two models. this was probably because the rna amount had already dropped to a very low level, and therefore the effects at the subsequent examined time points were less obvious (fig. 4c) . in all models, 30 nm etv treatment produced no effect on hbv transcription levels, because it specifically inhibits protein-priming activity [jones et al., 2013] . intracellular viral core protein was also determined using western blot assay in all three models, with the host protein b-actin as the internal reference. core protein level was decreased gradually following na 2-seo 3 treatment in a dose-dependent manner, whereas b-actin level remained unchanged (fig. s2) . but 30 nm etv treatment could not affect core expression in all models (fig. s2) . the result indicated that na 2 seo 3 influence specifically hbv transcription and expression. the fact that na 2 seo 3 repressed hbv protein synthesis and transcription in a dose-dependent and time-dependent manner prompted us to investigate whether it also influenced hbv dna levels. to address this issue, the encapsidated hbv dna was extracted from the cells treated with na 2 seo 3 or etv, and analyzed by southern blot assay. in all models, the viral nucleic acids were barely detectable after 30 nm etv treatment for three days (fig. 5a , c, and e) or four days (fig. 5b, d, and f) . considering that etv hardly changed the level of viral proteins or rnas, the dramatic inhibition of hbv dna was consistent with its specific suppression of hbv replication. a previous study also reported that 30 nm etv was sufficient to reduce hbv dna level by 90% [bader and korba, 2010] , which is in agreement with this result. in general, na 2 seo 3 treatment displayed a clear dose-dependent and time-dependent suppression of hbv dna level in all models (fig. 5) , although some slight differences still existed between the different models. treatment with na 2 seo 3 resulted in a very obvious repression of hbv dna in hepg2.2.15 cells. hepg2.2.15 and 1.1 or 1.3âhbv-transfected huh-7 were treated with na 2 seo 3 . partial samples were treated with various concentrations of na 2 seo 3 and harvested on day 3, and some samples were treated with a fixed concentration of 2.5 mm na 2 seo 3 , but harvested at different time points. mock-treated control and 30 nm etv-treated cells were collected on day 4. hbv rna levels were determined by northern blot assay. fig. 5 . suppression of hbv genome replication by na 2 seo 3 . hepg2.2.15 and 1.1 or 1.3âhbvtransfected huh-7 were treated with na 2 seo 3 . partial samples were treated with various concentrations of na 2 seo 3 and harvested on day 3, and some samples were treated with a fixed concentration of 2.5 mm na 2 seo 3 but harvested at different time points. mock-treated control and 30 nm etv-treated cells were collected on day 4. hbv dna levels were determined by southern blot assay. clear dna decline occurred with 0.5 mm na 2 seo 3 treatment on day 4, as well as with 2.5 mm na 2 seo 3 on day 1. the hbv dna levels then reduced progressively in line with increasing concentration and exposure time ( fig. 5a and b) . in huh-7 transfected with 1.1âhbv plasmid, na 2 seo 3 treatment resulted in a dose-dependent inhibition of hbv dnas, starting at 1.5 mm. time-dependent reduction of hbv dnas was also observed, but this was less obvious than that in hepg2.2.15 or 1.3âhbv plasmid-transfected huh-7. this was possibly due to the profound decrease in viral dna level observed as early as day 1 (fig. 5d) . in 1.3âhbv plasmid-transfected huh-7 cells, the decrease induced by na 2 seo 3 was weaker than in the other two tested models. the reduction in hbv dna was visible with 2.0 mm na 2 seo 3 treatment on day 3 (fig. 5e ) or with 2.5 mm on day 2 (fig. 5f ). to further investigate its anti-hbv activity, na 2-seo 3 was compared with clinically applied nucleos(t) ide analogs, including lamivudine (3tc), entecavir (etv), and adefovir (ade). after three or six days' treatment with single or combine drugs, encapsidated hbv dna levels were quantified using qpcr in hepg2.2.15. the result showed three days treatment with 2.5 mm na 2 seo 3 , 70 nm lamivudine, 9 nm entecavir, and 1.2 mm adefovir reduced viral dna levels to 48.97 ae 2.30%, 42.16 ae 2.70%, 17.62 ae 1.22%, and 13.15 ae 0.95%, respectively (fig. 6) . on day 6, the inhibitory effect on dna levels were further enhanced in na 2 seo 3 and lamivudine treated cells, with hbv dna levels decreasing to 24.53 ae 11.09% and 24.56 ae 0.07%, respectively (fig. 6) . moreover, combined drug treatment dramatically enhanced the inhibitory effect on hbv dna replication. on day 6, na 2 seo 3 plus lamivudine, entecavir, or adefovir reduced the encapsidated hbv dna levels to 5.49 ae 0.11%, 0.44 ae 0.04%, and 1.92 ae 0.07%, respectively (fig. 6) . hepatitis b virus, a prototype member of hepadnaviridae family, replicates by a unique protein-primed reverse transcription mechanism [nassal, 2008] . infectious virions contain the genome as a 3.2 kb relaxed circular dna (rc dna), which is transformed into a plasmid-like cccdna in the host cell nucleus [beck and nassal, 2007] . the two subsets of viral rnas, namely, the subgenomic rnas (pres/s mrnas and hbx mrna), and the greater-thangenome-length pregenomic rna (pgrna) and precore mrna, are transcribed by cellular rna polymerase ii using cccdna as the template [beck and nassal, 2007] . although se has been reported to influence multiple virus infections [rayman, 2000; beck et al., 2003; beck, 2007; harthill, 2011] , only limited information is available about whether it has any effect on hbv infection. one study has shown significantly lower concentrations of se in serum from patients with hbv infection compared with healthy individuals [khan et al., 2012] . moreover, according to an eight-year follow-up investigation of 130,471 individuals in qidong, china, se supplementation via table salt significantly reduced the incidence of primary liver cancer [yu et al., 1997] . however, none of the available studies showed solid evidence of the relationship between se and hbv replication. the present study is the first to demonstrate direct inhibitory effects of na 2 seo 3 on hbv at the cellular level, using different hbv cell models. both hbeag and hbsag are secreted to serum from hbv-infected hepatocytes, but appear at different phases of infection, and have different clinical implications [papatheodoridis et al., 2008; trepo et al., 2014] . hbsag is a hallmark of infection, although hbeag is usually associated with high levels of viral replication. although nucleoside/nucleotide analogues can inhibit hbv replication and result in obvious reductions in hbv dna and hbeag levels in serum, they have little impact on hbsag secretion [scaglione and lok, 2012] . the present study revealed evident declines in hbsag/ hbeag levels with na 2 seo 3 treatment in all three tested models (figs. 2 and 3) . however, it must be noted that the hcmv-promoter driven 1.1âhbv construct should not allow for the synthesis of hbeag because of the lack of intact precore region at the 5' end [nassal, 1992] . and hbeag detected in huh-7 transfected with 1.1âhbv plasmid might be core proteins due to antibody cross reactivity. so, intracellular core protein level was also determined in these models, and the result showed na 2 seo 3 treatment obviously reduced core expression in host cell, but not the internal reference protein (fig. s2) . these results prompted us to further investigate the reason of the viral antigen decreases at the hbv transcription level. many published research articles have revealed that se regulates various physiological activities of cells, such as gene transcription, signaling transduction, and cell apoptosis [mckenzie et al., 2002; fig. 6 . combined treatment of na 2 seo 3 with clinically applied nucleos(t) ide drugs. hepg2.2.15 were treated with 2.5 mm na 2 seo 3 (se) alone or in combination with lamivudine (3tc, 70 nm), entecavir (etv, 9 nm) or adefovir (ade, 1.2 mm) for three days and six days. intracellular dna of samples were extracted and viral dna was determined using qpcr. all data were normalized to the mock-treated control. ãã p < 0.01. zeng, 2009; sunde and raines, 2011] . na 2 seo 3 was shown by northern blot assay and qpcr to inhibit hbv transcription in a dose-dependent and timedependent manner (figs. 4 and s1). as hbv mrnas synthesis is primarily dependent on rna polymerase ii and the transcriptional regulation system of the host cells [levrero et al., 2009; quasdorff and protzer, 2010] , it is likely that the following cellular factors were possibly involved in the observed suppression. the first potential candidate is p53. na 2 seo 3 treatment may activate p53 by promoting its expression and phosphorylating multiple sites [smith et al., 2004] . both p53 and its homologue p73 have been reported to repress the activities of hbv promoters and enhancers [lee et al., 1998; buhlmann et al., 2008] . hence, p53 is a possible intermediary between na 2 seo 3 and hbv transcription. the second possible factor is specificity protein 1 (sp1), which binds to guanine-cytosine-rich dna elements and regulates a wide variety of genes in mammalian cells [chu, 2012] . at least three binding sites have been identified in the hbv genome, sp1 activates the transcription of hbv genes by binding to these sites [quasdorff and protzer, 2010] . se has been demonstrated to decrease sp1 expression and activity [husbeck et al., 2006] , and prevent activation of sp1-regulated hbv genes. therefore, se is likely to affect hbv transcription through a complicated regulatory network. finally, a trans-acting factor, hbx, encoded by the hbv genome, is a multifunctional viral protein and is broadly involved in transcription, signal transduction, cell cycle progress, apoptosis, genetic stability, and oncogenesis through protein-protein interaction [bouchard and schneider, 2004; tang et al., 2006; ng and lee, 2011; motavaf et al., 2013] . tang et al. reported that both hbv transcription and replication can be affected simultaneously by hbx, but the transcriptional activation of hbx may be critical for its augmenting effect on hbv replication [tang et al., 2006] . hbx also induces cytosolic calcium elevation and viral dna replication, but not viral transcription, by interacting with bcl-2 family members [geng et al., 2012] or by activating cytosolic calcium-dependent proline-rich tyrosine kinase-2 (pyk2) [bouchard et al., 2001] . although se has not been proved to have direct effects on hbx, it has been demonstrated to affect hbx-related host factors, including p53 and nuclear factor-kb, and to result in a significant decrease in cellular calcium release [uguz and naziroglu, 2012] . thus, hbx is another suspected viral factor that possibly mediates selenite-induced inhibition of hbv transcription and replication. the southern blot assay shown in figure 5 showed an evident decline in hbv dna level with na 2 seo 3 treatment in all three tested models. however, there were dissimilarities in selenite-induced suppression in the three models, possibly due to the differences in their cell lines and hbv-expressing systems. the progressive reduction in hbv dna displayed a similar trend to the decrease in hbv mrnas, which indicated that one potential mechanism of na 2 seo 3 in suppressing hbv replication is by inhibition of viral transcription. in addition to suppressing viral transcription, se may also inhibit hbv replication by cytosolic calcium release, which is related to hbx in host cells [bouchard et al., 2001; geng et al., 2012] . the hypothesis that selenite inhibits hbv transcription and replication via host and viral factors still needs to be verified, and the potential involved factor (s) need to be identified by further experiments. in addition, the anti-hbv efficacy of na 2 seo 3 was quantitatively compared with nucleos(t) ide analogues using qpcr, alone or in combination (fig. 6) . on day 6, na 2 seo 3 , in spite of a higher treating concertation, exhibited similar efficacy to the other drugs. the less potent of na 2 seo 3 on inhibiting hbv replication compared to the other tested drugs is probably due to its indirect effect on viral polymerase. however, as na 2 seo 3 and the nucleos(t) ide drugs affected different factors in the replication cycle of hbv, their combination showed more efficacy than either treatment alone did (fig. 6) . in conclusion, these data to date, for the first time, directly prove that na 2 seo 3 inhibits hbsag/hbeag expression, hbv transcription, as well as viral genome replication. this hbv suppressing effect has made se a potential therapeutic medication to control hbv infection. zc and xz performed the experiments. zc, fz, and kh designed the research. gs, gw, ws, yh, and xt provided experimental support. zc and fz drafted the manuscript. all authors read and approved the final manuscript for submission. simvastatin potentiates the anti-hepatitis b virus activity of fda-approved nucleoside analogue inhibitors in vitro hepatitis b virus replication selenium and vitamin e status: impact on viral pathogenicity selenium deficiency and viral infection the enigmatic x gene of hepatitis b virus calcium signaling by hbx protein in hepatitis b virus dna replication molecular mechanism of p73-mediated regulation of hepatitis b virus core promoter/enhancer ii: implications for hepatocarcinogenesis transcriptional regulation by post-transcriptional modification-role of phosphorylation in sp1 transcriptional activity a long hbv transcript encoding px is inefficiently exported from the nucleus evidence for multiple distinct interactions between hepatitis b virus p protein and its cognate rna encapsidation signal during initiation of reverse transcription hepatitis b virus x protein targets bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction review: micronutrient selenium deficiency influences evolution of some viral infectious diseases inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action noncompetitive inhibition of hepatitis b virus reverse transcriptase protein priming and dna synthesis by the nucleoside analog clevudine the possible role of selenium concentration in hepatitis b and c patients characterization of mammalian selenoproteomes liver-specific enhancer ii is the target for the p53-mediated inhibition of hepatitis b viral gene expression prediction models of long-term cirrhosis and hepatocellular carcinoma risk in chronic hepatitis b patients: risk scores integrating host and virus profiles control of cccdna function in hepatitis b virus infection hepatitis b virus infection selenium and the regulation of cell signaling, growth, and survival: molecular and mechanistic aspects natural and synthetic selenoproteins selenoproteins and their impact on human health through diverse physiological pathways hepatitis b virus-induced hepatocellular carcinoma: the role of the virus x protein the arginine-rich domain of the hepatitis b virus core protein is required for pregenome encapsidation and productive viral positive-strand dna synthesis but not for virus assembly hepatitis b viruses: reverse transcription a different way hepatitis b virus x gene and hepatocarcinogenesis therapeutic strategies in the management of patients with chronic hepatitis b virus infection from selenium to selenoproteins: synthesis, identity, and their role in human health control of hepatitis b virus at the level of transcription the importance of selenium to human health effectiveness of hepatitis b treatment in clinical practice selenomethionine: a review of its nutritional significance, metabolism and toxicity selenium compounds regulate p53 by common and distinctive mechanisms selenium regulation of the selenoprotein and nonselenoprotein transcriptomes in rodents molecular functions and biological roles of hepatitis b virus x protein gcn5 acetyltransferase inhibits pgc1alpha-induced hepatitis b virus biosynthesis hepatitis b virus infection effects of selenium on calcium signaling and apoptosis in rat dorsal root ganglion neurons induced by oxidative stress protective role of selenium against hepatitis b virus and primary liver cancer in qidong treatment of chronic hepatitis b selenium as an essential micronutrient: roles in cell cycle and apoptosis selenium as an anticancer nutrient: roles in cell proliferation and tumor cell invasion selenium-dependent glutathione peroxidase modules encoded by rna viruses we thank dr michael nassal for kindly providing the pch-9/3091 plasmid and dr yosef shaul for kindly providing the 1.3â hbv plasmid. key: cord-318570-wj7r6953 authors: xiao, yinzong; thompson, alexander j.; howell, jessica title: point-of-care tests for hepatitis b: an overview date: 2020-10-02 journal: cells doi: 10.3390/cells9102233 sha: doc_id: 318570 cord_uid: wj7r6953 despite the heavy disease burden posed by hepatitis b, around 90% of people living with hepatitis b are not diagnosed globally. many of the affected populations still have limited or no access to essential blood tests for hepatitis b. compared to conventional blood tests which heavily rely on centralised laboratory facilities, point-of-care testing for hepatitis b has the potential to broaden testing access in low-resource settings and to engage hard-to-reach populations. few hepatitis b point-of-care tests have been ratified for clinical use by international and regional regulatory bodies, and countries have been slow to adopt point-of-care testing into hepatitis b programs. this review presents currently available point-of-care tests for hepatitis b and their roles in the care cascade, reviewing evidence for testing performance, utility, acceptability, costs and cost-effectiveness when integrated into hepatitis b diagnosis and monitoring programs. we further discuss challenges and future directions in aspects of technology, implementation, and regulation when adopting point-of-care testing in hepatitis b programs. more than 257 million people, or 3.2% of the world's population, are estimated to be living with chronic hepatitis b virus infection, with the greatest disease burden in low-resource countries in the asia-pacific and sub-saharan africa [1] . without treatment, one in every four persons infected with chronic hepatitis b will develop liver cirrhosis over 20-30 years, and 2-5% of people with cirrhosis will develop liver cancer annually [2] . globally, over 800,000 deaths annually are attributable to hepatitis b infection [1, 3] . most of this disease burden is preventable by appropriate guideline-based treatment [4] [5] [6] [7] [8] . given the magnitude of the global public health burden from hepatitis b, the world health organization (who) has outlined ambitious hepatitis b elimination targets of a 65% reduction in mortality and a 90% reduction in incidence from baseline (2015) by 2030 [9] . however, current estimates suggest that we are a long way from achieving these goals unless investment and care cascade are scaled up [1, 10] . the hepatitis b vaccination has greatly contributed to preventing transmission and reducing hepatitis b incidence globally; however, vaccination coverage is still suboptimal in resource-limited regions [11] , and most countries in africa have been unable to implement the hepatitis b birth-dose vaccine due to multiple logistical and cost barriers [12, 13] . meanwhile, for people who are already living with hepatitis b, receiving early diagnosis and clinical care is the key to reducing morbidity and mortality. however, in 2016, the who estimated that only 11% of people living with hepatitis b were diagnosed, among whom only 17% of those eligible were on treatment [1]. the hepatitis b cascade of care involves multiple steps: screening, diagnosis, linkage to care, assessment of liver disease stage and treatment eligibility, then treatment and/or monitoring, including surveillance for hepatocellular carcinoma (hcc) (figure 1) . laboratory blood tests are required at every step of the care cascade, including blood tests for hepatitis b serology, quantitative hepatitis b virus (hbv) dna level by polymerase chain reaction (pcr) and liver function tests (figure 1 ). these tests require laboratory resourcing, technology and expertise beyond existing peripheral laboratory capabilities in many low-resource and geographically isolated regions [14] [15] [16] . in many countries, laboratory services are centralised due to high costs and limited skilled technician capacity; however, transport of blood samples from regional to centralised laboratories presents its own challenges in geographically isolated or insecure regions, particularly if cold chain supply must be preserved [14] . cost is another major limitation: price reductions for diagnostics have fallen slowly over time compared with medication costs, and hepatitis b diagnostic tests cost more than therapy in many low-income countries [16, 17] . moreover, the requirement for lifelong monitoring for most people living with hepatitis b that involves regular blood tests [4, 5] , combined with barriers to timely healthcare access such as hepatitis b-related stigma [18, 19] , healthcare costs for users and providers [20] and the logistics of accessing consistent, high-quality, affordable healthcare services in a timely manner are major barriers for people to receive guideline-based care [16] . these barriers lead to significant attrition from every step of the hepatitis b care cascade over time, and those lost from care represent missed opportunities for treatment and liver cancer prevention [16, 21] . laboratory-based blood tests are required at every stage of the hepatitis b cascade of care for diagnosis, assessment of liver disease stage, treatment eligibility and long-term monitoring of disease progression. diagnostic testing for hepatitis b involves detection of hepatitis b surface antigen (hbsag) in blood, which indicates active infection with the virus. standard laboratory electro-chemiluminescence immunoassay-based hbsag testing is performed on serum or plasma samples derived from whole blood. if active infection is confirmed, subsequent blood tests are performed to determine the stage of disease and need for treatment, including a hepatitis b virus (hbv) polymerase chain reaction (pcr)-based quantitative dna level or viral load, a hepatitis b eag and eab assay and liver function tests to determine whether an elevated aminotransferase (alt) indicative of liver inflammation or other signs of impaired liver function are present. further assessment for the presence of liver fibrosis and cirrhosis is also required, most commonly by transient elastography and/or liver biopsy. all patients irrespective of treatment require ongoing disease monitoring, including at minimum an hbv dna level, hbeag and hbeab (if not already seroconverted from hbeag positive to hbeab positive) and alt levels every 3-6 months. point-of-care tests (pocs; also known as rapid diagnostic tests, rdts) are simplified versions of laboratory-based tests that have the potential to circumvent major barriers people face to accessing hepatitis b blood-based testing in various settings. pocs usually require small amounts of body fluids (for example, a finger-prick blood sample or oral swab), short turn-around time, and are generally easy to use with minimal required training and therefore can be provided to people in a variety of community and outreach settings by a broad range of trained workers [22] and are scalable to rapidly reach large populations as has been seen with the highly successful egyptian national hepatitis c screening program [23] . the simple collection process (finger-prick or mouth swab) is also highly acceptable, feasible and attractive to people undergoing testing [22, 24, 25] . a key benefit of pocs in the field of hepatitis b is to engage hard-to-reach communities for testing, such as using hbsag poc tests for hepatitis b screening in remote areas, or harm reduction programs [24] [25] [26] [27] . pocs also have great potential for retaining patients in care when used in the community for chronic hepatitis b stage evaluation and disease monitoring [26, 27] . figure 2 outlines the key phases of disease in chronic hepatitis b infection and the indicators for blood testing in each stage. the who recommends that an ideal poc test needs to meet the assured criteria of being "affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users" [28] . since 1998, the who has implemented an evaluation and performance assessment program for all pocs in viral hepatitis to report on accepted quality parameters for widespread clinical use [29] . many pocs have been developed in the field of hepatitis b, particularly for screening and diagnosis; however, only three pocs for detecting hbsag have been prequalified by the who [29] . there is currently a lack of pocs for hepatitis b stage assessment or monitoring that have been endorsed to use by the who; however, several novel tests are now in clinical trials that may fill this important care delivery gap. typically, poc tests have lower accuracy than traditional laboratory-based tests, but they facilitate the triage of people who require more complex and expensive laboratory assays to confirm a positive poc test result and thereby reduce costs. regulatory and economic constraints are additional barriers to transferring pocs to field use. in different settings, they therefore require a comprehensive appraisal of factors including testing performance, feasibility (such as storage requirements, power supply), acceptability and cost-effectiveness when using pocs to scale up access to hepatitis b diagnosis and management under real-life conditions. in this review, we outline the accuracy of available poc tests for hepatitis b and explore the evidence for utility and cost-effectiveness when integrated into hepatitis b diagnosis and monitoring programs. we also describe future technologies and explore how poc tests might best be used to achieve who 2030 hepatitis b elimination goals. practically, the three key clinical requirements for poc hepatitis b assays in the field are for diagnosis of current infection, determining treatment eligibility and also monitoring, as well as diagnosis of hepatitis b immunity and the need for hepatitis b vaccination in the uninfected ( figure 2 ). detection of hepatitis b surface antigen (hbsag) is the primary step to diagnose current hepatitis b infection, and multiple hbsag pocs are commercially available. most are qualitative lateral-flow chromatographic immunoassays which are one-step, easy to use, can be used with a variety of different specimens (whole blood, serum and plasma) and provide rapid semiquantitative visible results (usually within 15-30 min). to date, three hbsag rapid tests (determine hbsag 2, alere medical co. ltd, chiba-ken, japan; vikia hbsag, biomérieux sa, marcy-l'étoile, france; and sd bioline wb, abbott diagnostics korea inc. giheung-gu, republic of korea) have met who prequalification criteria [29] , with multiple studies showing their high accuracy for determining hbsag positivity in various populations, particularly in moderate-high-prevalence populations (table s1) . determine hbsag poc test is the one of the most widely-used hbsag poc tests [30] with the most published data on clinical performance. a 2017 meta-analysis [31] including 9 studies with 7730 samples showed a pooled sensitivity of 90.8% and specificity of 99.1% using determine. though most studies [32] [33] [34] [35] [36] [37] [38] showed high clinical sensitivity of 89-100% in the general population, the reported sensitivity varied widely in hiv-infected populations (56-100%) [39] [40] [41] [42] [43] [44] . the cause of the reported lower sensitivity in hiv-coinfected populations [39, 40, 43, 44] is unclear, but potential reasons may include the cross reaction of hiv-reverse transcriptase inhibitors and hepatitis b virus, a higher rate of occult hepatitis b infection in early hiv cohorts, a higher reported rate of hbsag loss in both untreated and treated hiv-infected populations and the use of tenofovir-based hiv regimens that effectively suppress hepatitis b virus dna levels and a large decline in hbsag titres [31, 45, 46] . sd bioline hbsag [38, [47] [48] [49] and vikia hbsag poc test [32, 33, 38, 50, 51] have also been shown to have good sensitivity (above 90%) and excellent specificity (above 99%) in general populations; however, lower sensitivity was also reported in hiv-infected populations [40] . a common application of these hbsag pocs is to measure seroprevalence in general or specific subpopulations in low-resource settings [52] [53] [54] [55] [56] . they have also been used in mass screening programs for hepatitis b in both community outreach [24, 57] and health-facility-based screening [52, 53, [58] [59] [60] [61] [62] in low-resource settings and shown great public health benefits. for example, in a community-based outreach screening program conducted in 75 camps in southern india [63] , the "screen and vaccinate/linkage to care" strategy led to over 7700 vaccinations in the camps and 162 people with high viral load getting treatment. the program increased the accessibility of hepatitis b diagnostic testing in a low-resource setting, and the timely results of pocs contributed to people's engagement in post screening interventions [63] . the hbsag pocs were also used in programs to engage hard-to-reach populations such as people who inject drugs, sex workers [64] , disadvantaged groups or some ethnic groups [65, 66] by providing self-testing, community or health-facility-based testing services. in a randomised control study conducted in a clinic engaging mostly african immigrants in france [65] , people without health cover attending a clinic were provided free testing for hepatitis b, hepatitis c and hiv using either pocs or prescriptions of testing at a pathologist; a higher rate of testing and linkage to care was observed among people allocated to receive pocs. however, another multicentre randomised control study in france [66] found no difference in effectiveness of linkage to care using the approach of an hbsag rapid test plus a standard lab-based confirmatory serology test versus lab-based standard serology in five clinics. in this study, it was described that participants received testing results via mail or phone call, but it was unclear whether participants received testing results at the same visit if they were in the poc testing group [66] . other than the who-prequalified hbsag pocs, emerging new brands of hbsag have been reported in field studies including the drw-hbsag assay, diagnostics for the real world ltd., (ce-marked) [35, 67] , first response hbsag card test, premier medical corporation [68] (ce-marked), naosign(r) hbs poc strips, bioland [69] , and one step hbsag test, general biologicals corporation [70, 71] (non-exhaustive list). a study in mongolia [72] which tested 19 commercially available hbsag pocs using a serum sample showed the average sensitivity and specificity being 100% and 99%, respectively. whilst most hbsag pocs of various brands have shown promising clinical performance [35, 38, 67, 70, 73] , available validation data are limited and further studies with large sample size and in diverse populations including different ethnicity and hepatitis b prevalence populations and people living with hiv are needed. multiplex diagnostic pocs can be highly attractive for low-middle-resource settings with the capacity to detect multiple pathogens using a single testing strip. some multiplex pocs which detect hbsag are commercially available and some are ce-marked (hbsag/hcv/hiv/syphilis combo test, euro genomas; hbsag and hcv combo test, euro genomas; artron detect 3 hiv/hcv/hbv combo, artron laboratories; hiv, hbsag and hcv rapid test, maternova inc., providence, ri, usa), but none have been listed by who prequalification [16, 29, 74] . accuracy of hbsag detection using multiplex has been shown to be high [75] , but limited clinical validation data are available. innovations in sampling technique have provided more convenient specimen collection methods, such as using oral fluid as specimen collected by an oral swab [25, 48, 51] . the simplified process was highly acceptable to individuals [25, 48] , but testing accuracy is a challenge to overcome [48] , and it may additionally require trained technicians or lab-based enzyme immunoassays or equipment for sample preparation such as requiring a centrifuge for target analyte separation [51] . future development needs to consider combining sample preparation steps together with detection and readout into one single device, without sacrificing testing accuracy. although many studies of different hbsag pocs showed very good sensitivity [31, 38, 73] , false negativity is still among the biggest concerns: around one in ten negative test results on average could be hbsag positive [31] . most cases with false negative results were reported to have low titres of hbsag, such as in studies using determine/vikia hbsag pocs, where most false negative cases had hbsag titres lower than 30 iu/ml [32, 33] . as hbsag level does not correlate with severity of liver damage, there is a chance that people with advanced liver disease may be missed. other potential factors affecting the accuracy of hbsag pocs may include hbv dna level, different genotypes, co-infection with hepatitis c or hiv and hepatitis b variants with s gene mutations that are not detected by the poc hbsag test [31, 33, 76, 77] . as only a few studies have obtained comprehensive serological and genetic profiles of false negative cases, more data are needed to explore these associations and determine the implications for clinical practice. specimen type is unlikely to affect the efficacy of hbsag poc tests: a meta-analysis showed similar pooled sensitivity of studies using whole blood sample compared to plasma or serum [31] , and studies evaluating hbsag pocs using capillary whole blood collected by finger-prick all showed reasonably high sensitivity (88-90%) [32, 44, 78] . in practice, there is no absolute cut-off for testing performance when choosing pocs for hepatitis b programs, and the increased access to testing might mitigate the harm caused by reduced accuracy; however, sensitive pocs that have been validated in similar contexts to their planned use should be prioritised [79] . hepatitis b surface antibody (anti-hbs) is the key marker to determine an individual's immunity status to hepatitis b virus and triage the need for vaccination. a few anti-hbs pocs are commercially available, but most have poor reported sensitivity ranging from 20% to 70% [33, [80] [81] [82] . one study reported a sensitivity of 91.8% using an anti-hbs rapid test card among 1272 samples [70] ; however, these findings require further validation. a study [66] showed using hbsag/ anti-hbs pocs was not effective in increasing vaccination rate due to poor sensitivity of anti-hbs poc and high reliance on confirmatory enzyme immunoassay. though a poc test for anti-hbs can help with triage vaccination need, given hepatitis b vaccination is relatively cheap, context-specific cost-effectiveness analyses would be needed to determine settings where the use of pocs of anti-hbs would be cost-effective. treatment decisions in hepatitis b are guided by patient age, hepatitis b dna viral load and the degree of liver inflammation and fibrosis, as measured by alanine aminotransferase (alt) levels and either transient elastography or liver biopsy, respectively [4, 5, 83] . however, there are few poc tests currently available for these parameters, and none have been widely validated and who prequalification approved. hepatitis b virus (hbv) dna level is the critical indicator when deciding an individual's management plan as per clinical guidelines. polymerase chain reaction (pcr) platforms for nucleic acid detection are still the main technique of quantitative assessment hbv dna levels; conventional pcr platforms are usually built in laboratories and require high manual input and pose barriers for accessibility in remote areas and other resource-limited areas areas [12] . a rapid molecular test, xpert ® hbv viral load (cepheid inc., sunnyvale, ca, usa, ce-marked, approved by american fda and tga in australia), is commercially available for hbv dna quantification that provides test results in less than one hour [84, 85] . the test is a cartridge-based, real-time pcr assay which is run on the genexpert instrument, a molecular diagnostic platform. the processing unit of the system is around the size of a coffee machine, and it also runs a range of other rapid molecular tests such as who prequalified xpert hcv viral load, hiv-1 qual and hiv-1 viral load tests [29] , which poses an opportunity for hepatitis b viral load test to be adopted in areas with existing platforms at a low additional cost. so far, limited data are available on the analytical performance of the assay [84, 85] . two recent studies [84] using serum samples showed a good correlation between hbv dna quantification by using xpert hbv viral load assay with the results of the laboratory reference assay; they also have a low limit of detection (lod) of 7.5 iu/ml, which is similar to most commonly used hbv dna platforms (usually with lod of 10iu/ml) [84] . in practice, xpert testing for hbv dna led to a faster workflow with a mean time to result being 6-8 h, which provided a near-poc solution [84, 86] . however, as a new unit, genexpert facilities are still expensive; the operation requires uninterrupted power supply, as well as technician training and skills for system running, services and reagent maintenance. hepatitis e antigen (hbeag) is a key indicator to determine phase of chronic hepatitis b infection (figure 2) , treatment initiation and is used as a surrogate of hbv dna measurement for evaluating risks of maternal-to-child-transmission [2, 4, 83, 87] . several hbeag pocs are commercially available; however, published data show the accuracy of hbeag pocs has a wide range, with sensitivity of 30-82% and specificity of 67-100% [33, 80, 88] . similarly, anti-hbe pocs are reported to have poor sensitivity but excellent specificity in studies [33, 81] . given the high costs and challenges in accessing hbv dna testing in low-resource settings, the who recommends hbeag to triage treatment [83, 89, 90] ; therefore, the low testing accuracy of hbeag pocs is an urgent issue to be addressed. novel serum biomarkers such as hbv core antigen (hbcrag) have been shown to correlate with serum hbv dna levels and intrahepatic cccdna levels, a marker of hepatitis b-related hcc risk, and have therefore been explored as a potential indicator for treatment determination, off-therapy virologic suppression and hcc risk evaluation [91, 92] . however, there is no rapid lateral flow assay for hbcrag so far. another novel biomarker, serum hbv rna [93] , has been shown to be positively correlated with hbv dna level, and levels were higher than hbv dna in patients on nucleos(t)ide analogues and can thus be a potential marker for off-therapy hbv suppression. however, its clinical predictive utility is not yet well defined, and the measurement of serum hbv rna presents its own challenges even in routine lab-based testing; thus, further studies are needed to guide defining the clinical role and development of hbv rna pocs. alt is part of the liver function test assay panel and is a key marker of liver inflammation, used to determine hepatitis b treatment eligibility [4, 5, 83] (figure 2 ). alt has been proposed as an indicator for treatment in people with positive hbsag in low-resource settings where hbv dna testing is unavailable. a semiquantitative poc using alt 40u/l as the cut-off (biopoint ® alt-1) has been developed and manufacturer data suggest a high sensitivity of 94% and specificity of 85% [94] . several serological biomarkers have been combined in algorithms to offer indirect non-invasive assessment of liver fibrosis and have been validated to varying degrees in hepatitis b populations, such as the ast to platelet ratio index (apri) and fibrosis 4 index (fib-4) [95, 96] . these indices require quantitative testing results of ast, platelets with or without alt, unfortunately none of which are currently available in a poc test format. dried blood spot (dbs), while not a poc test, is a sampling method which offers viable solutions for mass screening or testing in low-resource settings where testing capacity or access are limited. in practice, a single finger-prick blood sample is applied to a chemically modified paper card which collects and store serological markers and nucleic acid; specimens obtained in the field can be transported to a laboratory at ambient temperatures, where the blood sample is processed following a dbs protocol and tested using immunoassays or molecular techniques [83, 97] . dbs samples have a relatively long shelf-life at ambient temperature without sample degradation [98] , which is attractive for regions that are geographically isolated or have varying security situations precluding rapid transport to a central laboratory. this method is now recommended by the 2017 who hepatitis b testing guidelines [83] in settings where no access to venous blood sampling or quality-assured testing assays is available. dbs testing has been used to detect hbsag, hbeag, anti-hbc, hbv dna and even for viral genotyping [99, 100] . a meta-analysis [101] evaluating dbs for hbv dna quantification showed pooled sensitivity of 95% (83-99%) and specificity of 99% (53-100%); however, most of the included studies used cold chain to store samples, which might limit the generalisation of the accuracy estimates in field conditions. although dbs testing increases testing access in low-resource and geographically isolated settings, it still requires high technical expertise and standard laboratory assays that may not be routinely available. cost-effectiveness and affordability are key considerations when adopting poc tests in hepatitis b programs. quoted costs of lateral flow-based hbsag pocs are generally lower than laboratory-based immunoassays, with the estimated procurement costs being us$0.2-0.95 and us$0.4 to 2.8 per test, respectively [72, 83] . conventional lab-based testing usually requires additional costs such as a reading machine, professional laboratory staff and technical training; therefore, the total costs for testing are often much higher than using pocs in low-resource settings. multiplex poc testing is expected to be cheaper than multiple poc tests. for example, the manufacturing costs of a hiv/hcv/hbsag poc is around us$1 [102] ; thus, using multiplex in high-risk populations who require broad spectrum pathogen screening is expected to be resource-saving. costs for conventional hepatitis nucleic acid testing are estimated to range from us$30 to 120 [15] , and the cost can be up to us$400 per assay in resource-limited countries and regions [12] . in 2018, a viral load testing program was introduced in sub-saharan african countries to access an integrated molecular diagnostics instrument (hologic panther system), at an all-inclusive ceiling price of us$12 per patient sample [103] . the foundation for innovative new diagnostics (find) has negotiated the price of xpert hbv viral load assay for 145 developing countries, and it costs us$14.9 per cartridge excluding shipment; however, the testing instrument costs between us$11,530 to us$64,350 depending on the throughput capacity of the processing unit [104, 105] . however, these costs may still be higher than what programs could afford in some settings. in addition, due to reduced accuracy compared with standard assays, diagnostic poc testing is often used as a screening tool to triage those requiring more expensive laboratory-based testing confirmation [15] , which means many of the costs for centralised laboratory services are only partially offset by poc test use. while novel poc testing may have increased testing performance, costs usually fall slowly due to patent protection laws [16] . even for countries that could afford these pocs, it may cost more than lab-based testing where well-established laboratory services are available; therefore, the main demand for pocs is limited to self-testing or outreach programs to improve testing uptake. the cost-effectiveness of using pocs for hepatitis b can therefore be different in different settings. using hbsag pocs as a screening tool was found to be cost-effective in community-based approach in hbv-endemic but low-resource settings. nayagam et al. [106] assessed a community-based hbv screening and treating program in the gambia where hbsag pocs were provided to adult participants door-to-door at a total screening cost of us$7.4 per person. the program was found to be highly cost-effective, with an icer of us$540 per daly averted compared to status quo where no publicly provided hbv screening or treatment was available. integrating low-cost hbv pocs into existed healthcare services such as antenatal screening [52, 58, 107] , blood donor screening [62] and hiv clinics [59] [60] [61] can be another solution to achieve scale-up of hbv testing [108, 109] . zhang et al. [109] showed the integration of hbv screening within the existing antenatal care in cambodia was highly cost-effective. in their model, the unit cost of hbsag and dna test (estimated to be us$1 and us$30) was one of the key parameters driving cost-effectiveness; in such cases, cheap pocs could potentially improve the cost-effectiveness of such an integration program even further. studies in low hbv endemicity countries showed programs offering hepatitis b screening followed by vaccination or linkage to clinical care among people with increased risks are likely to be cost-effective [110] ; however, there is a lack of programs adopting pocs in hepatitis b screening strategy, and thus, a lack of evidence suggesting economic impacts using pocs for hepatitis b in populations who have regular access to healthcare services. however, a few studies have shown rapid hepatitis c or hiv testing nested in harm reduction programs or among priority populations can be cost-effective [111] [112] [113] . more evidence on using pocs in hbv screening or monitoring programs in the field is needed, especially covering the implementation costs and the effects of broader testing access compared to standard testing services or no testing services (where there being no access to testing is the current practice). whilst poc testing theoretically circumvents many test access barriers, acceptability from targeted population remains a key determinant of successful implementation of hepatitis b programs. however, limited data are available on the satisfaction appraisal from users and stakeholders. in general, pocs are highly acceptable to customers due to their easy-to-use nature, short turnaround time, minimal bio sample requirement and provision of testing capacity to familiar staff in contexts where people want to be tested [114] . in a survey conducted among implementers and users of hep b and c testing services from 43 countries, almost half of respondents from low-and middle-income countries preferred a poc test method using capillary whole blood [83] . while there is no agreement on what accuracy would be considered acceptable, half of the respondents would accept an assay with a minimal sensitivity of 95% [83] . acceptability of rapid testing for hepatitis b or other blood-borne viruses and sexual transmitted diseases can be varied in different populations. a survey done in a prison setting showed hcv pocs were highly accepted [115] . another study showed that people may find it stressful when testing hiv, hcv and syphilis using a poc test [116] . when using pocs for blood-borne virus screening in public events or community outreach programs, the acceptance rate varied widely in customers with different socioeconomic status, ethnic or geographic backgrounds [63, 117, 118] . in health facility settings, healthcare providers find pocs generally speed up decision making and improve patients' compliance with chronic management plans requiring repeat testing over time [30] ; however, there are also general concerns such as suboptimal testing accuracy and increased workload for healthcare workers [119] . other than getting tested from healthcare providers or trained personnel, pocs also have the potential to be a self-testing tool with universal access. in some countries, rapid tests for hepatitis b and/or hiv and hepatitis c can be purchased online or over the counter. while self-testing offers a confidential testing solution for customers, a standard approach will be needed to ensure that people having accessible pre-and post-counselling, as well as pathways of linkage to care. a general limitation of pocs for hepatitis b is reduced accuracy compared to standard lab-based testing. there are also specific limitations for individual poc tests which were highlighted above (section 2), such as hbsag poc tests which were shown to have varied sensitivity in hiv-infected populations. in addition, there is still a lack of poc tests for liver cirrhosis and hbv dna levels required to determine treatment eligibility for patients with hepatitis b. there are also limitations in the aspects of regulatory process, procurement and storage management for poc tests, as well as costs when implementing pocs for hepatitis b in different settings. the who prequalification process for in vitro diagnostic tests for diseases with a high individual or public health risk, including hepatitis b, assesses both the test's performance and manufacturing quality. for countries without regulatory procedures in place, this provides a thorough review of potential diagnostic tests they could select based on specific needs; however, the process of getting prequalified approval by the who can be slow. for low-resource settings, stock-out and supply issues can be a barrier for use of poc tests; lack of scale may also mean they can be more expensive than high throughput assays in some settings; testing accuracy as well as instrument maintenance can be impacted by extreme weather conditions (heat, humidity) in the field; novel testing platforms such as genexpert can still be expensive, and the use of the instruments can be subject to field conditions such as power supply. on the other side, for high-income countries, a main challenge for the introduction and implementation of poc tests is the regulatory and reimbursement approval process for new diagnostics, which require demonstration of analytic and clinical validity, as well as clinical usefulness and cost-effectiveness data. as an example, the fda regulatory process can be long and expensive [120] , and the return for investment in high-income countries where poc tests will compete for market with standard diagnostic pathways can be challenging. increasing testing accuracy is the major challenge for poc tests that are already compact and easy to use. when developing poc diagnostics, features targeting resource-limited settings without basic infrastructures or cold chain need to be included; tests with high quality need to be validated across populations and specimen type. rapid affordable serology tests of high accuracy for novel biomarkers which could be alternatives for molecular testing are a major need. technology is needed to integrate convenient sampling and specimen preparation into a one-step testing assay. inter-user variability is another challenge to address if poc tests require technique training or multiple steps; a standard protocol or mobile apps can be used to overcome this problem where suitable. miniaturisation of testing instruments is the trend, especially for instruments that could perform molecular analysis such as portable hand-held devices, without sacrificing testing accuracy. dry blood spot kits for hiv and hepatitis c can already be ordered online to be sent to a home address as a private way to test for infection [121] . faecal occult blood tests are mailed out to all older adults in some regions as a public health initiative to screen for bowel cancer [122] . a similar approach could be evaluated to screen for hepatitis b among populations that are disengaged from traditional health services. in resource-limited settings, adopting hepatitis b testing in existing platforms or programs can be more cost-effective than starting a new initiative [109] . mobile phone technology has the potential to be used for screening and monitoring health conditions [123] . mobile phones are now being used around the world for contact tracing for sars-cov-2, an approach that is immediately applicable to hepatitis b. recently, google searches for anosmia have been linked to the epidemiology of sars-cov-2 [124] . there is a need for the streamlining of regulatory and reimbursement approval processes in high-income countries where the traditional approval process is expensive and slow, particularly for poc diagnostics suitable for use as public health tools to promote the engagement of marginalised individuals, including people who inject drugs, migrants and culturally and linguistically diverse communities affected by hepatitis b. in low-and middle-income countries where regulatory processes can be less demanding, the key is to ensure the quality and performance of tests as they come to market. more than 60 products have been prequalified since the who prequalification process started in 2010 [29] . it has been proposed that a model list of essential diagnostics be developed, comparable with the model list of essential medicines maintained by the who. such a list would help in the selection of diagnostic methods and would facilitate improvements in the regulation and affordability of in vitro diagnostic tests and in training in their use. the who has set ambitious goals for the elimination of hepatitis b as a public health threat by 2030. birth dose vaccination is the most important public health intervention to reduce incidence and will also reduce mortality level long term. for the individual already infected with hepatitis b, the key to preventing liver-related harm is the maintenance of sustained viral suppression. this requires diagnosis and linkage to care; in some people, antiviral therapy will be necessary. hepatitis b is typically asymptomatic until advanced disease has developed. therefore, screening is required. the risk factors and epidemiology of hepatitis b are well described, but screening rates are suboptimal and often occur in the context of opportunistic doctor-patient consultations following presentation with an unrelated problem. testing typically involves venesection followed by centralised testing in a laboratory with batch processing and automation to improve efficiency. this system works well for the engaged individual being cared for by a motivated health care practitioner. however, even in high-income countries, up to 80% of infected patients remain unaware of their infection [125] . thus, there is a need to scale up screening for hepatitis b in high-risk populations, and a need to reconsider current models of care for screening. now that hepatitis b treatment is cheap, safe and highly effective and durable, there is an urgent need to reconsider a public health approach to the management of hepatitis b. point-of-care tests provide a tool for mass screening in community settings. they also provide the opportunity to reduce the care cascade to one of same-day "test and treat". the effective employment of such strategies will be necessary for achievement of who elimination goals. natural history of chronic hepatitis b: special emphasis on disease progression and prognostic factors global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the global burden of disease study clinical practice guidelines on the management of hepatitis b virus infection update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld 2018 hepatitis b guidance long-term suppression of hepatitis b e antigen-negative chronic hepatitis b by 24-month interferon therapy the long-term benefits of nucleos(t)ide analogs in compensated hbv cirrhotic patients with no or small esophageal varices: a 12-year prospective cohort study the risk of hepatocellular carcinoma decreases after the first 5 years of entecavir or tenofovir in caucasians with chronic hepatitis b requirements for global elimination of hepatitis b: a modelling study third dose of hepatitis b vaccine. reported estimates of hepb3 coverage. secondary third dose of hepatitis b vaccine. reported estimates of hepb3 coverage hepatitis b in sub-saharan africa: strategies to achieve the 2030 elimination targets reported estimates of hepb_bd coverage. secondary hepb birth dose. reported estimates of hepb_bd coverage aiming for the elimination of viral hepatitis in australia, new zealand, and the pacific islands and territories: where are we now and barriers to meeting world health organization targets by 2030 the future of viral hepatitis testing: innovations in testing technologies and approaches accelerating the elimination of viral hepatitis: a lancet gastroenterology & hepatology commission diagnosis of viral hepatitis more than a virus: a qualitative study of the social implications of hepatitis b infection in china managing chronic hepatitis b: a qualitative study exploring the perspectives of people living with chronic hepatitis b in australia innovative strategies for the elimination of viral hepatitis at a national level: a country case series pdf;jsessionid=9deca1ff83bc4a8c41b74e3be2649662?sequence=12017 the rapid-ec study-a feasibility study of point-of-care testing in community clinics targeted to people who inject drugs in screening and treatment program to eliminate hepatitis c in egypt acceptability and feasibility of a screen-and-treat programme for hepatitis b virus infection in the gambia: the prevention of liver fibrosis and cancer in africa (prolifica) study implementation of rapid hiv and hcv testing within harm reduction programmes for people who inject drugs: a pilot study hepatitis b screening in an argentine emergency department: a pilot study to increase vaccination in a resource-limited setting point-of-care hepatitis c testing from needle and syringe programs: an australian feasibility study rapid tests for sexually transmitted infections (stis): the way forward helath organisation list of prequalified in vitro diagnostic products. secondary world helath organisation list of prequalified in vitro diagnostic products 2020 a practical guide to global point-of-care testing diagnostic accuracy of tests to detect hepatitis b surface antigen: a systematic review of the literature and meta-analysis validation of rapid point-of-care (poc) tests for detection of hepatitis b surface antigen in field and laboratory settings in the gambia, western africa performance of rapid tests for detection of hbsag and anti-hbsab in a large cohort evaluation of rapid diagnostic tests for the detection of human immunodeficiency virus types 1 and 2, hepatitis b surface antigen, and syphilis in ho chi minh city, vietnam. am evaluation of a new hepatitis b virus surface antigen rapid test with improved sensitivity evaluation of the performance of four rapid tests for detection of hepatitis b surface antigen in antananarivo, madagascar hepatitis b surface antigen seroprevalence among prevaccine and vaccine era children in bangladesh evaluation of four rapid tests for detection of hepatitis b surface antigen in ivory coast prevalence of infection with hepatitis b and c virus and coinfection with hiv in medical inpatients in malawi detection of highly prevalent hepatitis b virus coinfection among hiv-seropositive persons in ghana reliability of rapid testing for hepatitis b in a region of high hiv endemicity viral hepatitis and rapid diagnostic test based screening for hbsag in hiv-infected patients in rural tanzania prevalence and associations with hepatitis b and hepatitis c infection among hiv-infected adults in south africa field performance of the determine hbsag point-of-care test for diagnosis of hepatitis b virus co-infection among hiv patients in zambia occult hepatitis b and hiv infection hiv-hepatitis b virus coinfection prevalence of chronic hepatitis b virus infection before and after implementation of a hepatitis b vaccination program among children in nepal point of care and oral fluid hepatitis b testing in remote indigenous communities of northern australia evaluation of the analytical performance of six rapid diagnostic tests for the detection of viral hepatitis b and c in lubumbashi, democratic republic of congo performance of point of care assays for hepatitis b and c viruses in chronic kidney disease patients evaluating hbsag rapid test performance for different biological samples from low and high infection rate settings & populations hepatitis b virus sero-prevalence amongst pregnant women in the gambia seroprevalence of hbv among people living with hiv in anyigba assessment of hepatitis b immunization programme among school students in qatar seroprevalence of hepatitis b virus infection and associated factors among healthcare workers in northern tanzania seroprevalence of hepatitis b and c virus infections among diabetic patients in kisangani (north-eastern democratic republic of congo) eligibility for hepatitis b antiviral therapy among adults in the general population in zambia maternal hepatitis b infection burden, comorbidity and pregnancy outcome in a low-income population on the myanmar-thailand border: a retrospective cohort study the prevalence of hepatitis b virus among hiv-positive patients at kilimanjaro christian medical centre referral hospital sero-prevalence of hbv and associated risk factors among hiv positive individuals attending art clinic at mekelle hospital hepatitis b infection, viral load and resistance in hiv-infected patients in mozambique and zambia palliduminfections among blood donors and transfusion-related complications among recipients at the laquintinie hospital in douala prevalence of hepatitis b and hepatitis c infection from a population-based study in southern india prevalence estimates of hiv, syphilis, hepatitis b and c among female sex workers (fsw) in brazil simultaneous human immunodeficiency virus-hepatitis b-hepatitis c point-of-care tests improve outcomes in linkage-to-care: results of a randomized control trial in persons without healthcare coverage. open forum infect effectiveness of hepatitis b rapid tests toward linkage-to-care performance of a new rapid test for the detection of hepatitis b surface antigen in various patient populations hepatitis b vaccination in burkina faso: prevalence of hbsag carriage and immune response in children in the western region a simple and inexpensive point-of-care test for hepatitis b surface antigen detection: serological and molecular evaluation a simple and rapid test-card method to detect hepatitis b surface antigen and antibody: potential application in young children and infants evaluation of the performance of two rapid immunochromatographic tests for detection of hepatitis b surface antigen and anti hcv antibodies using elisa tested samples sensitivity and specificity of commercially available rapid diagnostic tests for viral hepatitis b and c screening in serum samples evaluation of performance testing of different rapid diagnostic kits in comparison with eias to validate detection of hepatitis b virus among high risk group in nigeria multi-disease diagnostics landscape for integrated management of hiv, hcv, tb and other coinfections analytical performances of simultaneous detection of hiv-1, hiv-2 and hepatitis cspecific antibodies and hepatitis b surface antigen (hbsag) by multiplex immunochromatographic rapid test with serum samples: a cross-sectional study performance evaluation of 70 hepatitis b virus (hbv) surface antigen (hbsag) assays from around the world by a geographically diverse panel with an array of hbv genotypes and hbsag subtypes comparative performance of three rapid hbsag assays for detection of hbs diagnostic escape mutants in clinical samples: table 1 point-of-care screening for hepatitis b virus infection in pregnant women at an antenatal clinic: a south african experience a guide to aid the selection of diagnostic tests evaluation of rapid diagnostic tests for assessment of hepatitis b in resource-limited settings poor sensitivity of rapid tests for the detection of antibodies to the hepatitis b virus: implications for field studies performance of rapid diagnostic tests for the detection of anti-hbs in various patient populations performance of the xpert hbv viral load assay versus the aptima quant assay for quantifying hepatitis b virus dna evaluation of the xpert hbv viral load for hepatitis b virus molecular testing rapid, random-access, quantification of hepatitis b virus using the cepheid xpert®hbv viral load assay prevention of materno-foetal transmission of hepatitis b in sub-saharan africa: the evidence, current practice and future challenges poor sensitivity of commercial rapid diagnostic tests for hepatitis b e antigen in senegal, west africa validation of the treat-b score for hepatitis b treatment eligibility in a large asian cohort: treat-b improves with age development of a simple score based on hbeag and alt for selecting patients for hbv treatment in africa hepatitis b core-related antigen (hbcrag): an alternative to hbv dna to assess treatment eligibility in africa serum hepatitis b virus rna: a new potential biomarker for chronic hepatitis b virus infection validation of a novel rapid point-of-care alt test in patients with viral hepatitis non-invasive assessment of liver fibrosis and prognosis: an update on serum and elastography markers comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and fibrosis-4 index for detecting liver fibrosis in adult patients with chronic hepatitis b virus infection: a systemic review and meta-analysis dried blood spots-preparing and processing for use in immunoassays and in molecular techniques assessment of dried blood spot samples as a simple method for detection of hepatitis b virus markers diagnostic accuracy of serological diagnosis of hepatitis c and b using dried blood spot samples (dbs): two systematic reviews and meta-analyses evaluation of the efficiency of dried blood spot-based measurement of hepatitis b and hepatitis c virus seromarkers diagnostic accuracy of detection and quantification of hbv-dna and hcv-rna using dried blood spot (dbs) samples-a systematic review and meta-analysis usefulness of simultaneous screening for hiv-and hepatitis c-specific antibodies and hepatitis b surface antigen by capillary-based multiplex immunochromatographic rapid test to strengthen prevention strategies and linkage to care in childbearing-aged women living in resource-limited settings breakthrough agreement will reduce costs and increase access to diagnostic technology for millions in low-and middle-income countries technologies to support diagnosis and linkage to care: knowing your status and getting care 2.0 secondary genexpert®negotiated prices cost-effectiveness of community-based screening and treatment for chronic hepatitis b in the gambia: an economic modelling analysis seroprevalence of hepatitis b virus surface antigen and factors associated among pregnant women in dawuro zone, snnpr, southwest ethiopia: a cross sectional study aile, i. the cost-effectiveness of predonation screening for transfusion transmissible infections using rapid test kits in a hospital-based blood transfusion centre integrated approach for triple elimination of mother-to-child transmission of hiv, hepatitis b and syphilis is highly effective and cost-effective: an economic evaluation the effectiveness and cost-effectiveness of screening for and vaccination against hepatitis b virus among migrants in the eu/eea: a systematic review cost-effectiveness of rapid hepatitis c virus (hcv) testing and simultaneous rapid hcv and hiv testing in substance abuse treatment programs cost-effectiveness of one-time hepatitis c screening strategies among adolescents and young adults in primary care settings cost-effectiveness of strategies for testing current hepatitis c virus infection community-based, point-of-care hepatitis c testing: perspectives and preferences of people who inject drugs time matters: point of care screening and streamlined linkage to care dramatically improves hepatitis c treatment uptake in prisoners in england stressful point-of-care rapid testing for human immunodeficiency virus, hepatitis c virus, and syphilis implementing rapid hiv testing in outreach and community settings: results from an advancing hiv prevention demonstration project conducted in seven u acceptability of hiv self-testing: a systematic literature review exploring the barriers and facilitators to use of point of care tests in family medicine clinics in the united states innovation under regulatory uncertainty: evidence from medical technology do you need a dbs test? secondary do you need a dbs test national bowel cancer screening program secondary national bowel cancer screening program will an innovative connected aidesmart! app-based multiplex, point-of-care screening strategy for hiv and related coinfections affect timely quality antenatal screening of rural indian women? results from a cross-sectional study in india use of google trends to investigate loss-of-smell-related searches during the covid-19 outbreak new virological tools for screening, diagnosis and monitoring of hepatitis b and c in resource-limited settings this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-331731-c2r0kfaz authors: anugwom, chimaobi m; aby, elizabeth s; debes, jose d title: inverse association between chronic hepatitis b infection and covid-19: immune-exhaustion or coincidence? date: 2020-06-05 journal: clin infect dis doi: 10.1093/cid/ciaa592 sha: doc_id: 331731 cord_uid: c2r0kfaz nan m a n u s c r i p t dear editor, we read with great interest the report by zhao et al, regarding a case of delayed immune response to sars-cov-2 in a patient with hiv and hcv co-infection [1] . the authors stipulate the previous hiv and hcv infection could confer immune dysfunction providing a differential immune response during covid-19 development. this report, as most initial reports, originate in china which has an intermediate-high prevalence of chronic hepatitis b (hbv) infection [2] . we evaluated all peer-reviewed articles, written in the english language, reporting cases of covid-19 infection and specifically defining rates of hbv infection and hospital admission, since december 1 st , 2019 until march 25 th , 2020 and found a surprisingly low prevalence of chronic hbv in covid-19 cases admitted to the hospital. indeed, of the 2054 cases that were reported with this information, only 28 patients (1.36%) were reported positive for hbv. several of these studies reported 0% incidence of hbv among individuals infected with covid-19. we matched the hbv-rates in covid-19 subjects to age-specific rates of hbv reported in the respective geographic areas of origin ( table 1 ). the median age of covid-19 infected individuals in the evaluated studies ranged between 47-51 years, corresponding to hbv rates ranging from 7-11% while the hbv rates of those with covid-19 remained between 0-1.3%. it is unclear whether this is a simple epidemiological "misconnection" or if being chronically infected with hbv impacts the chances of clinically significant infection with sars-cov-2 leading to less hospital admissions, in a similar fashion as that reported by zhao et al to hiv and hcv. in this regard, research has documented that, chronic hbv infection leads to a reduced or absent virus specific t-cell reactivity (although hbv-specific t cells). this phenomenon, a c c e p t e d m a n u s c r i p t known as "immune exhaustion", is manifested by an impaired ability of t-lymphocytes to produce appropriate cytokines secondary to years of continuous, yet inefficient, immune reaction to the virus [3] . immune exhaustion is also frequently observed in chronic hcv infection [4] . in this setting, it is plausible that the exhaustion of t-lymphocytes may affect their ability to respond to other viruses and reduce the degree of "cytokine storm" that has been noticed in covid-19 patients, thus culminating in a less severe disease. similar patterns of immune co-interaction with consequences in clinical presentation and prognosis have been reported in individuals infected with hbv and schistosomiasis [5] . a c c e p t e d m a n u s c r i p t early virus clearance and delayed antibody response in a case of covid-19 with a history of co-infection with hiv-1 and hcv prevalence of hepatitis b virus infection in molecular and transcriptional basis of cd4⁺ t cell dysfunction during chronic infection the path to cancer and back: immune modulation during hepatitis c virus infection, progression to fibrosis and cancer, and unexpected roles of new antivirals helminth-induced immune modulation influences the outcome of acute and chronic hepatitis b virus infection a comparative study on the clinical features of covid-19 pneumonia to other pneumonias clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical progression of patients with covid-19 in epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore we thank drs. lanjuan li (wuhan, china) and barnaby young (singapore, singapore) for providing information from their manuscripts regarding hbv. a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-313627-g1iqhsdk authors: zou, xiaojing; fang, minghao; li, shusheng; wu, liang; gao, bing; gao, hong; ran, xiao; yibian,; li, renjie; yu, shanshan; ling, jianmin; li, donghui; tian, deying; huang, jiao title: characteristics of liver function in patients with sars-cov-2 and chronic hbv co-infection date: 2020-06-15 journal: clin gastroenterol hepatol doi: 10.1016/j.cgh.2020.06.017 sha: doc_id: 313627 cord_uid: g1iqhsdk summary background and aims coronavirus disease 2019 (covid-19) is a major global health threat. we aimed to describe the characteristics of liver function in patients with sars-cov-2 and chronic hepatitis b virus (hbv) co-infection. methods we enrolled all adult patients with sars-cov-2 and chronic hbv co-infection admitted to tongji hospital from february 1 to february 29, 2020. data of demographic, clinical characteristics, laboratory tests, treatments, and clinical outcomes were collected. the characteristics of liver function and its relation with the severity and prognosis of disease were described. results of 105 sars-cov-2 and chronic hbv co-infected patients, elevated levels of liver test were seen in several patients at admission, including elevated levels of alanine aminotransferase (22,20.95%), aspartate aminotransferase (29, 27.62%), total bilirubin (7, 6.67%), gamma-glutamyl transferase (7, 6.67%) and alkaline phosphatase (1, 0.95%). the values of the indices mentioned above increased substantially during hospitalization (all p<0.05). 14 (13.33%) patients developed liver injury. most of them (10, 71.43%) recovered after 8 (range 6-21) days. notably, 4 (28.57%) patients rapidly progressed to acute-on-chronic liver failure. the proportion of severe covid-19 was higher in patients with liver injury (p= 0.042). complications including aclf, acute cardiac injury and shock happened more frequently in patients with liver injury (all p<0.05). the mortality was higher in individuals with liver injury (28.57% vs 3.30%, p=0.004). conclusion liver injury in patients with sars-cov-2 and chronic hbv co-infection was associated with severity and poor prognosis of disease. during the treatment of covid-19 in chronic hbv-infected patients, liver function should be taken seriously and evaluated frequently. in december 2019, pneumonia caused by severe acute respiratory syndrome 2 coronavirus 2 (sars-cov-2), now known as coronavirus disease 2019 (covid-19), 3 was first reported in wuhan, china. it has subsequently spread throughout china and 4 other countries. a total of 750,890 cases and 36, 405 deaths had been reported all over 5 the word by march 31, 2020. 1 it has emerged as a major global health threat. 6 according to recent reports, 2-11% of covid-19 patients had liver comorbidities and 7 14-35% cases have been reported with abnormal levels of alanine aminotransferase 8 (alt) and aspartate aminotransferase (ast) during disease progression. 2-5 however, 9 the exact cause of preexisting liver conditions had not been outlined in these studies. in this study, we aimed to describe the characteristics of liver function and its 3 relation with severity and prognosis in patients with sars-cov-2 and chronic hbv 4 co-infection, in order to provide evidence for the clinical treatment of these specific 5 patients and contribute to improving their prognosis. this is a single-center, retrospective study of 105 patients with sars-cov-2 and 9 chronic hbv co-infection hospitalized at tongji hospital. tongji hospital is one of the 10 major comprehensive medical treatment centers and assigned for the treatments for 11 severe covid-19 patients by the government. we recruited inpatients from february 12 1 to february 29, 2020, who had been diagnosed as covid-19 and chronic hbv 13 infection according to who interim guidance and aasld guidelines. 9, 10 all patients 14 had a history of chronic hbv infection and tested positive for hbsag at admission. laboratory confirmation of covid-19 was performed by the local health authority as 16 previously described. 7 the ethics committee of tongji hospital approved this study 17 (tj-irb20200225). data extraction was performed by a trained team of physicians using a standardized 20 form to collect data on demographic characteristics, duration from illness onset to 21 hospitalization, underlying chronic medical conditions, symptoms from onset to 22 admission, continuous laboratory tests, treatments, complications and outcomes from 1 electronic medical records. the information on anti-hbv treatment was collected by 2 medical history. hbv serological markers were tested using commercially available 3 microparticle enzyme immunoassay kits (axsym, abbott laboratories, abbott park, 4 il, usa). hbsag > 0.05 iu/ml was hbsag-positive. hepatitis b virus e antigen 5 (hbeag) <1 iu/ml and ≥1 iu/ml mean hbeag-negative and hbeag-positive 6 respectively. severe illness of covid-19 was defined as one of the following: respiratory rate > 8 30 breaths/min; severe respiratory distress; or spo2 ≤ 93% on room air. 9 liver test 9 abnormalities were defined by the abnormal of the following indices in serum: 10 alt>41 units/liter (u/l), ast>40 u/l, gamma-glutamyl transferase (γ-gt)>71 u/l, 11 alkaline phosphatase (alp)>130 u/l, or total bilirubin (tbil)>26 µmol/l. liver 12 injury was defined as alt and/or ast over 3 uln, and/or tbil over 2 uln. 11 acute-on-chronic liver failure (aclf) was defined as tbil ≥5 mg/dl (85 µmol/l) 14 and coagulopathy (international normalized ratio (inr) ≥1.5 or prothrombin activity 15 (pta)< 40%) complicated within 4 weeks by clinical ascites and/or encephalopathy 16 in a patient with previously diagnosed or undiagnosed chronic liver disease/cirrhosis, of 105 patients with sars-cov-2 and chronic hbv co-infection, 14(13.33%) had 11 liver injury and 4 (3.81%) developed aclf during the hospitalization. liver injury was more common in males than in females (92.86% vs. 46.15%, 5 p=0.001). the incidence of fever was higher in patients with liver injury (p=0.011). were still hospitalized; 55 (52.38%) patients had been discharged, and 7 (6.67%) 7 patients died. the mortality was significantly higher in individuals with liver injury 8 (28.57% vs. 3.30%, p=0.004) ( table 3) . in the present study, we found that liver test abnormalities were relatively common 1 in patients with sars-cov-2 and chronic hbv co-infection, and the values of alt, 2 ast, tbil, alp and γ-gt increased substantially during hospitalization. a small part 3 of patients developed liver injury. patients with liver injury were more likely to 4 develop severe illness and had a worse prognosis including higher mortality and 5 incidence of complications. 6 the present study reported evidence of liver injury in patients with sars-cov-2 7 and chronic hbv co-infection. several patients had various abnormal liver tests. according to previous studies, the incidence of alt and ast abnormalities were 9 14.34%-29.5% and 17.9%-35%, respectively, 2, 4, 5, 16 which were similar to ours. liver 10 injury occurred in 21.5% of patients with covid-19 during the hospitalization as cai 11 q et al. reported, 17 which was higher than that in our study (13.33%). several reasons 12 may explain it. firstly, the two studies used different criteria for liver injury. we 13 defined alt and/or ast over 3 uln, and/or tbil over 2 uln as liver injury 14 according to the protocol for prevention, diagnosis and treatment of liver injury in 15 covid-19, 11 while liver injury was defined as alt and/or ast over 3 uln, alp, 16 ggt, and/or tbil over 2 uln in the study of cai q et al. secondly, the interval from 17 onset to admission of patients in the present study was 10 days, which may lead to the 18 missed diagnosis of early liver injury for lack of data prior to admission. furthermore, 19 there is heterogeneity in the population characteristics included in the two studies. whether liver injury in covid-19 is worth taking seriously remains 21 controversial. 18, 19 a recent study showed the presence of abnormal liver tests and liver injury were associated with the progression to severe pneumonia. 17 in our study, 1 patients with liver injury were more likely to develop severe illness and had a worse 2 prognosis including higher mortality and incidence of complications such as aclf, 3 acute cardiac injury and shock. liver injury happened to most patients within one 4 week, and recovered normality after several days. however, four chronic 5 hbv-infected patients deteriorated rapidly after sars-cov-2 co-infection with 6 progressively elevated jaundice, coagulation dysfunction and ascites, and were 7 diagnosed as aclf. eventually, they all died of multi-organ failure. those with liver 8 injury but no coagulation dysfunction generally went on to recover. these findings 9 indicate that liver injury in patients with sars-cov-2 and chronic hbv co-infection 10 was associated with disease severity and worse prognosis. liver function should be 11 evaluated more frequently in these special individuals, especially within a week after 12 admission. once liver injury occurs, it should be treated timely in order to prevent 13 poor prognosis, particularly for those with coagulation dysfunction. 14 drug-induced liver injury has been paid more attention in recent years. intravenous 15 methylprednisolone was reported to be associated with acute liver injury, while data 16 on association between oral methylprednisolone and liver injury is insufficient. 20 in 17 the present study, more patients with liver injury received methylprednisolone. this study received these drugs. no difference was observed in the use of these drugs 22 between patients with and without liver injury, expect interferon atomization therapy, 1 which was more given to patients with liver injury. however, in the present study, not 2 all patients experienced liver injury after these treatments. 3 patients experienced liver 3 injury before methylprednisolone therapy and 2 patients experienced liver injury 4 before interferon atomization therapy. therefore, the association between these drugs 5 and liver injury could not be further analyzed. 20 lancet gastroenterol hepatol2020. epub ahead of print. death 7 (6.67) 4 (28.57) 3 (3.30) 0.004 data are median (iqr) or n (%). niv: non-invasive ventilation. imv: invasive mechanical ventilation. ards: acute respiratory distress syndrome. aclf: acute-on-chronic liver failure. we described the characteristics of liver function and its relation with severity and prognosis in patients with sars-cov-2 and chronic hepatitis b virus (hbv) co-infection. patients with sars-cov-2 and chronic hbv co-infection who developed liver injury were more likely to progress into severe illness and had a worse prognosis including higher mortality and incidence of complications such as acute-on-chronic liver failure, acute cardiac injury and shock. liver function should be evaluated more frequently in patients with sars-cov-2 and chronic hbv co-infection, especially within one week after admission. who. coronavirus disease 2019 (covid-19) situation report-71. available at: 15 clinical features of patients infected with 2019 18 novel coronavirus in wuhan epidemiological and clinical characteristics of 20 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study 10 we want to thank all participants in the study. we would like to thank all the 11 medical staffs at tongji hospital who contributed to the study. key: cord-003018-qrt07zmz authors: miyakawa, kei; matsunaga, satoko; yamaoka, yutaro; dairaku, mina; fukano, kento; kimura, hirokazu; chimuro, tomoyuki; nishitsuji, hironori; watashi, koichi; shimotohno, kunitada; wakita, takaji; ryo, akihide title: development of a cell-based assay to identify hepatitis b virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide date: 2018-05-04 journal: oncotarget doi: 10.18632/oncotarget.25348 sha: doc_id: 3018 cord_uid: qrt07zmz sodium taurocholate cotransporting polypeptide (ntcp) is a major entry receptor of hepatitis b virus (hbv) and one of the most attractive targets for anti-hbv drugs. we developed a cell-mediated drug screening method to monitor ntcp expression on the cell surface by generating a hepg2 cell line with tetracycline-inducible expression of ntcp and a monoclonal antibody that specifically detects cell-surface ntcp. using this system, we screened a small molecule library for compounds that protected against hbv infection by targeting ntcp. we found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface ntcp with an ic(50) of ~40 μm. we also found that glabridin could attenuate the inhibitory effect of taurocholate on type i interferon signaling by depleting the level of cell-surface ntcp. these results demonstrate that our screening system could be a powerful tool for discovering drugs targeting hbv entry. hepatitis b virus (hbv) is the causative agent of chronic hepatitis b (chb), which can lead to liver cirrhosis and hepatocellular carcinoma. the world health organization reported that over 240 million people worldwide have chronic hbv [1] . despite the effectiveness of the hbv vaccine, worldwide prevalence of the disease remains high, and chb is a major global health problem. current therapeutic regimens for chb include pegylated interferon (ifn) and nucleoside/ nucleotide analogues. both treatments aim to prevent progression of the disease to liver failure, cirrhosis, and hepatocellular carcinoma. however, these treatments have limited effectiveness for hbv clearance [2] . in fact, pegylated ifn maintains viral suppression only in approximately 25% of patients [3] . nucleoside and nucleotide analogues inhibit hbv replication by targeting viral dna polymerase, but long-term treatment is required to achieve clinical benefits. for example, a 12-month course of lamivudine achieves clearance of hepatitis b e antigen (hbeag) in approximately 30% of patients with chb [4] . moreover, long-term treatment can be associated with a higher risk of side effects and emergence of drug resistant viruses, resulting in treatment failure and disease progression. therefore, it is vital to research paper www.oncotarget.com develop new types of antiviral drugs for hepatitis b treatment. in the hbv life cycle, the hepatitis b surface antigen (hbsag) initially attaches to heparan sulfate proteoglycans on the host cell surface [5] . this attachment seems to be relatively low affinity and reversible, but is essential for the subsequent more specific interaction between hbs and the hepatocyte-specific bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) [6] . although various membrane proteins have been reported to be hbv entry receptors, accumulating evidence now suggests that ntcp is an essential receptor for hbv infection [7] . discovery of this receptor has dramatically increased our understanding of the molecular basis of hbv entry. viral entry is currently one of the most important targets in the search for new drugs to treat viral infections and identification of ntcp has kindled interest in exploring compounds that inhibit hbv entry. ntcp is exclusively expressed at the basolateral membrane of hepatocytes [8] [9] [10] , and is involved in reuptake of conjugated bile acids from the bloodstream into hepatocytes [11] . ntcp is one of the factors that highly restrict host tropism of hbv to hepatocytes. the specific interaction of the pres1 domain of hbsag with ntcp triggers hbv attachment and initiates entry into hepatocytes. the synthetic peptide drug myrcludex b exhibits significant anti-hbv activity by competitively inhibiting this interaction, both in vitro and in vivo, and is currently undergoing a phase ii clinical trial for chronically hbv-infected patients [12, 13] . physiological substrates of ntcp such as taurocholic and glycocholic acids can also inhibit hbv infection, suggesting the competitive binding of bile acids and pres1 to ntcp [14] . interestingly, pres1-ntcp binding could trigger type i ifn signaling, whereas bile acid treatment does not [15] . bile acid intake via ntcp was found to inhibit the ifn pathway in the physiologically relevant range of concentrations [16] . these observations support the possibility that ntcp also takes part in the ifn-mediated innate antiviral response. because high expression often causes cell cycle inhibition, there are very few conventional hepatocellular carcinoma cell lines with ectopic expression of high levels of ntcp. moreover, there are no commercially available monoclonal antibodies (mabs) specifically recognizing cell-surface ntcp due to the difficulty of producing full-length ntcp protein with native structure. in the current study, we generated hbv-susceptible hepg2 cells with a tetracycline-inducible ntcp gene (intcp cells) without affecting cell growth. we also generated a high quality mab (clone 9a8) to efficiently detect cell-surface ntcp. using these tools, we identified the compound glabridin, which significantly inhibits hbv infection through the downregulation of ntcp from the cell surface. endogenous ntcp expression is rarely detectable in hepatoma cell lines such as hepg2 and huh7, and these cells are not susceptible to hbv infection. therefore, we generated the intcp cell line, a hepg2based cell line harboring a tetracycline-inducible human ntcp gene ( figure 1a) . treatment of the intcp cell line with the tetracycline analogue doxycycline (dox) caused expression of ntcp in a dox-dependent manner, and ntcp expression was 20-to 100-fold higher than endogenous expression in differentiated heparg cells and primary hepatocytes, as revealed by western blot and quantitative reverse transcription-pcr ( figure 1b-1d) . to determine whether dox-induced ntcp protein localized to the plasma membrane, we performed a pres1 peptide binding assay. we found that the pres1 peptide detected dox-treated intcp cells but not untreated cells, indicating the presence of ntcp on the cell surface ( figure 1e ). consistent with a previous report [6] , a western blot of ntcp showed major bands of 60-80 kd that were shifted to a single band of 30-40 kd after treatment with peptide n-glycosidase (pngase), implying that ntcp was modified by n-glycosylation (supplementary figure 1a) . although ntcp expression has been reported to affect cell proliferation [17] , intcp cells showed no differences in cell cycle progression or cell expansion with or without dox treatment ( figure 1f, 1g) . we subsequently examined the susceptibility of intcp cells to hbv infection. at an moi of 6000 geq/cell, intcp cells showed high susceptibility to hbv infection (~80% infected) without dmso treatment ( figure 1h , supplementary figure 1b ). this infection was significantly inhibited by pres1 peptide treatment ( figure 1h ), which indicates that hbv infection was ntcp-mediated. taken together, these results show that dox-induced ntcp proteins are exposed on the cell surface and functionally interact with pres1. although the above results suggest that ntcp proteins localize on the cell surface, this could not be directly demonstrated due to the lack of suitable antibodies for flow cytometry or immunofluorescence microscopy analysis. therefore, we generated a monoclonal antibody (mab) for this purpose. because recombinant ntcp protein tends to form insoluble aggregates, we utilized the wheat germ cell-free system, which has been shown to have advantages for the production of membrane proteins [18, 19] . the synthesized ntcp proteins were purified and used to immunize mice, and more than 140 hybridoma clones were established (figure 2a ). using a www.oncotarget.com flow cytometer-based screening assay with dox-treated and untreated intcp cells, we identified a hybridoma clone producing anti-ntcp mab, clone 9a8 ( figure 2b ). the 9a8 mab could recognize endogenous ntcp in differentiated heparg cells (supplementary figure 2) . we performed immunofluorescence microscopy using the 9a8 mab in various cell lines and cell-surface ntcp was clearly observed ( figure 2c , 2d). because the three-dimensional organoid culture recapitulates cell-cell interactions and recent studies have indicated some advantages of hepatoma organoids in hepatitis virus infection [20] , we investigated the localization of ntcp in hepatoma organoids. we embedded intcp cells in hydrogel before performing immunofluorescence microscopy with the 9a8 mab to visualize ntcp. ntcp localization was widespread in the membrane of internal cells as well as on the surface of the organoid ( figure 2e ). epitope mapping using recombinant ntcp mutants revealed that the 9a8 mab recognizes amino acids 317-326 of ntcp ( figure 2f ). to test whether the 9a8 antibody can inhibit hbv infection, we pretreated intcp cells and primary human hepatocytes with 9a8 mab and subsequently infected cells with wild type hbv and hbv encoding a luciferase reporter gene (hbv-nl) [21] . the 9a8 mab failed to inhibit hbv infection ( figure 2g , 2h), suggesting that the interaction between 9a8 mab and ntcp neither blocks hbv-host cell interaction nor causes downregulation of ntcp from the cell surface. because the 9a8 mab binds ntcp but does not interfere with its localization and function, it can be used to screen for antiviral compounds that modulate the level of cell-surface ntcp. briefly, intcp cells were treated with 102 different low-molecular-weight chemical compounds from a library derived from natural plant extracts for 24 hours and we subsequently quantified cell viability and the amount of cell-surface ntcp using the 9a8 mab ( figure 3a ). we identified two compounds, geraldol and glabridin, that could decrease the amount of cell-surface ntcp without observable cytotoxicity ( figure 3b ). flow cytometry analysis using the 9a8 mab demonstrated that both geraldol and glabridin decrease the levels of cell-surface ntcp in a dose-dependent fashion ( figure 3c ), but further analysis revealed that geraldol negatively regulates the tetracycline-responsive promoter activity ( figure 3d ). microscale thermophoresis analysis of the biomolecular interaction [22] revealed that glabridin could weakly but directly interact with ntcp ( figure 3e ). therefore, we focused on glabridin for further functional analyses. flow cytometry analysis showed that glabridin downregulated the amount of cell-surface ntcp in hepg2-hntcp-c4 [23] and heparg cells with ic 50 values of 28 µm and 34 µm, respectively ( figure 4a ). notably, treatment with 50 µm glabridin had no effect on the level of ntcp mrna (supplementary figure 3a) , suggesting that glabridin affects ntcp protein rather than ntcp gene expression. consistently, treatment with glabridin rapidly (within three hours) modulated the membrane localization of ntcp, but not that of the transferrin receptor ( figure 4b ). similar results were obtained with cell-surface biotinylation analysis in which only cell-surface proteins were purified and detected by western blotting (supplementary figure 3b ). immunofluorescence microscopy using the 9a8 mab showed that in glabridin-treated cells, ntcp mainly accumulated in the cytoplasm ( figure 4c ). to test the role of endocytosis in ntcp localization, we treated cells concurrently with glabridin and inhibitors of clathrinmediated or caveolar endocytosis. we found that genistein, a specific inhibitor of caveolar endocytosis, completely disrupted ntcp internalization ( figure 4d ), suggesting that glabridin causes the internalization of ntcp through caveolar endocytosis. because internalized membrane proteins are typically trafficked to degradation or recycling pathways, we next performed a cycloheximide chase assay. interestingly, ntcp protein levels were relatively stable in control cells, but the half-life of ntcp was significantly shorter in glabridin-treated cells ( figure 4e ). this suggests that glabridin reduces the amount of ntcp on the cell surface by promoting its endocytosis and subsequent intracellular degradation. previous reports have indicated that glabridin induces apoptosis in certain cancer cells [24] , but we observed no negative effect on cell viability under our experimental conditions ( figure 4f ). we next assessed the impact of glabridin on hbv infection in hepatocytes. time-of-addition experiments demonstrated that 50 µm glabridin inhibited hbv infection when added early in infection but became less effective when added later ( figure 5a ), suggesting that it acts at an early stage of the viral life cycle. indeed, when glabridin was added to hbv-producing cells, it did not affect the amounts of core-associated viral dna and/or rna (supplementary figure 4) . consistent with these observations, intcp cells treated with glabridin three hours prior to infection showed a significant reduction in the percentage of infected cells and the amount of hbsag secretion ( figure 5b , 5c). we performed a parallel analysis with differentiated heparg cells and confirmed that glabridin blocked hbv infection and downregulated endogenous ntcp with an ic 50 value of 33 µm ( figure 5d ). these results suggest that glabridin inhibits hbv infection by removing ntcp from the cell surface. ntcp is a transporter for bile acid uptake. we investigated the effect of glabridin on ntcp-mediated www.oncotarget.com anti-ntcp mab generation. using a wheat germ cell-free protein production system, large amounts of ntcp were synthesized with high solubility. following mouse immunization, we established over 140 hybridoma clones. (b) selection of 9a8 clones producing anti-ntcp mab. culture supernatants of hybridoma clones were used as primary antibodies for flow cytometry analysis of dox-treated (red line) or -untreated intcp cells (blue line). (c-e) immunofluorescence staining of cell-surface ntcp by 9a8 mab on intcp cells (c), hepg2-hntcp-c4 cells (d), and intcp-derived spheroid (e). scale bars: 10 µm. (f) epitope mapping of 9a8 mab. recombinant wild-type or partially truncated ntcp proteins tagged with his were generated using wheat germ extracts and subjected to western blotting using anti-his or 9a8 antibodies. the predicted epitope of 9a8 mab is shown in pink. (g, h) 9a8 mab fails to inhibit hbv infection. intcp cells (g) and primary human hepatocytes (h) were infected with hbv or its reporter virus (hbv-nl) respectively, in the presence of 9a8 mab. anti-hbs mab (clone 33a4, which recognizes the pres1 domain) was used as a control. viral infectivity was determined by intracellular hbcag staining (g) or nanoluc activity (h) of infected cells. uptake of taurocholate (tca) in a sodium-containing condition and found that glabridin reduced tca uptake in a dose-and time-dependent manner ( figure 6a ). a previous study has shown that ntcp-mediated bile acid transport affects the expression of ifn stimulatory genes (isgs) in primary human hepatocytes [15] , so we investigated whether glabridin counteracts this function of bile acid. primary human hepatocytes were treated with type i ifn and tca in the presence or absence of glabridin. treatment with ifn upregulated various isg proteins including mx1 and bst2, which are known to inhibit hbv replication [25] [26] [27] [28] , while concurrent treatment with tca reduced this effect ( figure 6b ), in accordance with previous observations [15] . interestingly, the addition of 50 µm glabridin partially cancelled the effect of tca in isg expression ( figure 6b ). these findings suggest that glabridin enhances the innate immune response by suppressing bile acid uptake in hepatocytes through the downregulation of cell-surface ntcp. in this study, we generated intcp cells, which have high ntcp expression and high susceptibility to hbv infection, and also developed a monoclonal antibody (mab) that recognizes cell-surface ntcp. using these tools, we identified glabridin as a compound that inhibits hbv infection by downregulating levels of its entry receptor, ntcp. although primary hepatocytes express ntcp at low levels for the uptake of bile acids, endogenous ntcp in hepatocellular carcinoma cell lines is not sufficient to achieve successful infection with hbv in vitro. hepatocellular carcinoma cell lines stably expressing ntcp have been created, and exhibited increased susceptibility to hbv infection [29] , but it has been shown that continuous ntcp expression can induce cell cycle arrest at the g0/g1 phase [17] , indicating that ectopic ntcp expression may be unfavorable for the long-term culture. to overcome these issues, we created the intcp cell line, featuring inducible ntcp expression, using the third-generation tetracyclineinducible gene expression system in hepg2 cells. upon transient treatment with tetracycline derivatives, this cell line exhibits high expression of cell-surface ntcp and becomes highly susceptible to hbv infection without any observable cell cycle arrest. this unique feature of our newly developed cell line makes it a useful tool for further biological analysis of ntcp in hepatic cells. we created a new mab recognizing cell-surface ntcp by using a wheat germ cell-free protein production system to synthesize full-length recombinant ntcp protein, which was then used to immunize mice. generally, the quality of an antibody is determined largely by the antigen used for immunization. preparation of immunogens derived from highly insoluble membrane proteins is challenging, and ntcp tends to be insoluble in conventional cell expression systems due to its multiple transmembrane domains. there are several methods for preparing antigens derived from insoluble proteins, including use of synthetic peptides containing the predicted immunogenic epitope of extracellular domains [30] , but synthetic peptides are structurally linear and do not recapitulate the native structural features of membrane proteins. by contrast, the wheat germ cell-free protein production system utilizes a eukaryotic translation system to synthesize structurally intact and biologically active proteins similar to those expressed in mammalian cells [31, 32] . this approach enabled us to create a mab capable of detecting a spatial antigen within the region of ntcp exposed on the cell surface. our work demonstrates the advantage of the cell-free system in the production of proteins with multiple transmembrane domains [18, 19] . the three-dimensional structure of ntcp protein has not been well characterized and the number of transmembrane domains is controversial at present. several groups have predicted that ntcp has 7-9 transmembrane domains and that the c-terminus of ntcp is located in the cytoplasm [33, 34] . we found that our 9a8 mab recognizes amino acids 317-326 of ntcp on intact cells without membrane permeabilization, implying that this epitope is possibly exposed to the extracellular space. more precise structural biological studies should be carried out to elucidate the topology of the ntcp protein. immunofluorescence microscopy experiments using our 9a8 antibody revealed that ntcp localized on the plasma membrane, frequently at cell-cell contact sites. this localization was more obvious in a hepatocyte organoid culture system, suggesting that the localization of ntcp may depend on cell polarity. because the threedimensional culture system recapitulates cell polarity, it is useful for analyzing the function of ntcp in sodium taurocholate transport as well as hbv infection. recent studies demonstrated that the three-dimensional culture system is suitable for analyzing polarized hbv transmission [35, 36] . it is not well established whether ntcp plays a role in viral transmission in polarized cells, and our newly developed 9a8 mab may be useful for pursuing this intriguing question. several compounds have been shown to target ntcp. recent studies demonstrated that antagonists of retinoic acid receptor ro41-5253 [37] or the cytokine interleukin-6 [38] could reduce ntcp expression. a previous report also indicated that the green tea extract epigallocatechin-3-gallate (egcg) also inhibited hbv entry into primary human hepatocytes [39] . interestingly, egcg induced endocytosis of ntcp from the plasma membrane followed by protein degradation. in the current study, we found that glabridin, a compound from licorice extract, also causes ntcp receptor downregulation. although the precise mechanisms are unclear, our findings intcp cells were treated with glabridin (10, 25, and 50 µm) for three or 20 hours. after incubation with [ 3 h]-taurocholate (tca) for 15 minutes, cells were washed and intracellular radioactivity was quantified. cyclosporin a (csa, 10 µm) was used as a positive control in this assay. * p < 0.05, ** p < 0.01, two-tailed unpaired t-test. (b) glabridin counteracts bile acids to promote innate immune signaling. primary human hepatocytes were sequentially treated with ifn-β (100 u/ml), tca, and glabridin (50 µm), as shown in left panel. the expression of representative isgs, including mx1 and bst2, was quantified using qpcr. ns, not significant; ** p < 0.01, two-tailed unpaired t-test. www.oncotarget.com suggest that glabridin directly binds ntcp and causes its internalization by caveolar endocytosis. by estimating protein turnover using a cycloheximide chase assay, we were able to infer that glabridin promotes the intracellular degradation of ntcp. interestingly, glabridin is orally bioavailable and is known to rapidly accumulate in the liver [40] [41] [42] . since ic 50 value of glabridin for inhibiting of hbv entry is relatively high (~40 µm), this compound might have little translational benefit in hbv research. however, the data obtained from our study demonstrates the potential scope of our current methodology in drug discovery for hbv therapeutics. it is possible that the internalization of ntcp could inhibit its transporting activity thereby causing unfavorable effects on hepatocytes. however, people with i223t or s267f mutations in the ntcp gene show decreased surface expression and transporting activity of ntcp, but there have been no reports of serious diseases resulting from these mutations to date [43, 44] . the frequent occurrence of physiological downregulation of ntcp is also notable. this downregulation is typically controlled by transcription factors, such as hnf4α, under cholestasis conditions [45] . furthermore, recent papers have shown that ntcp is the main transporter for conjugated bile acids into the liver, but other auxiliary transporters, such as oatp, may compensate when ntcp is absent [46, 47] . although these reports hint that transient downregulation of ntcp might not induce immediate adverse effects on the liver, the effect should be further investigated in in vivo models to determine whether ntcp inhibition is a reasonable anti-hbv drug target. in conclusion, we identified as glabridin as a natural compound capable of inhibiting hbv infection by impairing viral entry into host cells, with an additional effect of enhancing the antiviral immune response. furthermore, our data demonstrate that our newly developed cell line and antibody will serve as powerful tools for drug discovery targeting hbv entry and for exploring molecular mechanisms underlying hbv spread. to generate intcp cells, a hepg2 tet-on advanced cell (clontech) parental cell line was transduced with a retroviral vector encoding the ntcp gene fused to a tetracycline-responsive element, then was selected with puromycin (1 µg/ml), and cultured with dmem (wako) supplemented with 10% fbs. unless otherwise indicated, intcp cells were treated with doxycycline (sigma-aldrich) for 24 hours before experiments. the intcp spheroid was made using the 3-d life dextran-cd hydrogel sg kit (cellendes) and cultured for seven days on a chamber slide (thermo fisher scientific). primary human hepatocytes (pxb-cells) were purchased from phoenixbio. heparg cells were purchased from biopredic international and differentiated according to the manufacturer's instructions. the chemical compound library from natural plant extracts was obtained from tokiwa phytochemical. nocodazole, cyclosporin a, genistein, and pitstop2 were purchased from sigma-aldrich. glabridin, staurosporine, and ifn-β were obtained from wako. a protemist xe robotic protein synthesizer (cellfree sciences) was used for the generation of full length ntcp and its truncated derivatives as previously described [48, 49] . immunization of balb/c mice with recombinant ntcp and generation of hybridoma cells producing anti-ntcp antibody were performed as previously described [48] . purification of antibodies in the culture supernatant of the hybridoma clones was performed by centrifugation at 8,000 rpm for 15 minutes and elution with acrosep hyper df columns (pall). samples were then concentrated using amicon ultra filters (merck millipore). immunoglobulin characterization was carried out using the isostrip mouse monoclonal antibody isotyping kit (roche). cells were fixed with 4% paraformaldehyde, blocked with 10% normal goat serum (thermo fisher scientific), and stained with either anti-ntcp mab (clone 9a8) or anti-hbcag polyclonal antibody (dako) and alexa fluor-conjugated secondary antibodies (thermo fisher scientific). alexa fluor 594-conjugated phalloidin (thermo fisher scientific) was used for f-actin staining. for intracellular staining, cells were permeabilized with 0.5% triton x-100 before blocking. for the pres1-peptide binding assay, intcp cells pretreated with 5 µg/ml dox for 24 hours were incubated with 400 nm fitc-conjugated pres1 peptide (the first 59 amino acid residues of small hbs domain fused with n-terminal myristoyl group and c-terminal fitc) at 37° c for two hours. cells were then washed with pbs and fixed with 4% paraformaldehyde. microscopic imaging was performed with an fv1000-d confocal laser scanning microscope (olympus) or bz-9000 fluorescence microscope (keyence). cells were detached with 5 mm edta in pbs and fixed with 4% formaldehyde before incubation with either anti-ntcp (9a8) or anti-transferrin receptor (genetex) antibodies at 4° c. cells were then stained with pe-conjugated secondary antibody and analyzed using a facscanto ii instrument (bd biosciences). for the drug screen, cells were treated with candidate compounds www.oncotarget.com (50 µm) 24 hours before analysis. data were analyzed with flowjo software (treestar). wild-type hbv was derived from the supernatants of hepg2.2.15 cells, which were stably transfected with a complete hbv genome. hbv reporter viruses (hbv-nl) were produced by transient transfection of hepg2 cells with puc1.2-hbv/nl and puc-hbv-d, as previously described [21] . the collected supernatants were filtered through a 0.45-μm filter (merck millipore), and concentrated approximately 100 times using a peg virus precipitation kit (biovision). cells were infected with wild-type hbv at a concentration of 5,000 genome equivalents per cell in the presence of 4% peg8000 for 16 hours. alternatively, cells in a 96-well plate were inoculated with 5 µl of hbv-nl in the presence of 4% peg8000 for 16 hours. hbv-infected cells were cultured in fresh medium for an additional 5-6 days and their infectivity was determined by intracellular hbcag staining or extracellular hbsag quantification, as previously described [28] . the infectivity of hbv-nl was quantified using the nano-glo luciferase system (promega), according to the manufacturer's instructions. samples in sds loading buffer were loaded onto 10% polyacrylamide gels, electrophoresed, and blotted onto pvdf membranes (merck millipore), as previously described [28] . membranes were probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (ge healthcare). for protein degradation analysis, cycloheximide (100 µg/ml) was added 2-6 hours before harvesting cells. the primary antibodies used were as follows: anti-ntcp, anti-vinculin, anti-α-tubulin (sigma-aldrich) and anti-his (genetex). detected proteins were visualized using a fluorchem digital imaging system (alpha innotech). band analysis was performed with imagej software (national institutes of health). messenger rna extraction and subsequent cdna synthesis was performed using trizol reagent (thermo fisher scientific) and revertra ace (toyobo), respectively, according to each manufacturer's instructions. gene expression was then analyzed by qpcr using sybr premix ex taq ii (takara) and a cfx96 real-time pcr detection system (bio-rad). the primer pairs used were 5′-ggacttcgagcaagagatgg-3′ and 5′-agcactgtgttggcgtacag-3′ for actb; 5′-atggaggcccacaacg cgtctgccc-3′ and 5′-cagaaggtggagcaggtggtcatcac-3′ for ntcp; 5′-ggctgtttaccagactccgaca-3′ and 5′-cacaaa gcctggcagctctcta-3′ for mx1; and 5′-tctcctgcaaca agagctgacc-3′ and 5'-tctctgcatccagggaagccat-3' for bst2. recombinant ntcp-gfp and gfp proteins were incubated with different concentrations of compounds in 50 mm potassium phosphate buffer (ph 7.0) containing 100 mm nacl, 0.2% bsa, and 0.005% ddm (n-dodecylβ-d-maltopyranoside) for one hour at room temperature. samples were loaded on monolith nt.115 standard treated capillaries (nano temper technologies) and analyzed with a monolith nt.115 blue red microscale thermophoresis instrument. cell cycle analysis was performed with a tali image-based cytometer (thermo fisher scientific). cell viability was measured with cell counting kit-8 (dojindo) or celltiter-glo assay (promega) according to the manufacturer's instructions. cells were treated with [ 3 h]-taurocholic acid (tca) in a sodium-free or sodium-containing buffer at 37ºc for 15 minutes to allow tca uptake into cells. cells were washed and intracellular radioactivity was measured using a liquid scintillation counter. all graphs represent means and standard deviations. the statistical significance of differences between two groups was tested using a two-tailed unpaired t test with prism 6 software (graphpad). km designed and performed research, analyzed data, and wrote the manuscript; sm, yy, md, and kf performed research and analyzed data; hk and tc analyzed data; hn, kw, ks, and tw contributed reagents and analyzed data; and ar designed and supervised the research, analyzed the data, and wrote the manuscript. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity virologic monitoring of hepatitis b virus therapy in clinical trials and practice: recommendations for a standardized approach pegylated interferon alfa-2b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial nucleoside analogues for chronic hepatitis b: antiviral efficacy and viral resistance entry of hepatitis b and hepatitis d virus into hepatocytes: basic insights and clinical implications sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for speciesspecific entry into hepatocytes characterization of cloned rat liver na(+)-bile acid cotransporter using peptide and fusion protein antibodies in situ localization of the hepatocytic na+/ taurocholate cotransporting polypeptide in rat liver chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas sinusoidal (basolateral) bile salt uptake systems of hepatocytes first-in-human application of the novel hepatitis b and hepatitis d virus entry inhibitor myrcludex b treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study kinetics of the bile acid transporter and hepatitis b virus receptor na+/taurocholate cotransporting polypeptide (ntcp) in hepatocytes solute carrier ntcp regulates innate antiviral immune responses targeting hepatitis c virus infection of hepatocytes bile acids modulate the interferon signalling pathway down-regulation of ntcp expression by cyclin d1 in hepatitis b virus-related hepatocellular www.oncotarget.com carcinoma has clinical significance production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system a cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters single particle imaging of polarized hepatoma organoids upon hepatitis c virus infection reveals an ordered and sequential entry process novel reporter system to monitor early stages of the hepatitis b virus life cycle molecular interaction studies using microscale thermophoresis evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells overexpressing a membrane transporter ntcp glabridin mediate caspases activation and induces apoptosis through jnk1/2 and p38 mapk pathway in human promyelocytic leukemia cells mxa inhibits hepatitis b virus replication by interaction with hepatitis b core antigen the interferon-inducible protein tetherin inhibits hepatitis b virus virion secretion identification of bst-2/tetherin-induced hepatitis b virus restriction and hepatocyte-specific bst-2 inactivation molecular dissection of hbv evasion from restriction factor tetherin: a new perspective for antiviral cell therapy cell culture models for the investigation of hepatitis b and d virus infection generation and identification of peptide-based monoclonal antibodies against vacuolar proton pyrophosphatase of toxoplasma gondii advances in genome-wide protein expression using the wheat germ cell-free system cell-free expression systems for eukaryotic protein production organization of the membrane domain of the human liver sodium/bile acid cotransporter cloning and functional characterization of human sodium-dependent organic anion transporter (slc10a6) concise review: organoids are a powerful tool for the study of liver disease and personalized treatment design in humans and animals cultivation of hepg2.2.15 on cytodex-3: higher yield of hepatitis b virus and less subviral particles compared to conventional culture methods dysregulation of retinoic acid receptor diminishes hepatocyte permissiveness to hepatitis b virus infection through modulation of sodium taurocholate cotransporting polypeptide (ntcp) expression interleukin 6 inhibits hbv entry through ntcp down regulation epigallocatechin-3-gallate inhibits entry of hepatitis b virus into hepatocytes determination of glabridin in human plasma by solidphase extraction and lc-ms/ms role of p-glycoprotein in limiting the brain penetration of glabridin, an active isoflavan from the root of glycyrrhiza glabra role of p-glycoprotein in the intestinal absorption of glabridin, an active flavonoid from the root of glycyrrhiza glabra ethnicity-dependent polymorphism in na+-taurocholate cotransporting polypeptide (slc10a1) reveals a domain critical for bile acid substrate recognition genetic polymorphisms in na+-taurocholate co-transporting polypeptide (ntcp) and ileal apical sodium-dependent bile acid transporter (asbt) and ethnic comparisons of functional variants of ntcp among asian populations hepatocyte nuclear factor-4alpha is a central transactivator of the mouse ntcp gene sodium taurocholate cotransporting polypeptide (slc10a1) deficiency: conjugated hypercholanemia without a clear clinical phenotype hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice wheat germ cell-free system-based production of hemagglutininneuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen www.oncotarget.com we thank naohito nozaki for antibody production and haruka sato, kyoko ohnishi, and sho nonoyama for their technical assistance. the authors declare that they have no competing interests. this work was supported in part by an amed grant-in-aid for the program on the innovative development and the application of new drugs for hepatitis b (jp17fk0310103 to ar and jp17fk0310104 to km), the creation of innovation centers for advanced interdisciplinary research areas program (to ar), and by a gsk japan research grant (to km). key: cord-280643-n8qjorqk authors: wu, kai-lang; zhang, xue; zhang, jianlin; yang, yongbo; mu, yong-xin; liu, mo; lu, lu; li, yan; zhu, ying; wu, jianguo title: inhibition of hepatitis b virus gene expression by single and dual small interfering rna treatment date: 2005-04-26 journal: virus res doi: 10.1016/j.virusres.2005.04.001 sha: doc_id: 280643 cord_uid: n8qjorqk rna interference (rnai) has been successfully applied in suppression of hepatitis b virus (hbv) replication. to circumvent the problem that mutation in hbv genome may result in resistance when sirna is further developed as an anti-viral drug, in this study, we established a dual small interfering rna (sirna) expression system, which could simultaneously express two different sirna molecules that can specifically target two genes. to test the effectiveness of this system, we applied this new approach to express simultaneously two different 21-bp hairpin sirna duplexes that specifically attack the hbs and hbx genes of hbv, respectively, in bel-7402 and hepg2.2.15 cells. results indicated that dual sirna could simultaneously inhibit the expression of hbs and hbx gene by 83.7% and 87.5%, respectively, based on luciferase assays. in addition, dual sirna molecules were able to significantly reduce the amount of hbv core associated dna, which is considered as an intracellular replicative intermediate, and the viral dna in culture supernatant. therefore, this dual sirna system provides a more powerful tool for the study of gene function and implicates a potential application in the treatment of viral infection. rna interference (rnai) is a natural process of eukaryotic cells by which double-stranded rna initiates and directs the degradation of homologous mrna (hannon, 2002) . this rna silencing mechanism was first described in caenorhabditis elegans and drosophila melanogaster (fire et al., 1988) . it has many similarities to the posttranscriptional gene silencing in plants. specific inhibition of cellular mrna by rnai can be triggered in mammalian cells by the introduction of synthetic 21-to 23-nucleotide double-stranded small interfering rna (sirna) (elbashir et al., 2001; paul et al., 2002) or, alternatively, by the transcrip-tion of sirna from a dna construct driven by the rna polymerase cassette (brummelkamp et al., 2002) . these findings open up a new field for the analysis and control of the processes of gene expression, and perhaps pathogen infection. the replication of a growing number of human pathogenic viruses in cell culture was shown to be inhibited by rnai, including poliovirus (coburn and cullen, 2002) , hiv-1 (jacque et al., 2002; lee et al., 2002) , flock house virus (fhv) (dector et al., 2002) , rous sarcoma virus (hu et al., 2002) dengue virus (adelman et al., 2002) , hepatitis c virus (hcv) (kapadia et al., 2003) replicons, influenza virus (ge et al., 2003) , hepatitis b virus (hbv) (hamasaki et al., 2003; mccaffrey et al., 2003) , hpv (jiang and milner, 2002) . recently, it was reported that rnai could also induce transcriptional silencing of sars coronavirus (he et al., 2003) . in most above studies, synthetic 21-nucleotide doublestranded sirnas were applied. however, vector based rnai techniques were used more frequently in recent studies. each vector expresses unique sirna that can degrade a specific target. eight genotypes (a-h) of hbv have been described. the number of hbv carriers worldwide has been estimated to be more than 400 million. these individuals have a 15-25% risk of developing liver diseases such as liver cirrhosis and hepatocellular carcinoma (kao and chen, 2002) . although a few drugs were developed against hbv infection, the success rate of these treatments, however, is low and frequently infections reoccur (carreno et al., 1992; lai et al., 1997) . the fact that rnai can be applied for blocking the replication of hbv in several reports provided insights into the field of controlling infectious human hepatitis. nevertheless, mutations in hbv genome may result in viral resistance to sirna. it has been reported that hiv-1 can escape from rnai-mediated inhibition due to nucleotide change in the genome (das et al., 2004) . one strategy to circumvent the problem is to choose target in the relatively conserved dna sequence. the other approach is to produce multiple sirnas that target different sites or genes on the viral genome. we here established a system that can express two sirna duplexes simultaneously and target the s and x genes of hbv, respectively. to study the effects of dual rnai on hbv gene expression in a cell culture model, we used a derivative of the human hepg2 hepatoma cell line, hepg2.2.15, which has been stably transformed with several copies of the hbv genome and used as an in vitro model for hbv replication. the effects of dual sirna system on hbv gene expression were investigated in this study. two human hepatoma cell lines, bel-7402 and hepg2.2.15 were maintained in dulbecco's modified eagle medium (gibco/brl) supplemented with 100 units/ml penicillin, 100 g/ml streptomycin and 10% heat-inactivated fetal bovine serum at 37 • c under 5% co 2 . cells were seeded onto 24-well plates at a density of 1.0 × 10 5 or 4.0 × 10 5 cells per 24-well plate or 6-well plate and grown to the confluence reaching approximately 60% at the time of transfection. cells were transfected with 0.1 or 0.4 g of plasmid pcmv-hbs together with 0.45 or 1.2 g psliencer-2.1-u 6 -sirna, using sofast tm transfection reagent (xiamen sunma biotechnology co. ltd., china) according to the protocol provided by the manufacturer. the cells were harvested 48 h after transfection. full-length hbv genomic dna (subtype ayw) was cloned into the hindiii and saci sites of pbluescript (stratagene) to generate the plasmid pblue-hbv. hbs gene was cloned into the hindiii and saci sites of vector pcmv-tag2a (stratagene) fig. 1 . schematic diagrams of luciferase fusion genes, sirna targeting sites and dual sirna expression cassettes. (a) diagram of the two reporter fusion vectors, which contain targeted sequences of hbs or hbx gene and the luciferase report gene driving by the cmv promoter. (b) locations of rnai targeted sites and structure of the hbv genome. downward arrows indicate the locations of rnai target sites within the four hbv transcripts. the 3.5-kb transcript is the pregenomic rna that serves as the template for hbv viral dna replication. the hbv open reading frames are shown below aligned with the hbv mrnas. pol, polymerase; core, hbcag; s1, large presurface antigen; s2, mid pre-surface antigen; s, hb-sag; x, x gene. the numbers above the arrows indicate the sirna target sites. 1 = hbs 1 sirna, 2 = hbs 2 sirna, 3 = hbs 3 sirna, 4 = hbs 4 sirna, 5 = hbs 5 sirna, 6 = hbx 1 sirna, 7 = hbx 2 sirna, 8 = hbx 3 sirna. (c) diagram of dual sirna expression cassettes. to yield plasmid pcmv-hbs. hbx gene was cloned into pcmv-tag2a at ecori and xhoi sites to generate pcmv-hbx. two-pair of primers 5 -ctgcgagatctatggagagc-tcacatcaggattc-3 (sense), 5 -gttaggtcgacaa-tgtatacccaaagacaaaaagaa-3 (antisense) or 5 -gatcatacgcgtaagcttttcatttattgatcat-3 (sense), 5 -gtcggggcttcattcactcgtctagaac-tgat-3 (antisense) were used to amplify the hbs and hbx gene, respectively. the pcr products were then cloned into sali and bglii sites of plucf to generate plasmid plucf-hbs and plucf-hbx (fig. 1a) , in which the hbs or hbx were fused in frame with the luciferase gene and the expression of the fusion gene was drove by the cmv promoter (fig. 1a ). five regions of the hbs gene and three regions of the hbx gene were selected as the targeted sequences of sirna in this study (fig. 1b) . to construct single sirna expression vector, two 64nt primers, each containing a 19nt target sequence in the sense and antisense forms from different regions of the hbs gene or hbx gene as indicated below, were systhesized (invitrogen): 5 -gctcccgcgtgtcttggcc-3 (hbs 1 sirna); 5 -ggtggacttctctcaattt-3 (hbs 2 sirna); 5 -gccaaaattcgcagtccc-3 (hbs 3 sirna); 5 -gttgctgtaccaaacctt-3 (hbs 4 sirna); 5 -gctcagtttactagtgcca-3 (hbs 5 sirna); 5 -gcacttcgcttcacctctg-3 (hbx 1 sirna); 5 -gcaatgtcaacgaccgacc-3 (hbx 2 sirna); 5 -gtttaaagactgggaggag-3 (hbx 3 sirna). sense and antisense primers were then cloned into psilence-2.1-u 6 plasmid (amibion) at bamhi and hindiii sites after annealing according to the manufacturer's instructions. to generate the dual sirna expression plasmid, two primers 5 -gctgatgacgtcagtggaaagacgcg-3 -(sense) and 5 -tcagcgaattcacgccaagcttttcc-3 (antisense) were designed to amplify a dna fragment containing u6 promoter and hbx 2 sirna expression cassette from recombinant plasmid psilencer-2.1-u6-hbx 2 . the pcr product was then cloned into aatii and ecori sites of plasmid psilencer-2.1-u6-hbs 2 to generate recombinant plasmid psilencer-2.1-u6-hbsx, which carries two independent sirna expression cassettes (fig. 1c ). bel-7402 cells were co-transfected with reporter plasmids and sirna expression plasmids. cells were washed with pbs and lysed with luciferase cell culture lysis reagent (promega). ten microliters of the cell lysates and 100 l of luciferase assay substrate (promega) were mixed and fluorescence intensity was detected by the luminometer (turner t20/20). assays were performed in triplicate, and expressed as means ± s.d. relative to vector control as 100%. bel-7402 cells and hepg2.2.15 cells were transfected with sirna expression plasmids, the level of hbsag protein in culture media from transfected cells were then determined by enzyme-linked immunosorbent assay using a hbv diagnostic kit (shanghai kehua biotech co. ltd.). assays were performed in triplicate independent experiments. bel-7402 cells and hepg2.2.15 cells were transfected with sirna expression plasmids, total rna were then extracted from transfected cells by trizol reagent (invitrogen) according to the method described in the manufacturer's manual. reverse transcription were performed with total rna as the template. the cdnas were synthesized with hbs or hbx gene specific primers, 5 -gcggggtttttcttgttgac-3 (sense), 5 -ctacgaaccactgaacaaat-3 (antisense) or 5 -cctgcgcgggacgtcctttg-3 (sense), and 5 -cagtctttgaagtatgcctc-3 (antisense). to assay the effect of sirnas on hbv replication, intracellular core-associated hbv dna was extracted by the method described previously (pugh et al., 1988) . briefly, 1 × 10 5 transfected hepg2.2.15 cells were lysed and centrifuged at 25 • c. magnesium chloride was added to the supernatant. dna not protected by hbv core was treated digested with deoxyribonuclease (dnase i). then the lysates were treated with proteinase-k and, after phenol/chloroform extraction; core-associated hbv dna was recovered by ethanol precipitation, and quantified by real time-pcr (rt-pcr) as described by the manufacturer (pg biotech, shenzhen, china). the hbv dna in the supernatants was also quantified following the procedure provided by the manufacturer (pg biotech, shenzhen, china). primers used in rt-pcr were: p1, 5 -atcctgctgctatgcc-tcatctt-3 and p2, 5 -acagtggggaaagcccta-cgaa-3 . the probe was 5 -tggctagtttactagtgc-cattttg-3 . pcr reaction was carried out and analyzed by a pe gene amp 7700 (perkin-elmer, usa). to efficiently screen sirna molecules, selected targeting dna sequences were fused in frame with that of luciferase gene, in which luciferase activity was supposed to represent the level of hbs or hbx mrna expression. cells were co-transfected with plucf-hbs or plucf-hbx and eight single sirna expression vectors, respectively. luciferase activities were then determined from those transfected cells. result showed that hbs 1 sirna, hbs 2 sirna and hbx 2 sirna strongly inhibited luciferase activities by 81.5%, 80.5%, and 76.5%, respectively, comparing to that of vector control ( fig. 2a and b) . these results indicated that the three sirnas could efficiently degrade the mrna of hbs-luciferase or hbx-luciferase fusion gene. to evaluate the effects of dual sirna expression plasmid on the inhibition of hbs-luciferase or hbx-luciferase fusion gene expression, cells were co-transfected with plucf-hbs or plucf-hbx and the dual sirna expression plasmid phb-sxsirna. result from luciferase activity assays indicated that there was a further reduction in luciferase activity by dual fig. 2 . quantitative analysis of luciferase activity in cells after transfected with sirna expression plasmids. (a) bel-7402 cells were co-transfected with plucf-hbs plasmid and psliencer-2.1-u 6 -sirna (hbs 1 sirna, hbs 2 sirna, hbs 3 sirna, hbs 4 sirna, hbs 5 sirna) plasmids; psliencer-2.1-u 6 plasmid was used as a control. (b) bel-7402 cells were co-transfected with plucf-hbx plasmid and psliencer-2.1-u 6 -sirna (hbx 1 sirna, hbx 2 sirna, hbx 3 sirna) plasmids; psliencer-2.1-u 6 vector was used as a control. (c) bel-7402 cells were co-transfected with plucf-hbs and psliencer-2.1-u 6 -sirna (hbs 2 sirna, hbx 2 sirna, hbsxsirna) plasmids; psliencer-2.1-u 6 was used as vector control. (d) bel-7402 cells were co-transfected with plucf-hbx and psliencer-2.1-u 6 -sirna (hbx 2 sirna, hbsxsirna, hbs 2 sirna); psliencer-2.1-u 6 was used as control. forty-eight hrs after transfection, cells were lysed and luciferase activities were determined by luminometer. sirna duplexes (hbsxsirna) comparing to that of single sirna expression vectors (hbs 2 sirna or hbx 2 sirna). the reduction rate of luciferase activity caused by hb-sxsirna was 83.7% to hbs and 87.5% to hbx, respectively ( fig. 2c and d) . to evaluate the influence of rnai on hbs gene expression, bel-7402 cells were transfected with psilence2.1-u6-sirna, pcmv-hbs or hbsxsirna and hepg2.2.15 cells were transfected with psilence2.1-u6-sirna or hbsx sirna. hbsag concentrations in the culture media of transfected and control cells were measured 2 days after transfection by elisa using hbv diagnostic kit. results showed that hbsag level was decreased in the bel-7402 cells after transfection with hbs 1 sirna, hbs 2 sirna or hbsxsirna with reduction rate of 91.5%, 88.5% ,and 83.7%, respectively ( fig. 3a and d) . in hepg2.2.15 cells, transfection with hbs 1 sirna, hbs 2 sirna, or hbsxsirna reduced hbsag level by 75.4%, 85.7%, and 87.6%, respectively ( fig. 3b and c) . in addition, transfection with hbx 2 sirna reduced the level of hbsag production by 65.3% in hepg2.2.15 cells (fig. 3e) . to determine whether sirnas specificly degrade hbs or hbx mrna, we used semi-quentitation rt-pcr analyses to determine the levels of hbs or hbx mrna in two different cell lines, bel-7402 ( fig. 4a and b) and hepg2.2.15 ( fig. 4c and d) , 2 days after transfection. results indicate that the levels of hbs mrna were significantly decreased by the treatment of hbsxsirna (fig. 4a, lane 1) , hbs 1 sirna (fig. 4a, lane 2) , hbs 2 sirna (fig. 4a, lane 3) in bel-7402. the levels of hbx mrna were also decreased by the treatment of hbsxsirna (fig. 4b, lane 1) , hbx 2 sirna (fig. 4b, lane 2) , but not by that of psliencer-2.1-u 6 or untreated cells (fig. 4b, lanes 3 and 4) . similar results were also obtained in hepg2.2.15 cell lines under the same conditions of sirna treatments ( fig. 4c and d) . results showed that the levels of hbs mrna were dramatically reduced in hepg2.2.15 cells after the treatment of hbsxsirna (fig. 4c, lane 6) , hbs 1 sirna (fig. 4c, lane 7) and hbs 2 sirna (fig. 4c, lane 8) , respectively. the levels of hbx mrna in hepg2.2.15 cells were also reduced by the treatment of hbsxsirna (fig. 4d, lane 2) , hbx 2 sirna (fig. 4d, lane 3) , but not by that of psliencer-2.1-u 6 or untreated cells (fig. 4d, lanes 1 and 4) . in addition, our results showed that the inhibition effects of dual sirna, hbsxsirna on the levels of hbs and hbx mrna (fig. 4a (lane 1) , b (lane 1), c (lane 6), and d (lane 2)) were more sever than that of single sirna (fig. 4a, (lanes 2 and 3) , b (lane 2), c (lane 7 and 8), and d (lane 3)). to determine the effectiveness of sirnas on viral dna replication, hbv core associated dna (as an intracellular replicative intermediate) and hbv dna were extracted from hepg2.2.15 cells transfected with hb-sxsirna, hbs1sirna, hbs2sirna, hbx2sirna, and vector, respectively. the levels of hbv core associated dna and hbv dna were determined by real time pcr. results indicated that the levels of hbv core associ-ated dna were significantly decreased in the cells transfected by hbsxsirna, hbs 1 sirna, hbs 2 sirna, and hbx 2 sirna with reduction rate of 90.2%, 85.7 %, 81.3%, and 60.4%, respectively, compared with that of vector control (fig. 5a) . in hepg2.2.15 cells, transfection with hb-sxsirna, hbs 1 sirna, hbs 2 sirna and hbx 2 sirna reduced the level of viral dna in supernatants media by 88.7%, 82.6%, 78.4%, and 58.3%, respectively (fig. 5b) . it has been attracted considerable attentions in the use of rnai as therapeutics to treat a variety of diseases, including tumors and viral infections. hamasaki et al. (2003) demonstrated that rnai could attenuate the replication of hbv genome in cell culture. shlomai and shaul (2003) used a similar approach to inhibit the replication and expression of hbv in hepg2.2.15 cell line, in which all hbv proteins could be expressed. recently, mccaffrey et al. (2003) went further in this field by showing that rnai were function well in transgenic mice. these reports demonstrate that sirna treatments can be used to suppress hbv in cell cultures and animal models as well as provided insights into the application of controlling infectious human hepatitis. in this study, we applied a different approach by designing a pair of 64nt primers that contain a specific 19nt target sequence from hbv genome to create recombinant psilencer-u6 plasmid. primers were annealed and cloned into bamhi-hindiii sites of the psilencer2.1-u6 vector. in order to construct a useful tool to choose the most effective sirna molecules, we created a quick screening vector plucf by fusing the targeted sequence and the reporter luciferase gene together to produce recombinant plucf plasmid, which could express hbs-luciferase or hbx-luciferase fusion mrnas. therefore, we can initially select the suitable sirna duplexes rapidly by simply analyzing the activities of lucifearse. by using this approach, we have identified two sirna molecules (hbs 1 sirna and hbs 2 sirna) having significant impact on the hbs-luciferase fusion gene expression and one sirna duplex (hbx 2 sirna) having effects on the expression of hbx-luciferase fusion gene. this provides a quick approach to select effective sirna in the study of gene expression and function analysis. to further study the effects of selected rnai molecules on hbv gene expression and viral replication in a cell culture models, we used a derivative of the human hepg2 hepatoma cell line, hepg2.2.15, which has been stably transformed with several copies of the hbv genome and used as an in vitro model for hbv replication. the effects of dual sirna system on hbv gene expression and viral replication were studied thoroughly by the analyzing the levels of viral protein production through enzyme-linked immunosorbent assay and the levels of viral rna expression by semi-quantitated rt-pcr analysis. all results indicated that hbs 1 sirna, hbs 2 sirna, and hbx 2 sirna had significant reduction effects on viral mrna expression, and viral protein production. the fact that mutation in hbv genome may result in resistance if sirna molecules were further developed as antiviral drugs raised our concerns. our strategies to address such potential problems are to choose targets in the relatively conserved dna sequences and to generate multiple sirna molecules that can target different sites or genes on the viral genome. to test our approach, in this study we established a system that can simultaneously express two sirna duplexes from a single vector that can attack the s and x genes of hbv, respectively. results from luciferase activity assay, enzyme-linked immunosorbent assay and semiquantitated rt-pcr analysis were consistently showed that the dual sirna molecules had synergetic effects or more efficient on the targeted viral protein production and hbs and hbx gene expression comparing to that of the single sirna molecules. more importantly, dual sirna could simultaneously inhibit the expression of hbs and hbx gene by 83.7% and 87.5%, respectively. therefore, this dual sirna system could provide a more powerful tool for the study of gene function and could be used as a potential application in the treatment of viral infection. in the last 20 years, hbv infection affects millions of people each year worldwide. current therapies of hbv infection including immune modulators such as interferon alfa, or nucleoside analogs such as lamivudine have provided some degree of cures, but the efficiency of treatment was limited. as a potential therapy, sirna seems to be a hopeful alternative strategy. we believe that our approach presented in this study could be broadly used. for example, it could be used to generate more than two sirnas duplexes that could silent more genes in order to study the interactions of genes and their functions. such strategies of constructing multiple-sirna vectors can confront the evading mechanism of virus infections. obviously, this cocktail approach would be benefit to application of sirna therapy in viral infections, especially to those viruses with high mutation rate. in addition, this approach could also used to deal with two or more viruses, which are especially useful in the treatment of co-infections by two or more pathogens, such as hbv-hiv and hcv-hiv. we are in the process of testing these approaches. rna silencing of dengue virus type 2 replication in transformed c6/36 mosquito cells transcribing an inverted-repeat rna derived from the virus genome asystem for stable expression of short interfering rnas in mammalian cells long-term follow-up of hepatitis b chronic carriers who responded to interferon therapy potent and specific inhibition of human immunodeficiency virus type 1 replication by rna interference human immunodeficiency virus type 1 escapes from rna interference-mediated inhibition rotavirus gene silencing by small interfering rnas duplexes of 21-nucleotide rnas mediate rna interference in cultured mammalian cells potent and specific genetic interference by doublestranded rna in caenorbabditis elegans rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription short interfering rna-directed inhibition of hepatitis b virus replication rna interference inhibition of sars-associated coronavirus infection and replication by rna interference inhibition of retroviral pathogenesis by rna interference modulation of hiv-1 replication by rna interference selective silencing of viral gene expression in hpv-positive human cervical carcinoma cells treated with sirna, a primer of rna interference global control of hepatitis b virus infection interference of hepatitis c virus rna replication by short interfering rnas lamivudine is effective in suppressing hepatitis b virus dna in chinese hepatitis b surface antigen carriers: a placebocontrolled trial expression of small interfering rnas targeted against hiv-1 rev transcripts in human cells inhibition of hepatitis b virus in mice by rna interference effective expression of small interfering rna in human cells duck hepatitis b virus(dhbv) particles produced by transient expreesion of dhbv dna in a human hepatoma cell line are infectious in vitro inhibition of hepatitis b virus expression and replication by rna interference this study was supported by the research funds from the ministry of education and wuhan university. key: cord-267709-i2loz1xb authors: li, tongya; ke, zunlong; liu, weiyong; xiong, ying; zhu, ying; liu, yingle title: human hepatitis b virus core protein inhibits ifnα-induced ifitm1 expression by interacting with baf200 date: 2019-05-09 journal: viruses doi: 10.3390/v11050427 sha: doc_id: 267709 cord_uid: i2loz1xb human hepatitis b virus core protein (hbc) is a structural protein of the hepatitis b virus (hbv) and contributes to hbv regulation of host-cell transcription. however, the mechanisms of transcriptional regulation remain poorly characterized. to dissect the function of hbc, a yeast two-hybrid was performed to identify hbc-binding proteins, and the c-terminal of brg1/hbrm-associated factors 200 (baf200c) was identified. then, the existence of hbc interactions with baf200c and full-length baf200 was confirmed via co-immunoprecipitation assays in 293t, hepg2 and hepg2-ntcp cells. furthermore, we show that the binding between hbc and baf200 was of vital importance to hbc mediated downregulation of interferon-induced transmembrane protein 1 (ifitm1) expression, and the mechanisms for the downregulation were disclosed as follows. basal level of ifitm1 expression depends on baf200, rather than the jak–stat1 pathway. the interaction of hbc with baf200 disturbs the stability of the polybromo-associated baf (pbaf) complex and results in the suppression of iftm1 transcription. finally, the antiviral effects of ifitm1 on cell proliferation and hbv replication were found to be partially restored when hbc was co-transfected with baf200. collectively, our findings indicate that hbc plays a role in hbv resistance against the antiviral activities of ifnα, providing details about hbv evasion of host innate immunity. the human hepatitis b virus (hbv) is a double stranded dna virus in the hepadnaviridae family [1] . hbv infection could cause acute and chronic hepatitis b (chb), which can progress to cirrhosis and hepatocellular carcinoma, leading to high mortality rates worldwide. antiviral therapy with interferon aims to induce permanent immune control of hbv infection through stimulation of the hosts' innate immune response. nevertheless, experimental data from hbv infected chimpanzees and urokinase-type plasminogen activator/severe combined immunodeficiency (upa-scid) mice have shown that hbv infection does not induce an intrahepatic innate immune response that can be detected [2, 3] . this is because early in infection it acts like a stealth virus, remaining undetected and spreading until the onset of the adaptive immune response several weeks later [4] . besides acting as a stealth pathogen, recent developments have shown that hbv can avoid recognition by the host innate immune system. however, the precise mechanisms are largely unknown. human hepatitis b virus core protein (hbc) is 183 amino acids in length and dimeric in solution [5] . hbc dimers assemble into t = 4 (120 copies) or t = 3 (90 copies) capsids of hbv, with t = 4 capsids being the predominant form in vivo [6] . hbc is composed of an assembly domain (aa 1-149) and a nucleic acid-binding domain (aa 150-183) (figure 1a) , moreover, it not only acts as a structural protein of hbv, but also works as an essential regulator in viral replication [5, 7] . the nucleic acid-binding harbors a nuclear localization sequence (nls), which mediates the transport of hbc into the nucleus [7, 8] . in vitro studies have shown that hbc binds directly to the covalently closed circular dna (cccdna) upon entering the nucleus, such as the camp response element of hbv, enh i [9] , and the nuclear factor kappa b binding site of hbv, enh ii [10] , to regulate hbv transcription. in addition, hbc appears to regulate the activities of host cells by interacting directly with the host genome [11] . however, the details of hbc function in host transcriptional regulation are not well understood. human swi/snf (mating-type switching (swi) and sucrose non-fermenting (snf)) complexes regulate the expression of numerous interferon (ifn)-inducible genes by mediating atp-dependent chromatin remodeling, exposing the binding sites to the transcriptional machinery. swi/snf complexes are critical for proliferation, differentiation, tumorigenesis, and dna repair [12] . there are two forms of swi/snf complexes: brg1/hbrm-associated factors (baf) and polybromo-associated baf (pbaf). only pbaf can facilitate the ligand-dependent transcriptional activation by interacting with the nuclear receptors [13] . baf and pbaf complexes share most of their subunits and are distinguished by the presence of two specific subunits-baf180 and baf200-both of which only exist in pbaf [14] . furthermore, baf200, but not baf180, is essential for the stability of pbaf, and the depletion of baf200 leads to the complete inactivation of pbaf [13] . baf200 is encoded by arid2. besides the n-terminal at-rich interactive domain (arid), baf200 contains multiple lxxll motifs, which have been shown to participate in the regulation of protein-protein interaction [15] . additionally, baf200 has been reported to be a potential tumor suppressor and involved in the ifn signal pathway [16] . ifns are essential components of the innate immune response and act as the first line of defense against invading microorganisms or pathogens. interestingly, hbv escapes the host innate immune response merely by preventing the induction of ifns [17] . ifns modulate host defenses against microbial infection through the induction of ifn-stimulated genes (isgs) by the janus kinase (jak)-signal transducer and activator of transcription (stat) signaling pathway. among these isgs, interferon-induced transmembrane protein (ifitm) 1, 2, and 3, which are a cluster of genes encoding membrane proteins, exhibit antiviral capabilities mainly through the inhibition of virus entry [18] . ifitm1 restricts the infection of various viruses, including type 1 human immunodeficiency virus [19] , hepatitis c virus [20] , severe acute respiratory syndrome (sars) coronavirus [21] , and influenza a virus [21, 22] . however, whether ifitms inhibit hbv infection has not been reported. in this study, we start with the discovery that hbc can interact with baf200. then, we focus on functions of the interaction and disclose that overexpressed hbc downregulates the baf200-dependent expression of ifitm1 via disruption of pbaf complex stability. finally, our data demonstrates that the antiviral effects of ifitm1 on cellular proliferation and hbv replication are partially restored when hbc is co-expressed with baf200 in hbv-infected cells. these findings enrich details about how hbv counteracts human natural immunity, revealing a potential target for novel therapeutic strategies of hbv infection. baf200c (4255-5319 nt of arid2) was amplified from the fragment screened by the yeast two-hybrid system using primers-sense, 5'-gatccatggcaaactcgacggggaa-3' and antisense, 5'-aattctcactgcagcatttctga-3'-and inserted into a pcmv-flag vector (stratagene, san diego, ca, usa). baf200 (full-length arid2) and hbc cdnas (taxonomy id: 489463) were synthesized by genscript co. ltd. (nanjing, china). pgc-fu-flag and the phbv1.31 vector were kindly gifted by prof. r xiang (xiangya school of medicine of central south university, china). the pgc-fu-flag vector was ligated and constructed with baf200 at restriction enzyme site agei. the full-length of hbc was cloned into the pgbkt7 (clontech, clontech laboratories, inc., mountain view, ca, usa) vector with primers-sense, 5'-acttccagacttctagggagac-3' and antisense, 5'-ctgccctgtgacggaattga-3'-and cloned into the pcmv-ha vector (clontech) with primers-sense, 5'-gatccatggacattgaccactataaa-3' and antisense, 5'-tcgacctaacattgagattcccgaga-3'. the matchmaker gal4 two-hybrid system 3 (clontech) was used for the screening of a human fetal brain cdna library (clontech) with pgbkt7-hbc as bait. co-transformants were selected on synthetic dropout (sd), media lacking leucine, and tryptophan (sd/-leu/-trp) and were validated by growth on sd, media lacking leucine, tryptophan, adenine, and histidine (sd/-leu/-trp/-ade/-his) and containing 5-bromo-4-chloro-3-indolyl-α-d-galactoside (x-α-gal). then, positive colonies were sequenced (invitrogen, carlsbad, ca, usa). hepg2 cells and 293t cells, purchased from cctcc, were cultured in dulbecco's modified eagle medium (dmem, gibco, thermo fisher scientific, waltham, ma, usa), supplemented with 10% (v/v) fetal bovine serum ((fbs, gibco), 1% penicillin and 0.1 mg/ml streptomycin in a humidified incubator maintained at 37 • c with 5% co 2 . hepg2.2.15 cells, obtained from prof. r xiang, were cultured in 1640 medium (gbico) in the presence of g418 (200 µg/ml, sigma-aldrich, st. louis, mo, usa) in a humidified incubator maintained at 37 • c with 5% co 2 . hepg2-ntcp cells and hepaad38 cells, provided by prof. y zhu (wuhan university, china), were cultured in dmem (gibco), supplemented with 10% heat-inactivated fetal calf serum (gibco), 100 u/ml penicillin, and 100 µg/ml streptomycin sulfate at 37 • c in 5% co 2 . all the transfection reactions were performed on indicated cells (2 × 10 6 ) in log phase, using lipofectamine 2000 (invitrogen) according to the manufacturer's protocol. co-transfection was performed using a total of 5 µg of plasmids or vectors in a 1:1 (w/w) ratio: the pcmv-ha-hbc or pcmv-ha vectors were co-transfected with pcmv-flag-baf200c or pgc-fu-flag-baf200 into indicated cells. the medium was refreshed with serum-free dmem/1640 6 h after transfection. the supernatant was harvested 36 h post incubation for co-immunoprecipitation or western blot analyses. the supernatants of hepaad38 cells were concentrated 100-fold by ultracentrifugation as hbv inoculums. hbv stock titer (genome equivalents [geq] per milliliter) was measured by using qpcr. for infection, hepg2-ntcp cells of a low passage number were seeded onto precooling collagen i-coated plates and incubated in dmem for 6 h, then the medium was replaced by primary hepatocyte maintenance medium (pmm) with 2% fbs (gibco) for 12 h. after this, cells were infected with 1000 geq per cell of hbv in pmm containing 4% (w/v) polyethylene glycol 8000 (peg 8000) for 16 h. after the virus-containing medium was removed, cells were washed several times and cultured in fresh pmm. the medium was changed every other day [23, 24] . pmm is williams' e medium supplemented with an insulin-transferrin-selenium solution (thermo, waltham, ma, usa), 10 ng/ml of human epidermal growth factor (egf, peprotech, rocky hill, nj, usa), 2 mm l-glutamine (thermo), 18 µg/ml of hydrocortisone, 2% dimethyl sulfoxide (dmso), 40 ng/ml of dexamethasone, 100 µg/ml of streptomycin, and 100 u/ml of penicillin. after transfection, cells were washed with ice-cold pbs after 36 h, harvested by scraping, then lysed using an ripa lysis buffer (50 mm tris-hcl [ph 7.4], 150 mm nacl, 1% np40, 0.1% sds) supplemented with a protease inhibitor cocktail (thermo scientific). after centrifugation at 13,000 rpm at 4 • c for 5 min, supernatants were incubated with primary antibody or igg (santa cruz, santa cruz, ca, usa) and protein g agarose beads (ge healthcare, chicago, il, usa) at 4 • c overnight. immunoprecipitates were washed with a washing buffer (50 mm tris-hcl [ph 7.4], 150 mm nacl) three times, then whole cell lysate and immunoprecipitated fractions were used for western blot analysis. the following primary antibodies were used: anti-ha (catalog no. h6908, sigma-aldrich), anti-flag (catalog no. f1804, sigma-aldrich), anti-baf200 (catalog no. a302-230a, bethyl), anti-hbc (catalog no. b0586, dako). in order to make sure the loading amounts of the protein were comparable, the protein concentration was quantified by bradford assay [25] . thirty micrograms of protein was separated by sds polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (pvdf, thermo scientific) membranes. after blocking with 5% gelatin in tbst, the membranes were incubated with the indicated primary antibodies at 4 • c overnight. the membranes were washed three times, incubated with secondary antibodies (1:8000) conjugated with horseradish peroxidase (hrp) for 1 h at room temperature, and visualized with the ecl system (biorad, hercules, ca, usa) according to manufacturer's instructions. blots were probed with hrp-conjugated secondary antibodies. the following antibodies were used: anti-ifitm1, 2, 3, (catalog no.13126, 13530, 59212, cell signaling technology, danvers, ma, usa), anti-baf180 (catalog no. a301-591a, bethyl), anti-stat1 (catalog no. 9172, cell signaling technology), and anti-phospho-stat1 (catalog no. 9167, cell signaling technology), and β-actin (catalog no. 60008-1-ig, proteintech group). the gray density of the western blots was measured by using imagej software (national institutes of health, bethesda, md, usa). rt-pcr assays were performed to determine the relative mrna levels. total rna was extracted by trizol (invitrogen) according to the manufacturer's instructions. the quantity of the rna samples was detected by nanodrop 2000 (thermo scientific). one microgram of rna was reverse transcribed using random hexamer primers (fermentas, waltham, ma, usa) and m-mulv reverse transcriptase. the levels of ifitms mrna and intracellular hbv genomic dna were determined by rt-qpcr analysis using sybr green premix (takara, tokyo, japan) on a lightcycler®480ii (roche, basel, switzerland) system. the expression of the target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (gapdh) by the ∆∆ct method. β-actin was used as control. primers were as follows: ifitm1, forward: 5'-ccccaaagccagaagatgcacaaggag-3', reverse: 5'-cgtcgccaaccatcttcctgtccctag-3'; ifitm2, forward: 5'-catcatcatcccagtgttgg-3', reverse: 5'-gataaagggctgatgcagga-3'; ifitm3, forward: 5'-caaggaggagcacgagg-3', reverse: 5'-ttgaacagggaccagacg-3'; β-actin, forward: 5'-ctcttccagccttccttcct-3', reverse: 5'-agcactgtgttggcgtacag-3'; gapdh, forward: 5'-gatggcaagatcttctgcgtg-3', reverse: 5'-ccgtcgactcacaggaaatagtcggc-3'. the sirnas were designed using oligo 6.0 software, and synthesized by genscript co. ltd. (nanjing, china) as follows: siifitm1: sirna1: 5'-aaaccuucacucaacacuuccuu-3', sirna2: 5'-aaacuuaagagaaauacacacuu-3'; sihbc: sirna1: 5'-aaacuuuacugggcuuuauucuu-3', sirna2: 5'-aagagaaacuguccuugaguauu-3'; viruses 2019, 11, 427 5 of 15 sicontrol: 5'-guauauaagcaagcauuacuu-3'. all the sirnas were transfected using rnaimax (invitrogen) according to the manufacturer's instructions. one hundred microliters of hepg2 or hepg2.2.15 cells of the same passage number were seeded into 96-well plates at a density of 2 × 10 3 cells per well. after culturing for 24 h, cells were transfected with pgc-fu-flag vector, sirna, or pgc -fu-flag-baf200 and incubated for 24 h, then treated with 0, 200, 400, 600, 800, or 1000 u/ml ifnα for 24 h. afterwards, cell viability was assessed using the mtt assay. the medium was refreshed and 5 mg/ml 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt, sigma-aldrich) was added at 20 µl per well. after incubating for 2 h, the medium was removed, and cells were lysed in 100 µl dmso. finally, absorbance was measured by a microplate reader (thermo scientific) at 490 nm. to ensure the results of the mtt assays, a trypan blue exclusion assay was performed. after the transfection and treatment of ifnα, cells were harvested. then trypan blue (sigma-aldrich) was added to the cell suspension to a final concentration of 0.04% (w/v), and the mixture incubated at room temperature for 5 min. ten microliters of the suspension was transferred to a hemocytometer and viable cells were counted. the test was repeated at least three times. hepg2 cells were transfected with phbv1.31 and pgc-fu-flag-baf200, or together with pcmv-hbc-ha, in a 1:1 ratio. after treatment with ifnα for 72 h, the supernatant was collected to detect the levels of the hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) using a commercial elisa kit (neobioscience, hangzhou, china) and the hbv dna copy number was quantified by real-time pcr with a commercial pcr-fluorescence quantification kit (bioer, hangzhou, china). for the extraction of the nucleic acid, hepg2 cells were collected at 96 h post-transfection and lysed in a precooling lysis buffer (0.5% np-40, 50 mm tris-hcl [ph 7.0]). after centrifugation at 10,000× g for 1 min, the nuclei were pelleted, and the supernatant was adjusted with 10 mm mgcl 2 and treated with dnase i for 1 h at 37 • c to remove the free dna. the mixture was incubated at 75 • c for 15 min in the presence of 10 mm edta to inactivate the enzymes, then cultured with proteinase k in the presence of 1% sds to digest proteins. at last, the nucleic acids were purified by phenol-chloroform extraction and ethanol precipitation. for the extraction of extracellular encapsidated hbv dna, free dna and enzymes of 10 µl cell culture supernatant were removed according to the methods mentioned above. then, the mixture was added to 100 µl of a lysis buffer (20 mm edta, 20 mm tris-hcl, 0.5% sds, and 50 mm nacl) containing proteinase k and incubated at 50 • c overnight. after this, hbv dna was isolated by phenol-chloroform extraction and ethanol precipitation. hbv dna was subjected to real-time by pcr using primers (5'-agaaacaacacatagcgcctcat-3' and 5'-tgccccatgctgtagatcttg-3') and probe (5'-tgtgggtcaccatattcttggg-3'). data were presented as mean ± standard deviation (sd). all experiments were repeated three times. the statistical analysis was assessed using the student's t-test. differences were considered statistically significant at p < 0.01 (**). to dissect the function of hbc (figure 1a ), we screened a human fetal brain cdna library for novel hbc-interacting proteins using a yeast two-hybrid system. co-transformants were selected on sd/-leu/-trp and were validated by growth on sd/-leu/-trp/-ade/-his/x-α-gal ( figure 1b) . then, positive colonies were sequenced. finally, the c-terminal of baf200 (baf200c, aa 1419-1773) was identified as one of the strongest binding partners of hbc in ah109. baf200c contains znf domains and can bind to dna, rna, or proteins [15] (figure 1a ). because this interaction might play an important role in hbv-infected hepatocytes, our studies focused on the function of hbc interaction with baf200. to verify the two-hybrid results, co-ip assays were performed. first, baf200c was co-transfected with either empty vectors or hbc into 293t cells, then the whole cell lysate was immunoprecipitated by an anti-flag antibody and then subjected to western blot by anti-ha antibodies to detect the interacting proteins. the results indicate that the expressed c-terminal of baf200 co-precipitated with hbc ( figure 1c ). then, hbc interaction with full-length baf200 was further assessed in hepg2 cells. ip was performed against ha-tagged hbc and the co-precipitation was detected with the anti-flag antibody. the data showed that overexpressed baf200 co-precipitated with hbc ( figure 1d ). to investigate the endogenous interaction of baf200 and hbc, hepg2-ntcp cells were infected with or without hbv by inoculation with or without the supernatants of hepaad38 cells. exogenous expression of sodium taurocholate co-transporting polypeptide (ntcp) in human hepg2 cells (hepg2-ntcp) rendered them susceptible to hbv/hdv infection [24] . hepg2-ntcp cells were infected with hbv by inoculation with or without the supernatants of hepaad38 cells [23, 24] . the lysates of hepg2-ntcp cells were immunoprecipitated by hbc antibodies or baf200 antibodies, then subjected to western blot by baf200 antibodies or hbc antibodies to detect the interacting proteins ( figure 1e ). the result indicated that endogenous baf200 also co-precipitated with endogenous hbc, as seen in overexpressed proteins. collectively, the co-ip results confirm the binding between hbc and baf200. and can bind to dna, rna, or proteins [15] (figure 1a ). because this interaction might play an important role in hbv-infected hepatocytes, our studies focused on the function of hbc interaction with baf200. to verify the two-hybrid results, co-ip assays were performed. first, baf200c was cotransfected with either empty vectors or hbc into 293t cells, then the whole cell lysate was immunoprecipitated by an anti-flag antibody and then subjected to western blot by anti-ha antibodies to detect the interacting proteins. the results indicate that the expressed c-terminal of baf200 co-precipitated with hbc ( figure 1c) . then, hbc interaction with full-length baf200 was further assessed in hepg2 cells. ip was performed against ha-tagged hbc and the co-precipitation was detected with the anti-flag antibody. the data showed that overexpressed baf200 coprecipitated with hbc ( figure 1d) . to investigate the endogenous interaction of baf200 and hbc, hepg2-ntcp cells were infected with or without hbv by inoculation with or without the supernatants of hepaad38 cells. exogenous expression of sodium taurocholate co-transporting polypeptide (ntcp) in human hepg2 cells (hepg2-ntcp) rendered them susceptible to hbv/hdv infection [24] . hepg2-ntcp cells were infected with hbv by inoculation with or without the supernatants of hepaad38 cells [23, 24] . the lysates of hepg2-ntcp cells were immunoprecipitated by hbc antibodies or baf200 antibodies, then subjected to western blot by baf200 antibodies or hbc antibodies to detect the interacting proteins (figure 1e ). the result indicated that endogenous baf200 also co-precipitated with endogenous hbc, as seen in overexpressed proteins. collectively, the co-ip results confirm the binding between hbc and baf200. hbc-interacting partners were tested for β-galactosidase activity on an sd/-leu/-trp/-ade/-his/x-α-gal plate. vector: pgadt7; positive control: pgbkt7-53 and pgadt7-t co-transformant; negative control: pgbkt7-lam and pgadt7-t transformant. (c) 293t cells were co-transfected with pcmv-flag-baf200c and pcmv-ha vector or pcmv-ha-hbc, and co-ip assays were performed with anti-flag antibody or control igg. immuno-complexes were detected by western blot assays using the anti-ha antibody or anti-flag antibody (control). (d) hepg2 cells were co-transfected with the pgc-fu-flag-baf200 and pcmv-ha vectors or pcmv-ha-hbc, and co-ip assays were carried out with anti-ha antibody or igg. immuno-complexes were detected by western blot assays using the anti-flag antibody or anti-ha antibody (control). (e) hepg2-ntcp cells were incubated with supernatants isolated from the supernatants of hepaad38 cells cultures (containing hbv) at 1000 geq for 16 h or not (mock). co-ip assays were performed with hbc antibody or baf200 antibody, immuno-complexes were detected by western blot assays using baf200 antibody or hbc antibody. input control assays were performed in whole cell lysates (wcl). because baf200 was reported to be a critical regulator of ifn signaling [13] , we first focused on the effect of baf200 on the expression of ifitms, which are ifnα effectors. the impact of overexpressed baf200 on ifitm1, 2, and 3 was examined via protein ( figure 2a ) and mrna (figure 2b ) levels. the results demonstrated that baf200 up-regulated ifitm1 protein expression (figure 2a) and significantly increased the mrna expression ( figure 2b ). however, no apparent effect of baf200 was observed on the expression of ifitm2 and ifitm3 (figure 2b) . furthermore, we observed that the ifitm1 expression level was suppressed 2.1-fold when endogenous baf200 was silenced in hepg2 cells (figure 2c) . the data suggest that baf200 specifically mediates basal level expression of ifitm1. next, the effect of the hbc interaction with baf200 on ifitm1 expression was further investigated. we co-transfected hbc and baf200 into hepg2 cells, treated the cells with 500 u/ml ifnα, and detected the expression of ifitm1 by western blot (figure 2d ). the results indicate that baf200 enhanced ifitm1 expression, while the enhancement was partially reduced when hbc was co-transfected. to further examine the effect of ifnα on hbc function, we treated the transfected cells with ifnα at various concentrations from 200 u/ml to 1000 u/ml for 24 h and examined the ifitm1 mrna levels ( figure 2e ). as expected, hbc impaired the enhancement of ifitm1 mrna expression by baf200. however, compared to the control, expression was inhibited to a similar degree upon ifnα stimulation with different concentrations. taken together, the data demonstrates that hbc can down-regulate ifitm1 expression via binding to baf200, whereas the binding is irrelevant to the stimulation of ifnα. ifnα at various concentrations from 200 u/ml to 1000 u/ml for 24 h and examined the ifitm1 mrna levels (figure 2e ). as expected, hbc impaired the enhancement of ifitm1 mrna expression by baf200. however, compared to the control, expression was inhibited to a similar degree upon ifnα stimulation with different concentrations. taken together, the data demonstrates that hbc can down-regulate ifitm1 expression via binding to baf200, whereas the binding is irrelevant to the stimulation of ifnα. furthermore, the mechanism about how hbc regulated ifitm1 expression was explored. since the jak-stat1 signaling pathway has been reported to be essential to ifnα-induced antiviral activities [26] , we examined whether it was involved in baf200-mediated ifitm1 expression. since the phosphorylation of stat1 is a necessary step for jak-stat1 signaling, we used western blot assays to detect phosphorylated stat1 (pstat1) in hepg2 cells with or without baf200 (figure 3a) . the results showed that pstat1 appeared only upon ifnα stimulation, independent of the presence of baf200. it has been shown that baf200 is essential for the stability of pbaf, and the depletion of baf200 leads to the complete inactivation of pbaf [13] . hence, we predicted that the interaction of hbc with baf200 would interrupt the stability of pbaf complexes, leading to potential baf200-dependent ifitm1 expression. to verify this hypothesis, co-ip assays were carried out to analyze the effect of hb on baf180-baf200 immuno-complexes in hepg2 cells (figure 3b) . ip was performed against flag-baf200 and the co-precipitation of baf180 was probed. it demonstrated that hbc co-transfection along with baf200 profoundly reduced the co-precipitation of baf180-baf200 immuno-complexes when compared with the baf200 transfected alone condition. endogenous baf180-baf200 interaction was further assessed in hbv infected hepg2-ntcp cells (figure 3e) . the co-ip assay showed that the concentration of baf180-baf200 complexes was reduced more than 50-fold compared to the non-infected mock control. these results imply that hbc de-stabilizes the pbaf complex by preventing the baf180-baf200 interaction, probably by competitive binding to baf200. we next determined whether overexpression of hbc regulated ifitm1 transcription in vitro. flag-baf200 was transfected with or without ha-hbc into hepg2 cells. cells were later treated with ifnα (500 u/ml) and total mrna was extracted to detect transcription levels of ifitm1, 2, and 3 by rt-qpcr. the data indicates that, upon ifnα stimulation, the suppression of ifitm1 expression by hbc is specific (figure 3c ) and statistically significant (figure 3d ) in vitro. collectively, the data demonstrates that hbc interacts with baf200, and the hbc-baf200 interaction prevents the baf180-baf200 interactions that might abolish pbaf complex stability, which in turn regulates the suppression of ifitm1 transcription. the pcmv-flag vector and pcmv-flag-baf200 were transfected into hepg2 cells, with or without ifnα (500 u/ml) treatment for 24 h. phosphorylation of stat1 was detected via western blot using antibodies against phosphorylated stat1 (pstat1). meanwhile, stat1 and baf200 were detected using anti-flag and anti-stat1 antibodies as control. (b) hepg2 cells were co-transfected with pgc-fu-flag-baf200 and pcmv-ha vector or pcmv-ha-hbc, with or without ifnα (500 u/ml) treatment for 24 h. co-ip assays were performed with anti-flag antibody, then baf200-baf180 immuno-complexes were detected by western blot assays using anti-baf180 antibody or anti-flag antibodies (control). input control assays were performed in wcl using anti-ha or anti-baf180 antibodies. meanwhile, total mrna was extracted to examine the effect of baf200 and hbc combination on ifitm1, 2, 3 transcriptions by rt-pcr (c). β-actin was detected as control. ifitm1 transcription level was determined using rt-qpcr and normalized to the gapdh mrna level. (d) the flag-baf200 transfection with ifnα treatment was designated as 1. *, p < 0.05; **, p < 0.01. nd = not detected. (e) hepg2-ntcp cells were incubated with supernatants isolated from the supernatants of hepaad38 cells cultures (containing hbv) at 1000 geq for 16 h or not (mock). co-ip assays were performed with baf200 antibodies, and immuno-complexes were detected by western blot assays using baf180 antibodies or baf200 antibodies. input control assays were performed in wcl using hbc antibody or baf180 antibody. finally, we evaluated the effect of hbc on the antiviral activities of iftim1 in hbv-infected cells. firstly, the impact of hbc on the anti-proliferation action of ifitm1 was investigated via sirnamediated knockdown in hepg2 cells and hepg2.2.15 cells. proliferation of indicated cells was assessed by mtt assays and viable cell counting. indeed, in hepg2 cells and hepg2.2.15 cells, the data from direct cell counting were consistent with those obtained from mtt assays (figure 4a, 4c, 4d, 4e) . in hepg2 cells, the cell viability increased with the time of ifnα stimulation (figure 4a) and decreased with the concentration of ifnα (figure 4c) . unter treatment of 500 u/ml ifnα, baf200 enhanced the inhibitory effect of ifnα, and overexpression of hbc partially recovered the cell the pcmv-flag vector and pcmv-flag-baf200 were transfected into hepg2 cells, with or without ifnα (500 u/ml) treatment for 24 h. phosphorylation of stat1 was detected via western blot using antibodies against phosphorylated stat1 (pstat1). meanwhile, stat1 and baf200 were detected using anti-flag and anti-stat1 antibodies as control. (b) hepg2 cells were co-transfected with pgc-fu-flag-baf200 and pcmv-ha vector or pcmv-ha-hbc, with or without ifnα (500 u/ml) treatment for 24 h. co-ip assays were performed with anti-flag antibody, then baf200-baf180 immuno-complexes were detected by western blot assays using anti-baf180 antibody or anti-flag antibodies (control). input control assays were performed in wcl using anti-ha or anti-baf180 antibodies. meanwhile, total mrna was extracted to examine the effect of baf200 and hbc combination on ifitm1, 2, 3 transcriptions by rt-pcr (c). β-actin was detected as control. ifitm1 transcription level was determined using rt-qpcr and normalized to the gapdh mrna level. (d) the flag-baf200 transfection with ifnα treatment was designated as 1. *, p < 0.05; **, p < 0.01. nd = not detected. (e) hepg2-ntcp cells were incubated with supernatants isolated from the supernatants of hepaad38 cells cultures (containing hbv) at 1000 geq for 16 h or not (mock). co-ip assays were performed with baf200 antibodies, and immuno-complexes were detected by western blot assays using baf180 antibodies or baf200 antibodies. input control assays were performed in wcl using hbc antibody or baf180 antibody. finally, we evaluated the effect of hbc on the antiviral activities of iftim1 in hbv-infected cells. firstly, the impact of hbc on the anti-proliferation action of ifitm1 was investigated via sirna-mediated knockdown in hepg2 cells and hepg2.2.15 cells. proliferation of indicated cells was assessed by mtt assays and viable cell counting. indeed, in hepg2 cells and hepg2.2.15 cells, the data from direct cell counting were consistent with those obtained from mtt assays (figure 4a,c,d,e) . in hepg2 cells, the cell viability increased with the time of ifnα stimulation (figure 4a) and decreased with the concentration of ifnα (figure 4c) . unter treatment of 500 u/ml ifnα, baf200 enhanced the inhibitory effect of ifnα, and overexpression of hbc partially recovered the cell viability (figure 4a) , however, the sirna mediated knock down of iftim1 increased the cell viability robustly, about two-four-fold compared to the control (figure 4c) . the results suggest that ifitm1 makes major contributions to ifnα inhibitory activities for hbv replication. next, we focused on the role of hb. in hepg2.2.15 cells, which contain a stably integrated hbv genome and produce endogenous hbc, overexpression of baf200 promoted the inhibition of ifnα (figure 4d ), however when hbc was silenced by sihbc, cell viability was reduced substantially (figure 4e) . consequently, the results demonstrate that hbc antagonizes the suppression of iftim1 and the proliferation of hbv-infected cells in vitro. next, the hbc effects on the inhibition of ifitm1 on hbv replication were measured. in figure 4e , phbv1.31, which contains 1.3-fold hbv genome and can produce endogenous hbc, was co-transfected with empty vector flag-baf200 or ha-hbc into hepg2 cells. after treating the cells with 500 u/ml ifnα for 72 h, supernatants were collected to determine the hbv replication level by detecting the quantitation of hbsag, hbeag, and hbv dna copy numbers (figure 4f ). in the control, empty vector and phbv1.31 were co-transfected, and it was observed that ifnα stimulation inhibited the hbv replication level significantly. in contrast to the control, when baf200 was overexpressed, the concentration of hbsag and hbeag in the supernatant was reduced about one-fold, and hbv dna copies were also suppressed up to five-fold. as expected, the ifnα inhibition was restored when hbc was co-transfected with baf200. notably, hbsag and hbeag levels increased to similar levels as the control, with a slight elevation in the hbv dna copy numbers. collectively, the results illustrate that hbv evades ifnα mediated antiviral activity in vitro by downregulating the expression of ifnα effector. however, overexpressed hbc had little observed impact on the recovery of hbv dna levels, implying that ifitm1 is not the only primary ifnα-inducible isg that suppresses hbv dna replication. hbv replication is not directly cytotoxic to cells, while the host immune responses in infected hepatocytes are the main determinants of hepatocellular injury and hbv pathogenesis. innate , then treated with ifnα in indicated concentration for 24 h. afterwards, the cell viability was measured using mtt assay and direct cell counting via trypan blue exclusion assay (a,c-e). (f) hepg2 cells were transfected with phbv1.31 and pgc-fu-flag-baf200, or co-transfected with pcmv-ha-hbc, then treated with 500 u/ml ifnα for 72 h. hbv replication levels were determined in the supernatants by measuring hbsag and hbeag concentration using elisa and hbv dna copy numbers using rt-qpcr. the empty vector and phbv1.31 were co-transfected in the non-ifnα induced condition as control. the control without ifnα treatment was designed as 100% (a,c-f). *, p < 0.05, **, p < 0.01, ns = non-significant. hbv replication is not directly cytotoxic to cells, while the host immune responses in infected hepatocytes are the main determinants of hepatocellular injury and hbv pathogenesis. innate immunity is important in controlling viral spread immediately after infection and initiates efficient development of an adaptive immune response. the early phase of a viral infection is mainly characterized by the production of cytokines, type i ifn, and natural killer (nk) cells [27] . type i ifns can be triggered directly by virus replication through the detection of viral rna or dna, and play a critical role in the immune recognition of hbv. they can be triggered directly by viral replication via detecting the presence of viral rna or dna and induce a large number of effectors working in combination to achieve a fully functional antiviral state [4, 28] . ifn effectors target different steps of the viral life cycle, limiting the propagation and spread of the virus and restricting viral infection. therefore, interferon therapy remains one of the therapeutic strategies for hbv infection. in fact, the majority of chb patients treated with ifnα cannot acquire a long-lasting sustained response, especially those with a high viral load. data from animal models have shown that ifn effectors were rapidly induced upon infection with hbv [28] . however, the hbv-induced ifn responses are weak [29] , and that is why acute hbv infections usually show a lack of clinical symptoms. studies have shown that the clinical outcomes in chb patients are primarily determined by the interaction between hbv replication and host immune responses [30] . as an important effector of type i ifns, mxa exhibits strong anti-viral activity against hbv, however, hbv down-regulates mxa expression significantly via the interactions between hbc and the interferon-inducible mxa promoter [31, 32] . in addition, reports have indicated that under the stimulation of ifnα, the ifn-signaling pathway is generally blocked, however the formation of stat1 and isgf3 were evenly enhanced in hepg2.2.15 cells [33] . here, our study shows that hbc inhibits the ifnα-induced ifitm1 transcription through disturbing the stability of pbaf complex in vitro, independent of the jak-stat1 pathway (figure 4a ). moreover, hbc exhibits anti-apoptosis activity by inhibiting trail-induced expression of the pathway-related death receptor in hepatocytes [34] . hbc acts as a tumor suppressor as well, by blocking the transcription of the human tumorigenesis-associated genes, ifnβ and p53 [11, 35] . notably, our data indicates that the inhibitory effect of ifitm1 on hbv dna replication seems weak. a potential explanation is hbv integration into the host genome. the hbv genome can present as non-integrated cccdna, which serves as a template for replication and can persist even after hbsag loss [36, 37] . hbv manages to escape immunological recognition in early infection, remaining undetected and spreading for nearly five weeks [38] . the use of cccdna as a transcriptional template in the nucleus likely contributes to hbv's capacity to limit detection in hepatocytes [37] . in addition, hbv evades the host response through a complex combination of processes that include signaling interference, effector modulation, and continual viral genetic variation [37] . reports have shown that hbv polymerase and hbx protein directly inhibit the cellular machinery that detects replication intermediates [29, 33] . here, our study provides novel evidence that hbv counteracts ifnα antiviral activities through effector modulation, i.e., the downregulation of ifitm1 expression by hbc. the mechanism of the downregulation is illustrated as follows. under normal conditions, ifnα induction activates pbaf complexes, which consist of baf200, baf180, and other subunits. the activation of the pbaf complex triggers histone sliding on chromatin dna, leading to the exposure of interferon-stimulated response elements (isre). in this way, pbaf complexes facilitate the initiation of ifitm1 transcription (figure 5a) . actually, baf200 is essential for the stability of the pbaf complex. under hbv-infected condition, the interaction of hbc with baf200 competes against the binding between baf180 and baf200, resulting in the loss in pbaf complex stability. thus, the efficient process of the pbaf complex is interrupted, leading to the suppression of ifitm1 transcription (figure 5b ). it provides details for the mechanism of hbv escape from ifnα-induced immune elimination. moreover, our future work will continue to explore the intrahepatic effect of hbc on hbv replication using hbv-transgenic mice. were evenly enhanced in hepg2.2.15 cells [33] . here, our study shows that hbc inhibits the ifnαinduced ifitm1 transcription through disturbing the stability of pbaf complex in vitro, independent of the jak-stat1 pathway (figure 4a) . moreover, hbc exhibits anti-apoptosis activity by inhibiting trail-induced expression of the pathway-related death receptor in hepatocytes [34] . hbc acts as a tumor suppressor as well, by blocking the transcription of the human tumorigenesis-associated genes, ifnβ and p53 [11, 35] . taken together, our data demonstrate that hbc downregulates ifitm1 expression through interactions with baf200, rather than the jak-stat1 pathway. the results also show that hbc partially restores ifitm1 antiviral effects on cell proliferation and hbv replication in infected cells in vitro by disturbing the stability of pbaf. it enriches the mechanism that hbv counteracts the innate immunity of ifnα, revealing a potential target for novel therapeutic strategies of hbv infection. nuclear import of hepatitis b virus capsids and genome genomic analysis of the host response to hepatitis b virus infection efficacy of nvr 3-778, alone and in combination with pegylated interferon, vs entecavir in upa/scid mice with humanized livers and hbv infection innate immune responses in hepatitis b virus (hbv) infection full-length hepatitis b virus core protein packages viral and heterologous rna with similarly high levels of cooperativity morphological irregularities in dane particle cores hepatitis b virus core protein phosphorylation sites affect capsid stability and transient exposure of the c-terminal domain nuclear export and import of human hepatitis b virus capsid protein and particles the hepatitis b virus (hbv) core protein enhances the transcription activation of cre via the cre/creb/cbp pathway hepatitis b viral core protein activates the hepatitis b viral enhancer ii/pregenomic promoter through the nuclear factor kappab binding site hepatitis b viral core protein disrupts human host gene expression by binding to promoter regions exome sequencing identifies frequent mutation of the swi/snf complex gene pbrm1 in renal carcinoma pbaf chromatin-remodeling complex requires a novel specificity subunit, baf200, to regulate expression of selective interferon-responsive genes selectivity of chromatin-remodelling cofactors for ligand-activated transcription expressing the human genome arid2: a new tumor suppressor gene in hepatocellular carcinoma noncytolytic control of viral infections by the innate and adaptive immune response ifitms restrict the replication of multiple pathogenic viruses ifitm proteins restrict hiv-1 infection by antagonizing the envelope glycoprotein ifitm1 is a tight junction protein that inhibits hepatitis c virus entry distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus ifitm proteins mediate the innate immune response to influenza a h1n1 virus, west nile virus and dengue virus hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding ifn-alpha inhibits hbv transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccdna minichromosome viruses and interferon: a fight for supremacy interferon-stimulated genes: a complex web of host defenses temporal analysis of early immune responses in patients with acute hepatitis b virus infection innate and adaptive immune responses in chronic hepatitis b virus infections: towards restoration of immune control of viral infection mxa inhibits hepatitis b virus replication by interaction with hepatitis b core antigen human mxa protein participates to the interferon-related inhibition of hepatitis b virus replication in female transgenic mice interferon-alpha response in chronic hepatitis b-transfected hepg2.2.15 cells is partially restored by lamivudine treatment hepatitis b virus core protein inhibits trail-induced apoptosis of hepatocytes by blocking dr5 expression transcriptional repression of the human p53 gene by hepatitis b viral core protein (hbc) in human liver cells persistence of cccdna during the natural history of chronic hepatitis b and decline during adefovir dipivoxil therapy hepatitis b virus infection and the immune response: the big questions viral clearance without destruction of infected cells during acute hbv infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we are grateful to r xiang for kindly providing hepg2.2.15 and phbv3.1 plasmids, a francis and r dillard for their invaluable comments on the manuscript. the authors declare no conflict of interest. key: cord-002282-ldfa616a authors: joung, young hee; park, se hee; moon, ki-beom; jeon, jae-heung; cho, hye-sun; kim, hyun-soon title: the last ten years of advancements in plant-derived recombinant vaccines against hepatitis b date: 2016-10-13 journal: int j mol sci doi: 10.3390/ijms17101715 sha: doc_id: 2282 cord_uid: ldfa616a disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. every year more than 100 million children are vaccinated with the standard world health organization (who)-recommended vaccines including hepatitis b (hepb). hepb is the most serious type of liver infection caused by the hepatitis b virus (hbv), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. to date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently hepb and influenza. more inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. in this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis b surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed. hepatitis b (hepb) is an infection with the hepatitis b virus (hbv), which attacks the liver and can cause both acute and chronic disease. the world health organization (who) estimates that 240 million persons are chronically infected with hbv and that more than 780,000 people die every year due to complications of hepb, including cirrhosis and liver cancer [1] . the point that needs the most attention is the high rates of chronic infection found in the sub-saharan africa and east asia, where between 5% and 10% of the adult population is infected, as well as in the amazon and the southern parts of eastern and central europe. otherwise, less than 1% of the population in western europe and north america is chronically infected [1] . hbv is a hepatotropic dna virus that replicates by reverse transcription. the human hbv is a small circular dna molecule of 3.2 kb [2] . its genome consists of four partially overlapping open reading frames (orfs), namely the envelope gene (pre-surface/surface (pre-s/s)), the core gene (pre-core/core (pre-c/c)), the polymerase gene (pol) and the transactivating protein x (x). the orf pre-s/s encodes pre-s1, pre-s2 and surface (s) proteins that form the surface antigen (hbsag), and hbsag protein is the main antigen to elicit virus-neutralizing and protective antibodies by the immune system [3, 4] . understanding the hbv genome and structure is an essential prerequisite for preventive or therapeutic vaccination against hepb. a vaccine against hepb has been available since 1982. this first licensed anti-hbv vaccine containing subviral particles of hbv purified from the inactivated serum of carriers revealed very high efficacy [5] , and a subsequent subunit vaccine made using the small surface antigen (s-hbsag) was developed in the early 1980s [6] . the yeast system for the recombinant antigen was to ensure safety and low cost. yeast-derived s-hbsag assembled into virus-like particles (vlps) were as immunogenic as natural subviral particles, and highly effective vaccines containing s-hbsag have been widely used as prophylactic vaccines against hbv infection [7] . however, some groups of vaccines do not develop protective immunity against the virus and immunosenescence frequently occurs in adults [8] . additionally, high cost limit and the necessity of accompanying infrastructure for the cold chain distribution and intravenous administration still constituted a barrier to vaccination approaches in developing countries. in order to successfully solve these problems, many research projects have been undertaken to develop more efficacious, easily administrated, and thermostable vaccines. a new recombinant hbv vaccine containing the pre-s/s has greater immunogenic potential than the conventional s antigen-based vaccines in terms of antibody induction and cellular immune response. middle (pre-s2 + s, m-hbsag) or large (pre-s1 + pre-s2 + s, l-hbsag) surface antigens [9] have been used as components of specific immunotherapeutic vaccines for chronic hbv carriers [10, 11] . additionally, chimeric protein created by fusing the hbv core antigen (hbcag) to the pre-s1 showed strong anti-hbc and moderate anti-pre-s1 immune responses [12] . although vaccination is one of the most powerful and cost-competitive achievements, some vaccines may still have certain limitations related to maintenance of the cold chain, downstream processing costs, administration risk, and expensive scalability [13] [14] [15] [16] [17] . from these reasons, the use of plant cells as alternative production platforms have received considerable attention in terms of intrinsic safety, scalability, and posttranslational modification of target proteins [17, 18] . plant systems can be scaled up quickly to generate large quantities of the protein product, are not susceptible to contamination with known human or mammalian pathogens and are resistant to enzymatic digestion in the gastrointestinal tract. in addition, transgenic plants can be engineered to express and translate multiple proteins concurrently with appropriate folding and assembly into multimeric proteins, especially the posttranslational adjustments of antibodies. not all recombinant antigens will benefit from plant-based systems, but the best production system for each recombinant protein should be chosen using a case-by-case approach [19] . merlin et al. [19] proposed that plants are the most the beneficial for the production of four major categories of pharmaceutical proteins: ones that are required in large quantities, that need to be rapid-response, that require complex posttranslational modifications, or that are intended for oral delivery. within these categories, they suggested appropriate candidates to meet a spectrum of research, development, commercial needs, such as human glutamic acid decarboxylase, norwalk virus-like particles, monoclonal antibody 2g12, and human interleukin-6. those plant-made antigens have already been tested in clinical trials, with successful outcomes ( table 1 ). the enzyme glucocerebrosidase for gaucher's disease, the first pmf-derived enzyme "elelyso™", has been approved and marketed by protalix in 2012. elelyso™ is based on the use of carrot cells to produce recombinant taliglucerase alpha, which is used in enzyme replacement therapy to treat adult patients. this special food and drug administration (fda) approval case was fast tracked based on its applicability to a rare genetic disease and the bioreactor production under stringent conditions [20, 21] . medicago has ongoing phase ii clinical trials for a plant-derived vlp quadrivalent seasonal influenza vaccine and an h5 pandemic influenza vaccine [22] . they are focusing on vlp vaccine development. vlps are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome and thus are not infective. another important advantage as emerging vaccine is the more effective activation of key aspects of the immune response to achieve potent immune stimulation and to provide immunological memory for long-lasting protection [22, 23] plant-based platforms including whole plant, organs or cell and expression technology to produce target antigens of interest are diverse [38] [39] [40] . representative plant species expressing the oral vaccine are potato, tomato, and tobacco; additionally, maize, rice, carrot, and soybean are also applied in this field [41] [42] [43] [44] [45] [46] [47] [48] [49] . those plants are mainly focused on traditional and usually eaten crops in human, because it is known that inexperienced plants sometimes have problems with certain plant allergies. target antigen proteins were expressed by a plant cell nuclear genome expression system in these plant species. edible plant vaccines are based on different parts of plants, such as fruits, seeds, and root vegetables. such food vaccines are prepared directly without expensive purification of the antigens, which is essentially required for parenteral administration of vaccines [50] . therefore, the lyophilization of organs expressing stable antigens would facilitate their processing, purification and storage, reducing costs and allowing more practical vaccines. although stable transformation into transgenic plants is commonly accepted, the low production level of the resultant recombinant protein remains an issue of concern. an efficient alternative to nuclear transformation for vaccine antigens and other therapeutic proteins is plastid transformation [51] . the highest expression of transgenes, up to more than 70% of total soluble protein, are reported in chloroplast transformation [52,53] otherwise, the universal expression level in most studies has been 1% of total soluble protein (tsp) or 50 µg/g fresh leaf tissue [54, 55] . chloroplast technology can also avoid the controversy related to transgene containment [40] and express multigenes as single operon [56] . waheed et al. [40] reviewed recent vaccine antigens against human diseases expressed via plastid genome since 2011. two plant species, tobacco (15 different antigens) and lettuce (four different antigens), have mainly been used in plastid transformation. these results suggest that more industrial interest is needed to strengthen the research/academia-industry linkages in the chloroplast-based vaccine market. stable transformation has its own advantages such as reliable harvest of target proteins over multiple generations and optimized protocols for delivery of foreign genes into various plant species. although there are problems with the time required, seed resources can be grown anywhere with minimal cost and labor once the plant has been developed for the first time [21] . most clinical trials, except for the three cases of eleyso, prx-102, and recombinant human intrinsic factor, have used a tobacco-based transient expression system (table 1 ). in recent years, interest in transient expression has increased due to the containment of the system and the possibility of rapid upscaling due to the short interval between transformation and expression, which are attractive features for the industrial scale production and approval of the expressed products, e.g., the mass production of tobacco by medicago and kentucky bioprocessing. pogue et al. [57] reviewed plant-based transient expression systems for the production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product. transient expression provides a safe and environmentally friendly system for both indoor and outdoor application with high speed and low cost of the genetic manipulation, rapid manufacturing cycles, and economical production. transient production using an agrobacterium tumefaciens-mediated transfer-dna delivery system (agro-infiltration) and/or virus-based replicating systems, the two dominant approaches, guarantees both the quality of the resulting purified products and the speed of development [57, 58] . spiegel et al. [59] demonstrate the application of the classical nicotiana benthamiana/a. tumefaciens transient expression system to accelerate the development of a malaria vaccine candidate, with screens for expression, solubility, and stability using fluorescent fusion proteins. in marin viegas et al. [60], a transient expression system for the production of human tg2 in n. benthamiana leaves was optimized, and the reactivity of plant-produced tg2 in a cd screening test was evaluated. hence, transient expression performed in contained facilities satisfies good manufacturing practices, and quick expression can avoid the time-consuming stable transformation [58, 61] . in a comparison of productivity in terms of biomass production, hiatt and pauly [62] reported that grams of product may take only two weeks plus a few weeks more. in large-scale biomanufacturing systems, recombinant proteins can be produced at levels of 200-1000 mg/kg fresh weight tissue in as little as three months [57] . the transient expression of human interleukin-6 in n. benthamiana (7.8% tsp) produces 80-fold more than stable expression in tobacco plants (0.3 mg/g fresh weight (fw)) [63]. conversely, hgad65mut is expressed at higher levels in stable tobacco plants (143.6 µg/g fw) than in n. benthamiana (96.6 µg/g fw) [64] . this result suggests that expression potential or level varies case by case, depending on the target protein. in last decade, there has been a considerable increase in the use of transgenic plants to generate recombinant proteins for medical and veterinary use ( table 2) . research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently hepatitis b and influenza. in the case of hepatitis b, both stable and transient expression systems have been developed in various plants, including potato, lettuce, tobacco, tomato, carrot, and arabidopsis for stable expression and n. benthamiana for transient expression. a detailed review of hepatitis b will be presented in the next part of this article. influenza is also a main target of this field because it is a widely distributed viral infection of humans and animals, and a new epidemic strain appears every one to two years. this pattern requires the production of new vaccines at the same frequency, and a promising solution is to establish a rapid, flexible, and safe system. the production of various antigens such as hemagglutinin (ha) and the extracellular domain of matrix protein 2 (m2e) has mainly focused on transient expression systems using n. benthamiana leaves. using the medicago "proficia™" system, vaccine production can be initiated within less than three weeks from the identification of the genetic sequence of a pandemic or seasonal influenza strain [155]. vamvaka et al. [74] reported the development of transgenic rice plant expressing the hiv-neutralizing antibody 2g12 in the endosperm (42 µg/g dry seed weight) to evaluate the potential of rice seeds as a vehicle for inexpensive microbicide production. production is higher than the initial achievement of maize-derived 2g12 (30 µg/g) by rademacher et al. [156] . rubio-infante et al. [75] demonstrated the immunogenic potential of tobacco chloroplast-derived multi-hiv in an oral immunization scheme and proposed it as a vaccine prototype capable of inducing broad immune responses as it carries various b and t cell epitopes from several hiv strains. dengue has become a significant public health problem, and the threat of dengue fever is now increasing in temperate regions due to dramatic climate change. the rice codon-optimized consensus domain iii of dengue virus envelope glycoprotein (e) has been fused to the m cell-binding peptide via agroinfiltration with a plant virus-based expression system [157] [158] [159] . carrying these results a step further, kim et al. [159] generated an ebola ric-based denv vaccine in tobacco plants using a geminiviral vector expression system and reported its immunogenic properties as a self-adjuvanting dengue vaccine candidate. previously, phoolcharoen et al. [160] reported that plant-expressed ebola ric protected mice against a lethal ebola virus challenge. the expression of subunit vaccines for animal viral diseases, such as avian influenza [98, 161] , foot-and-mouth disease (fmd) [100] [101] [102] , and diarrhea [123, 126, 127] , which are considered to be the most important causes of economic losses in plants, has been frequently reported. the commercialization of veterinary vaccine is relatively easy compared with that of human vaccines. to date, four cases in clinical trials are ready to enter the market. [162] suggest that the highly immunogenic vp8 epitopes produced in n. benthamiana are candidates for a subunit vaccine, specifically for the g9p rotavirus strain. the greatest problem of plant-derived vaccine development is the extremely low expression level of the foreign protein in plants. for this reason, many researchers have studied how to improve protein expression levels in plants. in the case of plant-derived hbv vaccines, the first report was on the expression of the small hepatitis b surface antigen (s-hbsag) in transgenic tobacco plants. in this report, the hbsag produced in transgenic tobacco was antigenically and physically similar to the hbsag particles derived from human serum and recombinant yeast [163] . afterward, many research groups attempted hbsag expression in different tissues and plant species, such as tobacco, potato, lettuce, soybean, lupine, maize, tomato, peanut and laminaria japonica (table 3 ). in the transgenic tobacco plant transformed with the s-hbsag gene controlled by the 35s promoter, expression levels were very low: less than 0.01% total soluble protein and less than 10 ng/g fresh weight in leaf tissues. the expression levels of s-hbsag in other plant species were not significantly higher; in some species, expression levels were even lower than in tobacco. to improve vaccine production in plants, the most widely used strategies involve: (1) suitable promoters, such as strong constitutive promoters, tissue-specific promoters and promoters that are inducible by environmental factors; (2) targeting systems to specific organelles; (3) optimized codon usage; (4) alternative polyadenylation signals; (5) increased translation efficiency using leader sequences; and (6) different vector systems. many hbsag-overexpressing transgenic plants have been developed using strong constitutive promoters, such as the 35s promoter with enhancer [164] [165] [166] . in addition to tissue-specific promoters, the patatin promoter for potato tuber [165, 167] , globulin promoter for maize seed [168] and fruit-specific promoters [169, 170] were used. specific organelle-, endoplasmic reticulum (er)-, vacuole-and chloroplast-targeted strategies have also been tried [167, 171] . the hbsag has been expressed in non-edible plants, such as tobacco, using four different expression cassettes: the hbsag gene without er retention signal (hbs), the hbsag gene with er retention signal (her), and each gene controlled by the ubiquitin promoter (ubq) or ethylene forming enzyme promoter (efe) [172] . in this report, the maximum expression level (19.4 ng/g fw of leaves) was observed in efe-hbs transformed plant growth in vitro, but a higher proportion of the particulate form of the antigen was observed when it was expressed with an er retention signal. the efe promoter is more effective in in vitro-cultured plantlets, whereas the ubq promoter is more effective in greenhouse-grown plantlets. the maximum expression level was 2 µg/g fw in the ubq-her transformed nt-l cell suspension culture [173] . the expression level was increased up to 8 µg/g fw using hbsag fused with the 3 region from the soybean vegetative storage protein gene and was controlled by a chimeric ocs-mas promoter. upon transformation into a soybean cell culture using the same vector, the maximum expression level was 74 µg/g fw [174] . soybean cell/transgenic pmsi164/eha105 ubq3/er 700 ng/g fw n.a. [190] banana/transgenic pbi121/eha105 efe er 38 ng/g fw (leaf) n.a. [191] lupin/transgenic prok/c58 35s/n.a. 150 ng/g fw (callus) oral, igg antibodies in serum (maximum 19 miu/ml) [187] lupin/transgenic prok/gv3101, eha105, lba4404 35s/n.a. 2.5-6 µg/g fw (callus) n.a. [192] maize/transgenic n.a./eha101 3xglb1/cell wall 0.51% of tsp (seed) n.a. [168] maize/transgenic n.a./eha101 glob1/cell wall 0.46% of tsp (seed) oral (germ, bioencapsulated), iga and igg antibodies in serum (maximum 4632 miu/ml) [193] maize/transgenic n.a./eha101 3kbglb1/cell wall 0.41% of tsp (seed) oral (germ, wafer feeding), iga and igg antibodies in serum [194] maize/transgenic n.a./eha101 glob1/cell wall 0.46% of tsp (seed) oral (germ, wafer feeding), iga and igg antibodies in serum [195] cherry tomatillo/transgenic pcambia1301/eha105 35s/n.a. 10 ng/g fw (fruit) oral, igg antibodies in serum [196] tomato/transient pbi121/eha105 efe/er 0.5 µg/g dw (leaf) n.a. [169] tomato/transgenic pbinplus-ars/lba4404 35s/er n.a. n.a. [191] tomato/transgenic pbm/lba4404 d35s/n.a. 0.01%-0.05% of tsp (leaf) n.a. [181] tomato/transgenic pbinplus-ars/lba4404 35s/n.a. 0.3 µg/g dw (fruit) oral, iga and igg antibodies in serum (maximum 300 miu/ml) [193] carrot cell/transgenic ppcv812/n.a. mas/n.a. 25 ng/g fw n.a. [198] laminaria japonica/transgenic pcat/n.a. sv40/n.a. 0.05%-0.25% of tsp n.a. [199] peanut oral, igg antibodies in serum (maximum 800 miu/ml), oral, iga and igg antibodies in serum (maximum 558 miu/ml) [167, 202] lettuce/transgenic pgptv-bar/eha105 35s/n.a. 2-23 µg/g fw n.a. [201] tomato/transgenic pbinplus-ars/lba4404 35s/n.a. 0.002% of tsp (fruit) n.a. [203] tomato/transgenic pbinplus-ars/aglo 35s/n.a. 0.003%-0.021% of tsp (fruit) oral (freeze-dried material), igg antibodies in serum [204, 205] carrot/transgenic pbinplus-ars/n.a. 35s/er 42 ng/g fw (leaf) n.a. [206] l-hbsag hbsag has been expressed in vegetative crops, such as potato, tomato, soybean and lettuce. the expression level of transgenic potato tubers was 1-11 µg/g fw. the highest expression in a tuber was developed using a construct driven by the camv 35s promoter with dual enhancers, the tobacco etch virus 5 -utr, and the 3 region from the soybean vegetative storage protein gene [165] . expression level of hbv-protein in potato was little increased when controlled by the tuber specific promoter [167] . target dna is inserted into a genomic dna as a random event when a using agrobacterium-mediated transformation. for this reason, it is difficult to conclude what is the best method for increase of hbv-protein expression level because differences in the expression level between the transgenic lines even with the same vector construction. expression in tomato fruit has been reported at 0.5 µg/g dry weight. to achieve a higher level of expression, several strong and inducible promoters, such as the enhanced dual 35s, ubq and efe promoters, were tested, as well as organelle targeting sequences. the greatest improvement resulted from the hbsag gene with an er retention signal controlled by efe promoter [169] . sunil kumar et al. [170] reported hbsag transformation in banana. the maximum expression in banana leaves has been reported at 38 ng/g fw. the expression levels in banana fruits were not presented in this report, but the expression level was presumably lower than in the leaf tissue. as with leafy vegetables, a variety of expression technologies have not yet been applied. in lettuce, the maximum expression level was 60 µg/g fw, which is the maximum anti-hbsag antibody titer of 300 miu in immunized mice serum [188, 189, 201] . upon transformation into a soybean cell culture using a construct of hbsag fused with the 3 region from the soybean vegetative storage protein gene and as controlled by a chimeric ocs-mas promoter, the maximum expression level was 74 µg/g fw [174] . grains are a further option for the expression of candidate vaccine antigens. they have long stability of expressed recombinant proteins with low water content [212] . in maize seed, the maximum expression has been reported at 0.51% of total soluble protein (approximately 80 µg/g fw). this level of expression was achieved using a barley alpha amylase signal sequence-fused s-hbsag gene with a 3× globulin1 promoter [168] . all of the results suggest that the expression levels of hbsag are highly variable and depend on plant species, tissue types and culture conditions. the major recombinant hepatitis b vaccines contain s-hbsag; therefore, the expression of this protein has been the focus in plants. the proteins pres2-s, m-hbsag, and pres1-pres2-s, l-hbsag, have been much less studied than s-hbsag. m-hbsag and l-hbsag have been transformed into potato, tomato, and tobacco (table 3) . although the expression was optimized using suitable promoters, leader sequences and targeting signals, the expression levels of m-/l-hbsag were lower than for s-hbsag. however, hbcag induces a heightened immune response [213, 214] and spontaneously assembles into capsid-like particles [215] . for these reasons, efforts devoted to the production of an anti-hbv vaccine have focused on hbcag in the last few years. especially, hbcag has been abundantly produced using transient expression systems mediated by icon binary vectors [208] or viral vector systems [209] (table 3 ). transgenic tobacco plants-derived hbsag was antigenically and physically similar to the human serum and recombinant yeast derived-hbsag particles [163] . to analyze the immunological response in vivo, tobacco-expressed hbsag was purified and injected into balb/c mice. the anti-hepb response to the tobacco-derived hbsag was qualitatively similar to the response obtained by immunizing mice with commercialized yeast-derived hbsag vaccine [175] . these results showed a possibility of developing injected vaccines using plant-expressed hbsag. due to differences of the manufacturing processes between companies, the amount of hbsag protein per dose differs among the various hbv vaccine products [216] . for this reason, there is no international standard for the hbsag protein quantity in vaccines, but there is a standard based on protective efficacy of vaccination related to the anti-hepb antibodies induction. an anti-hbsag of ≥10 miu/ml measured 1-3 months after the last dose of the vaccine are considered to be immune to hepb. although no international standard of antigen concentration is defined, considering the feasibility and cost-effectiveness of the injected vaccine, the concentration of antigen should be over 40 µg/ml [217] . despite many attempts to increase hbsag expression in transgenic plants, the expression level remains too low for use as an injected vaccine. the currently used hbv vaccine contains hbsag and is produced by yeast cells. the yeast-derived hbv vaccine can be supplied inexpensively ($1-20 per single dose [218] ); therefore, it is difficult for plant-derived vaccines to have a competitive price. however, plant suspension cultures may be used as an alternative to yeast to produce antigens for purification. expression levels have approached 74 µg/g fw (22 mg/l culture medium) in transgenic soybean culture [174] . although the expression level (8 µg/g fw) was lower in transgenic tobacco cell suspension culture than in soybean [174] , the former has been used to secrete hbsag into the culture medium, with a six-fold increase in secretion in response to jasmonic acid or salicylic acid treatment during cell culture, and the amount of antigen secreted was 180 µg/l medium [170] . another breakthrough regarding expression problems was achieved through the utilization of virus-based transient expression systems for the robust production of hbv antigens, such as s-hbsag and hbcag, with yields as high as 2 mg/g fw [208, 209, 211] . tobacco-derived proteins showing the maximum anti-hbsag antibody titer of 1165 miu in immunized mice serum [211] are preferred the application of injection after purification process, rather than oral administration in order to remove many toxic alkaloids and phenolic substances which have a tobacco plant [219] . however, improvements of several orders of magnitude are still needed for plant cell culture systems to be competitive, particularly given the slow growth rates of plant cells compared with yeast. in transgenic suspension cell culture, the formation of vlps by hbv antigens made it possible to exploit relatively inexpensive protein purification techniques, such as the sucrose gradient [174, 182, 184] or cesium chloride gradient ultracentrifugation [173] . the highest expressed soybean cell culture was used for antigen purification, and the antigen was suitable for injection [174] , but the yield remained unsatisfactory and was not cost-effective. the biggest advantage of edible plant-derived vaccines is their easy application to oral delivery. the benefits of plant-derived edible vaccines are as follows: (1) during oral delivery, plant-derived vaccines are protected in the stomach by plant cell wall and slow release in the gut; (2) the plant tissue expressing antigen may be used as raw or dried food; (3) capsules can also be made from partially or fully purified vaccine proteins; (4) no need for cold chain systems for storage and delivery of the plant tissues or extracts; and (5) the plant-derived vaccines are cost efficient compared with traditional vaccines. edible plant-derived hbv antigens have been administered by oral injection or feeding in mice with/without adjuvants [41, [165] [166] [167] 193 ]. an oral vaccine candidate has also been administered to human volunteers in small-scale clinical trials without adjuvants. the first trial was administered to three human volunteers in row lettuce leaves in two doses (0.5-1 µg of s-hbsag/dose) without the use of an adjuvant. all volunteers responded, with two of them having serum responses in excess of the protective minimum level (10 miu/ml of serum). however, the antibody levels declined rapidly [187, 220] . in the second trial, previously vaccinated human volunteers were fed two or three doses of 100 g of raw potato tubers (approximately 1 mg of the s-hbsag/dose). more than half of the subjects showed increased antibody titers [221] . the animal experiments and trials showed the potential for plant-derived hbv antigens to be used as an oral vaccine for the prevention of hbv, but there remain many problems to be solved for practical application, such as the administration of bulky plant material, declining long-term responses, individual differences in the immune response and the difficulty of defining the antigen dose [222] . the expression level of plant-derived hbv antigen is only 1/20-1/25 of the expression of yeast-derived hbv antigen; however, the expression yield and plant production scale are still increasing [223, 224] . tomato is possible intake without any processing or cooking. therefore, tomato fruit is a very attractive crop to develop an oral vaccine. according to the study to date, the expression level of hbv antigen was very low as 10 ng/g fw ( table 3 ). the maximum titers of anti-hbsag antibody in serum is 300 miu using oral application. this antibody yield was high compared to the expression level of hbv antigen in tomato fruits [225] . hbv antigen expression in maize produced much higher levels of antigen, and the palatability and digestibility were better than for potato. that is, cereal crops can easily transport or storage in dry state. in addition, the maize system induced a strong immune response with 4632 miu of maximum titer by both injection and oral administration [193, 194] . this result suggests the possibility of providing a raw material for thermostable formulation at $0.01 per dose [193] . plant components such as saponin, flavonoids, and plant oils also function as adjuvants [226] [227] [228] and help maintain the immune response in the long term [195] . the lyophilization method is an excellent way to increase the stability and shelf life of the plant-derived vaccines. in the previous study, the storage stability of lyophilized powder form was limited at 4 • c [189] . in a recent study, successful long-term storage at 37 • c was achieved though improvements in the process [229] . it is easier to control the concentration and standardize antigen doses and process the antigen into a tablet or capsule form using a powdered tissue instead of freeze-drying [188] . despite over 20 years of effort, no commercial plant-based anti-hbv vaccine has been developed. to commercialize a plant-derived hbv vaccine, several points should be considered. first, the greatest barrier is the low expression levels of hbv antigen in plants; however, expression yield and plant production scale can still be increased using plant expression vector optimization, which should be focused on the target plant. the process can also be more competitive by improving the plant-derived antigen to increase the immune response to the vaccine. second, an hbv antigen expressed in an edible plant has the advantage of being usable as an oral vaccine without processing. it is first necessary to analyze the characteristics of the target plants and the expressed protein for the development of an oral vaccine because plant components, secondary metabolites and foreign protein expression characteristics vary with plant species. to obtain feasible and cost-effective vaccines, the target plants for edible vaccines should have a long shelf life, be heat stable and be edible as a raw material. candidate grain crops are maize and rice; candidate vegetative crops are tomato and banana. third, consideration should be made of the public's acceptance of gm crops, especially plant-derived edible vaccines. for injected vaccine development, the most cost-effective method is a suspension culture in a closed environment, according to the regulations of good manufacturing practice. further safety of plant-derived vaccines can be obtained by following the same regulations established for traditional vaccines. in addition, for oral vaccines produced from gm crops, environmental risk assessment and human risk assessment should be performed. for these reasons, plant-derived oral vaccines cannot be called cost efficient compared with traditional vaccines, and the current concern over the use of gm plants is now affecting research in this field. world health organization (who) compact organization of the hepatitis b virus genome. hepatotlogy structural and functional analysis of full-length hepatitis b virus genomes in patients: implications in pathogenesis overview of hepatitis b virus mutations and their implications in the management of infection the newly licensed hepatitis b vaccine: characterics and indications for use human hepatitis b vaccine from recombinant yeast recombinant hepatitis b triple antigen vaccine: hepacare comparative immunogenicity of a pres/s hepatitis b vaccine in non-and low responders to conventional vaccine hepatitis b virus morphogenesis specific vaccine therapy in chronic hepatitis b: induction of t cell proliferative responses specific for envelope antigens induction of strong hepatitis b virus (hbv) specific t helper cell and cytotoxic t lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic hbv infection recombinant hepatitis b core antigen carrying pres1 epitopes induce immune response against chronic hbv infection medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants biological safety concepts of genetically modified live bacterial vaccines role of genetic factors and environmental conditions in recombinant protein production for molecular farming evolution of plant-made pharmaceuticals a perspective on the use of pleurotus for the development of convenient fungi-made oral subunit vaccines immunological aspects of using plant cells as delivery vehicles for oral vaccines comparative evaluation of recombinant protein production in different biofactories: the green perspective carrot cells: a pioneering platform for biopharmaceuticals production plants as factories for human pharmaceuticals: applications and challenges molecular pharming-vlps made in plants heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein malaria vaccine candidate antigen targeting the pre-erythrocytic stage of plasmodium falciparum produced at high level in plants plant-based production of recombinant plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate a plant-produced pfs25 vlp malaria vaccine candidate induces persistent transmission blocking antibodies against plasmodium falciparum in immunized mice transgenic tomato plants expressing the antigen gene pfcp-2.9 of plasmodium falciparum production, characterisation and immunogenicity of a plant-made plasmodium antigen the 19 kda c-terminal fragment of plasmodium yoelii merozoite surface protein 1 a plant-produced pfs230 vaccine candidate blocks transmission of plasmodium falciparum production and characterization of an orally immunogenic plasmodium antigen in plants using a virus-based expression system rice endosperm produces an underglycosylated and potent form of the hiv-neutralizing monoclonal antibody 2g12 a plant-derived multi-hiv antigen induces broad immune responses in orally immunized mice oral delivery of plant-derived hiv-1 p24 antigen in low doses shows a superior priming effect in mice compared to high doses chloroplast expression of an hiv envelop-derived multiepitope protein: towards a multivalent plant-based vaccine biochemical and immunological characterization of the plant-derived candidate hiv-1 mucosal vaccine ctb-mpr anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants expression of hiv-1 antigens in plants as potential subunit vaccines rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of m2 protein linked to flagellin in plants using viral vectors high-yield expression of m2e peptide of avian influenza virus h5n1 in transgenic duckweed plants oral immunization of haemaggulutinin h5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza a virus infection intranasal vaccination with a plant-derived h5 ha vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge. hum. vaccines immunother biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants plant-derived h7 vlp vaccine elicits protective immune response against h7n9 influenza virus in mice and ferrets safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine derived from influenza a (h1n1) pdm09 virus: a phase i dose-escalation study in healthy adults human antibody response to n-glycans present on plant-made influenza virus-like particle (vlp) vaccines. vaccine a plant-based system for rapid production of influenza vaccine antigens. influenza other respir the human potential of a recombinant pandemic influenza vaccine produced in tobacco plants setting up a platform for plant-based influenza virus vaccine production in south africa formulation development of a plant-derived h1n1 influenza vaccine containing purified recombinant hemagglutinin antigen. hum. vaccines immunother plant-produced recombinant influenza vaccine based on virus-like hbc particles carrying an extracellular domain of m2 protein preclinical and clinical development of plant-made virus-like particle vaccine against avian h5n1 influenza a plant-produced influenza subunit vaccine protects ferrets against virus challenge. influenza other respir plant-expressed ha as a seasonal influenza vaccine candidate highly pathogenic avian influenza as a zoonotic agent transient expression of the ectodomain of matrix protein 2 (m2e) of avian influenza a virus in plants a transgenic plant cell-suspension system for expression of epitopes on chimeric bamboo mosaic virus particles development of a new vector using soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans expression of vp1 protein of serotype a and o of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs immunogenicity of foot-and-mouth disease virus structural polyprotein p1 expressed in transgenic rice foliar extracts from transgenic tomato plants expressing the structural polyprotein, p1-2a, and protease, 3c, from foot-and-mouth disease virus elicit a protective response in guinea pigs induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes optimization of ammonium sulfate concentration for purification of colorectal cancer vaccine candidate recombinant protein ga733-fck isolated from plants optimization of elisa conditions to quantify colorectal cancer antigen-antibody complex protein (ga733-fck) expressed in transgenic plant expression of ga733-fc fusion protein as a vaccine candidate for colorectal cancer in transgenic plants plant-derived epcam antigen induces protective anti-cancer response apple latent spherical virus vector as vaccine for the prevention and treatment of mosaic diseases in pea, broad bean, and eustoma plants by bean yellow mosaic virus generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine a plant based protective antigen [pa (div)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines production of a plant-derived immunogenic protein targeting apob100 and cetp: toward a plant-based atherosclerosis vaccine a plant-produced antigen elicits potent immune responses against west nile virus in mice suppression of inhibitor formation against fviii in a murine model of hemophilia a by oral delivery of antigens bioencapsulated in plant cells effects of plant cultivation density and light intensity on the production of a vaccine against swine edema disease in transgenic lettuce production of double repeated b subunit of shiga toxin 2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease immunogenicity of eit chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against escherichia coli o157:h7 induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin b subunit vaccine without plant-associated sugar modification oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (as16) of ascaris suum fused with cholera toxin b subunit synthesis and assembly of an adjuvanted porphyromonas gingivalis fimbrial antigen fusion protein in plants transient expression of vp2 in nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus vp8 antigen produced in tobacco transplastomic plants confers protection against bovine rotavirus infection in a suckling mouse model greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression oral administration of plant-based rotavirus vp6 induces antigen-specific igas, iggs and passive protection in mice efficacy of a bvdv subunit vaccine produced in alfalfa transgenic plants immunocompetent truncated e2 glycoprotein of bovine viral diarrhea virus (bvdv) expressed in nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines the effect of plant tissue and vaccine formulation on the oral immunogenicity of a model plant-made antigen in sheep use of the wound-inducible ntqpt2 promoter from nicotiana tabacum for production of a plant-made vaccine the release and induced immune responses of a plant-made and delivered antigen in the mouse gut plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (prrsv) extraction, purification and characterization of the plant-produced hpv16 subunit vaccine candidate e7 ggg plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 l1 antigen fused with ltb as adjuvant: transplastomic plants show pleiotropic phenotypes anti-cancer activity of plant-produced hpv16 e7 vaccine production of human rotavirus and salmonella antigens in plants and elicitation of fljb-specific humoral responses in mice piri, i. isolation of ompa gene from salmonella typhimurium and transformation into alfalfa in order to develop an edible plant based vaccine expression of helicobacter pylori tonb protein in transgenic arabidopsis thaliana: toward production of vaccine antigens in plants expression of an immunogenic f1-v fusion protein in lettuce as a plant-based vaccine against plague biopharmaceutical protein production under controlled environments: growth, development, and vaccine productivity of transgenic tomato plants grown hydroponically in a greenhouse plant-derived recombinant fl, v, and f1-v fusion antigens of yersinia pestis activate human cells of the innate and adaptive immune system effective plague vaccination via oral delivery of plant cells expressing f1-v antigens in chloroplasts a plant-produced plague vaccine candidate confers protection to monkeys plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice expression and immunogenicity of the mycobacterial ag85b/esat-6 antigens produced in transgenic plants by elastin-like peptide fusion strategy superexpression of tuberculosis antigens in plant leaves oral immunogenicity of a plant-made, subunit, tuberculosis vaccine continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures toward the development of a plant-based vaccine against reovirus generation of plant-derived recombinant dtp subunit vaccine analysis and stability of the respiratory syncytial virus antigen in a t3 generation of transgenic tomato plants immunogenic properties of plant-derived recombinant smallpox vaccine candidate pb5 accumulation of recombinant sars-cov spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines clinical safety and immunogenicity of tumor-targeted, plant-made id-klh conjugate vaccines for follicular lymphoma recombinant antibody 2g12 produced in maize endosperm efficiently neutralizes hiv-1 and contains predominantly single-glcnac n-glycans m cell-targeting ligand and consensus dengue virus envelope protein domain iii fusion protein production in transgenic rice calli expression of a cholera toxin b subunit and consensus dengue virus envelope protein domain iii fusion gene in transgenic rice callus novel vaccination approach for dengue infection based on recombinant immune complex universal platform a nonreplicating subunit vaccine protects mice against lethal ebola virus challenge immunization with plant expressed hemagglutinin protects chickens from lethal highly pathogenic avian influenza virus h5n1 challenge infection engineering and expression of a human rotavirus candidate vaccine in nicotiana benthamiana expression of hepatitis b surface antigen in transgenic plants expression and characterization of hepatitis b surface antigen in transgenic potato plants production of hepatitis b surface antigen in transgenic plants for oral immunization oral immunization with hepatitis b surface antigen expressed in transgenic plants expression of the hepatitis b surface s and pres2 antigens in tubers of solanum tuberosum production of highly concentrated, heat-stable hepatitis b surface antigen in maize transient and stable expression of hepatitis b surface antigen in tomato (lycopersicon esculentum l.) expression of hepatitis b surface antigen in transgenic banana plants expression of the human hepatitis b virus large surface antigen gene in transgenic tomato plants hepatitis b surface expression in transgenic tobacco (nicotiana tabacum) plants using four different expression cassettes expression of hepatitis b surface antigen in transgenic banana plants and nt-1 cell line of tobacco hepatitis b surface antigen (hbsag) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form immunogenicity of transgenic plant-derived hepatitis b surface antigen analysis of transgenic tobacco plants carrying the gene for the surface antigen of the hepatitis b virus immunogenicity of parenterally delivered plant-derived small and medium surface antigens of hepatitis b virus oral administration of low doses of plant-based hbsag induced antigen-specific igas and iggs in mice, without increasing levels of regulatory t cells tissue specific expression of hepatitis b virus surface antigen in transgenic plant cells and tissue culture immunogenicity and tolerance following hiv-1/hbv plant-based oral vaccine administration production of marker-free plants expressing the gene of the hepatitis b virus surface antigen virus-like particles expression and assembly in plants: hepatitis b and norwalk viruses secretion of hepatitis b surface antigen in transformed tobacco cell suspension cultures a plant signal peptide-hepatitis b surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells process options in hepatitis b surface antigen extraction from transgenic potato study of the immunogenicity of hepatitis b surface antigen synthesized in transgenic potato plants with increased biosafety a plant-derived edible vaccine against hepatitis b virus low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis b surface antigen for prototype plant-derived vaccine tablet formulation freeze-drying of plant tissue containing hbv surface antigen for the oral vaccine against hepatitis b analysis of the limitations of hepatitis b surface antigen expression in soybean cell suspension cultures obtaining tomato plants transgenic for the pres2-s-hdel gene, which synthesize the major hepatitis b surface antigen agrobacterium tumefaciens-mediated transformation of yellow lupin to generate callus tissue producing surface antigen of hbv in a long-term culture bioencapsulation of the hepatitis b surface antigen and its use as an effective oral immunogen supercritical fluid extraction provides an enhncement to the immune response for orally-delivered hepatitis b surface antigen oral delivery of wafers made from hbsag-expressing maize germ induces long-term immunological systemic and mucosal responses oral immunization of animals with transgenic cherry tomatillo expressing hbsag construction of a plant effective expression vector containing the gene of hepatitis b virus surface antigen the integration of a major hepatitis b virus gene into cell-cycle synchronized carrot cell suspension cultures and its expression in regenerated carrot plants expression of hepatitis b surface antigen gene (hbsag) in laminaria japonica (laminariales, phaeophyta) transforming hbsag into peanut and detection of its immunogenecity plant expression, lyophilisation and storage of hbv medium and large surface antigens for a prototype oral vaccine formulation oral immunogenicity of potato-derived hbsag middle protein in balb/c mice retention of the ability to synthesize hiv-1 and hbv antigens in generations of tomato plants transgenic for the tbi-hbs gene synthesis of hepatitis b virus surface antigen in tomato plants transgenic for the pres2-s gene candidate mucosal vaccine against hepatitis b based on tomatoes transgenic for the pres2-s gene comparative analysis of hbv m-antigen production in leaves of individual transgenic carrot plants application of the human hepatitis b virus core antigen from transgenic tobacco plants for serological diagnosis high-level production of hepatitis b core antigen in plant leaf and its immunogenicity in mice extremely high-level and rapid transient protein production in plants without the use of viral replication a dna replicon system for rapid high-level production of virus-like particles in plants tandem fusion of hepatitis b core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins seed-based expression systems for plant molecular farming the use of viral vectors to produce hepatitis b virus core particles in plants hbv core particles as a carrier for b cell/t cell epitopes envelopment of the hepatitis b virus nucleocapsid weekly epidemiological record; hepatitis b position paper world health organization (who) toward the global elimination of hepatitis b virus transmission optimization of elastin-like polypeptide fusions for expression and purification of recombinant proteins in plants plant-based edible vaccine against hbv oral immunization of human with transgenic lettuce expressing hepatitis b surface antigen the twenty-year story of a plant-based vaccine against hepatitis b: stagnation or promising prospects? recombinant hepatitis b vaccines: disease characterization and vaccine production. in production of recombinant proteins. novel microbial and eukaryotic systems plants as bioreactors for the production of vaccine antigens immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis b and human immunodeficiency viruses saponin-adjuvanted particulate vaccines for clinical use immunomodulatory properties of vitamins, flavonoids and plant oils and their potential as vaccine adjuvants and delivery systems activation of antigen-presenting cells by immunostimulatory plant dna: a natural resource for potential adjuvant stability of s-hbsag in long-term stored lyophilised plant tissue key: cord-350393-j80k2v21 authors: chen, liping; huang, shaoping; yang, jingmao; cheng, xin; shang, zhiyin; lu, hongzhou; cheng, jilin title: clinical characteristics in patients with sars‐cov‐2/hbv co‐infection date: 2020-07-15 journal: j viral hepat doi: 10.1111/jvh.13362 sha: doc_id: 350393 cord_uid: j80k2v21 covid‐19 has become a global pandemic and garnered international attention. although the clinical features of covid‐19 related liver injury have been investigated, there have been no reports and studies on the clinical characteristics of covid‐19 patients co‐infected with hepatitis b virus (hbv). this study aimed to evaluate whether sars‐cov‐2/hbv co‐infection could influence liver function and the disease outcome. all 326 confirmed covid‐19 cases in shanghai public health clinical center (the covid‐19 designated hospital in shanghai, china) from january 20, 2020 to february 24, 2020 were enrolled and followed up until february 29 in this study. the clinical, laboratory data and the length of stay were collected and analyzed retrospectively. 20 patients with hbv co‐infection (6.1%) and 306 patients (93.9%) without hbv infection showed no differences in the level of liver function parameters. however, compared with hbsag‐ patients [145.4 mg/l (103.9‐179.2)], hbsag+ patients had a lower level of prealbumin [(102.3 mg/l (76.22‐160.2), p=.0367]. there were also no significant differences for the discharge rate and the length of stay between two groups. taken together, we found no evidence that sars‐cov‐2/hbv co‐infection could aggravate liver injury or extend duration of hospitalization. since the first covid-19 case was reported in wuhan, hubei province, china in december 2019, coronavirus pneumonia caused by sars-cov-2 infection has become prevalent globally 1 . so far, there have been almost 2 million patients infected by sars-cov-2 2 , becoming a huge threat to global health. in addition to fever, dry cough, weakness, and breathing difficulty, abnormal liver function may occur in considerable proportion of sars-cov-2 infected patients (14.8%-76.3%) [3] [4] [5] [6] [7] . although the exact mechanism of covid-19 related liver damage is still unknown, the abnormal liver function was associated with severe disease and mortality risk in covid-19 patients [5] [6] [7] . hepatitis b virus (hbv) has a worldwide distribution and remains a leading public health problem, with a high prevalence of hbsag at about 6.0% in china 8 . huang reported sars patients with hbv infection were more prone to develop higher degree of liver injury and severe hepatitis, however, the data of the prevalence of sars-cov-2/hbv co-infection in covid-19 patients is still absent 9 . in our previous study 7 (138 cases), there were only 9 (6.1%) cases (too small) with underlying liver diseases, so no further analysis was made accepted article over the clinical features of covid-19 patients with hbv infection. in view of the current pandemic of sars-cov-2, the clinical characteristics of sars-cov-2 coinfection with hbv should be identified. in this retrospective study, we expanded the sample size and aimed to evaluate the influence of sars-cov-2/hbv co-infection on the clinical characteristics including liver function and disease outcome. this is a retrospective, single-center study. a total of 326 patients with sars-cov-2-positive results, admitted to shanghai public health clinical center (shphc) from january 20, 2020 to february 24, 2020 were included and followed up until february 29. the clinical criteria of diagnosis and di scharge refer to the standards for "diagnosis and treatment scheme of new coronavirus infected pneumonia" (trial version 6). this study was approved by the ethics committee of the shanghai public health clinical center (2019-s047-02, review date: jan 13, 2020) and was exempted from the need for informed consent from patients. the basic information and clinical characteristics of patients, including demographic, clinical, laboratory data and the length of stay were collected by electronic medical records. the continuous data were presented as the mean ± standard deviation (sd), median and interquartile range (iqr), as appropriate. the independent sample t test or mann-whitney u test were used to compare the differences between two groups; the categorical variables were expressed by frequency and percentage, and compared by the chi-square test or fisher exact test. p < 0.05 was determined as with statistically significant differences. statistical analysis software graphpad prism 6 was used for all analyses in this study. of the 326 patients, 20 cases (6.1%) were hbv infected (hbsag-positive, hbeag-negative with undetectable hbv viral load (vl<100 iu/ml; icycler device, bio-rad, usa; lower limit of quantification: 100 iu/ml). as shown in table 1 , 20 patients with hbv co-infection (6.1%) and 306 patients (93.9%) without hbv infection didn't show any differences regarding age and gender distribution (p>.05). there were also no differences in the level of liver function in hbsag-patients, 245 cases (80%) were discharged with the median hospital stays of 14 days (11) (12) (13) (14) (15) (16) (17) (18) (19) . neither the discharge rate nor length of stay show any difference between the two groups. infection, there is still a presumption that several cell types in other humoral organs can be attacked by sars-cov-2. as for liver, the abundant protein levels of ace-2, a functional receptor of sars-cov-2, in bile ducts have been observed 10 . in addition, a recent study based on the analysis of online single cell rna sequencing date from healthy liver tissues also confirmed that the level of ace-2 mrna in bile duct cells is comparable to atii cells, but not in hepatocytes. these results suggest that abnormal liver function may be caused by sars-cov-2 preferentially binding to cholangiocytes. however, according to recent reports, the bile duct injury related specific index, such as alp, was not frequently elevated in covid-19 patients 1 . in general, whether sars-cov-2 can directly infect liver cells and leads to the abnormal liver function is a controversial issue due to the absence of histological evidence of covid-19 patients. this article is protected by copyright. all rights reserved according to our recent study 7 , we found elevated alt, ast were more common in covid-19 patients with abnormal liver function and liver damage can also occur in mild covid-19 patients without any related medications before hospitalization. there's reason to believe sars-cov-2 may attack the liver. taken into consideration that viral co-infection can exacerbate liver injury thus have a big impact on disease progression and outcome 11, 12 , we investigated the prevalence of hbv infection in covid-19 patients and found that there was a comparable rate of sars-cov-2/hbv co-infection to that of general population (6.1% vs 6%). these observations suggested that sars-cov-2/hbv co-infection is common in covid-19 patients, although we couldn't assess whether existence of hbv infection increases susceptibility to sars-cov-2 infection. as for liver function parameters, there were no significant differences in related indices, except for the prealbumin, which is frequently measured as indicators of liver reserve and more sensitive than other indicators. the level of prealbumin was lower in patients with co-infection than that in the control group, which indicates that the liver reserve capacity was weak in covid-19 patients with hbv co-infection. so in our study, no evidence showed coexistence of hbv infection increases the liver injury in covid-19 patients. the outcomes of covid-19 patients with/without hbv infection were also compared in our current study，and sars-cov-2/hbv co-infection had no effect on the course and prognosis of covid-19, including the rates of severe/critically ill, mortality and discharged and hospital stays. however, previous study 12 this article is protected by copyright. all rights reserved although this study is a single-center, retrospective study, it is convincing as this center is the only designated hospital for covid-19 treatment in shanghai, china and a cohort of more than 300 cases were analyzed. moreover, the positive rate of hbsag in covid-19 patients were also close to the that in the general population. taken together, our study is the first to elaborate on the clinical characteristics of sars-cov-2/hbv co-infection patients and demonstrate that the coinfection with hbv slightly affect liver function, showing no impact on the covid-19 outcome. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china coronavirus disease 2019 liver injury during highly pathogenic human coronavirus infections. liver international : official journal of the international association for the study of the covid-19 in a designated infectious diseases hospital outside hubei province longitudinal association between markers of liver injury and mortality in covid-19 in china covid-19: abnormal liver function tests clinical features of covid-19-related liver functional abnormality. clinical gastroenterology and hepatology : the official clinical practice journal of the american gastroenterological association 2020 hepatitis b virus serological screen in a general hospital in beijing from 2008 to 2018, and challenges to our vaccination policy liver injury in covid-19: management and challenges tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis hepatitis b virus coinfection in human immunodeficiency virus-infected patients: a review study of the relationship sars and hepatitis virus b accepted article key: cord-252586-fuaoelgb authors: phillips, sandra; chokshi, shilpa; chatterji, udayan; riva, antonio; bobardt, michael; williams, roger; gallay, philippe; naoumov, nikolai v. title: alisporivir inhibition of hepatocyte cyclophilins reduces hbv replication and hepatitis b surface antigen production date: 2014-10-08 journal: gastroenterology doi: 10.1053/j.gastro.2014.10.004 sha: doc_id: 252586 cord_uid: fuaoelgb background & aims: cyclophilins are host factors required for hepatitis c virus replication. cyclophilin inhibitors such as alisporivir have shown strong anti–hepatitis c virus activity in vitro and in clinical studies. however, little is known about whether hepatocyte cyclophilins are involved in the hepatitis b virus (hbv) life cycle. we investigated the effects of 2 cyclophilin inhibitors (alisporivir and nim811) on hbv replication and hepatitis b surface antigen (hbsag) production in cell lines. methods: liver-derived cell lines producing full-length hbv and hbsag particles, owing to stable (hepg2215) or transient (huh-7) transfection, or infected with hbv (heparg cells; invitrogen [carlsbad, ca]), were incubated with alisporivir or nim811 alone, or alisporivir in combination with a direct antiviral (telbivudine). the roles of individual cyclophilins in drug response was evaluated by small interfering rna knockdown of cyclophilin (cyp)a, cypc, or cypd in hepg2215 cells, or cypa knockdown in huh-7 cells. the kinetics of antiviral activity were assessed based on levels of hbv dna and hbsag and southern blot analysis. results: in hepg2215, huh-7, and heparg cells, alisporivir reduced intracellular and secreted hbv dna, in a dose-dependent manner. knockdown of cypa, cypc, or cypd (reduced by 80%) significantly reduced levels of hbv dna and secreted hbsag. knockdown of cypa significantly reduced secretion of hbsag, leading to accumulation of intracellular hbsag; the addition of alisporivir greatly reduced levels of hbsag in these cells. the combination of alisporivir and telbivudine had greater antiviral effects than those of telbivudine or alisporivir alone. conclusions: alisporivir inhibition of cyclophilins in hepatocyte cell lines reduces replication of hbv dna and hbsag production and secretion. these effects are potentiated in combination with direct antiviral agents that target hbv-dna polymerase. t he cyclophilins are a group of cellular proteins with peptidyl-prolyl isomerase enzymatic activity, which catalyze the cis to trans conversion of proline-containing peptides and facilitate protein folding. 1, 2 there are 7 main cyclophilins in human beings: cyclophilin a (cyp)a, cypb, cypc, cypd, cype, cyp40, and cyp natural killer (nk), 1 which are localized in different cellular compartments. for example, cypa and cyp40 are present in the cytosol, cypb and cypc reside in the lumen of the endoplasmic reticulum, cypd is present in the mitochondria, and cype is localized in the nucleus. cyclophilins are involved in the life cycle of a wide range of viruses including hepatitis c virus (hcv), human immunodeficiency virus, vaccinia virus, coronaviruses, and polyomavirus bk, acting as host co-factors essential for virus replication. [3] [4] [5] [6] [7] [8] cypa is the main cyclophilin that is involved directly in the life cycle of hcv 2-4 and inhibition of its peptidyl-prolyl isomerase activity with cyclophilin inhibitors was shown to interfere at multiple sites of the hcv life cycle in hepatocytes, affecting not only replication but also the hcv secretion from infected cells. [9] [10] [11] [12] [13] the role of cellular cyclophilins in the hepatitis b virus (hbv) life cycle, however, is poorly understood. in the present study we investigated whether hepatocyte cyclophilins are involved in hbv replication, hepatitis b surface antigen (hbsag) production and secretion, and the effects of nonimmunosuppressive cyclophilin inhibitors alone and in combination with a direct antiviral agent targeting hbv polymerase. four human hepatoma cell lines that produce full hbv virions and hbsag subviral particles were used in this study: (1) huh-7 cells (japan health science research resources bank, osaka, japan), and (2) huh-7 with stable knockdown (kd) of cypa using a short hairpin, 11 both cell lines were transfected with hbv dna; (3) hepg2215 cells, which are stably transfected with hbv dna; and (4) heparg cells (invitrogen, carlsbad, ca), which were infected with hbv. the huh-7 and hepg2215 cells were cultured at 37 c and 5% co 2 in dulbecco's modified eagle's medium (dmem; life technologies, paisley, uk) with 10% fetal calf serum (fcs) (life technologies), as described previously. 14 the huh-7 cypa kd cells were cultured in dmem/10% fcs plus 1â nonessential amino acids and 1 mg/ml of the selection marker puromycin (life technologies). heparg cells are terminally differentiated and were purchased from invitrogen (carlsbad, ca). the cells were cultured according to the manufacturer's instructions (heparg cell user guide; invitrogen) on collagen i-coated plates. initially, cells were grown in william's medium e with heparg thaw, plate&general purpose medium supplement and gluta-max (invitrogen), and prepared in a standardized 2-step process. after 7 days, cells were maintained in william's medium e with heparg maintenance medium supplement plus glutamax. huh-7 and huh-7 cypa kd cells were transfected with a hbv plasmid, psm2 (kindly provided by professor hans will), which contained a head-to-tail hbv-dna dimer. 15 briefly, huh-7 cells, at a density of 1.5 â 10 5 /well, were seeded onto 24-well plates and cultured for 48 hours at 37 c to reach confluency. the cells then were transfected with 0.5 mg of psm2 with fugene 6 (roche, burgess hill, uk) according to the manufacturer's instructions. the transfection efficiency was determined using a b-gal staining kit (life technologies). heparg cells were infected with hbv derived from 5-day culture supernatants of ad38 cells, as described. 16 hepg2215 and hbv-infected heparg cells were seeded at 1 and 0.3 â10 6 cells, respectively, onto 24-well plates and maintained at 37 c. the cell lines subsequently were treated as described later. hepg2215 cells were transfected with cypa, b, c, or d small interfering rna (sirna) (sigenome smart pool; thermo scientific dharmacon, epsom, uk). to optimize the knockdown effect of cyclophilin expression, preliminary experiments with a range of sirna concentrations, specific for each cyclophilin, were conducted by testing 30, 60, 100, and 150 nmol/l over 96 hours (see the supplementary materials and methods section). the cyclophilin messenger rna (mrna) expression at baseline and at different time points after sirna transfection was assessed by realtime polymerase chain reaction (pcr) with primers specific for each cypa, b, c, and d. after these optimization experiments, sirna stock solutions of 20 mmol/l were diluted in 50 ml opti-mem medium (life technologies) for a final concentration of 60 nmol/l for cypa and cypd sirna, and 100 nmol/l for cypc. lipofectamine 2000 (invitrogen, life technologies) was diluted into 50 ml opti-mem medium and incubated for 5 minutes at room temperature. combined diluted sirna and diluted lipofectamine were incubated for 30 minutes at room temperature. hepg2215 cells (1 â 10 5 cells per well) then were treated with the sirna-lipofectamine complex and incubated for 48 hours at 37 c. nontargeting scrambled sirna was used as a negative control; glyceraldehyde-3-phosphate dehydrogenase sirna and siglo (thermo scientific dharmacon) green were used as positive controls for sirna delivery and transfection, respectively. the specificity of sirna silencing was crosschecked using real-time pcr and primers specific for cypa, cypc, cypd mrna with hepg2215 cells transfected with cypa, cypc, or cypd sirna, respectively. this confirmed that each sirna selectively decreased only targeted cyclophilin mrna (by approximately 80%), whereas the other cyclophilin (cypa, cypc, or cypd) mrna expression was comparable with the control (scramble sirna). the knock-down effect for cypa protein was tested further by western blotting 11 and by enzyme-linked immunosorbent assay (elisa), as described later. alisporivir and nim811 are nonimmunosuppressive cyclophilin inhibitors 9 ; telbivudine, a nucleoside analog, is a potent inhibitor of hbv-dna polymerase. 17 all compounds were provided by novartis (basel, switzerland). in a series of experiments, the cells used (stably transfected hepg2215, transiently transfected huh-7, huh-7 cypa kd cells, and hbv-infected heparg cells) were cultured alone and with several concentrations of alisporivir or nim811. alisporivir and nim811 stock solutions were prepared as 2000â stocks in 100% dimethyl sulfoxide (dmso) and the final working concentrations of alisporivir and nim811 (0.25, 1, 5, and 20 mg/ml) were prepared daily in dmem/10% fcs with a final dmso concentration of 0.05%. the control also contained 0.05% dmso. the culture media were replaced every 24 hours with 1.5 ml of fresh alisporivir-containing or nim-containing medium. supernatants and cells were collected at baseline (bl), and at 24, 48, and 72 hours. hbvinfected heparg cells were treated with alisporivir or nim811 for 7 days, cells and supernatants were collected at bl, and at 24, 72, 120, and 168 hours (supplementary figure 1 ). for all conditions and time points, hepg2215 and huh-7 cells were tested in at least 3 independent experiments with each condition run in triplicate wells. heparg and huh-7 cypa kd cells were tested in duplicates. because the culture medium was replaced every 24 hours, the graphic representations of the results for viral particles secreted in supernatants are shown with each time point starting from zero after each medium change. cytotoxicity was assessed by microscopic observation of the cells, trypan blue exclusion, and/or by lactate dehydrogenase (ldh) release in supernatants using the ldh-cytotoxicity assay kit (biovision research products, mountain view, ca). before treating hepg2215 cells with the combination of alisporivir (alv) and telbivudine, we first tested the antiviral activity of a range of telbivudine concentrations in the current model. stock solutions with 2000â telbivudine in 100% dmso were used to prepare 6 working concentrations daily, ranging from 0.04 to 20 mmol/l telbivudine (supplementary figure 2) . the cell culture medium containing these working concentrations was replaced daily and samples were collected at bl, and at 24, 48, and 72 hours. based on this experiment, in the subsequent combination experiments telbivudine was used at 10 mmol/l (2.42 mg/ml), which corresponds to the maximum concentration levels observed in clinical studies (see supplementary materials and methods section). to test the antiviral activity of alv in combination with telbivudine, hepg2215 cells were treated for 72 hours with the following: alv 0.25 mg/ml/telbivudine 10 mmol/l, alv 1 mg/ml/telbivudine 10 mmol/l, alv 5 mg/ml/telbivudine 10 mmol/l, and alv 20 mg/ml/telbivudine 10 mmol/l. intracellular, nucleocapsid-associated viral dna was extracted from transfected huh-7 and 2215 cells or hbv-infected heparg cells, as described previously. 14, 18 the culture supernatants were treated with dnase i for 1 hour at 37 c, 10 minutes at 75 c, and then placed on ice for 1 minute. the supernatants were centrifuged at 10,000 rpm and the pellets were discarded. the dna was extracted from the supernatants using the qiaamp dna mini qiagen kit (qiagen, sussex, uk). hbv dna was quantitated by taqman real-time polymerase chain reaction (applied biosystems 7500; applied biosystems, paisley, uk) using the eurohep hbv standard and hbv-dna plasmid. 14, 18 for southern blot analysis, 20 mg of hind iii-treated intracellular dna was analyzed on a 1% agarose gel and transferred to a hybond-xl (ge healthcare life sciences, pittsburgh, pa) membrane, as described. 19 radioactive hbvspecific probes were generated from a sac i-hind iii fragment derived from psp65-ayw-1.3 by nick translation and purified by spin column chromatography. prehybridization, hybridization, and washes were as described previously. 20 quantitation of hbsag, cypa, and cypb by elisa hbsag secreted during cell culture (shbsag) and intracellular hbsag (ihbsag) were quantitated using a commercially available hbsag elisa (abazyme, needham, ma). hbsag levels were quantitated in triplicates of the culture supernatants and cytoplasmic extracts of stably and transiently transfected cells. a standard curve was established by using a serial dilution of a known quantity of purified hbsag protein (american research products, belmont, ma). the intracellular cypa and cypb levels in supernatants were quantitated by elisa, as described previously. 21 statistical analyses were performed using the spss package (ibm, new york, ny). one-way analysis of variance was used, followed by dunnett's post hoc test to compare one treatment group with the nontreated group and the tukey post hoc test was used for multiple comparisons between groups. 22 a p value less than .05 was considered statistically significant. alisporivir treatment of hepg2215 cells resulted in a progressive reduction of secreted and intracellular, nucleocapsid-associated hbv dna dependent on both drug concentration and time of drug exposure ( figure 1a and b). the secreted viral dna was reduced by 52% after 72 hours of treatment with alisporivir 20 mg/ml, compared with cells incubated with medium only (p < .01). the level of intracellular hbv dna also was reduced by approximately 60% after 72 hours of treatment with 5 and 20 mg/ml of alisporivir (p < .01). nim811 treatment of hepg2215 and of heparg cells also reduced the secreted and intracellular nucleocapsidassociated hbv dna, however, its antiviral effect was lower than alisporivir (supplementary figure 3) . the difference between nim811 and alisporivir in reducing hbv-dna level was particularly apparent with hepg2215 cells at 5 mg/ml: 6% vs 34% hbv-dna reduction at 48 hours. in huh-7 cell cultures, the control experiments for transfection efficiency (b-gal staining) showed hbv replication in 30%-40% of cells. alisporivir treatment of hbvtransfected huh-7 cells also resulted in a dose-dependent reduction of secreted and intracellular hbv dna. the most profound reduction was observed after 72 hours of treatment with alisporivir at 5 mg/ml (58% and 73%, respectively) and 20 mg/ml (64% and 58%, respectively) ( figure 1c and d) . overall, the alisporivir antiviral activity was greater in transiently transfected huh-7 cells in comparison with stably transfected hepg2215 cells (p ¼ .038 for secreted hbv dna and p < .001 for cytoplasmic hbv dna). as stated earlier, alisporivir at 5 mg/ml reduced intracellular hbv-dna levels by 73% and 58% in huh-7 and hepg2215 cells, respectively, after 72 hours of treatment ( figure 1b and d) . this difference was even more apparent at earlier time points. after 24 hours, the intracellular hbv-dna reduction in hepg2215 cells and huh-7 cells was 16% and 64%, respectively ( figure 1b and d) . importantly, the southern blot analysis of intracellular dna supports the quantitative real-time pcr results outlined earlier because alisporivir reduced intracellular hbv dna in a dose-dependent manner ( figure 1e ). hbv-infected heparg cells were treated with alisporivir or nim811 for 7 days, starting 16 hours after hbv inoculation (supplementary figure 1) . also in this model, the cyclophilin inhibitors reduced hbv-dna level ( figure 1f and supplementary figure 3) . after 120 and 168 hours of treatment with the highest concentration of alisporivir, intracellular nucleocapsid-associated hbv dna was reduced by 80% and 90%, respectively, similar to the effect of nim811: 70% and 90% reduction, respectively. in hepg2215 cells, there were minimal changes in shbsag or ihbsag levels (figure 2a and b) . the magnitude of reduction of shbsag by both alisporivir and nim811 was lower than the reduction of hbv-dna level ( supplementary figures 3 and 4) . in huh-7 cells, significant reductions in both shbsag and ihbsag were observed with the two highest alisporivir concentrations ( figure 2c and d) . the most significant change was seen after 72 hours of treatment with 20 mg/ml alisporivir, in which shbsag was reduced by 69% and ihbsag was reduced by 53%. in all experiments, alisporivir treatment was not cytotoxic for hepg2215 and hbv-transfected huh-7 cells, as shown by ldh release assay (supplementary figure 5) . to determine the relative involvement of different cyclophilins in hbv replication, the expression of cypa, b, c, and d were silenced selectively with a respective sirna. the figure 6) . the efficiency of the transfection and the sirna delivery were assessed with siglo and glyceraldehyde-3-phosphate dehydrogenase, respectively (supplementary figure 6d and e) . the expression of cypa, c, or d mrna in hepg2215 cells was reduced markedly (by !80%) in comparison with the controls treated with nontargeting, scrambled sirna for the duration of the experiment (supplementary figure 7a) . the cypa gene silencing also efficiently reduced the intracellular cypa protein when tested by elisa (supplementary figure 7b) , or by western blot (supplementary figure 7c) . both secreted and intracellular hbv-dna levels were reduced by 80% and 97%, respectively, in cypa-silenced cells ( figure 3a and 3b) . secreted hbsag was reduced by 65% in the same cells ( figure 3c ). the cypa-silenced cells were treated with alisporivir and compared with the cypasilenced cells without alisporivir (cypa þ no alisporivir) to determine whether alisporivir inhibition of other cellular cyclophilins would enhance the antiviral effect of silencing cypa. the addition of alv did not result in any further decrease in the levels of secreted and intracellular nucleocapsid-associated hbv dna and shbsag (figure 3) . we also used huh-7 cells with stable cypa kd (supplementary figure 7d) and assessed the impact on hbv replication and hbsag. the secreted hbv dna was reduced by 50% in these cells in comparison with normal huh-7 cells (figure 4a ), and adding alisporivir had only a marginal effect. intracellular nucleocapsid-associated hbv dna was affected even more in this cell line with a 90% reduction ( figure 4b ). secreted hbsag also was reduced alisporivir inhibits hbv replication 407 significantly (90%) whereas intracellular hbsag accumulated in these cells ( figure 4c and d) . interestingly, treatment of the cypa kd cells with alisporivir (20 mg/ml) profoundly decreased intracellular hbsag levels. in cypcsilenced cells, secreted and intracellular nucleocapsidassociated hbv dna were reduced by 61% and 93%, respectively, compared with negative controls (figure 5a and b) and hbsag levels were reduced by 30% ( figure 5c ). the result of silencing cypd was similar to silencing cypcas secreted and intracellular nucleocapsid-associated hbv dna and shbsag, which were reduced by 62%, 96%, and 30%, respectively ( figure 5d -f). cypa silencing with sirna resulted in a greater reduction in secreted hbv dna and shbsag compared with sirna silencing of cypc or cypd. as stated earlier, at 72 hours treatment, the silencing of cypa reduced secreted hbv dna by 80% ( figure 3a ) compared with 60% in cypc or cypd-silenced cells ( figure 5a and d) . at the same time point, shbsag was reduced by 65% ( figure 3c ) compared with 30% in cypc or cypd silenced cells ( figures 5c and f, respectively) . interestingly, adding alisporivir to cypc and cypd-silenced cells further reduced the 3 viral parameters measured ( figure 5 ). this increased effect in the presence of alisporivir was seen only when the levels of secreted and intracellular dna as well as shbsag were still increased in the cyclophilin-silenced cells such as at 24 hours for intracellular nucleocapsid-associated dna and 48 and 72 hours for shbsag. this additional effect of alisporivir, however, disappeared when the levels of secreted and intracellular nucleocapsid-associated hbv dna became very low in cyclophilin-silenced cells. because cypa represents a key target for cyclophilin inhibitors, we analyzed the changes of intracellular cypa levels in untreated and alisporivir-treated hepg2215 and huh-7 cells. in hepg2215 cells, the cypa levels were 16 times lower than in huh-7 cells (figure 6a and b) and were not affected by the alisporivir treatment, whereas in huh-7 cells the alisporivir treatment resulted in a dose-dependent decrease of intracellular cypa levels. the corresponding levels of cypb in culture supernatants of huh-7 cells increased steadily, dependent on time and alisporivir concentration ( figure 6c ), in accordance with the previous findings of cypb release from alisporivir-treated cells. 21 in heparg cells, the cypa and cypb levels were similar to those in huh-7 (data not shown). a significantly greater reduction of hbv replication was observed with alisporivir in combination with a direct antiviral agent, telbivudine, compared with alisporivir alone. this was more apparent at the lower concentrations of alisporivir: 0.25 and 1 mg/ml (figure 7) . secreted viral dna and intracellular nucleocapsid-associated hbv dna were reduced by 39% and 62%, respectively, in cells treated with 0.25 mg/ml alisporivir plus 10 mmol/l telbivudine vs 9% and 0% in cells treated with alisporivir alone at 48 hours ( figure 7a and b compared with figure 1a and b) . furthermore, at 48 hours, the reduction of secreted hbv dna was more apparent with alisporivir plus telbivudine, compared with telbivudine alone ( figure 7a ). the combination of alisporivir plus telbivudine had a similar effect on shbsag and ihbsag levels compared with alisporivir alone ( figure 7c and d compared with figure 2a and b). hbv is the smallest human dna virus with a unique genomic organization and replication mechanism. 23 viral replication takes place in the cytoplasm of infected hepatocytes with 3 subsets of hbv-rna transcripts: pregenomic rna, envelope pres/s rnas, and hbx mrna, encoding all structural and nonstructural viral proteins. characteristic of hbv infection are the spheric and filamentous subviral particles composed exclusively of viral envelope proteins and host-derived lipids, which are produced 10 3 -to 10 6 -fold in excess to full virions and secreted from hepatocytes. 23 by using 3 different in vitro hbv models: stably transfected (hepg2215), transiently transfected (huh-7) , and hbvinfected (heparg) cells, the present study shows that alisporivir inhibits hbv replication 411 cellular cyclophilins have a major role in the hbv life cycle in hepatocytes. blocking cyclophilins' enzymatic activity with small molecules, alisporivir or nim811, or the selective silencing of individual cyclophilins with sirna, markedly reduced hbv-dna replication, as well as hbsag production and secretion from cells. cypa is one of the most abundant cytosolic proteins (approximately 0.1% of cell proteins) 2 and the present study shows the major utilization of cyclophilin a both in hbv replication and in hbv envelope protein secretion from hepatocytes. the experiments using huh-7 cells with stable cypa kd, and the findings that alv enhanced hbv dna and hbsag reduction in cypc and cypd-silenced cells show that cypa is important for the formation of intracellular, nucleocapsid-associated hbv dna and viral secretion, as well as for hbsag secretion from cells. the quantitation of intracellular cypa showed a greater amount in huh-7 than in hepg2215 cells, which is the likely explanation for the much greater effect of alisporivir on hbsag secretion from huh-7 compared with hepg2215 cells. importantly, cyclophilin inhibitors, such as alisporivir and nim811, block all cellular cyclophilins and in hepg2215 cells there was a dose-dependent reduction in hbv dna. the data thus suggest that, in this model, the antiviral effect was primarily a result of alisporivir and nim811 targeting other cyclophilins (eg, cypd and/or cypc), leading to the observed hbv-dna reduction in these cells. the results from the present study together with previously published data indicate that the mechanisms by which alisporivir and nim811 impact hbv replication and hbsag production are likely to involve interference at multiple sites of the hbv life cycle. first, the finding that cyclophilin inhibition reduces intracellular nucleocapsidassociated hbv dna in the cytoplasm suggests an antiviral effect on the production of new virions and a reduction in the recirculation of hbv nucleocapsids from the cytoplasm to the nucleus, which is a key mechanism to replenish the viral template. we found a marked reduction of intracellular hbv dna in cells with stable cypa kd, along with intracellular hbsag accumulation. previous studies have shown the intracellular interaction between cypa and hbsag and their close association during secretion from liver cells. 24 thus, cyclophilin inhibitors will disrupt the cypa/hbsag complex and reduce envelope protein secretion. second, hepatitis b x antigen (hbxag), which is essential for hbv replication, associates with the outer membrane of mitochondria, and by regulating cytosolic calcium and signaling it drives hbv replication. [25] [26] [27] nim811 was shown to block cytosolic calcium signaling and the opening of mitochondrial permeability transition pores as a result of its inhibition of cypd. 28 therefore, blocking cypd interferes with hbxag regulation of calcium and will impact hbv replication negatively. third, cypa is an important co-factor for lipids and apolipoprotein b (apob) trafficking and nim811 was shown to decrease apob secretion, as well as the egress of hcv particles from jfh1-hcv-infected cells. 13 because cellular lipids are part of the hbv envelope proteins, the impact of cyclophilin inhibition on apob secretion is likely to interfere with the intracellular formation and secretion of lipoproteins part of hbv viral and subviral particles. fourth, recent findings have shown that the sodium taurocholate cotransporting polypeptide, a membrane bile acid transporter expressed in the liver, is a functional receptor for hbv and hepatitis delta virus, via pre-s1 binding. 29, 30 moreover, cyclosporin a and nonimmunosuppressive analogues, such as alisporivir, recently were shown to inhibit hbv entry into cells by blocking the sodium taurocholate cotransporting polypeptide, which is a cyclophilin-independent mechanism. 31, 32 the ultimate treatment goal of patients with chronic hepatitis b is to prevent the development of cirrhosis, liver failure, and hepatocellular carcinoma. hbsag is the hallmark of hbv infection and hbsag loss, with or without seroconversion to antibody to hepatitis b surface antigen, is the ideal treatment end point, is associated with improved longterm outcome, [33] [34] [35] shows markedly enhanced antiviral tcell reactivity, 36 and it allows therapy to be stopped with a minimal risk of relapse. although the nucleos(t)ide polymerase antivirals are potent inhibitors of hbv replication and result in a profound reduction of circulating hbv, they have little effect on hbsag transcription/subviral particle production. hbsag loss has been reported to occur in 2%-8% of hbeag-positive patients and in 0%-5% of hbeagnegative patients after 3-5 years of continuous treatment. 33, 37 the clinical implication of the present study is that it provides in vitro evidence supporting a new therapeutic approach in chronic hepatitis b with a combination of a direct antiviral agent (such as the hbv-dna polymerase inhibitors) with a host-targeting antiviral (such as alisporivir), which is likely to increase the rate of hbsag clearance. alisporivir is a host-targeting antiviral agent currently in development for the treatment of chronic hepatitis c. analyses of the clinical database, involving more than 2000 patients, show that alisporivir is well tolerated and has a markedly better safety profile when given as interferon-free treatment than with interferon-containing treatment. 38 a combination treatment with an hbv nucleos(t)ide polymerase inhibitor plus alisporivir could be beneficial because of the interference at multiple sites of the hbv life cycle, especially the additional effects in reducing hbsag production and secretion, plus blocking hbv entry into noninfected hepatocytes. in the present study, the combination of alisporivir plus telbivudine showed greater impact on hbsag and supports the potential utility of this combination, which deserves to be tested in clinical studies. in conclusion, cyclophilins are involved in multiple steps of the hbv life cycle in infected hepatocytes and blocking their enzymatic activity using nonimmunosuppressive cyclophilin inhibitors reduces viral replication and hbv envelope protein production and secretion. the combination of alisporivir, a host-targeting agent, and an hbv polymerase inhibitor, was found to decrease hbsag production markedly, as well as hbv replication, indicating a potential utility for a more effective therapy in chronic hepatitis b. note: to access the supplementary material accompanying this article, visit the online version of gastroenterology at www.gastrojournal.org, and at http://dx.doi.org/10.1053/ j.gastro.2014.10.004. the cyclophilins curing a viral infection by targeting the host: the example of cyclophilin inhibitors multiple cyclophilins involved in different cellular pathways mediate hcv replication completion of the entire hepatitis c virus life cycle in genetically humanized mice redistribution of cyclophilin a to viral factories during vaccinia virus infection and its incorporation into mature particles cyclophilin a and viral infections cyclophilin a and nuclear factor of activated t cells are essential in cyclosporinemediated suppression of polyomavirus bk replication the sarscoronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors profile of alisporivir and its potential in the treatment of hepatitis c debio 025, a cyclophilin binding molecule, is highly efficient in clearing hepatitis c virus (hcv) replicon-containing cells when used alone or in combination with specifically targeted antiviral therapy for hcv (stat-c) inhibitors the isomerase active site of cyclophilin a is critical for hepatitis c virus replication essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics inhibition of cyclophilins alters lipid trafficking and blocks hepatitis c virus secretion cd8(þ) t cell control of hepatitis b virus replication: direct comparison between cytolytic and noncytolytic functions effect of interferon alpha on hepatitis b virus replication and gene expression in transiently transfected human hepatoma cells persistence of the hepatitis b virus covalently closed circular dna in heparg human hepatocyte-like cells telbivudine, a nucleoside analog inhibitor of hbv polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir apobec and inos are not the main intracellular effectors of ifn-gammamediated inactivation of hepatitis b virus replication detection of specific sequences among dna fragments separated by gel electrophoresis molecular cloning: a laboratory manual the cyclophilin inhibitor debio-025 shows potent anti-hepatitis c effect in patients coinfected with hepatitis c and human immunodeficiency virus multiple comparison analysis testing in anova new insight in the pathobiology of hepatitis b virus infection hepatitis b virus (hbv) surface antigen interacts with and promotes cyclophilin a secretion: possible link to pathogenesis of hbv infection calcium signaling by hbx protein in hepatitis b virus dna replication hepatitis b virus x protein is essential to initiate and maintain virus replication after infection hepatitis b virus hbx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the hbx protein involved in hepatitis b virus replication hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilin-independent interference with the ntcp receptor cyclosporin a and its analogs inhibit hepatitis b virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (ntcp) asian-pacific consensus statement on the management of chronic hepatitis b: a 2008 update easl clinical practice guidelines: management of chronic hepatitis b virus infection chronic hepatitis b: update 2009 kinetics of hepatitis b surface antigen decline during 3 years of telbivudine treatment in hepatitis b e antigen-positive patients effectiveness of hepatitis b treatment in clinical practice interferon (ifn)-free alisporivir has a better overall safety profile compared to ifn-containing treatment: a pooled analysis of the alv development program supplementary figure 6. dose-dependent inhibition of cellular cyclophilin expression by cyclophilin-specific sirna. inhibition of glyceraldehyde-3-phosphate dehydrogenase (gapdh) by gapdh-specific sirna and transfection efficiency (siglo) 150 nmol/l for 24, 48, 72, and 96 hours. (a) cypa, (b) cypc, and (c) cypd expressions were measured in the cyclophilin sirna-treated cells by real-time pcr. as controls. hepg2215 cells were transfected with gapdh sirna and siglo transfection indicator for 48 hours. (d) gapdh expression was measured in gapdh and cypa sirna-treated cells at bl (48 h after transfection), and at 24, 48, and 72 hours by real-time pcr. (e) transfection efficiency was measured by flow cytometry at bl and 72 hours. nd, not done. the mean values and the sd of 2 independent experiments are shown. fitc the authors thank professor hans will (hamburg, germany) for providing the psm2 plasmid and the hepg2215 cells. these authors disclose the following: shilpa chokshi has received a research grant from novartis, and nikolai naoumov is an employee of novartis pharma ag, basel, switzerland. the remaining authors disclose no conflicts. this work was supported in part by a research grant from novartis pharma ag, basel, switzerland. the sirna oligonucleotides for cypa, cypb, cypc, and cypd (m-004979-01-0005; m-004606-00-0005; m-008819-00-0005; m-009708-00-0005; sigenome smart pool; thermo scientific dharmacon) were prepared in opti-mem medium at the following concentrations: 300, 600, 1000, and 1500 nmol/l. lipofectamine was diluted in opti-mem medium at a ratio of 1:50 for 5 minutes. each sirna concentration was mixed with the lipofectamine complex, gently mixed, and incubated for 30 minutes. hepg2215 cells seeded at a density of 1 â 10 5 cells per well were treated to this sirna:lipofectamine complex and incubated for 24, 48, 72, and 96 hours at 37 c. the cells also were transfected at these different time points with siglo green to assess the transfection efficiency. the cellular rna was extracted from the cells, reverse-transcribed, and quantitated by real-time pcr as previously described. 1 the primers used were commercially available (hsppia_1_sg; hsppib_1_sg; hsppic_1_sg; hsppif_1_sg; and b-actin as the housekeeping gene hb-actb_1_sg; qiagen). telbivudine stock solutions of 2000â were made in 100% dmso and diluted to prepare the following working concentrations: 0.04 mmol/l (9.6 â 10 -3 mg/ml), 0.16 mmol/l (0.038 mg/ml), 0.65 mmol/l (0.157 mg/ml), 2.5 mmol/l (0.605 mg/ml), 10 mmol/l (2.42 mg/ml), and 20 mmol/l (4.84 mg/ml). hepg2215 cells were treated with these working solutions of telbivudine at bl, and at 24, 48, and 72 hours. the telbivudine-containing media were replaced every 24 hours. the supernatants were collected at every time point and the hbv dna extracted from the supernatants was quantitated by real-time pcr. key: cord-015941-4fz79wzf authors: hu, yuan title: molecular techniques for blood and blood product screening date: 2018-11-10 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-3-319-95111-9_2 sha: doc_id: 15941 cord_uid: 4fz79wzf blood product safety is a high priority for manufacturing industries, hospitals, and regulatory agencies. an important step in ensuring safety is the screening of donated blood for infectious diseases. molecular technologies for screening infectious diseases have improved remarkably over the years. molecular biological assay significantly reduced the risk of transfusion-transmitted infections. unlike previous methods, molecular technologies for screening infectious diseases are specific, efficient, easy to use, and economical. a new era in molecular biology is coming to the field of blood safety. direct detection of viral antigens and virus-specific antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. there are some limitations. for direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts sufficiently detectable in the body by an antibody-mediated assay. for indirect virus detection by virus-specific antibodies [e.g., an immunofluorescence assay or enzyme immunoassay (eia), etc.], there is a problem in that shortly after infection by a pathogenic virus, and there is a window period in which antibody generation is insufficient for detection [4] . to reduce this window period of low detection, direct nucleic acid tests are needed. through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. the nucleic acid tests can also provide evidence for genetic variation in viruses. molecular methods include the use of nucleic acid probes as well as amplification-based and dna sequence-based techniques. an increasing number of molecular diagnostic methods are now available commercially [2] . in comparison to classical methods, molecular biological methods are superior in terms of rapidness, specificity, and sensitivity. the current nucleic acid detection methods in the field may be grouped into two major classes: amplifying techniques such as pcr and non-amplifying techniques such as southern blot hybridization. amplifying techniques are more sensitive than non-amplifying techniques. there are two different types of amplifying methods [5] , target amplification methods and signal amplification methods. target amplifying techniques include pcr, nucleic acid sequence-based amplification (nasba) [6, 7] , self-sustaining sequence amplification (3sr), transcription-based amplification (tas), transcription-mediated amplification (tma), strand displacement amplification (sda), and ligase chain reaction (lcr). signal amplification methods include branched dna signal amplification (bdna) [8] , cleavage-based signal amplification (cycling probe technologies and invader assay), qß replicase, hybrid capture, cycling probe technologies (cpt), and rolling-circle amplification (rca) [9] . to further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing [2] [3] [4] [5] . southern blotting [10] was named after edward m. southern who developed this procedure at edinburgh university in the 1970s. this technique is used to detect specific sequences within mixtures of dna, which is size fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. after blocking of non-specific binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the specific dna sequence of interest. should specific dna be present on the blot, it will combine with the labeled probe and be detectable. in 1983, dr. kary mullis at cetus corporation conceived of polymerase chain reaction [11] . there is not a single technique that has had a greater impact on the practice of molecular biology than pcr. with this technique, we can detect infectious disease agents at an extremely low level. it is based on the ability of sense and antisense dna primers to hybridize to a dna of interest. following extension from the primers on the dna template by dna polymerase, the reaction is heat denatured and allowed to anneal with the primers once again. another round of extension leads to a multiplicative increase in dna products. therefore, a minute amount of dna can be efficiently amplified in an exponential fashion to result in larger amounts of dna that are more easily manipulated. by including critical controls, the technique can be made quantitative. the current level of the sensitivity and detection limit is as low as 10-50 copies per ml blood in hiv testing [1, 12, 13] . important clinical examples of the use of pcr are detection of hiv and hcv [14] [15] [16] . pcr techniques have evolved into different branches. some of them are now widely in use for virus detection in clinical diagnostics. these are real-time pcr by taqman (roche), lightcycler (roche), smartcycler (cepheid), in situ pcr, nested pcr, nested real-time pcr [17] , broad-range pcr, multiplex pcr, rt-pcr, arbitrarily primer pcr, long pcr, and quantitative pcr. real-time sequence technology will be coming soon for more detailed detection. in the past, identification of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (rflp). with real-time sequence technology, we will be able to detect a virus early as well as to obtain the viral sequence. microarrays were developed at stanford university by schena and co-workers in the early 1990s [18] . for medical applications, a microarray analysis offers a very accurate screening technology. it allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. it promises to be a fast and accurate diagnostic tool in the field of clinical microbiology and virology. applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. compared with existing technologies, it allows for a wider variety of specific tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. with the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identification of many types of pathogenic contaminants. in the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. it can provide vast information about the identity of blood-borne pathogens as well as their gene expression profiles [19] . to ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents [20] . all of the above tests are referred to as screening tests and are designed to detect as many infectious agents as possible. because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. in order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more specific tests called confirmatory tests. thus, confirmatory tests help determine whether a donor is truly infected. if any one of these tests fails, affected blood products are considered unsuitable for transfusion [20] . nucleic acid testing (nat) employs testing technology that directly detects the genomes of viruses. because nat detects a virus's genetic material instead of waiting for the body's response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [21] , thus further improving blood safety. nucleic acid testing is becoming the gold standard because of greater sensitivity compared to antibody tests [2] . since 1999, nat has been approved by the fda and used to detect hiv-1 and hcv; this technology currently is under investigation for detecting other infectious disease agents. we know that for many viral infections, viral rna appears very early in the infection, in 1 to 2 weeks, but the antibody doesn't appear until 10-12 weeks, e.g., hiv and hcv [21] . in order to virtually prevent infection by all the transfusion-associated viruses, we need to detect the viruses in their window period, and a nat or gene-based testing method is needed. nat also provides an opportunity for the viral, e.g., hiv or hcv, infected donor to seek early treatment. on the other hand, nat is not only a sensitive method but also a rapid method, which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical. the hepatitis b virus (hbv) is a highly infectious and often non-symptomatic virus that is transmitted primarily through blood and blood-derived fluids and is a leading cause of liver infection worldwide [22] . the world health organization (who) estimates that 2 billion people worldwide have been infected with hbv and 350,000,000 people are chronically infected. chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1000,000 deaths each year. each year up to 200,000 people become newly infected in the united states alone. since screening for hbv began in 1969, the rate of infection through blood transfusions has greatly decreased. however, as of 2000, hbv is still transmitted through blood transfusions in 1 out of 137,000 units of blood. one reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. serologic tests for hepatitis b virus include hepatitis b surface antigen (hbsag) and hepatitis b core antibody (hbcab). hbv, which mainly infects the liver, has an inner core and an outer envelope (the surface). the hbsag test detects the outer envelope, identifying an individual infected with the hepatitis b virus. this virus can cause inflammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. fortunately, most patients recover completely and test negative for hbsag within a few months after the illness. a small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. chronically infected people can develop severe liver disease as time passes and need to be followed carefully by an experienced physician. to reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hepatitis b surface antigen by the most sensitive and specific assays. blood donations that are found to be reactive in the hbsag test are automatically confirmed by the hbsag confirmatory assay. if the specimen is neutralizable in the confirmatory test, the specimen is considered positive for hbsag. hepatitis b surface antigen testing of donated blood has begun in 1975 (table 1) . currently, all blood donors are screened for hbsag, but occasional transmission of hbv still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). detection of early hbv infection of blood donors is still a major problem of blood transfusion. the current third-generation licensed hbsag tests (mostly radioimmunoassay and enzyme immunoassays) cannot detect hbv in the window period for hbv infection. this is a strong motivation for introducing molecular detection techniques to the field [23] . there are some commercially available test methods for detecting hbv dna in the market now, such as chiron's quantiplex hbv dna [24] , digene's hybrid capture, abbott's hbv dna assay, and roche's amplicor hbv monitor. using these commercial hybridization or pcr-based assays, hbv dna can be detected 1-3 weeks before the appearance of hbsag [25] . some chronically infected patients who have lost their hbsag remain hbv dna positive but are disqualified as potential blood donors. molecular detection of hbv dna is more sensitive than current methods employed for hbsag screening [22] [23] [24] [25] . determination of anti-hbc (total) is also used to monitor the progress of the hepatitis b viral infection. determination of anti-hbc (igm) is employed to distinguish an acute hepatitis b infection from a chronic infection. the anti-hbc test developed in 1987 detects an antibody to the hepatitis b virus that is produced during and after infection. if an individual has a positive anti-hbc test, but the hbsag test is negative, it may mean that the person once had hepatitis b but has recovered from the infection. of the individuals with a positive test for anti-hbc, many have not been exposed to the hepatitis b virus; thus, there is a frequent problem of false positives. although the individual may be permanently deferred from donating blood, it is unlikely that the person's health will be negatively affected. (note: this antibody is not produced following vaccination against hepatitis b [26] . the hepatitis c virus (hcv) is a member of the flaviviridae family of viruses, which are associated with both human and animal diseases [27] . hepatitis caused by hcv is the most common chronic blood-borne infection in the united states. over 4 million americans are believed to be infected. hcv can also be transmitted through blood transfusion. hcv causes inflammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver inflammation, cirrhosis, cancer, and death. eventually, up to 20% of people with hcv may develop cirrhosis of the liver or other severe liver diseases. as in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. because of the risk of serious illness, people with hcv need to be followed closely by a physician with experience evaluating this infection. since the full-length hcv cdna was first cloned in 1989, significant progress has been made in characterizing its molecular biology [13] . but, the natural history of hcv infection is still evolving, and current treatment options for patients are either limited or expensive [27] . there is no vaccine for hcv, and the current treatment includes a combination of alpha interferon and ribavirin as well as the combination of the nucleotide polymerase inhibitor sofosbuvir and the ns5a inhibitor velpatasvir [28] . although the former is efficacious in only a minority of patients [29] , the latter has been shown to be effective in a broad range of patients [28] . the life cycle of the hcv continues to be poorly understood due to the lack of an efficient cell culture system [30] . there is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hepatitis c virus. we recently established a cell culture system for the replication of hcv by using human t and b leukemia cell lines [31] . this model should represent a valuable tool for the detailed study of the initial steps of the hcv replication cycle and for the evaluation of evolving antiviral molecules. currently, appropriate vaccine strategies for hcv have not been developed. early detection and prevention of hcv infection are most important for blood safety. it is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the hcv genome because of a high mutation rate in this genomic region. however, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of hcv. in 1990, the first specific test for hepatitis c virus, the major cause of "non-a, non-b" hepatitis, was introduced. now, a third-generation elisa kit is available to detect antibodies to hcv, and screening blood for hcv antibodies is recommended. these assays are based on detection of serum antibody to various hcv antigens because these antibodies are nearly universally present in patients who are chronically infected with hcv [32] . the hcv screening tests are known to have significant limitations, and positive samples should be further tested by hcv confirmatory tests. guidelines provided by the cdc recommend that hcv antibody screening testpositive samples should be confirmed with serologic or nucleic acid supplemental testing. hcv confirmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. in this way, the response to individual antigens can be recognized, and some false-positive elisa results can be eliminated (e.g., riba, chiron hcv 3.0, and pcr assay) (e.g., roche cobas amplicor hcv test, version 2.0). laboratories can choose to perform this testing on all positive specimens or based on screening test-positive (signal to cutoff) ratios. the positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened. hcv antibodies are not generally detectable for at least 6 weeks and may not appear for several months. acute hcv infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. high-sensitivity detection of hcv during the window period is a long-term technical challenge in the field. tests for hcv rna genome detection based on the pcr or other highly sensitive rna detection systems have been used for the diagnosis of acute hepatitis [26] . sensitive detection of hcv rna based on rt-pcr or other nucleic acid amplification techniques can be readily accomplished with kits that are now available commercially. for example, in 1999 the fdaapproved roche's amplicor hiv-1 monitor ultrasensitive quantitative assay. it can measure hiv levels at as few as 50 copies/ml, and another commercial kit, the lcx hiv rna quantitative assay from abbott laboratories, also has a detection limit at 50 copies/ml. some studies even showed a sensitivity limit at one copy [33] . in fact, a qualitative assay should be much more sensitive than a quantitative assay for hiv/ hcv screening. a sensitive qualitative hcv molecular detection assay will possibly interdict and virtually prevent all transfusion-associated hiv/hcv. the current sensitivity standard for clinical diagnostics is 100 copies per ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies per ml. hiv-1 and/or hiv-2 virus cause acquired immunodeficiency syndrome or aids. the test is designed to detect antibodies directed against antigens of the hiv-1 or hiv-2 viruses. hiv-1 is much more common in the united states, whereas hiv-2 is prevalent in western africa. donors are tested for both viruses because both are transmitted by infected blood, and a few cases of hiv-2 have been identified in us residents. in 1985, the first blood screening eia test to detect hiv was licensed and quickly implemented by blood banks to protect the blood supply. in 1992, testing of donor blood for both hiv-1 and hiv-2 antibodies (anti-hiv-1 and anti-hiv-2) was implemented. in 1996, hiv p24 antigen testing of donated blood was mandated. now, the p24 antigen testing is going to be compared with a pcr-based test for their ability to detect hiv in the window period. htlv retroviruses are endemic in japan and the caribbean but relatively uncommon in the united states [34] . they cause adult t-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. the infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. in rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. htlv-ii infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. disease associations with htlv-ii have been hard to confirm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections or rare cases of neurological disease. in 1989, human t-lymphotropic virus antibody testing of donated blood was begun. blood is now routinely screened for antibodies to htlv-i/ii. these test screens for antibodies directed against epitopes of the htlv-i and htlv-ii viruses. several commercial assays based on the enzyme-linked immunosorbent assay (elisa) or particle agglutination formats are used for screening of htlv antibodies, followed by confirmatory assays using western blotting. in some infected individuals, the serologic response to htlv infection is very low. these problems have been solved by the application of pcr amplification of specific sequences in the virus genome. pcr can be used to detect htlv-i/ii proviruses and is now the method of choice for detection of htlv dna directly from blood and many other tissues. commercial pcr kits for htlv are available [34] . the west nile virus (wnv) is a single-stranded rna virus of the flaviviridae family and is one of the most recent emerging infectious disease threats to public health and, potentially, to the safety of our blood supply [35] [36] [37] . in 2002, wnv was identified as transfusion transmissible. it is transmitted by mosquitoes to birds and other animals through a mosquito bite. the virus can infect people, horses, many types of birds, and some other animals. wnv was shown in 2002 to be transmissible by blood [35] , with an estimated mean risk of 2/10,000-5/10,000 in outbreak regions in the united states. the most common symptoms of transfusion-transmitted cases of wnv were fever and headache. detection of wnv includes either a measurement of wnv antibodies or of wnv nucleic acid (detecting genetic material from the virus itself). there are two types of wnv antibody testing: igm and igg. in most individuals, igm antibodies will be present within 8 days after the initial exposure to wnv, followed by igg production several weeks later. but, the antibodies tested to detect wnv are not expedient for donor blood screening. nucleic acid testing involves amplifying and measuring the west nile virus's genetic material to detect the presence of the virus in the blood or tissue. wnv nat will be negative in the blood once clinical illness has occurred. in this situation, both nat and igm antibody testing may be needed. nucleic acid tests to screen blood for wnv are commercially available and in current use. but, the viral yield for wnv infection is much lower than other viruses. consequently, a more sensitive wnv nat system for donor blood screening will be required, which could further reduce the risks of transfusiontransmitted wnv. serum samples from all blood units should be subjected to either the vdrl (venereal disease research laboratory) test or a treponemal test, such as the treponema pallidum hemagglutination (tpha) test before transfusion. any unit found positive should be discarded as per standard safety procedures. this test is done to detect evidence of infection with the spirochete that causes syphilis. blood centers began testing for this shortly after world war ii, when syphilis rates in the general population were much higher. the risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors and because the spirochete is fragile and unlikely to survive blood storage conditions. sensitivity and specificity of serologic tests vary depending on the type of test performed and the stage of the disease. if the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. syphilis serological tests for donors have less clinical significance. a nucleic acid test for accurately detecting syphilis is needed. it can be used to determine whether a blood donor is currently or has recently been infected with the spirochete. in recent years, numerous infectious agents found worldwide have been identified as potential threats to the blood supply, and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. even if the hepatitis virus test is negative for all known a-e hepatitis agents, there are some unidentified hepatitis viruses, called non-a-e hepatitis viruses that can still be transmitted by blood transfusion. in the future, advances in nat may allow rapid discovery of the unknown hepatitis viruses. hepatitis delta virus (hdv) is a small rna virus that can infect only individuals who have hbv; worldwide more than 15 million people are coinfected [38] . hdv is clinically important because it generally makes hbv infections more damaging to the liver. increased understanding of the molecular virology of hdv will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. pcr and real-time pcr methods are available for hdv rna detection [39] . tt virus (ttv) [40] , named for the patient from whom it was first isolated with non-a-e and g posttransfusion hepatitis in japan in 1997, is a newly discovered transfusion-transmitted, single-stranded and circular dna virus [41] . ttv is nonenveloped, and its entire sequence of ~3.9 kb has been determined. it is also often interpreted as a transfusion-transmitted virus [42] . at least 16 genotypes have been identified, and ttv is now found all over the world. ttv infection was sought by detection of ttv dna in serum by polymerase chain reaction using primers generated from a conserved region of the ttv genome, e.g., the utr region [42] . donor blood and blood product can be screened for ttv dna by using pcr or real-time pcr. the significance of positive findings is still unclear, because high-level ttv carriers in healthy populations are currently found [42] [43] [44] . whether ttv actually causes hepatitis remains to be determined. cytomegalovirus (cmv) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. donor blood is not routinely tested for cmv, and the prevalence of cmv antibody ranges from 50 to 80% of the population. but, blood contaminated with cmv can cause problems in neonates or immunocompromised patients. it also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis, and colitis. potential problems in selected patient populations can be prevented by transfusing cmv negative blood or frozen, deglycerolized red blood cells. serologic tests for antibody to cmv are useful for determining whether a patient had cmv infection in the past, a determination of great clinical importance for organ and blood donors and in the pretransplant evaluation of prospective transplant recipients [45] . commercial nat kits are available for cmv [5] , and these include the amplicor pcr cmv monitor test and hybrid capture system cmv dna test. chagas disease is caused by the blood-borne parasite, trypanosoma cruzi, which is transmitted to humans through insects. in the united states, chagas disease is considered one of the neglected parasitic infections, a group of five parasitic diseases that have been targeted by cdc for public health action [46] . commercial anti-t. cruzi assay kits are available for the qualitative detection of antibodies, trypanosoma cruzi (t. cruzi), the causative agent of chagas disease in human serum and plasma specimens by abbott diagnostics [47] . sensitive screening tests for malaria are neither commercially available nor officially approved yet. the most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria [48] . donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. because there are no practical laboratory tests available to test donor blood, donors traveling to high-risk malaria areas are excluded from donating blood for 6 months. however, there is a need to develop suitable screening tests, especially for use in an endemic area. a number of clinical research approaches have been developed for the extraction, amplification, and detection of malaria parasite dna from blood products [49] . variant creutzfeldt-jakob disease [50] (vcjd, a rare but fatal brain infection) was first described in 1996 in the united kingdom. vcjd is strongly linked with exposure to the bovine spongiform encephalopathy (bse) agent. bse is a transmissible spongiform encephalopathy (tse) affecting cattle and was first reported in the united kingdom in 1986. it has different clinical and pathologic characteristics from classic cjd. each disease also has a particular genetic profile of the prion protein gene [51] . in recent years, questions have been raised concerning the potential risk of variant creutzfeldt-jakob disease for recipients of plasma-derived clotting factors, including the united states licensed factor viii (pdfviii), factor ix (pdfix), and other plasma-derived products such as immune globulins and albumin. in the past 10 years, there have been some reported cases of probable variant creutzfeldt-jakob disease (vcjd) transmission by red blood cell transfusions in the united kingdom [52] . prion infections are associated with long and clinically silent incubations [50, 51] . the number of asymptomatic individuals with vcjd prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. in order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vcjd prion infection [52] . recently research papers have shown that sensitivity detection methods are available for vcjd prion [53] [54] [55] . however, commercial detection kits are not yet available. the dengue virus (denv) is a member of the virus family flaviviridae and is transmitted to people through the bite of an infected mosquito [56] . the dengue virus has been shown to have four subtypes. these subtypes are different strains of dengue virus that have 60-80% homology between each other. dengue has emerged as a worldwide problem only since the 1950s. with more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. according to cdc, as many as 100 million people are infected yearly [57] . dengue is caused by any one of the four related viruses transmitted by mosquitoes. there are not yet any vaccines to prevent denv infection, and the most effective protective measure is to avoid mosquito bites. there have been healthcare-related transmissions, including transmission by blood products [58] . dengue infection has a viremic phase that lasts 4-8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [58] . there are currently no tests for direct detection of dengue virus, but there are, however, commercial elisa tests to detect antibodies of the dengue virus in blood samples from patients [59] . recently, research papers have shown that pcr detection methods are available for any dengue virus strain [57, 60] . babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis [61] . babesiosis is a malaria-like parasitic disease [62] , and there are over 100 species of babesia identified [63] . in the united states, babesia microti is the agent most commonly reported to cause human infection. clinical confusion between human babesiosis and malaria is often reported in literature [62] . babesia infection can also be acquired by blood transfusion [64, 65] . in fact, there have been many cases of transfusion-induced babesiosis documented [64, 65] . risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. current standards issued by the american association of blood banks (aabb) require the indefinite deferral of a blood donor with a history of babesiosis [65] . there is a need to develop methods for identification babesia microti in order to reduce the risk of transmission of babesiosis by transfusion. diagnosis depends upon finding parasites on blood film examination which can be detected 2-4 weeks after a tick bite. hamster inoculation and serology have also been used for diagnosis. the indirect fluorescent antibody test (ifat) is available for b. microti and is the most useful serological test for early diagnosis [66] . also, the pcr screen tests for babesiosis are technically available in the field [67] . chagas disease is named after the brazilian physician carlos chagas, who discovered the disease in 1909 [68] . chagas disease is spread mainly by blood-sucking insects infected with trypanosoma cruzi. chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. national screening of the blood supply [69] was instituted in early 2007 by fda, and more than 1000 donors with t. cruzi infection have been identified within the past 3 years of testing. "screening for t. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of chagas disease," says karen midthun, m.d., acting director of the fda's center for biologics evaluation and research. currently, serological elisa tests are available to diagnose chronic chagas disease [70] . pcr test is not a tool for diagnosis of chronic chagas disease in clinical practice yet, although some research results have showed that pcr is a very sensitive parasitological test for chagas disease in active transmission regions [71] . more studies are needed for the development of this molecular method. coronavirus is an rna virus known to be associated with respiratory disease. severe acute respiratory syndrome (sars) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from asia in 2003. there has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with sars is present in the blood of people who are sick, and it is possible that the virus could be present in the blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit sars through a blood donation. to help determine whether or not an individual might be infected with sars, a blood collection facility will ask a potential donor orally or in writing about any travel to a sars-affected country or a history of sars or possible exposure to sars. enzyme-linked immunoassays for detection of specific igg and igm antibodies and rt-pcr for detection of sars coronavirus-specific rna in the sars patients have been developed. rapid, sensitive, and specific identification of sars and other novel coronaviruses by molecular methods will be very important in the future. ebola virus disease (evd) is a rare and deadly disease caused by infection with one of the ebolavirus species [72] . the recent outbreak in 2014-2015 is the largest ebola outbreak since the ebola virus was first discovered in 1976, first in yambuku, democratic republic of congo, and then in nzara, south sudan. the virus family filoviridae includes three genera: cuevavirus, marburgvirus, and ebolavirus. there are five species that have been identified: zaire, bundibugyo, sudan, reston, and taï forest. the first three, bundibugyo ebolavirus, zaire ebolavirus, and sudan ebolavirus, have been associated with large outbreaks in africa. the virus responsible for causing the 2014 west african outbreak belongs to the zaire species (who) [72] . samples from patients are an extreme biohazard risk. currently, a number of approaches have been developed and are available for diagnoses of ebola virus disease: (1) antibody-capture enzyme-linked immunosorbent assay (elisa), (2) antigen-capture detection tests, (3) serum neutralization test, (4) reverse transcriptase polymerase chain reaction (rt-pcr) assay, (5) electron microscopy, and (6) virus isolation by cell culture tests. the who and fda are working to help expedite the development and availability of medical products -such as treatments, vaccines, diagnostic tests, and personal protective equipment -with the potential to help bring the ebola epidemic in west africa under control as quickly as possible (fda) [73] . based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non-a-e, g, and ttv posttransfusion hepatitis. it is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. in recent years, numerous new infectious agents found worldwide have been identified through time-consuming procedures. by the time a new virus, such as hcv, hiv, and sars, is found, many people are infected, and there could be a large number of fatalities. there is an urgent need to develop methods for rapid identification and characterization of previously unknown pathogenic viruses. the most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (rda), and broad-range polymerase chain reaction (br-pcr). but they are all time-consuming approaches. the new unrecognized and uncharacterized viral agents can be rapidly identified by some of the new molecular approaches, e.g., subtraction hybridization [74] and dna microarray. ensuring the safety and efficacy of blood and blood products is a critical regulatory challenge. the high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. it will be achieved by introducing more updated nucleic acid tests to the field of blood safety [2, 3] . nucleic acid testing is a method of testing blood that is more sensitive and specific than conventional tests that require the presence of antibodies to trigger a positive test result. also, nat works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. this improved sensitivity should enable us to significantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. we are not only to protect the blood supply from known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents [4, 36, 37, 75] . the nat methods are more sensitive and specific compared with non-nat. in the future, nat technology, such as pcr, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety. progress in blood supply safety advances in testing technology to ensure transfusion safety -nat and beyond the safety of the blood supply -time to raise the bar emerging infectious disease issues in blood safety in vitro nucleic acid amplification techniques evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents detection of piscine nodaviruses by real-time nucleic acid sequence based amplification (nasba) molecular-based methods for quantifying hiv viral load molecular diagnostics of infectious diseases 50 years of the double helix: from the concept of molecular hybridization to microarrays in vitro nucleic acid amplification techniques. diagnostic molecular microbiology principles and applications. mayo foundation multicenter evaluation of the performance characteristics of the nuclisens hiv-1 qt assay used for quantitation of human immunodeficiency virus type 1 rna serological and molecular biology screening techniques for hcv infection clinical evaluation of an hiv-1 and hcv assay and demonstration of significant reduction of the hcv detection window before seroconversion roche amplicor hiv-1 monitor ultrasensitive quantitative assay quantitative assay from abbott laboratories nested real-time pcr for hepatitis a detection introduction to microarray analysis. microarray analysis development and validation of a diagnostic dna microarray to detect quinolone-resistant escherichia coli among clinical isolates food & drug administration. testing requirements for communicable disease agents detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-amplification testing update on hepatitis b virus infection challenges in hepatitis b detection among blood donors assessment of hepatitis b virus dna stability in serum by the chiron quantiplex branched-dna assay hepatitis b virus. section two: specific virus families immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infection control practices advisory committee (hicpac) hepatitis c virus: virology, diagnosis and treatment sofosbuvir and velpatasvir for hcb genotype 1, 2, 4, 5, and 6 infection recent advances in prevention and treatment of hepatitis c virus infections the scientific challenge of hepatitis c detection of extrahepatic hcv replication by a novel highly sensitive single tube nested-pcr chapter 34: hepatitis c viruses. section two: specific virus families new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunodeficiency virus type 1 rna in plasma chapter 58: human t-cell leukemia virus types 1 and 2. section two: specific virus families estimated risk of transmission of the west nile virus through blood transfusion in the us transfusion-transmitted emerging infectious diseases: 30 years of challenges and progress the potential treat to blood transfusion safety of emerging infectious disease agents hepatitis delta virus. the lancet, early online publication quantification of hepatitis delta virus rna in serum by consensus real-time pcr indicates different patterns of virological response to interferon therapy in chronically infected patients ttv-a virus searching for a disease a novel unenveloped dna virus (tt virus) associated with acute and chronic non-a to g hepatitis role of transfusion-transmitted virus in acute viral hepatitis and fulminant hepatic failure of unknown etiology a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology chapter 77: cytomegalovirus. section two: specific virus families evaluation of a prototype trypanosoma cruzi antibody assay with recombinant antigens on a fully automated chemiluminescence analyzer for blood donor screening transfusion-transmitted malaria in countries where malaria is endemic: a review of the literature from sub-saharan africa detection and species identification of malaria parasites by isothermal thda amplification directly from human blood without sample preparation 18 years of research and surveillance prions: beyond a single protein crerutzfeldt-jakob disease and blood transfusion: updated results of the uk transfusion medicine epidemiology review study detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay preclinical detection of variant cjd and bse prions in blood quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus threat of dengue to blood safety in dengueendemic countries comparison of a commercial igm capture elisa with dengue antigen focus reduction microneutralization test and the centers for disease control dengue igm capture-elisa pcr detection of nearly any dengue virus strain using a highly sensitive primer cocktail do babesiosis and malaria share a common disease process? babesia: a world emerging babesia infection through blood transfusions: reports received by the us food and drug administration transfusion-transmitted babesia spp.: bull's-eye on babesia microti continuous in vivo culture and indirect fluorescent antibody test for zoonotic protozoa of babesia microti detection of babesia species from infected dog blood by polymerase chain reaction silver spring, m.d. 20993. complete list of donor screening assays for infectious agents and hiv diagnostic assays elisa versus pcr for diagnosis of chronic chagas disease: systematic review and meta-analysis pcr-based diagnosis for chagas' disease in bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis virus disease updated ebola response updates from fda rapid approach to identify an unrecognized viral agent emerging infectious disease agents and their potential threat to transfusion safety key: cord-002706-m3y35ozx authors: guo, fang; zhao, qiong; sheraz, muhammad; cheng, junjun; qi, yonghe; su, qing; cuconati, andrea; wei, lai; du, yanming; li, wenhui; chang, jinhong; guo, ju-tao title: hbv core protein allosteric modulators differentially alter cccdna biosynthesis from de novo infection and intracellular amplification pathways date: 2017-09-25 journal: plos pathog doi: 10.1371/journal.ppat.1006658 sha: doc_id: 2706 cord_uid: m3y35ozx hepatitis b virus (hbv) core protein assembles viral pre-genomic (pg) rna and dna polymerase into nucleocapsids for reverse transcriptional dna replication to take place. several chemotypes of small molecules, including heteroaryldihydropyrimidines (haps) and sulfamoylbenzamides (sbas), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic rna and viral dna polymerase. interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that haps and sbas differentially modulate the biosynthesis of covalently closed circular (ccc) dna from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded dna-containing progeny nucleocapsids in the cytoplasm. specifically, the mistimed cuing of nucleocapsid uncoating prevents cccdna formation during de novo infection of hepatocytes, while transiently accelerating cccdna synthesis from cytoplasmic progeny nucleocapsids. our studies indicate that elongation of positive-stranded dna induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind cpams and triggers its disassembly. understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccdna synthesis and cure chronic hepatitis b. hepatitis b virus (hbv) is a small dna virus that chronically infects 240 million people worldwide and causes approximately 686,000 deaths annually due to various severe liver diseases, including cirrhosis, hepatocellular carcinoma (hcc) and liver failure [1] . currently approved direct-acting antiviral agents against hbv are six nucleos(t)ide analogues that inhibit viral dna polymerase with varying potency and barriers to drug resistance [2] . although those viral dna polymerase inhibitors significantly reduce viral load and prevent liver disease progression, they rarely cure hbv infection due to their inability to eradicate cccdna [3] . hbv core protein is a small polypeptide of 183 amino acid residues. it exists in infected hepatocytes as several distinct quaternary structures and plays multiple roles in the viral replication cycle [4] . the best characterized function of core protein is the assembly of pre-genomic (pg) rna and viral dna polymerase complex into nucleocapsids where hbv dna synthesis takes place [5] . moreover, temporally and spatially regulated disassembly (or uncoating) of nucleocapsids is essential for delivery of viral relaxed circular (rc) genomic dna into the nuclei of infected hepatocytes [6, 7] , where it is converted to covalently closed circular (ccc) dna [8, 9] . furthermore, it has also been suggested that core proteins may associate with cccdna minichromosomes, in an as-yet undefined structural manner, to regulate its transcription [10] . interestingly, it was also reported that core protein can be hijacked by host immune responses to recruit cytokine-induced dna cytosine deaminase apobec3a to cccdna minichromosomes, which results in cytosine deamination and decay of cccdna [11, 12] . due to their unique structures and essential roles in viral replication, disruption of, or interference with, nucleocapsid assembly and/or disassembly with small molecular core protein allosteric modulators (cpams) represents a new frontier in development of novel antiviral agents against hbv [13, 14] . over the last two decades, at least five chemotypes of cpams have been reported [13] . those compounds bind to a hydrophobic pocket, designated as the hap pocket, at the dimer-dimer interface near the c-termini of core protein subunits [15, 16] . binding of these molecules in the hap pocket induces large scale allosteric conformational changes in core protein subunits and alters the capsid assembly kinetics and pathways [4, 17] . while heteroaryldihydropyrimidines (haps), such as bay 41-4109 and gls4, misdirect capsid assembly to form non-capsid polymers of core proteins [17, 18] , all other chemotypes of cpams, including sulfamoylbenzamides (sbas) and phenylpropenamides (ppas), represented by enan-34017 and at-61, respectively (s1 fig), induce the formation of morphologically "normal" empty capsids with distinct quaternary and/or tertiary structural changes and thus, preclude viral dna replication [19, 20] . thus far, several haps and sbas have been shown to inhibit hbv replication in animal models and are currently under preclinical or clinical development [14, 21] . inspired by the observation that a small molecule compound targeting the capsid protein of dengue virus has dual effects on both the assembly and disassembly (or uncoating) of the viral capsids [22] , we hypothesized that hbv cpams may not only disrupt capsid assembly, but also alter the structure and function of assembled nucleocapsids and consequentially affect viral dna replication and/or cccdna synthesis. indeed, we have now obtained evidence showing that haps and sbas, but not ppas, induce disassembly of nucleocapsids from virions as well as double-stranded dna-containing cytoplasmic progeny nucleocapsids and consequentially interfere with cccdna biosynthesis from de novo infection and intracellular amplification pathways. discovery of human sodium taurocholate cotransporting polypeptide (hntcp) as the bona fide receptor for hbv infection of hepatocytes allows for establishment of convenient hepg2derived hbv infection cell culture systems [23, 24] . we have thus established a novel ntcpexpressing human cell line, designated as c3a hntcp , and demonstrated its susceptibility to hbv infection. the parental cell line, c3a, is a subclone of hepg2 cells that exhibits strong contact inhibition of growth and metabolic features that are more similar to normal hepatocytes [25] . as shown in s2 fig, cccdna became detectable as early as 1 day and reached maximum levels at 2 day post infection by qpcr and conventional southern blot assays. hbv pgrna and core-associated viral dna replication intermediates as well as core protein also accumulated sequentially in infected cultures. hbsag was readily detectable by elisa as a product of hbv infection in the culture media. in order to investigate the effects of cpams on hbv infection, particularly cccdna synthesis from a de novo infection, c3a hntcp cells were mock-treated or treated with representative hap (bay 41-4109, gls4) or sba (enan-34017) and control compounds entecavir (etv) or myrcludex b (myrb), starting at 24 h before hbv infection until harvesting at day 3 and day 6 post infection. as expected (fig 1) , myrb, an acylated peptide derived from the hbv large envelope protein blocks virus entry [26] , inhibited cccdna formation and consequential accumulation of pgrna and core dna. also as anticipated, etv, an hbv dna polymerase inhibitor, did not affect the synthesis of cccdna and accumulation of pgrna, but inhibited the synthesis of core dna [27] . interestingly, gls4, bay 41-4109 and enan-34107 significantly reduced the amounts of cccdna. viral pgrna and core dna were also proportionally reduced. a more detailed time course study spanning the first four days post infection revealed that the three cpams significantly inhibited cccdna formation, whereas etv and ifn-α did not (s3 fig) (fig 1d, lanes 5 and 9) . however, while etv did not reduce viral rna, ifn-α reduced the levels of viral pgrna and 2.4/2.1 kb mrna, presumably due to suppression of cccdna transcription [28, 29] . to further characterize the inhibitory effect of cpams on cccdna biosynthesis, time-ofaddition and dose response experiments were performed. in agreement with its mode of action, myrb treatment starting at 24 h before or at the time of infection efficiently blocked cccdna formation, whereas delayed treatment starting at 24 h post infection completely failed to inhibit cccdna formation (fig 2a) . consistent with the kinetics of cccdna formation in this cell culture system (s3 fig), while cpam treatment starting at 24 h before or at the time of infection reduced cccdna formation at similar efficiency, their inhibitory effects were significantly reduced when the treatment started at 24 h post infection. moreover, all the three cpams inhibited cccdna formation in a concentration dependent manner (fig 2b) . those results are in agreement with a report published during the preparation of this manuscript that bay 41-4109 and sba derivative jnj-632 inhibited hbv cccdna synthesis during hbv infection of human primary hepatocytes [30] . to investigate the possibility that the observed inhibition of cpams on cccdna formation is due to inhibition of ntcp-mediated hbv entry, we examined their effects on hepatitis d virus (hdv) infection of c3a hntcp cells. the results demonstrated that while myrb efficiently and cytoplasmic core dna (c) were quantified by real-time pcr assays. differences in viral cccdna, core dna or pgrna between mock-treated control and treated groups were statistically analyzed (t-test, * p <0.05, ** p <0.01, *** p <0.001). (d) hybridization analyses of hbv replication intermediates in cells harvested at 6 dpi. upper panel, hirt dna extracted from the cells harvested at 6 dpi were denatured at 88˚c for 5 min to denature dp-rcdna to single-stranded dna and followed by restriction with ecori to convert cccdna into unit-length double stranded linear dna and detected by southern blot hybridization (labeled as ccc/ecori). unit-length hbv linear dna served as a molecular weight marker. lower panel, hbv rnas, pre-genomic rna (pgrna), 2.4 and 2.1kb mrna specifying envelope proteins were determined by northern blot hybridization. 28s and 18s ribosomal rna (rrna) served as loading controls. https://doi.org/10.1371/journal.ppat.1006658.g001 reduced genomic and antigenomic hdv rna by more than 3 log, etv and ifn-α as well as bay-41-4109 and enan-34017 did not apparently alter the amounts of hdv rna in the infected cultures (s4 fig). the results thus imply that inhibition of cccdna synthesis by the cpams is due to their interaction with hbv-specific components, most possibly nucleocapsids, but not the host cellular factor(s) in the entry pathway shared by hbv and hdv. cpams accelerate intracellular cccdna synthesis from cytoplasmic progeny nucleocapsid rcdna besides being synthesized from incoming virion dna in the de novo infection, cccdna in infected hepatocytes can also be amplified by delivery of rcdna from cytoplasmic progeny nucleocapsids into the nucleus and conversion to cccdna [31] [32] [33] . this intracellular cccdna amplification pathway has been demonstrated to function in cultured cells and in vivo in duck hepatitis b virus (dhbv)-infected ducks, and is regulated by the large envelope protein and host cellular factors [33, 34] . because cpams inhibit pgrna encapsidation and thus preclude viral dna replication and intracellular amplification of cccdna, their effects on intracellular progeny nucleocapsids and cccdna amplification cannot be investigated in cells directly treated with those compounds [35] . to circumvent this problem, as depicted in fig 3a, we first arrested hbv dna replication by culturing hepad38 cells in medium without tetracycline, but containing the reversible viral dna polymerase inhibitor foscarnet (trisodium phosphonoformate, or pfa for short) for four days, which arrested hbv dna replication predominantly at the stages of incomplete and complete minus-strand dna (fig 3b) [36] . the cells were then cultured in the presence of tetracycline to stop hbv pgrna transcription from integrated transgene in cellular chromosome, and also in the absence of pfa to allow viral dna replication and cccdna synthesis to resume. at the time of pfa withdrawal, mock treatment or treatment with etv, bay-41-4109, enan-34017 was initiated and cells were harvested at the indicated time points for analyses of hbv core dna and cccdna. synthesis and identity of cccdna in this cell culture system were confirmed by its resistance to heat denaturing and conversion into 3.2kb unit-length dna after heat denaturing and ecori digestion (s5 fig). as shown in fig 3b, upon removal of pfa from culture medium, the incomplete and complete minus strand, partial double-strand and complete double strand hbv dna species (including rcdna) sequentially increased from 3 to 12 h (fig 3b) . deproteinized rcdna (dp-rcdna) and cccdna became readily detectable at 9 and 12 h, respectively ( fig 3c) . as expected, etv treatment inhibited the elongation of arrested hbv dna species and prevented the production of rcdna as well as dp-rcdna and cccdna. interestingly, although bay-41-4109 or enan-34017 treatment did not inhibit elongation of minus-strand and partial double-strand dna in the cytoplasmic nucleocapsids, the rcdna was not accumulated in those cells ( fig 3b) . however, analysis of hirt dna indicated that the amount of dp-rcdna was not apparently reduced by cpam treatment and to our surprise, the cccdna was readily detected as early as 9 h after the removal of pfa in cpam-treated cells. experiments with additional cpams and extended treatment duration further supported the notion that the selected hap and sba compounds reduced the accumulation of cytoplasmic rcdna-containing nucleocapsids (s6 fig cpams do not inhibit completion of positive strand dna synthesis, but confer dnase i sensitivity of mature forms of viral dna the apparent contradicting effects of cpams on cytoplasmic rcdna-containing nucleocapsid accumulation and kinetics of cccdna synthesis in hepad38 cells prompted us to investigate whether those compounds inhibited the completion of rcdna synthesis and/or destabilized the nucleocapsids containing mature forms of double-stranded dna. this latter possibility may explain the observed acceleration of cccdna formation from intracellular progeny nucleocapsids [7, 37] . to this end, hbv capsids were purified from the cytoplasmic fraction of pfatreated cells and an endogenous dna polymerase assay were performed in vitro in the absence or presence of cpams. to probe the integrity of nucleocapsids, the accessibility of viral dna to dnase i digestion were tested at the completion of endogenous dna polymerase reaction, but before extraction of viral dna. southern blot analysis of viral dna species indicated that rcdna can be efficiently synthesized in the in vitro endogenous dna polymerase reaction when dntps were provided ( fig 5a) . furthermore, the presence of bay 41-4109, enan-34017 or at-61 did not inhibit rcdna synthesis. however, the rcdna synthesized in the presence of bay 41-4109 and enan-34017, but not at-61, was susceptible to dnase i digestion (fig 5a and 5b ). hence, our results indicate that cpams do not inhibit hbv dna synthesis, but favor a hypothesis that the selected cpams specifically alter the structure of, and destabilize mature rcdna-containing nucleocapsids and facilitate rcdna nuclear delivery and synthesis of cccdna. because the mature rcdna-containing nucleocapsids only constitute a small fraction of total cytoplasmic capsids [38] , it is not surprise that the cpam treatment did not alter the amounts and migration mobility of capsids, as revealed by a native agarose gel electrophoresis-based particle gel assay (fig 5a and 5b , lower panels) [35, 39] . the differential effects of cpams on cccdna synthesis from the de novo infection and intracellular amplification pathways argues that the compounds may have a different effect on the nucleocapsids in virions and in the cytoplasm. to investigate this possibility, we purified hbv virion particles from the blood of a chronic hbv carrier and examined the effects of cpams on rcdna synthesis and integrity of nucleocapsids in an in vitro endogenous dna polymerase reaction as described above. similar to the results obtained with cytoplasmic nucleocapsids, the presence of bay 41-4109, enan-34017 or gls4 did not inhibit rcdna synthesis from partially double-stranded virion dna, but conferred susceptibility of virion rcdna to dnase i digestion in a concentration-dependent manner ( fig 6a) . to further investigate whether cpams can directly induce structural change of the partially double-stranded dna-containing nucelocapsids, virion particles prepared from the patient serum were treated with the cpams in an endogenous dna polymerase reaction without dntp and followed by dnase i treatment prior to dna extraction. the results showed that each of the three tested cpams rendered all virion dna species susceptible to dnase i digestion in a concentration-dependent manner (fig 6b) . hence, the results indicate that irrespective to the maturation stage of viral dna, the nucleocapsids derived from virion particles are sensitive to cpam-induced structural changes that expose viral dna for dnase i digestion. elongation of positive-stranded dna gradually increases the vulnerability of cytoplasmic nucleocapsids to cpams encouraged by the observation that the cpam-induced virion dna susceptibility to dnase i did not depend on active dna polymerase reaction or ongoing dna chain elongation ( fig 6b) , we further examined the effects of cpams on capsids purified from the cytoplasm of hepad38 cells in an endogenous dna polymerase reaction buffer without dntps. as shown in fig 7a, while rcdna became susceptible to dnase i digestion at lower concentrations of [35, 39, 40] . a kinetics study further revealed that while up to 6 h incubation was required for enan-34017 to induce significant exposure of rcdna, 1 to 2 h exposure was sufficient for gls4 and bay 41-4109 to induce an extensive exposure of rcdna ( fig 7b) . to determine the kinetics of cpams induction of rcdna exposure in cells, hepad38 cells cultured in tet-free medium for 6 days were treated with gls4 for the indicated periods of time. the cytoplasmic lysates were mock-treated or treated with dnase i before extraction of dna. as shown in s8 fig, treatment of gls4 for 6 h induced extensive rcdna exposure for dnase i digestion. cpams promote uncoating of mature nucleocapsids although results presented above indicate that cpam treatment induces the exposure of rcdna in nucleocapsids to dnase i digestion, the extent of disruption on the structure of mature nucleocapsids by the different cpams remains to be determined. we therefore examined whether the mature viral dna species, rcdna and double-stranded linear (dsl) dna, were still associated with capsids after cpam treatment. the assumption is that if cpam treatment severely disrupts the mature nuclocapsids and results in releasing of viral dna, we anticipate that rcdna and dsldna species will not co-sediment with any form of capsids. however, if cpam treatment only mildly disrupts the mature nucleocapsids and causes the exposure of viral dna that is still associated with capsid structure, we anticipate to see the rcdna and/or dsldna species will co-sediment with capsids. to this end, hbv capsids prepared from hepad38 cells were mock treated or incubated with enan-34017, bay 41-4109 or gls4 in endogenous dna polymerase reactions without dntps. the reactions were then fractionated by sucrose density gradient ultracentrifugation. the amounts of total capsids and capsid-associated dna in each of the fractions were analyzed by a 1.5% native agarose gel electrophoresis-based particle gel assay [39] . as shown in the upper panels of fig 8 a to 8d , compared to mock-treated control, treatment with any of the three cpams did not alter the sedimentation profile of capsids and capsid associated hbv dna. these results are consistent with the observation that cpams only affect the mature nuclocapsids that constitute only a small portion of total capsids (fig 7 and s7 fig) . however, analysis of viral dna by southern blot hybridization in each of the fractions revealed that rcdna and dsldna were lost or reduced from capsids sedimenting to 21% to 25% sucrose after bay 41-4109 or gls4 treatment, but the majority of rcdna and dsldna still co-sediment with capsids after enan34017 treatment (fig 8 a to 8d, lower panels) . due to the small amounts of rcdna and dsldna and low sensitivity of southern blot hybridization assay, we were not able to identify the location or distribution of the disassociated dna species in the gradients. nevertheless, those results indicate that while bay 41-4109 or gls4 treatment most likely induced structural changes that were significant enough to cause loss of rcdna and dsldna from majority of the mature nucleocapsids, the less active compound enan-34017 might only induce structural changes that render the dna content susceptible to dnase digestion. these two distinct degrees of conformational alteration may correspond to a subtle increase in "capsid breathing" for enan-34017, versus extensive rearrangement of the capsid subunit organization for bay-41-4109 and gls4 [41] . the genomes of all viruses are wrapped with capsid protein(s) to form nucleocapsids. unlike viral enzymes that often have host cellular homologues, host cells do not encode proteins that are structurally and functionally similar to viral capsid proteins. therefore, viral capsid proteins are ideal and highly selective antiviral targets [42] . in addition to serving as a vehicle for transmission of viral genomes between host cells, hbv nucleocapsids have other unique functions in the viral life cycle [4, 9, 27] . as illustrated in fig 9, first, hbv dna replication occurs exclusively within the cytoplasmic nucleocapsids, by reverse transcription of viral pgrna first into negative-strand dna and then double-stranded rcdna. second, unlike all other viruses where the progeny nucleocapsids have only one destination, i.e., to be secreted as virions, the hbv progeny nucleocapsids can also deliver their rcdna into the nuclei to synthesize cccdna. due to superinfection exclusion [43, 44] , it is reasonable to consider that the activity of the intracellular amplification pathway is the key determinant of cccdna pool size in infected hepatocytes [45] . due to the large amounts of progeny nucleocapsids in the cytoplasm, this intracellular cccdna amplification pathway must be, and actually is, tightly controlled by viral and host factors [7, 34, 37, 46] . finally, the exclusive replication in nucleocapsids and the spatially-controlled disassembly and delivery of rcdna into nuclei at nuclear pore complexes protect the viral dna from recognition by cytoplasmic dna sensors, and thus favor the persistent infection by hbv [8, 9, 47] . hence, it has been speculated that targeting hbv core protein may disrupt multiple steps of hbv replication and activate innate immune response in virally infected cells. however, as mentioned, while several distinct chemotypes of hbv cpams have been shown to disrupt viral nucleocapsid assembly and consequentially inhibit viral genome replication, their effects on other aspects of nucleocapsid function have not been investigated. herein, we provide evidence suggesting that selected haps and sbas are able to interact with nucleocapsids from virions and mature forms of rcdna-containing nucleocapsids in the cytoplasm and subsequently interfere with their function of delivering viral rcdna to the nuclei for cccdna synthesis. although the structural basis for the cpams to target the highly selected sub-populations of nucleocapsids remains to be determined, our results are consistent with previous findings that mature hbv nucleocapsids are intrinsically unstable or fragile [48] , probably due to the stiffness of rc and dsldna, which imposes bending energy and electrostatic repulsion to the capsid shell [49] . moreover, viral dna synthesis in the nucleocapsids assembled from mutant dhbv core proteins with a serial n-terminal inertions destabilized mutant nucleocapsids, rendering mature viral dna selectively sensitive to nuclease digestion [50] . therefore, it is possible that the intrinsic instability of mature nucleocapsids, due to the completion of double-stranded dna, confers the selective sensitivity for cpams to induce disassembly. considering the result that cpam treatment induces the accessibility of all forms of virion dna, either rcdna or partially double-stranded dna, to dnase i digestion, a possible explanation is that like many other viruses, structural shifts or maturation of nucleocapsids may occur during or after virion assembly and secretion, and confer susceptibility to cpams [51] . moreover, a recent report showed that the carboxyl-terminal domain of core proteins is hypophosphorylated in dna-containing virions, but remains hyperphosphorylated in intracellular dna-containing nucleocapsids [52] . although the c-terminal arginine-rich domain of hbv core protein is not directly involved in the binding of cpams [39] , the dephosphorylation may alter the interaction between core protein and viral dna [53] , and thus affect the nucleocapsid structure in a manner distinct from that of the hyperphosphorylated state [54] . in addition, the sensitivity of partially double-stranded dna-containing cytoplasmic nucleocapsids to the increased concentrations of cpams strongly suggests that elongation of positive-stranded dna toward rcdna induces incremental, or gradual, structural changes favoring specific interaction with cpams. structural biology studies with purified double stranded dna-containing capsids may reveal those important structure differences. however, it is technically challenging to purify sufficient amounts rcdna-containing hbv capsids for cryo-em analyses. although our study revealed correlations between altered cccdna synthesis and induction of nucleocapsid destablization by cpams, the molecular mechanisms on how the cpaminduced nucleocapsid structural changes disrupt cccdna formation in de novo infection, but accelerate cccdna synthesis from progeny intracellular nucleocapsids remain to be determined. however, reasonable speculations can be made based on current knowledge. as illustrated in fig 9, on one hand, in de novo infection, cpams may interact with nucleocapsids in virions during endocytic entry or immediately after their release into the cytoplasm to induce viral dna exposure or release from nucleocapsids. the premature disassembly of nucleocapsids results in viral dna release and decay by cytoplasmic dnases before its arrival to the nuclear pore complex for nuclear import and subsequent cccdna synthesis. on the other hand, due to the shorter distance traveled as compared to the de novo infection, the intracellular progeny rcdna-containing nucleocapsids may reach nuclear pore complexes before a certain stage of their disassembly and cpam-induced uncoating actually accelerates the release of rcdna into the nuclei and thus cccdna formation. of course, a fraction of rcdna decay in the cytoplasm may also occur. in fact, this interpretation is in agreement with recent findings that enhanced destabilization of mature rcdna containing nucleocapsids, due to unidentified host cellular factors in mouse hepatocytes or a single amino acid substitution (i126a) in core protein, significantly reduced the accumulation of rcdna, but increased the dp-rcdna and cccdna synthesis [7, 37] . it had not escaped our attention that the released or exposed rc/dsldna in the cytoplasm may activate innate dna sensors [55] [56] [57] [58] . however, cytokine response was not detected in cpam-treated cells. whether this is due to the deficiency of cyclic gmp-amp synthase (cgas)-stimulator of interferon genes (sting) pathway in hepatocytes and hepatoma cells [59] [60] [61] or efficient digestion of the released viral dna by cytoplasmic nucleases [62] is currently under investigation. while inhibition of cccdna synthesis from de novo infection is obviously a beneficial therapeutic effect, the acceleration of intracellular cccdna amplification is detrimental. however, the potent inhibition of pgrna encapsidation by cpams will quickly block viral dna replication and production of mature progeny nucleocapsids and consequential cccdna synthesis. therefore, the only chance for cpams to accelerate cccdna synthesis during antiviral therapy is at the initial period of treatment to promote cccdna synthesis from pre-existing mature nucleocapsids in hbv infected cells. fortunately, treatment of hepad38 cells supporting steady-state hbv dna replication with etv and cpams to mimic the initial stage of antiviral treatment in vivo did not observe a significant increase of cccdna accumulation within first 24 h of treatment. instead, similar to etv, cpams prevented amplification of cccdna pool during a prolonged treatment, as compared with mock-treated control (s9 fig). in fact, those results are consistent with the observation that cpam treatment of primary human hepatocytes after establishment of hbv infection did not alter the amount of cccdna [30] . taking together, the results imply that cpams only significantly increase the amount of cccdna by acceleration of ongoing cccdna synthesis, such as under the condition of fast cccdna synthesis after release from pfa arrested hbv dna replication, but cannot accelerate the very low (or negligible) rate of cccdna synthesis to significantly increase the amount of cccdna in cells with established hbv infection. mechanistically, as stated above, it is possible that only when cpam-induced disassembly of mature nucleocapsids occurs at or near the nuclear pore complex may facilitate the import of rcdna into the nuclus for cccdna synthesis, but the mature nucleocapsids at such status may not exist in a significant amount in hepatocytes with established infection. nevertheless, careful monitoring of cccdna synthesis during the early phase of cpam treatment in vivo in animals and in clinical trials and pretreatment or combination with viral dna polymerase inhibitors might be considered. the hepad38 cell line (obtained from dr. christoph seeger at fox chase cancer center, philadelphia) supporting hbv pgrna transcription and subsequent viral dna replication in a tetracycline (tet)-inducible manner was maintained as previously described [63] . c3a cell line [a derivative of hepg2 (atcc hb-8065)] (atcc crl-10741) was maintained in dmem/f-12 (1:1) medium supplemented with 10% fbs (gemini bio-products), 100 u/ml penicillin, 100 μg/ml streptomycin. entecavir (etv) is a gift from dr. william s. mason at fox chase cancer center, philadelphia [64] . foscarnet was purchased from sigma. capsid assembly modulators enan-34017 and bay41-4109, gls4 and at-61 were described previously [35, 65] . myrcludex b is a gift of dr. stephan urban at heidelberg university, germany [26] . alpha-interferon (ifn-α) was purchased from pbl assay science. rabbit anti-hbc antibody was obtained from dako (b0586). human sodium taurocholate cotransporting polypeptide (ntcp) gene coding sequence was amplified from a cdna clone purchased from origene (sc118232). a carboxyl-terminal c9 tag was added by pcr amplification with the primers harboring c9 tag sequence and noti & bam hi restriction enzyme sites. the purified pcr fragments were digested with restriction enzymes not i and bam hi and subsequently cloned into a pqcxip vector (clontech). vsv g protein pseudotyped retroviruses were packaged in gp2-293 cells as previously described [66] . c3a hntcp cell line stably expressing human ntcp was established by infection of c3a cell line with the pseudotyped retroviruses and selected with medium containing 2 μg/ml of puromycin. puromycin-resistant cells were expanded into cell line and designated as c3a hntcp . proper expression of ntcp was confirmed by immunofluorescence and western blot assays. c3a hntcp cells were seeded into collagen-coated 24-well plates at a density of 4×10 5 cells per well and cultured in complete dmem medium containing 3% dimethyl sulfoxide (dmso). one day later, the cells were infected with hbv prepared from hepad38 cell culture media at a moi of 500 genome equivalents per cell in dmem containing 4% peg-8000. the inoculums were removed at 24 h post infection and the cultures were maintained in complete dmem medium containing 3% dmso until harvesting. extraction and analyses of hbv dna and rna by hybridization and realtime pcr assays hbv core dna and rna extraction from infected c3a hntcp or hepad38 cells, southern blot hybridization, and real-time pcr analyses were performed as described previously [35, 65] . hbv cccdna from hbv-infected c3a hntcp cells and hepad38 cells were extracted by a modified hirt dna extraction procedure [33] . a fraction of hirt dna preparation was digested with 1 unit of plasmid-safe adenosine triphosphate (atp)-dependent deoxyribonuclease (psad) (epicentre technologies) in a 25 μl reaction for 1 h at 37˚c to remove rcdna. the dnase was inactivated by incubation of the reactions for 30 min at 70˚c. cccdna in the psad-treated samples were quantified by a real time pcr assay with primer sequences ggggcgcacctctcttta (forward) and ccacccaggtagctagagtcattag (reverse). the real-time pcr was performed using the sybr premix ex taq on a lightcycler 480 ii (roche) as the following reaction procedure: 95˚c for 10 min then 45 cycles of 95˚c for 30 s, 60˚c for 5 s, and 72˚c for 30 s. the amount of hbv cccdna in a dna preparation was determined by real-time pcr using a plasmid containing hbv genotype d genome as the standard. for southern blot hybridization, the hirt dna samples were denatured at 88˚c for 5 minutes and chilled in ice. this procedure completely denatures deproteinized rc-dna (dp-rcdna) into single stranded dna, whereas cccdna will remain as double stranded circular dna [67] . the denatured hirt dna samples without or with further digestion with restriction enzyme eco ri to linearize cccdna into double-stranded linear dna were resolved in 1.5% agarose gel and transferred onto hybond-xl membrane. the membrane was probed with α-32 p-utp labeled minus strand specific full-length hbv riboprobe. hdv production, infection and rna quantification. cell culture derived hdv particles were generated as described previously [68] . briefly, huh7 cells (obtained from dr. christoph seeger at fox chase cancer center, philadelphia) were co-transfected with a plasmid containing three copies of hdv cdna sequence (psvld3) and a plasmid expressing hbv envelope proteins l, m and s (psv45h) [69] . hdv particles were harvested from the media between 6 to 9 and 9 to 12 days post transfection. after clarification by centrifugation, particles were precipitated in the presence of 10% peg-8000 and the pellet was dissolved in tan buffer (50 mm tris-hcl, ph 8.0 and 100 mm nacl) at 1% volume of the original culture medium. for infection, c3a hntcp cells were seeded into collagen-coated 24-well plates at a density of 4×10 5 cells per well and cultured in complete dmem medium containing 3% dimethyl sulfoxide (dmso). one day later, the cells were infected with hdv at a moi of 500 genome equivalents per cell in dmem containing 4% peg-8000. the inoculums were removed at 24 h post infection and the cultures were maintained in complete dmem medium containing 3% dmso until harvesting. intracellular hdv genomic and antigenomic rnas were quantified by qrt-pcr using strand-specific primers as described previously [68] . purification of hbv virions and cytoplasmic capsids. hbv virions were prepared from a hbv carrier's serum (from bioreclamation ivt, the complete resources for all biologicals) by sucrose gradient centrifugation. briefly, 1 ml of human sera was layered onto a 2-ml 20% (wt/vol) sucrose cushion in 0.15 m nacl, 0.02 m tris-hcl, ph 7.4, and centrifuged for 3 h at 45,000 rpm in sw55 rotor at 4˚c. the supernatant fluid was removed and pellet was dissolved in tne buffer (0.15 m nacl; 0.01 m tris-hcl, ph 7.4; and 0.1 mm edta). to prepare intracellular hbv capsids, hepad38 cells were cultured in tetracycline-free medium with or without 2 mm foscarnet (pfa) for 6 days with daily medium change. the cells were then lysed with chilled lysis buffer containing 10 mm tris-hcl (ph 8.0), 1 mm edta, 0.1% nonidet p-40 and 8% sucrose on ice for 10 minutes, the lysate was centrifuged at 10,000 g for 5 min to remove the nuclei and cell debris. the clarified supernatant was overlaid onto a 10-55% (w/w) sucrose gradient and centrifuged at 24,000 rpm for 16 h at 4˚c using a beckman sw28 rotor. nineteen 2-ml fractions were collected from the bottom of the cushion. ten microliters of each fraction was dot-blotted onto the nitrocellulose membrane and hbv core protein was detected by incubation with a rabbit antibody against hbv core protein (dako, b0586). the bound antibody was detected by irdye secondary antibodies and visualized by li-cor odyssey system. hbv core protein-positive fractions were pooled together and the sucrose concentration was adjusted to 10% by addition of tne buffer. the diluted sample was overlaid on a 20% sucrose cushion and centrifuged at 45,000 rpm for 3 h at 4˚c using beckman sw55 rotor. the pellet was resuspended in tne buffer. endogenous dna polymerase reaction. an endogenous dna polymerase reaction (epr) mixture was assembled with 20 μl of hbv virion or capsid preparation, 25 μl of 2x epr buffer which consisted of 0.15 m nacl, 0.1 m tris-hcl (ph 8.0), 20 mm mgcl2, 2 mm dithiothreitol, 0.2% (vol/vol) nonidet p-40. dntp and capsid assembly modulators were added, as indicated, to 0.1 mm or a specified final concentration, respectively. water was added to bring the reaction volume to 50 μl. after incubation at 37˚c for an indicated period of time, the reaction was subjected for extraction of viral dna with or without prior dnase i digestion at 37˚c for 30 min. the viral dna were resolved in 1.5% agarose gel and transferred onto hybond-xl membrane. the membrane was probed with α-32 p-utp labeled minus strand specific fulllength hbv riboprobe. particle gel assay. the cytoplasmic hbv capsids were analyzed by a native agarose gel electrophoresis-based assay. briefly, ten microliters of endogenous dna polymerase reaction or fraction of sucrose gradient centrifugation were resolved by electrophoresis through a 1.5% agarose gel and transferred to a nitrocellulose membrane by blotting with tne buffer (10 mm tris-hcl, ph 7.5, 150 mm nacl, and 1 mm edta). hbv capsids on the membrane were detected with a procedure described previously [65] . ultracentrifugation analysis of hbv capsid integrity. two hundred microliters of the purified hbv capsids were mixed with 250 μl of reaction buffer containing 0.15 m nacl, 0.1 m tris-hcl (ph 8.0), 20 mm mgcl 2 , 2 mm dithiothreitol, 0.2% (vol/vol) nonidet p-40. capsid assembly modulators were added as indicated and water was provided to bring the reaction volume to 500 μl. after incubation at 37˚c for the indicated period of time, the reaction was loaded onto a 15% to 30% linear sucrose gradient in tne buffer and spun at 27,000 rpm for 4 hr (beckman, rotor sw28). 1.5ml fractions were collected from the bottom of the centrifugation tube by blood collection set. sucrose concentration of each fraction was measured by refractor (mettler toledo). the rest of each fraction was mixed with 3ml tne buffer and pelleted by centrifugation at 46,000 rpm for 3 hr. pellet was dissolved in 40 μl tne buffer. 20μl of capsid solution were fractionated in a 1.5% agarose gel electrophoresis and transferred to a nitrocellulose membrane to detect hbv capsid by probing with anti-hbcag antibody. viral dna was extracted from the remaining sample of reactions and detected by southern blot hybridization with α-32 p-utp-labeled minus strand-specific full-length hbv riboprobe. and cytoplasmic core dna (c) were quantified by real-time pcr assays. hbsag in culture medium was measured by using an elisa assay kit (autobio) (d). (e) the infected cells were fixed at 3 and 8 days post infection (dpi) and hbcag was detected by indirect immunofluorescent staining (red). the cell nuclei were stained with dapi (blue). the images were captured with a nikon x71 microscopy. (f) detection of hbv replication intermediates by hybridization assays. top panel, hirt dna extracted from the cells was denatured at 88˚c for 5 min to denature dp-rcdna into single stranded dna and followed by restriction with ecori to convert cccdna into unit-length dsldna (labeled as ccc/ecori) and detected by southern blot hybridization. unit-length hbv linear dna served as a molecular weight marker. middle panel, hbv pgrna and mrnas specifying envelope proteins (envrnas) were detected by northern blot hybridization. 28s and 18s ribosomal rna (rrna) served as loading controls. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity hepatitis b virus resistance to nucleos(t)ide analogues therapeutic strategies for a functional cure of chronic hepatitis b virus infection core protein: a pleiotropic keystone in the hbv lifecycle molecular biology of hepatitis b virus infection nuclear import of hepatitis b virus capsids and release of the viral genome alteration of mature nucleocapsid and enhancement of covalently closed circular dna formation by hepatitis b virus core mutants defective in complete-virion formation hbv cccdna: viral persistence reservoir and key obstacle for a cure of chronic hepatitis b metabolism and function of hepatitis b virus cccdna: implications for the development of cccdna-targeting antiviral therapeutics structural organization of the hepatitis b virus minichromosome specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna interferon-gamma and tumor necrosis factor-alpha produced by t cells reduce the hbv persistence form, cccdna, without cytolysis the current status and future directions of hepatitis b antiviral drug discovery present and future therapies of hepatitis b: from discovery to cure high-resolution crystal structure of a hepatitis b virus replication inhibitor bound to the viral core protein small-molecule effectors of hepatitis b virus capsid assembly give insight into virus life cycle a heteroaryldihydropyrimidine activates and can misdirect hepatitis b virus capsid assembly hepatitis b virus capsids have diverse structural responses to small-molecule ligands bound to the heteroaryldihydropyrimidine pocket assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis b virus capsid tertiary and quaternary structure heteroaryldihydropyrimidine (hap) and sulfamoylbenzamide (sba) inhibit hepatitis b virus replication by different molecular mechanisms discovery and pre-clinical characterization of third-generation 4-h heteroaryldihydropyrimidine (hap) analogues as hepatitis b virus (hbv) capsid inhibitors a novel inhibitor of dengue virus replication that targets the capsid protein sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus. elife 1: e00049 hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes reversal of fulminant hepatic failure using an extracorporeal liver assist device prevention of hepatitis b virus infection in vivo by entry inhibitors derived from the large envelope protein dna polymerase kappa is a key cellular factor for the formation of covalently closed circular dna of hepatitis b virus ifn-alpha inhibits hbv transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccdna minichromosome alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccdna minichromosomes capsid assembly modulators have a dual mechanism of action in primary human hepatocytes infected with hepatitis b virus in hepatocytes infected with duck hepatitis b virus, the template for viral rna synthesis is amplified by an intracellular pathway formation of hepatitis b virus covalently closed circular dna: removal of genome-linked characterization of the intracellular deproteinized relaxed circular dna of hepatitis b virus: an intermediate of covalently closed circular dna formation hepadnavirus envelope proteins regulate covalently closed circular dna amplification sulfamoylbenzamide derivatives inhibit the assembly of hepatitis b virus nucleocapsids conditional replication of duck hepatitis b virus in hepatoma cells hepatitis b virus covalently closed circular dna formation in immortalized mouse hepatocytes associated with nucleocapsid destabilization secretion of genome-free hepatitis b virus -single strand blocking model for virion morphogenesis of para-retrovirus discovery and mechanistic study of benzamide derivatives that modulate hepatitis b virus capsid assembly preclinical characterization of gls4, an inhibitor of hepatitis b virus core particle assembly weak protein-protein interactions are sufficient to drive assembly of hepatitis b virus capsids capsid proteins of enveloped viruses as antiviral drug targets superinfection with woodchuck hepatitis virus strain whvny of livers chronically infected with strain whv7 superinfection exclusion in duck hepatitis b virus infection is mediated by the large surface antigen formation of the pool of covalently closed circular viral dna in hepadnavirus-infected cells production and function of the cytoplasmic deproteinized relaxed circular dna of hepadnaviruses the innate immune response to hepatitis b virus infection: implications for pathogenesis and therapy maturation-associated destabilization of hepatitis b virus nucleocapsid differential assembly of hepatitis b virus core protein on single-and double-stranded nucleic acid suggest the dsdna-filled core is spring-loaded duck hepatitis b virus nucleocapsids formed by n-terminally extended or c-terminally truncated core proteins disintegrate during viral dna maturation the structural biology of hiv assembly capsid phosphorylation state and hepadnavirus virion secretion hepatitis b virus core protein phosphorylation sites affect capsid stability and transient exposure of the c-terminal domain complete and incomplete hepatitis b virus particles: formation, function, and application mita/sting and its alternative splicing isoform mrp restrict hepatitis b virus replication inhibition of hepatitis b virus replication by activation of the cgas-sting pathway the cyclic gmp-amp synthetase-sting signaling pathway is required for both the innate immune response against hbv and the suppression of hbv assembly viral dna-dependent induction of innate immune response to hepatitis b virus in immortalized mouse hepatocytes lack of immunological dna sensing in hepatocytes facilitates hepatitis b virus infection activation of sting in hepatocytes suppresses the replication of hepatitis b virus hepatitis b virus evades innate immunity of hepatocytes but activates cytokine production by macrophages trex1 regulates lysosomal biogenesis and interferon-independent activation of antiviral genes inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication the amount of hepatocyte turnover that occurred during resolution of transient hepadnavirus infections was lower when virus replication was inhibited with entecavir interferons accelerate decay of replication-competent nucleocapsids of hepatitis b virus interferon induction of ifitm proteins promotes infection by human coronavirus oc43 hepatitis b virus e antigen production is dependent upon covalently closed circular (ccc) dna in hepad38 cell cultures and may serve as a cccdna surrogate in antiviral screening assays assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes effect of alpha interferon on the hepatitis c virus replicon we thank dr. eain a. murphy for critical reading and comments on the manuscript. conceptualization: fang guo, jinhong chang, ju-tao guo. key: cord-321481-vrfwczve authors: watashi, koichi; urban, stephan; li, wenhui; wakita, takaji title: ntcp and beyond: opening the door to unveil hepatitis b virus entry date: 2014-02-19 journal: int j mol sci doi: 10.3390/ijms15022892 sha: doc_id: 321481 cord_uid: vrfwczve chronic hepatitis b virus (hbv) infection, affecting approximately 240 million people worldwide, is a major public health problem that elevates the risk of developing liver cirrhosis and hepatocellular carcinoma. given that current anti-hbv drugs are limited to interferon-based regimens and nucleos(t)ide analogs, the development of new anti-hbv agents is urgently needed. the viral entry process is generally an attractive target implicated in antiviral strategies. using primary cells from humans and tupaia belangeri, as well as heparg cells, important determinants of viral entry have been achieved. recently, sodium taurocholate cotransporting polypeptide (ntcp) was identified as an hbv entry receptor and enabled the establishment of a susceptible cell line that can efficiently support hbv infection. this finding will allow a deeper understanding of the requirements for efficient hbv infection, including the elucidation of the molecular entry mechanism. in addition, pharmacological studies suggest that ntcp is able to serve as a therapeutic target. this article summarizes our current knowledge on the mechanisms of hbv entry and the role of ntcp in this process. hepatitis b virus (hbv) infection constitutes a serious public health problem, affecting approximately 240 million carriers worldwide [1] . chronic hbv infection significantly elevates the risk for developing liver cirrhosis and hepatocellular carcinoma. currently, conventional interferon-α (ifnα) or pegylated-ifnα and nucleos(t)ide analogs are available as anti-hbv agents [2, 3] . however, ifn-based therapies, which cause significant side effects, yields long-term clinical benefits in less than 40% of treated patients [4] . nucleos(t)ide analogs suppress an essential step in virus replication and thereby provide biochemical and histological improvement, but some of the early drugs give rise to drug-resistant viruses, which adversely affect long-term clinical outcome. thus, in order to approach curative treatments, new anti-hbv agents targeting different molecules involved in hbv infection and propagation are needed. nucleos(t)ide analogs suppress hbv replication mainly by inhibiting the reverse transcription process in the viral lifecycle ( figure 1 ) [3] . ifn functions as an immunomodulator and is also reported to directly interfere with hbv replication at multiple steps of the lifecycle [5] . given that hbv encodes only one viral protein carrying enzymatic activity, polymerase, in its genome, strategies for inhibiting viral enzyme are limited. although capsid or envelope protein assembly and the regulatory x-protein are possible future targets, it is critical for developing new classes of anti-hbv agents to identify cellular factors serving as possible drug targets. in general, the viral entry step is an attractive target for the development of antiviral agents [6] [7] [8] . the early hbv lifecycle, including the entry step, has gained significant attention very recently with regard to molecular mechanisms, triggered by the identification of sodium taurocholate cotransporting polypeptide (ntcp) as a cellular entry receptor [9] . this article summarizes the molecular evidence related to hbv entry, mainly focusing on recent findings, and its implications. hbv infection into host hepatocytes follows a multiple step process: (1) initially, hbv reversibly attaches to host cell surface proteoglycans with a low affinity; (2) this is followed by the process involving more specific receptor(s) with high affinity to mediate the early entry step; and (3) after endocytosis-mediated internalization, the virus fuses with the cellular membrane compartment, probably in an endosomal compartment, although the mechanisms are not fully understood. both initial attachment and, probably more importantly, specific receptor recognition contribute to host specificity and tissue tropism [10] . the initial attachment step is at least partly mediated by heparan sulfate proteoglycans [11] [12] [13] [14] . the third internalization step is reported to involve caveolae-, clathrin-or macropinocytosis-dependent endocytosis, depending on the cell types and experimental systems [15] [16] [17] [18] . however, cellular factors involved in the high-affinity binding and the early entry process remained to be elucidated until recently. the hbv surface proteins are composed of three proteins, termed the large (lhbs), middle (mhbs) and small (shbs) surface proteins, and include the pres1, pres2 and s regions: lhbs encompasses the pres1, pres2 and s regions; mhbs encompasses the pres2 and s; and shbs comprise the s region [19, 20] . the molecular requirement of hbv envelope proteins for hbv infection has been studied for more than a decade using primary hepatocytes from humans and tupaia belangeri, as well as heparg cells [21] . a series of analyses using neutralizing antibodies and introduced point mutations suggested that the s and pres1, but not the pres2, regions play a significant role in hbv infection [22] [23] [24] [25] [26] [27] . in a direct approach, the pres1 region in the lhbs has been shown to be essentially involved in the hbv infection process. this was demonstrated by the introduction of mutations in the viral context, infection competition with anti-pres1 antibodies and with peptides mimicking this region [28] [29] [30] [31] [32] [33] [34] . a myristoylated peptide encompassing amino acids 2-48 of the pres1 region turned out to be the most efficient in infection inhibition of hbv and also the envelope protein-related hepatitis d virus (hdv) [30, 31] . such a peptide has been used as a tool for characterizing the early infection step, including the identification of ntcp as an entry receptor [9] and as a lead substance (myrcludex-b) presently in the clinical development (see below) [10, 35, 36] . one of the recent milestones in the field in hbv molecular biology is the identification of ntcp as a host entry receptor, as reported by yan and zhong et al. in late 2012 [9] . by affinity purification and mass spectrometry analysis using an hbv pres1-derived lipopeptide as bait, they identified tupaia belangeri ntcp (tsntcp) as a cellular factor interacting with this lipopeptide. ntcp is a transporter residing in the basolateral membrane of hepatocytes and is involved in the hepatic uptake of mostly conjugated bile salts (see below). the lipopeptide was confirmed to specifically bind human ntcp (hntcp), as well as tsntcp, but surprisingly not crab-eating monkey ntcp (mkntcp), which correlated with the species specificity of hbv infection: hbv is able to efficiently infect humans and tupaia, but not crab-eating monkey [9] . interestingly, this result also correlated with the in vitro binding activity of the peptide to the respective primary hepatocytes [10] and their in vivo hepatotropism [37] . the role of ntcp in the viral infection of hbv, its satellite virus, hdv, and a closely related primate hepadnavirus wooly monkey hbv was further examined by knockdown and overexpression analyses [9, 38, 39] . sirna-mediated knockdown of ntcp in primary human hepatocytes (phh), primary tupaia hepatocytes and differentiated heparg cells reduced hbv and hdv infection, while ectopic expression of ntcp conferred hbv susceptibility in hepg2 cells, which originally did not support efficient infection [9] . this strongly argues that ntcp is an essential factor for hbv infection. the expression of ntcp in different cells was consistent with the hbv susceptibility, as it was significantly expressed in hbv-susceptible cells, phh and differentiated heparg cells, but was weakly expressed or absent in hepg2, huh-7, flc4 and hela cells, which show little to no infection [40] [41] [42] . the introduction of ntcp into huh-7 and undifferentiated heparg cells conferred hbv infection to these cells to some extent [38] . although the total expressions in these transduced cells were comparable, hntcp-expressing hepg2 cells showed much higher infection efficiency when compared with other human hepatocyte cell lines [38, 43, 44] . in the initial study, infection efficiency was ~10% in ntcp-overexpressing hepg2 cells cultured with medium containing 2% dimethyl sulfoxide (dmso) [9] . subsequent analysis showed that increasing the dmso concentration to more than 2.5%~3% augmented infection efficiency to 50%~70%, as evaluated by immunofluorescence of hbv proteins, although the virus inoculum was different in these studies [38, 43] . the speculations include that dmso augmented the gene expression of ntcp, promoted the membrane localization of ntcp and changed the post-translational modification of ntcp, but the detailed molecular mechanisms for dmso-mediated promotion of hbv infection is open for further studies. it remains unknown why not all of the cells were infected with hbv in these reports, but it is possible that the ntcp function for supporting hbv entry is reflected by post-translational modification, subcellular localization or other factors that are governed by cell conditions or by more general conditions, such as the cell cycle, cellular microenvironment or architecture. another open question is on the high susceptibility for hdv, but not hbv, in huh-7 cells overexpressing hntcp [9, 38] . future analysis of this issue is necessary in order to establish a cell culture model that is 100% susceptible to hbv infection. crucial amino acid sequences in ntcp involved in hbv infection have been analyzed. by sequence comparison between hntcp and mkntcp, replacement of amino acids 157-165 of hntcp with the respective sequence from mkntcp abrogated the ability to support hbv pres1-binding and, subsequently, infection, while mkntcp carrying a conversion to this region from hntcp conferred hbv susceptibility. thus, amino acids 157-165 of ntcp are crucial for ntcp-mediated hbv binding and infection [9, 45] . it has also been shown that hntcp bearing a substitution of the 84-87 aa from the mouse counterpart was able to bind pres1, but was not functional for hbv infection, while replacing these residues in mouse ntcp (mntcp) with the human counterparts supported the infection [38, 44] . these data indicate that the 84-87 aa residues are a determinant for ntcp function as an hbv entry receptor. it remains to be elucidated why mntcp does not support hbv infection, but mntcp was shown to support specific binding of the pres1-lipopeptide on the cell surface, although the binding capacity of mntcp to the pres1 region appears to be weaker than that of hntcp [44] . it is possible that the binding of hbv to ntcp is not sufficient and requires an additional molecule or mechanism to trigger the following early infection process. hdv is a virusoid-like particle, which depends on hbv for assembly and propagation [46] . hdv shares the hbv envelope proteins, lhbs, mhbs and shbs, and its attachment/early entry mechanism seems to be very similar to that for hbv. due to its completely different replication strategy, it is very likely that it depends on different cellular factors and follows different pathways after membrane fusion. intriguingly, hdv infection can be observed by complementing hntcp in either mouse-derived hepa1-6, mmhd3 and hep56.1d cells, rat hepatocyte tc5123 cells or non-hepatocyte hela, cho and vero cells. this is in stark contrast to hbv, which cannot infect these cells [38, 44] . this suggests that hbv requires additional host factors for infection or is restricted at a post-entry step prior to covalently closed circular dna (cccdna) formation. it is of particular interest to clarify the molecular mechanisms underlying the different cellular requirements between infection by hbv and hdv, especially when trying to establish a susceptible mouse model in the future. it is presently unclear whether there are additional cellular factors besides ntcp required for viral infection and determining the tissue and species tropism of hbv. these include factors essentially involved in the viral lifecycle during attachment, internalization, endocytosis, membrane fusion, uncoating, nuclear translocation and cccdna formation and those affecting post-entry restriction. overexpression of hntcp in mouse hepatocyte cell lines, such as hepa1-6 and mmhd3 cells, did not confer susceptibility to hbv infection, in contrast to the hbv infection observed after ntcp introduction into hepg2 cells [44] . hntcp conferred efficient hbv infection in hepg2 cells, but only a low efficiency of infection was observed in huh-7 and undifferentiated heparg cells and no detectable infection to mouse and rat hepatoma cells, including hep56.1d, hepa1-6 and tc5123 cells [38] . we also showed that different hepg2 clone isolates that similarly expressed high levels of ectopic ntcp, but were likely to have different cellular genetic backgrounds, had diverse efficiencies of hbv infection [43] . these observations favor the existence of additional host factors determining susceptibility to hbv infection. for hepatitis c virus (hcv) infection, multiple cellular factors are required for efficient viral entry, including low density lipoprotein receptor (ldlr), scavenger receptor class b type i (sr-bi), cd81, occludin (ocln) and claudin-1 (cldn-1) as viral entry receptors and niemann-pick c1-like 1 (npc1l1), epidermal growth factor receptor (egfr) and ephrin a2 (epha2) as other factors involved in entry [21, 47, 48] . it has been reported that the complementation of both hcd81 and hocln are required for rendering high hcv susceptibility in mice [49] . furthermore, in the case of duck hepatitis b virus (dhbv), multiple factors are suggested to be essential for efficient viral infection. carboxypeptidase d was confirmed to bind the dhbv envelope and function in viral attachment and entry [50] . however, overexpression of this protein alone in huh-7 cells did not support dhbv infection [51] . carboxypeptidase d was able to bind to dhbv and heron hbv, which did not infect primary duck hepatocytes, and this protein is also expressed in non-liver tissues [52] . thus, additional factors are likely to be required to explain dhbv susceptibility, one candidate of which can include duck ntcp [53] . these examples in viruses that utilize multiple receptors favor pursuing the identification of additional cellular factors crucial for hbv entry. ntcp, also designated as solute carrier family 10a1 (slc10a1), is a member of the slc10 transporter gene family. the slc10 family consists of seven members (slc10a1-7). among these, ntcp and apical sodium-dependent bile salt transporter (asbt), also known as slc10a2, are sodium-dependent transporters for bile acids [54] . ntcp is mainly distributed at the basolateral membrane of hepatocytes and plays a major role in the hepatic influx of conjugated bile salts from portal circulation [55, 56] . ntcp on the plasma membranes in hepatocytes binds two sodium ions together with one molecule of preferentially conjugated bile salt for uptake. in addition to bile salts, ntcp, like other transporters, binds and/or transports other molecules, including steroid hormones, thyroid hormones, drug-conjugated bile salt and a variety of xenobiotics [57, 58] . hntcp is a 349 aa protein with an apparent mass of 56 kda and includes a putative seven or nine transmembrane domains with a predicted topology of n-terminal extracellular and c-terminal intracellular ends [59] [60] [61] . while the structure of ntcp has not been resolved, the crystal structures of the asbts from neisseria meningitis (asbt nm ) and yersinia frederiksenii (asbt yf ) were recently reported [62, 63] . asbt nm shows a ten transmembrane domain and a hydrophobic inward-facing binding cavity. this structure is different from the model for hasbt currently favored based on bioinformatic prediction and experimental data, which carry seven to nine transmembrane helices with n-terminal extracellular and the c-terminal cytoplasmic domain [60, 64, 65] . a structural analysis of asbt yf proposed two conformations, inward-and outward-open structures of bile salt transporters by rotating two core helices transmembrane (tm)-4 and tm9 [63] . because asbt nm and asbt yf has only 26% and 22% homology, respectively, with hasbt and even lower homology with hntcp [62, 63] , it is uncertain whether the structural features of asbt nm or asbt yf are useful for designing drugs targeting hasbt and hntcp. several single nucleotide polymorphisms (snps) that alter the transporter activity of ntcp have been reported [66, 67] . as non-synonymous snps, i223t, a variant seen in 5.5% of allele frequencies in african americans, decreased plasma membrane-localized ntcp and reduced its transporter activity. the s267f variant, seen in 7.5% of allele frequencies in chinese americans, exhibited almost complete loss of function for bile acid uptake, but possessed normal transport activity for the non-bile acid substrate, estrone sulfate. another report showed that the a64t and s267f variants, carried by 1.0% and 3.1% of allele frequencies in koreans, respectively, decreased the uptake of taurocholic acid. these polymorphisms are dependent on ethnicity. however, there have been no reports of serious diseases associated with defects in the ntcp gene. no reports describing ntcp knockout mice have been published to date. thus, it is difficult to draw conclusions on whether the physiological roles of ntcp are complemented by other factors that share the redundant physiological function and whether ntcp inhibition is able to safely serve as an anti-hbv drug target. importantly, it was very recently reported that molecular determinants for the transporter function of ntcp overlapped with those for the ability to support hbv entry [68] . ntcp mutations in amino acids that were critical for bile salt binding (n262a, q293a/l294a) abrogated both the binding to pres1 peptide and the infection of hbv. the s267f variant of ntcp could neither bind to the pres1 region nor support hbv infection in cell culture. in general, the viral entry process is an attractive target for the development of antiviral agents. as noted above, the 2-48 aa region of pres1 in the lhbs protein is important for hbv infection [31] . myrcludex-b, which is an optimized synthetic lipopeptide consisting of the myristoylated 2-48 aa region of pres1, is able to strongly inhibit hbv infection in both cell culture and an in vivo mouse model [36] . the ic 50 in a cell culture model was reported to be approximately 100 pm [35] . following the successful clinical development of enfuvirtide as the first peptidic hiv entry inhibitor mimicking the region derived from the viral gp41 envelope glycoprotein [69] , myrcludex-b is now under clinical development in phase ib/iia [70] . mechanistically, myrcludex-b binds hntcp and inactivates its receptor function for hbv and hdv ( figure 1 ). remarkably, ic 50 to the transporter activity of ntcp was approximately 4 nm [38] , showing that binding saturation is not required for receptor inactivation, thus allowing a therapeutic window for infection inhibition without a complete abrogation of bile salt transportation [38] . thus, agents targeting ntcp are expected to be potent candidates that act as anti-hbv drugs. cyclosporin a (csa) is the first line of such compounds revealed to inhibit hbv infection by targeting ntcp [42, 45] . csa is known to be an immunosuppressant classified as a calcineurin inhibitor and is clinically used for the suppression of the immunological failure of xenografts after tissue transplantation [71] . in cell culture analyses, csa was also reported to suppress the replication of numerous viruses, including hiv, hcv, influenza virus, severe acute respiratory syndrome coronavirus, human papillomavirus, flaviviruses and hbv [72] [73] [74] [75] [76] [77] [78] [79] . in most of these cases, cyclophilins (cyps), cellular peptidyl prolyl cis-trans isomerases that catalyze conformational changes in proteins and are the primary cellular target for csa, were critical for efficient viral replication, and cyp inhibition by csa was responsible for antiviral activity. however, the anti-hbv entry activity of csa was not mediated by the inhibition of cyp, but rather, via direct targeting of ntcp. csa bound to ntcp on the plasma membrane and inhibited transporter activity ( figure 1 ) [42, 45] . it also inhibited binding between lhbs and ntcp in vitro (figure 1 ) [42] . this suggests that csa interacted with ntcp, thus inhibiting the recruitment of lhbs of incoming hbv to ntcp on the plasma membrane and blocking hbv entry. the anti-hbv activity of csa was pan-genotypic [42] . moreover, our derivative analysis identified a series of csa analogs having a stronger anti-hbv entry activity with a submicromolar ic 50 [42] . notably, non-immunosuppressive csa analogs may be potent anti-hbv agents. given that non-immunosuppressive csa analogs, including alisporivir (debio 025) and scy-635, have significant activity in decreasing hcv viral load in clinical trials and are regarded as promising anti-hcv drug candidates [80, 81] , further derivative analysis of csa may be a reasonable approach for drug development. as other examples, compounds known to be ntcp inhibitors, including progesterone, propranolol and bosentan, have been shown to block hbv infection (figure 1 ) [42] . ntcp substrates, such as taurocholate, tauroursodeoxycholate and bromosulfophthalein, also inhibited hbv infection [38, 42, 68 ]. an anticholesteremic drug, ezetimibe, has been shown to block hbv entry [82] , and this drug was reported to inhibit the ntcp transporter [83] . these results indicate that compounds modulating ntcp function could substantially inhibit hbv infection. hepg2 cells engineered to overexpress ntcp are also useful for high-throughput screening to identify compounds targeting ntcp and inhibiting hbv infection. one example identified in such chemical screening is the oxysterols, which are oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis [43] . host-targeting antivirals are generally expected to have significant advantages, including a much lower frequency drug resistance, universal antiviral effects beyond viral genotypes and complementary mechanisms of action that might act in a synergistic manner with currently available antiviral agents [48] . more importantly, they offer an additional therapeutic choice, given that only ifns and nucleoside analogs are currently available as anti-hbv agents. identification of ntcp as an hbv entry receptor has accelerated the understanding of hbv molecular biology and offered useful experimental systems to analyze the hbv and hdv lifecycle, including the identification of host restriction and dependency factors. ntcp also represents a new therapeutic target in the development of new anti-hbv agents. further analyses using a new cell culture system are necessary in order to clarify the molecular mechanisms underlying ntcp-mediated hbv infection and to establish an in vivo small animal model that fully supports hbv infection. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity optimal management of chronic hepatitis b patients with treatment failure and antiviral drug resistance hepatitis b: reflections on the current approach to antiviral therapy hepatitis b surface antigen levels: association with 5-year response to peginterferon alfa-2a in hepatitis b e-antigen-negative patients ifn-alpha inhibits hbv transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccdna minichromosome entry inhibitors and their use in the treatment of hiv-1 infection gulping rather than sipping: macropinocytosis as a way of virus entry hepatitis c virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus myristoylated pres1-domain of the hepatitis b virus l-protein mediates specific binding to differentiated hepatocytes viral and cellular determinants involved in hepadnaviral entry proteoglycans act as cellular hepatitis delta virus attachment receptors role of glycosaminoglycans for binding and infection of hepatitis b virus hepatitis b virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans clathrin-mediated endocytosis and lysosomal cleavage of hepatitis b virus capsid-like core particles hepatitis b virus may enter hepg2 cells complemented with human ntcp via macropinocytosis entry of hepatitis b virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis hepatitis b virus requires intact caveolin-1 function for productive infection in heparg cells structural relationships between minor and major proteins of hepatitis b surface antigen large surface proteins of hepatitis b virus containing the pre-s sequence entry of hepatitis b and c viruses-recent progress and future impact entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis b virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange n-terminal myristoylation-dependent masking of neutralizing epitopes in the pres1 attachment site of hepatitis b virus neutralization of hepatitis b virus infectivity by a murine monoclonal antibody: an experimental study in the chimpanzee the pre-s2 domain of the hepatitis b virus is dispensable for infectivity but serves a spacer function for l-protein-connected virus assembly a function essential to viral entry underlies the hepatitis b virus "a" determinant structural characterization of viral neutralizing monoclonal antibodies to hepatitis b surface antigen mapping of the hepatitis b virus pre-s1 domain involved in receptor recognition characterization of a hepatitis b and hepatitis delta virus receptor binding site mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres1 lipopeptides and tupaia hepatocytes efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein in vivo neutralization of hepatitis b virus infection by an anti-pres1 humanized antibody in chimpanzees infection process of the hepatitis b virus depends on the presence of a defined sequence in the pre-s1 domain fine mapping of virus-neutralizing epitopes on hepatitis b virus pres1 fine mapping of pre-s sequence requirements for hepatitis b virus large envelope protein-mediated receptor interaction prevention of hepatitis b virus infection in vivo by entry inhibitors derived from the large envelope protein hepatitis b virus hepatotropism is mediated by specific receptor recognition in the liver and not restricted to susceptible hosts hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis b virus infection of tupaia hepatocytes expression and transport function of drug uptake transporters in differentiated heparg cells molecular and functional characterization of bile acid transport in human hepatoblastoma hepg2 cells cyclosporin a and its analogs inhibit hepatitis b virus entry into cultured hepatocytes through targeting a membrane transporter ntcp evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells overexpressing a membrane transporter ntcp molecular determinants of hepatitis b and d virus entry restriction in mouse sodium taurocholate cotransporting polypeptide cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilin-independent interference with the ntcp receptor hepatitis delta virus the ins and outs of hepatitis c virus entry and assembly host-targeting agents for prevention and treatment of chronic hepatitis c-perspectives and challenges a genetically humanized mouse model for hepatitis c virus infection ganem, d. gp180, a host cell glycoprotein that binds duck hepatitis b virus particles, is encoded by a member of the carboxypeptidase gene family carboxypeptidase d (gp180), a golgi-resident protein, functions in the attachment and entry of avian hepatitis b viruses avian hepatitis b virus infection is initiated by the interaction of a distinct pre-s subdomain with the cellular receptor gp180 virus entry mediated by hepatitis b virus envelope proteins the solute carrier family 10 (slc10): beyond bile acid transport the human na + -taurocholate cotransporting polypeptide gene is activated by glucocorticoid receptor and peroxisome proliferator-activated receptor-gamma coactivator-1alpha, and suppressed by bile acids via a small heterodimer partner-dependent mechanism characterization of cloned rat liver na(+)-bile acid cotransporter using peptide and fusion protein antibodies the slc10 carrier family: transport functions and molecular structure bile acid transporters in health and disease molecular cloning, chromosomal localization, and functional characterization of a human liver na + /bile acid cotransporter organization of the membrane domain of the human liver sodium/bile acid cotransporter topography of the membrane domain of the liver na + -dependent bile acid transporter crystal structure of a bacterial homologue of the bile acid sodium symporter asbt structural basis of the alternating-access mechanism in a bile acid transporter membrane insertion scanning of the human ileal sodium/bile acid co-transporter topology scanning and putative three-dimensional structure of the extracellular binding domains of the apical sodium-dependent bile acid transporter (slc10a2) ethnicity-dependent polymorphism in na + -taurocholate cotransporting polypeptide (slc10a1) reveals a domain critical for bile acid substrate recognition genetic polymorphisms in na + -taurocholate co-transporting polypeptide (ntcp) and ileal apical sodium-dependent bile acid transporter (asbt) and ethnic comparisons of functional variants of ntcp among asian populations viral entry of hepatitis b and d viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide potent suppression of hiv-1 replication in humans by t-20, a peptide inhibitor of gp41-mediated virus entry the new front-line in hepatitis b/d research: identification and blocking of a functional receptor cyclophilin and viruses: cyclophilin as a cofactor for viral infection and possible anti-viral target cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes target cell cyclophilins facilitate human papillomavirus type 16 infection cyclosporin a inhibits the influenza virus replication through cyclophilin a-dependent and -independent pathways human immunodeficiency virus type 1 gag protein binds to cyclophilins a and b the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral ns5 protein cyclophilin a modulates the sensitivity of hiv-1 to host restriction factors activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the hbx protein involved in hepatitis b virus replication alisporivir, a cyclosporin derivative that selectively inhibits cyclophilin, for the treatment of hcv infection cyclophilin inhibitors for hepatitis c therapy ezetimibe blocks hepatitis b virus infection after virus uptake into hepatocytes structure-activity relationship for fda approved drugs as inhibitors of the human sodium taurocholate cotransporting polypeptide (ntcp) the authors are grateful to all of the members of the department of virology ii, national institute of infectious diseases, the department of infectious diseases, molecular virology, university hospital heidelberg, and the li lab at the national institute of biological sciences, beijing, for their research, technical support and discussions. funding was provided by the ministry of health, labor and welfare, japan, the ministry of education, culture, sports, science and technology, japan, the japan society for the promotion of science, the deutsche zentrum für infektionsforschung (dzif), the deutsche krebshilfe, the deutsche forschungsgemeinschaft (dfg) ur72/7-1, for1202/ur72/5-1, the ministry of science and technology, china (2010cb530101), and the national science and technology major project, china (2013zx09509102). all of the authors wrote the paper. the authors declare no conflict of interest. key: cord-017948-fqhl1qb4 authors: hu, yuan title: molecular techniques for blood and blood product screening date: 2012-04-05 journal: advanced techniques in diagnostic microbiology doi: 10.1007/978-1-4614-3970-7_28 sha: doc_id: 17948 cord_uid: fqhl1qb4 the food and drug administration (fda) is responsible for ensuring the safety of the more than 15 million units of blood and blood components donated each year in the united states. “blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries,” said david a. kessler, md, former fda commissioner [1]. screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. the united states has the safest blood supply in the world [1] and the fda is striving to keep it safe by decreasing the risk of infectious disease transmission. the regulatory agency is continuously updating its requirements and standards for collecting and processing blood. as mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases. in the united states, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (table 28.1). the field of clinical microbiology and virology are now focusing on molecular technology. currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. it is time for all blood safety procedures to include molecular detection techniques. this approach can significantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. this chapter reviews the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety. the food and drug administration (fda) is responsible for ensuring the safety of the more than 15 million units of blood and blood components donated each year in the united states. "blood banking has become a manufacturing industry, an industry that must conform to high standards and quality control requirements comparable to those of pharmaceutical companies or other regulated industries," said david a. kessler, md, former fda commissioner [ 1 ] . screening donated blood for infectious diseases that can be transmitted through blood transfusion is a very important step in ensuring safety. the united states has the safest blood supply in the world [ 1 ] and the fda is striving to keep it safe by decreasing the risk of infectious disease transmission. the regulatory agency is continuously updating its requirements and standards for collecting and processing blood. as mentioned earlier, an important step in ensuring safety is the screening of donated blood for infectious diseases. in the united states, tests for infectious diseases are routinely conducted on each unit of donated blood, and these tests are designed to comply with regulatory requirements (table 28 .1 ). the fi eld of clinical microbiology and virology are now focusing on molecular technology. currently, nucleic acid testing techniques have been developed to screen blood and plasma products for evidence of very recent viral infections that could be missed by conventional serologic tests. it is time for all blood safety procedures to include molecular detection techniques. y. hu (*) u.s. food and drug administration, northeast regional laboratory , 158-15 liberty avenue , jamaica , ny 11433 , usa e-mail: yuan.hu@fda.hhs.gov no of fi cial support or endorsement of this article by the food and drug administration is intended or should be inferred. this approach can signi fi cantly aid in blood safety to reduce the risk of transmission of serious disease by transfusion. this chapter reviews the current antigen/antibody-based technology, molecular biological technology, and published regulatory policy data for blood safety. direct detection of viral antigens and virus speci fi c antibodies has been a common tool for the diagnosis of virus infections in the past 40 years. there are some limitations. for direct detection of virus antigens, shortly after virus infection, only a few viruses release antigens in amounts suf fi ciently detectable in the body by an antibodymediated assay. for indirect virus detection by virus speci fi c antibodies (e.g., an immuno fl uorescence assay or enzyme immunoassay (eia), etc.), there is a problem in that shortly after infection by a pathogenic virus, there is a window period in which antibody generation is insuf fi cient for detection [ 2 ] . to reduce this window period of low detection, direct nucleic acid tests are needed. through the application of molecular biology, biological and biochemical analyses have been revolutionized, and nucleic acid, gene-based techniques have been developed to screen blood and plasma donations for evidence of very recent and earlier viral infections that might otherwise be missed by conventional serologic testing. the nucleic acid tests can also provide evidence for genetic variation in viruses. molecular methods include the use of nucleic acid probes as well as ampli fi cation-based and dna sequence-based techniques. an increasing number of molecular diagnostic methods are now available commercially. in comparison to classical methods, molecular biological methods are superior in terms of rapidness, speci fi city, and sensitivity. the current nucleic acid detection methods in the fi eld may be grouped into two major classes: amplifying techniques such as pcr and nonamplifying techniques such as southern blot hybridization. amplifying techniques are more sensitive than nonamplifying techniques. there are two different types of amplifying methods [ 3 ] , target ampli fi cation methods and signal ampli fi cation methods. target amplifying techniques include pcr, nucleic acid sequence-based ampli fi cation (nasba) [ 4, 5 ] , self-sustaining sequence ampli fi cation (3sr), transcription-based ampli fi cation (tas), transcription-mediated ampli fi cation (tma), strand displacement ampli fi cation (sda), and ligase chain reaction (lcr). signal ampli fi cation methods include branched dna (bdna) signal ampli fi cation [ 6 ] , cleavage-based signal ampli fi cation (cycling probe technologies and invader assay), qß replicase, hybrid capture, cycling probe technologies (cpt), and rolling-circle ampli fi cation (rca) [ 7 ] . to further insure the safety of blood products, it is of importance to further improve these and other types of nucleic acid testing. southern blotting [ 8 ] was named after edward m. southern who developed this procedure at edinburgh university in the 1970s. this technique is used to detect speci fi c sequences within mixtures of dna, which is size-fractionated by gel electrophoresis and then transferred by capillary action to a suitable membrane. after blocking of nonspeci fi c binding sites, the nitrocellulose replica of the original gel electrophoresis experiment is then allowed to hybridize with an oligonucleotide probe representing the speci fi c dna sequence of interest. should speci fi c dna be present on the blot, it will combine with the labeled probe and be detectable. by coelectrophoresing dna fragments of known molecular weight, the size(s) of the hybridizing band(s) can then be determined. southern blotting hybridization technology is one of the major tools that have already helped clinical staffs worldwide interpret genomic information. other competing methodologies include in situ hybridization and solution hybridization. important clinical examples of the use of this technology are dna fi ngerprinting and the ability to detect dna gene rearrangements. in 1983, dr. kary mullis at cetus corporation conceived of polymerase chain reaction [ 9 ] . there is not a single technique that has had a greater impact on the practice of molecular biology than pcr. with this technique, we can detect infectious diseases agents at an extremely low level. it is based on the ability of sense and antisense dna primers to hybridize to a dna of interest. following extension from the primers on the dna template by dna polymerase, the reaction is heat-denatured and allowed to anneal with the primers once again. another round of extension leads to a multiplicative increase in dna products. therefore, a minute amount of dna can be ef fi ciently ampli fi ed in an exponential fashion to result in larger amounts of dna that are more easily manipulated. by including critical controls, the technique can be made quantitative. the current level of the sensitivity and detection limit is as low as 10-50 copies per ml blood in hiv testing [ 1, 10, 11 ] . important clinical examples of the use of pcr are detection of hiv and hcv [12] [13] [14] . pcr techniques have evolved into different branches. some of them are now widely in use for virus detection in clinical diagnostics. these are real-time pcr by taqman (roche), light cycler (roche) and smart cycler (cepheid), and in situ pcr, nested-pcr, nested-real time pcr [ 15 ] , broad-range pcr, multiplex pcr, rt-pcr, arbitrarily primer pcr, long pcr, and quantitative pcr. real-time sequence technology will be coming soon for more detailed detection. in the past, identi fi cation of viral serotypes was restricted to investigative methods using antibody detection and restriction fragment length polymorphism (rflp). with real-time sequences technology, we will be able to detect a virus early as well as to obtain the viral sequence. microarrays were developed at stanford university by schena and coworkers in the early 1990s [ 16 ] . for medical applications, a microarray analysis offers a very accurate screening technology. it allows hundreds or thousands of nucleic acid hybridization reaction to be performed on a solid substrate. it promises to be a fast and accurate diagnostic tool in the fi eld of clinical microbiology and virology. applied to infection safety for blood and blood products, it will be able to screen for the presence of viral pathogens by matching genetic sequences. compared with existing technologies, it allows for a wider variety of speci fi c tests to be carried out simultaneously to determine the quality of the blood and will provide consumers with extra safety. with the use of molecular biology protocols, the microarray will permit the detection of lower concentrations of microorganisms in the blood and the accurate identi fi cation of many types of pathogenic contaminants. in the near future, progress can be expected in the application of microarray technology for screening of donated blood for infectious agents. it can provide vast information about the identity of bloodborne pathogens as well as their gene expression pro fi les [ 17 ] . to ensure a safe blood supply for those who may need a transfusion, an important step in ensuring safety is the screening of donated blood for infectious agents. after donation, each unit of donated blood undergoes a series of tests for bloodborne agents such as human immunode fi ciency virus (hiv)-1, hiv-2, hepatitis b virus (hbv), hepatitis c virus (hcv), human t-cell lymphotropic virus (htlv)-1 and htlv-ii, west nile virus (wnv), and treponema pallidum , the agent of syphilis. all of the above tests are referred to as screening tests, and are designed to detect as many infectious agents as possible. because these tests are so sensitive, some donors may have a false-positive result, even when the donor has never been exposed to the particular infection. in order to sort out true infections from such false-positive test results, screening tests that are reactive may be followed up with more speci fi c tests called con fi rmatory tests. thus, con fi rmatory tests help determine whether a donor is truly infected. if any one of these tests fails, affected blood products are considered unsuitable for transfusion [ 18 ] . nucleic acid testing (nat) employs testing technology that directly detects the genomes of viruses. because nat detects a virus's genetic material instead of waiting for the body's response, the formation of antibodies, as with many current tests, it offers the opportunity to reduce the window period during which an infecting agent is undetectable by traditional tests [ 19 ] , thus further improving blood safety. nat will become the gold standard because of greater sensitivity compared to antibody tests. since 1999, nat has been approved by the fda and used to detect hiv-1 and hcv; this technology currently is under investigation for detecting other infectious disease agents. we know that for many viral infections, viral rna appears very early in the infection, in 1-2 weeks, but the antibody does not appear until 10-12 weeks, e.g., hiv and hcv [ 20 ] . in order to virtually prevent infection by all the transfusion associated viruses, we need to detect the viruses in their window period, and a nat or gene-based testing method is needed. nat also provides an opportunity for the viral, e.g., hiv or hcv, infected donor to seek early treatment. on the other hand, nat is not only a sensitive method, but also a rapid method which is suitable for a blood bank laboratory because the turnaround time for maintaining blood donations is extremely critical. the hbv is a highly infectious and often nonsymptomatic virus that is transmitted primarily through blood and blood-derived fl uids and is a leading cause of liver infection worldwide. the world health organization (who) estimates that two billion people worldwide have been infected with hbv and 350,000,000 people are chronically infected. chronic infection results in a high risk for liver cancer and cirrhosis of the liver, which cause about 1,000,000 deaths each year. each year up to 200,000 people become newly infected in the united states alone. since the beginning of screening for hbv in 1969, the rate of infection through blood transfusions has greatly decreased. however, as of 2000, hbv is still transmitted through blood transfusions in 1 out of 137,000 units of blood. one reason for this is that currently available blood screening technologies detect core antibodies or surface antigens, which appear up to 8 weeks after infection. serologic tests for hbv include hepatitis b surface antigen (hbsag) and hepatitis b core antibody (hbcab). hbv, which mainly infects the liver, has an inner core and an outer envelope (the surface). the hbsag test detects the outer envelope, identifying an individual infected with the hbv. this virus can cause in fl ammation of the liver, and in the earliest stage of the disease, infected people may feel ill or even have yellow discoloration of the skin or eyes, a condition known as jaundice. fortunately, most patients recover completely and test negative for hbsag within a few months after the illness. a small percentage of people become chronic carriers of the virus, and in these cases, the test may remain positive for years. chronically infected people can develop severe liver disease as time passes, and need to be followed carefully by an experienced physician. to reduce the occurrence of posttransfusion hepatitis, it is essential to screen all blood donations for hbsag by the most sensitive and speci fi c assays. blood donations that are found to be reactive in the hbsag test are automatically con fi rmed by the hbsag con fi rmatory assay. if the specimen is neutralizable in the con fi rmatory test, the specimen is considered positive for hbsag. hbsag testing of donated blood has begun in 1975 (table 28.1 ) . currently, all blood donors are screened for hbsag, but occasional transmission of hbv still occurs due to the inclusion of window period donations (i.e., blood from recently infected donors who are antibody negative but still viremic). detection of early hbv infection of blood donors is still a major problem of blood transfusion. the current third-generation licensed hbsag tests (mostly radioimmunoassay and enzyme immunoassays) can not detect hbv in the window period for hbv infection. this is a strong motivation for introducing molecular detection techniques to the fi eld. there are some commercially available test methods for detecting hbv dna in the market now, such as chiron's quantiplex hbv dna [ 21 ] , digene's hybrid capture, abbott's hbv dna assay, and roche's amplicor hbv monitor. using these commercial hybridization or pcr-based assays, hbv dna can be detected 1-3 weeks before the appearance of hbsag [ 22 ] . some chronically infected patients who have lost their hbsag remain hbv dna positive, but are disquali fi ed as potential blood donors. molecular detection of hbv dna is more sensitive than current methods employed for hbsag screening. determination of antibodies to the hepatitis b core antigen (anti-hbc) (total) is also used to monitor the progress of the hepatitis b viral infection. determination of anti-hbc (igm) is employed to distinguish an acute hepatitis b infection from a chronic infection. the anti-hbc test developed in 1987 detects an antibody to the hbv that is produced during and after infection. if an individual has a positive anti-hbc test, but the hbsag test is negative, it may mean that the person once had hepatitis b, but has recovered from the infection. of the individuals with a positive test for anti-hbc, many have not been exposed to the hbv; thus, there is a frequent problem of false positives. although the individual may be permanently deferred from donating blood, it is unlikely that the person's health will be negatively affected. (note: this antibody is not produced following vaccination against hepatitis b). the hcv is a member of the flaviviridae family of viruses, which are associated with both human and animal diseases. hepatitis caused by hcv is the most common chronic bloodborne infection in the united states. over four million americans are believed to be infected. hcv can also be transmitted through blood transfusion. hcv causes in fl ammation of the liver, and up to 80% of those exposed to the virus develop a chronic infection, which can lead to liver in fl ammation, cirrhosis, cancer, and death. eventually, up to 20% of people with hcv may develop cirrhosis of the liver or other severe liver diseases. as in other forms of hepatitis, individuals may be infected with the virus, but may not realize they are carriers since they do not have any symptoms. because of the risk of serious illness, people with hcv need to be followed closely by a physician with experience evaluating this infection. since the fi rst cloning of fulllength hcv cdna in 1989, signi fi cant progress has been made in characterizing its molecular biology [ 11 ] . but, the natural history of hcv infection is still largely unclear and current treatment options for patients are limited. there is no vaccine for hcv, and the only available treatment, a combination of alpha interferon and ribavirin, is ef fi cacious in only a minority of patients [ 23 ] . the life cycle of the hcv is poorly understood due to the lack of an ef fi cient cell culture system [ 24 ] . there is an urgent need to develop a highly sensitive detection method for studying possible extrahepatic sites for the replication of hcv. we have recently established a cell culture system for the replication of hcv by using human t and b leukemia cell lines [ 25 ] . this model should represent a valuable tool for the detailed study of the initial steps of the hcv replication cycle and for the evaluation of antiviral molecules. currently, appropriate vaccine strategies for hcv have not been developed. early detection and prevention of hcv infection are most important for blood safety. it is a formidable task to design primers and probes for sensitive nucleic acid level diagnostic assays throughout the open reading frame of the hcv genome because of a high mutation rate in this genomic region. however, the untranslated region of about 341 nucleotides contains highly conserved domains which allows for stable primer design for qualitative and quantitative diagnostic tests which have equivalent sensitivity against the known six various genotypes of hcv. in 1990, the fi rst speci fi c test for hcv, the major cause of "non-a, non-b" hepatitis was introduced. now, a third generation elisa kit is available to detect antibodies to hcv and screening blood for hcv antibodies is recommended. these assays are based on detection of serum antibody to various hcv antigens because these antibodies are nearly universally present in patients who are chronically infected with hcv [ 26 ] . the hcv screening tests are known to have signi fi cant limitations and positive samples should be further tested by hcv con fi rmatory tests. guidelines provided by the cdc recommend that hcv antibody screening test positive samples should be con fi rmed with serologic or nucleic acid supplemental testing. hcv con fi rmatory tests include the recombinant immunoblot assay in which several recombinant peptide antigens are applied on a strip that is then probed with the patient's serum. in this way, the response to individual antigens can be recognized, and some false-positive elisa results can be eliminated (e.g., riba, chiron hcv 3.0, and pcr assay) (e.g., roche cobas amplicor hcv test, version 2.0). laboratories can choose to perform this testing on all positive specimens or based on screening test positive (signal to cutoff) ratios. the positive predictive values (s/co) can vary depending on the prevalence of infection in the population being screened. hcv antibodies are not generally detectable for at least 6 weeks and may not appear for several months. acute hcv infections are relatively rare among blood donors, but the antibody tests often fail to detect these patients in the window period between the time of infection and the time of appearance of antibody detectable by the above assays. high sensitivity detection of hcv during the window period is a longterm technical challenge in the fi eld. tests for hcv rna genome detection based on the pcr or other highly sensitive rna detection systems have been used for the diagnosis of acute hepatitis [ 26 ] . sensitive detection of hcv rna based on rt-pcr or other nucleic acid ampli fi cation techniques can be readily accomplished with kits that are now available commercially. for example, in 1999 the fda approved roche's amplicor hiv-1 monitor ultra sensitive quantitative assay. it can measure hiv levels at as few as 50 copies/ml and another commercial kit, the lcx hiv rna quantitative assay from abbott laboratories, also has a detection limit at 50 copies/ml. some studies even showed a sensitivity limit at 1 copy [ 27 ] . in fact, a qualitative assay should be much more sensitive than a quantitative assay for hiv/hcv screening. a sensitive qualitative hcv molecular detection assay will possibly interdict and virtually prevent all transfusion-associated hiv/hcv. the current sensitivity standard for clinical diagnostics is 100 copies/ml, but since there has been an improvement in technology, this would be the time to change sensitivity standard to 50 copies/ml. hiv-1 and/or hiv-2 virus cause acquired immunode fi ciency syndrome, or aids. the test is designed to detect antibodies directed against antigens of the hiv-1 or hiv-2 viruses. hiv-1 is much more common in the united states, whereas hiv-2 is prevalent in western africa. donors are tested for both viruses because both are transmitted by infected blood, and a few cases of hiv-2 have been identi fi ed in us residents. in1985, the fi rst blood-screening eia test to detect hiv was licensed and quickly implemented by blood banks to protect the blood supply. in 1992, testing of donor blood for both hiv-1 and hiv-2 antibodies (anti-hiv-1 and anti-hiv-2) was implemented. in 1996, hiv p24 antigen testing of donated blood was mandated. now, the p24 antigen testing is going to be compared with a pcr-based test for their ability to detect hiv in the window period. htlv retroviruses are endemic in japan and the caribbean but relatively uncommon in the united states. they cause adult t-cell leukemia/lymphoma and a neurological disorder similar to multiple sclerosis. the infection can persist for a lifetime but rarely causes major illnesses in most people who are infected. in rare instances, the virus may, after many years of infection, cause nervous system disease or an unusual type of leukemia. htlv-ii infections are usually associated with intravenous drug usage, especially among people who share needles or syringes. disease associations with htlv-ii have been hard to con fi rm, but the virus may cause subtle abnormalities of immunity that lead to frequent infections, or rare cases of neurological disease. in 1989, human-t-lymphotropic-virus-antibody testing of donated blood was begun. blood is now routinely screened for antibodies to htlv-i/ii. these test screens for antibodies directed against epitopes of the htlv-i/ii viruses. several commercial assays based on the enzyme-linked immunosorbent assay (elisa) or particle agglutination formats are used for screening of htlv antibodies, followed by con fi rmatory assays using western blotting. in some infected individuals, the serologic response to htlv infection is very low. these problems have been solved by the application of pcr ampli fi cation of speci fi c sequences in the virus genome. pcr can be used to detect htlv-i/ii proviruses and is now the method of choice for detection of htlv dna directly from blood and many other tissues. commercial pcr kits for htlv are available [ 28 ] . the wnv is a single-stranded rna virus of the flaviviridae family and is the most recent emerging infectious disease threat to public health and, potentially, to the safety of our blood supply. in 2002, wnv was identi fi ed as transfusion transmissible. it is transmitted by mosquitoes to birds and other animals through a mosquito bite. the virus can infect people, horses, many types of birds, and some other animals. wnv was shown in 2002 to be transmissible by blood [ 29 ] , with an estimated mean risk of 2/10,000-5/10,000 in outbreak regions in the united states. the most common symptoms of transfusion-transmitted cases of wnv were fever and headache. detection of wnv includes either a measurement of wnv antibodies or of wnv nucleic acid (detecting genetic material from the virus itself). there are two types of wnv antibody testing: igm and igg. in most individuals, igm antibodies will be present within 8 days after the initial exposure to wnv, followed by igg production several weeks later. but, the antibodies tested to detect wnv are not expedient for donor blood screening. nat involves amplifying and measuring the wnv's genetic material to detect the presence of the virus in blood or tissue. wnv nat will be negative in the blood once clinical illness has occurred. in this situation, both nat and igm antibody testing may be needed. nucleic acid tests to screen blood for wnv are commercially available and in current use. but, the viral yield for wnv infection is much lower than other viruses. consequently, a more sensitive wnv nat system for donor blood screening will be required, which could further reduce the risks of transfusion transmitted wnv. serum samples from all blood units should be subjected to either the venereal disease research laboratory (vdrl) test or a treponemal test, such as the t. pallidum haemagglutination (tpha) test before transfusion. any unit found positive should be discarded as per standard safety procedures. this test is done to detect evidence of infection with the spirochete that causes syphilis. blood centers began testing for this shortly after world war ii, when syphilis rates in the general population were much higher. the risk of transmitting syphilis through a blood transfusion is exceedingly small (no cases have been recognized in this country for many years) because the infection is very rare in blood donors, and because the spirochete is fragile and unlikely to survive blood storage conditions. sensitivity and speci fi city of serologic tests vary depending on the type of test performed and the stage of the disease. if the donor has spirochetemia, their serologic tests are usually negative, and if the donors are antibody positive, their blood is not infectious. syphilis serological tests for donors have less clinical signi fi cance. a nucleic acid test for accurately detecting syphilis is needed. it can be used to determine whether a blood donor is currently or has recently been infected with the spirochete. in recent years, numerous infectious agents found worldwide have been identi fi ed as potential threats to the blood supply and among these are several newly discovered hepatitis viruses that present unique challenges in assessing possible risks. even if the hepatitis virus test is negative for all known a-e hepatitis agents, there are some unidenti fi ed hepatitis viruses, called non a-e hepatitis viruses that can still be transmitted by blood transfusion. in the future, advances in nat may allow rapid discovery of the unknown hepatitis viruses. hepatitis delta virus (hdv) is a small rna virus that can infect only individuals who have hbv; worldwide more than 15 million people are coinfected [ 30 ] . hdv is clinically important because it generally makes hbv infections more damaging to the liver. increased understanding of the molecular virology of hdv will identify novel therapeutic targets for this most severe form of chronic viral hepatitis. pcr and real-time pcr methods are available for hdv rna detection [ 31 ] . tt virus (ttv) [ 32 ] , named for the patient from whom it was fi rst isolated with non-a-e and g posttransfusion hepatitis in japan in 1997, is a newly discovered transfusion transmitted, single-stranded and circular dna virus [ 33 ] . ttv is nonenveloped and its entire sequence of ~3.9 kb has been determined. it is also often interpreted as a transfusion-transmitted virus [ 32 ] . at least 16 genotypes have been identi fi ed, and ttv is now found all over the world. ttv infection was sought by detection of ttv dna in serum by polymerase chain reaction using primers generated from a conserved region of the ttv genome, e.g., the utr region [ 34 ] . donor blood and blood product can be screened for ttv dna by using pcr or real-time pcr. the signi fi cance of positive fi ndings is still unclear, because high level ttv carriers in healthy populations are currently found [ 35, 36 ] . whether ttv actually causes hepatitis remains to be determined. cytomegalovirus (cmv) is a virus belonging to the herpes group that is rarely transmitted by blood transfusion. donor blood is not routinely tested for cmv, and the prevalence of cmv antibody ranges from 50 to 80 % of the population. but, blood contaminated with cmv can cause problems in neonates or immunocompromised patients. it also remains a major pathogen for solid-organ transplant recipients causing febrile syndromes, hepatitis, pneumonitis, retinitis and colitis. potential problems in selected patient populations can be prevented by transfusing cmv negative blood or frozen, deglycerolized red blood cells. serologic tests for antibody to cmv are useful for determining whether a patient had cmv infection in the past, a determination of great clinical importance for organ and blood donors, and in the pretransplant evaluation of prospective transplant recipients [ 37 ] . commercial nat kits are available for cmv [ 3 ] , and these include the amplicor pcr cmv monitor test and hybrid capture system cmv dna test. sensitive screening tests for malaria are neither commercially available nor of fi cially approved yet. the most effective way of screening donors is to take a proper history of malaria or of fever that could be due to malaria. donor selection criteria should be designed to exclude potentially infectious individuals from donating red blood cells for transfusion. because there are no practical laboratory tests available to test donor blood, donors traveling to high risk malaria areas are excluded from donating blood for 6 months. however, there is a need to develop suitable screening tests, especially for use in an endemic area. a number of clinical research approaches have been developed for the extraction, ampli fi cation and detection of malaria parasite dna from blood products [ 37 ] . variant creutzfeldt-jakob disease (vcjd-a rare but fatal brain infection) [ 38 ] was fi rst described in 1996 in the united kingdom. vcjd is strongly linked with exposure to the bovine spongiform encephalopathy (bse) agent. bse is a transmissible spongiform encephalopathy (tse) affecting cattle and was fi rst reported in the uk in 1986. it has different clinical and pathologic characteristics from classic vcjd. each disease also has a particular genetic pro fi le of the prion protein gene. in recent years, questions have been raised concerning the potential risk of vcjd disease for recipients of plasma-derived clotting factors, including united states licensed factor eight (pdfviii), factor nine (pdfix), and other plasma-derived products such as immune globulins and albumin. in the past 10 years, there have been some reported cases of probable vcjd transmission by red blood cell transfusions in the united kingdom. prion infections are associated with long and clinically silent incubations. the number of asymptomatic individuals with vcjd prion infection is unknown, posing risk to others through blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. in order to decrease the risk, there is a need to establish a blood-based molecular assay for detection of vcjd prion infection. recently research papers have shown that sensitivity detection methods are available for vcjd prion [ 39 ] . however, commercial detection kits are not yet available. the dengue virus (denv) is a member of the virus family flaviviridae and is transmitted to people through the bite of an infected mosquito. the dengue virus has been shown to have four subtypes. these subtypes are different strains of dengue virus that have 60-80 % homology between each other. dengue has emerged as a worldwide problem only since the 1950s. with more than one-third of the world's population living in areas at risk for transmission, dengue infection is a leading cause of illness and death in the tropics and subtropics. according to cdc, as many as 100 million people are infected yearly. dengue is caused by any one of four related viruses transmitted by mosquitoes. there are not yet any vaccines to prevent denv infection, and the most effective protective measure is to avoid mosquito bites. there have been healthcare-related transmissions, including transmission by blood products [ 40 ] . dengue infection has a viremic phase that lasts 4-8 days, and blood collected during this phase may be infective when transfused into susceptible hosts [ 40 ] . there are currently no tests for direct detection of dengue virus, but there are however, commercial elisa tests to detect antibodies of the dengue virus in blood samples from patients. recently, research papers have shown that pcr detection methods are available for any dengue virus strain [ 41 ] . babesia is a protozoan parasite of the blood that causes a hemolytic disease known as babesiosis. babesiosis is a malaria-like parasitic disease, and there are over 100 species of babesia identi fi ed. in the united states, babesia microti is the agent most commonly reported to cause human infection. clinical confusion between human babesiosis and malaria is often reported in literature [ 42 ] . babesia infection can also be acquired by blood transfusion. in fact, there have been many cases of transfusioninduced babesiosis documented [ 43 ] . risk of developing this clinical infection is increased for elderly, asplenic, or immunosuppressed patients. current standards issued by the american association of blood banks (aabb) require the inde fi nite deferral of a blood donor with a history of babesiosis. [ 44 ] there is a need to develop methods for identi fi cation b. microti in order to reduce the risk of transmission of babesiosis by transfusion. diagnosis depends upon fi nding parasites on blood fi lm examination which can be detected 2-4 weeks after a tick bite. hamster inoculation and serology have also been used for diagnosis. the indirect fluorescent antibody test (ifat) is available for b. microti and is the most useful serological test for early diagnosis. also, the pcr screen tests for babesiosis are technically available in the fi eld [ 45 ] . chagas disease is named after the brazilian physician carlos chagas, who discovered the disease in 1909. chagas disease is spread mainly by blood-sucking insects infected with trypanosoma cruzi . chagas disease can also be spread through blood transfusion, organ transplants, and from a mother to an unborn child. national screening of the blood supply was instituted in early 2007 by fda, and more than 1,000 donors with t. cruzi infection have been identi fi ed within the past 3 years of testing. "screening for t. cruzi is an important safety measure to help protect our blood supply and to help prevent the spread of chagas disease," says karen midthun, m.d., acting director of the fda's center for biologics evaluation and research. currently, serological elisa tests are available for diagnose chronic chagas disease. pcr test is not a tool for diagnosis of chronic chagas disease in clinical practice yet, although some research results have showed that pcr is a very sensitive parasitological test for chagas' disease in active transmission regions [ 46 ] . more studies are needed for the development of this molecular method. coronavirus is an rna virus known to be associated with respiratory disease. severe acute respiratory syndrome (sars) is a newly recognized coronavirus whose genome sequence does not belong to any of the known coronavirus groups and which quickly spread all over the world from asia in 2003. there has been no evidence that this infection is transmitted from blood donors to transfusion recipients, but the virus associated with sars is present in the blood of people who are sick, and it is possible that the virus could be present in blood immediately before a person gets sick, so that an individual with infection but no symptoms possibly could transmit sars through a blood donation. to help determine whether or not an individual might be infected with sars, a blood collection facility will ask a potential donor orally or in writing about any travel to a sars-affected country or a history of sars or possible exposure to sars. enzyme-linked immunoassays for detection of speci fi c igg and igm antibodies and rt-pcr for detection of sars coronavirus speci fi c rna in the sars patients has been developed. rapid, sensitive, and speci fi c identi fi cation of sars and other novel coronaviruses by molecular methods will be very important in the future. based on past history, it is not just a hypothetical risk that many people have been infected with unrecognized viruses, for example, many patients with symptoms of non a-e, g, and ttv posttransfusion hepatitis. it is still possible that unexplained cases of posttransfusion hepatitis may be caused by a new, undiscovered pathogen. in recent years, numerous new infectious agents found worldwide have been identi fi ed through time-consuming procedures. by the time a new virus, such as hcv, hiv and sars, is found, many people are infected and there could be a large number of fatalities. there is an urgent need to develop methods for rapid identi fi cation and characterization of previously unknown pathogenic viruses. the most recent technologies for detecting and identifying previously unrecognized pathogens are expression library screening, representational difference analysis (rda), and broad-range polymerase chain reaction (br-pcr). but they are all time-consuming approaches. the new unrecognized and uncharacterized viral agents can be rapid identi fi ed by some of the new molecular approaches, e.g., subtraction hybridization [ 47 ] and dna microarray. ensuring the safety and ef fi cacy of blood and blood products is a critical regulatory challenge. the high safety level of the blood supply is the result of continued improvements in blood donor screening and testing. it will be achieved by introducing more updated nucleic acid tests to the fi eld of blood safety. nat is a method of testing blood that is more sensitive and speci fi c than conventional tests that require the presence of antibodies to trigger a positive test result. also, nat works by detecting the low levels of viral genetic material present when an infection occurs but before the body develops an immune response to a virus. this improved sensitivity should enable us to signi fi cantly decrease the infection window period, allowing for earlier detection of the infection and diminishing the chances for transmission of the agent via transfusion. we are to protect the blood supply from not only known pathogens but also the emergence of new and unrecognized and uncharacterized infectious agents. the nat methods are more sensitive and speci fi c compared with non-nat. in the future, nat technology, such as pcr, may allow routine screening of donors for all the known and unknown pathogens of concern to blood safety. progress in blood supply safety emerging infectious disease issues in blood safety white tj (eds) molecular microbiology: diagnostic principles and practice evaluation of a new nasba assay for the qualitative detection of hepatitis c virus based on the nuclisens basic kit reagents detection of piscine nodaviruses by real-time nucleic acid sequence based ampli fi cation (nasba) molecular-based methods for quantifying hiv viral load molecular diagnostics of infectious diseases dna, 50 years of the double helix: from the concept of molecular hybridization to microarrays in vitro nucleic acid ampli fi cation techniques multicenter evaluation of the performance characteristics of the nuclisens hiv-1 qt assay used for quantitation of human immunode fi ciency virus type 1 rna serological and molecular biology screening techniques for hcv infection clinical evaluation of an hiv-1 and hcv assay and demonstration of signi fi cant reduction of the hcv detection window before seroconversion hiv-1 monitor ultrasensitive quantitative assay. roche, nutley 14. lcx hiv rna quantitative assay from nested real-time pcr for hepatitis a detection introduction to microarray analysis development and validation of a diagnostic dna microarray to detect quinolone-resistant escherichia coli among clinical isolates testing requirements for communicable disease agents detection of hiv-1 and hcv infections among antibody-negative blood donors by nucleic acid-ampli fi cation testing immunization of health-care workers: recommendations of the advisory committee on immunization practices (acip) and the hospital infection control practices advisory committee (hicpac) assessment of hepatitis b virus dna stability in serum by the chiron quantiplex branched-dna assay section two: speci fi c virus families recent advances in prevention and treatment of hepatitis c virus infections the scienti fi c challenge of hepatitis c hirsh fi eld i (2003) detection of extrahepatic hcv replication by a novel highly sensitive single tube nested-pcr section two: speci fi c virus families new real-time reverse transcriptase-initiated pcr assay with single-copy sensitivity for human immunode fi ciency virus type 1 rna in plasma human t-cell leukemia virus types 1 and 2 (chap. 58) estimated risk of transmission of the west nile virus through blood transfusion in the us hepatitis delta virus quanti fi cation of hepatitis delta virus rna in serum by consensus real-time pcr indicates different patterns of virological response to interferon therapy in chronically infected patients ttv-a virus searching for a disease a novel unenveloped dna virus (tt virus) associated with acute and chronic non-a to g hepatitis role of transfusion-transmitted virus in acute viral hepatitis and fulminant hepatic failure of unknown etiology a novel dna virus (ttv) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology tt virus section two: speci fi c virus families emerging infectious disease agents and their potential threat to transfusion safety detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay threat of dengue to blood safety in dengue-endemic countries pcr detection of nearly any dengue virus strain using a highly sensitive primer cocktail do babesiosis and malaria share a common disease process? babesia infection through blood transfusions: reports received by the us food and drug administration transfusion-transmitted babesia spp.: bull's-eye on babesia microti detection of babesia species from infected dog blood by polymerase chain reaction pcr-based diagnosis for chagas' disease in bolivian children living in an active transmission area: comparison with conventional serological and parasitological diagnosis rapid approach to identify an unrecognized viral agent complete list of donor screening assays for infectious agents and hiv diagnostic assays key: cord-264488-989t9ld1 authors: park, il-hyun; kwon, young-chan; ryu, wang-shick; ahn, byung-yoon title: inhibition of hepatitis b virus replication by ligand-mediated activation of rnase l date: 2014-02-06 journal: antiviral res doi: 10.1016/j.antiviral.2014.01.021 sha: doc_id: 264488 cord_uid: 989t9ld1 rnase l is a cellular endoribonuclease that is activated by 2′,5′-linked oligoadenylates (2–5a), which are unique and specific ligands synthesized by a family of interferon-inducible, dsrna-activated enzymes named oligoadenylate synthetases. in the typical antiviral pathway, activated rnase l degrades viral and cellular rnas, thus limiting viral replication and spread. although the antiviral activity of rnase l has been demonstrated for several rna viruses, there is little evidence regarding its role against dna viruses. in the present study, the potential antiviral activity of rnase l against hepatitis b virus (hbv) was explored utilizing the recently reported infection protocol based on human hepatoma hepg2 cells stably complemented with the virus entry factor ntcp. viral replication and expression in this cell type was markedly inhibited by poly(i:c)or 2–5a-mediated activation of rnase l; however, the inhibition was significantly reversed by rnase l knockdown. further analysis in hbv1.2-transfected huh-7 hepatoma cells indicated that the antiviral activity of rnase l depends on its ribonuclease function. we also provide evidence for the specific roles of oas family members in this process. these results suggest that hbv replication can be regulated through interferon-mediated rna decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for hbv infection. rnase l is a latent endoribonuclease that is constitutively expressed in almost all mammalian cells. this enzyme is activated upon the binding of a specific ligand, 2 0 ,5 0 -linked oligoadenylate (2-5a), which is synthesized by a family of enzymes named oligoadenylate synthetases (oass). oas expression is induced by type i interferons (ifn-a/b), but the enzymes require dsrna for the activation of their catalytic activity (kristiansen et al., 2011) . in the typical antiviral pathway, viral dsrna binds to an oas, which results in the activation of the oas and the synthesis of 2-5a. rnase l, activated upon binding to 2-5a, degrades viral and cellular rnas, thus limiting viral replication and spread (chakrabarti et al., 2011) . in addition to the direct antiviral effect, rnase l can activate intracellular signaling pathways through the small rna cleavage products that it generates, leading to the induction of type i ifn production (malathi et al., 2007) . the antiviral role of rnase l has been established for a number of rna viruses. rnase l-deficient mice exhibited increased susceptibility to infection by these viruses (e.g., the picornavirus emcv and the flavivirus wnv) (silverman, 2007) . recent findings of viral functions that antagonize oas or rnase l, most notably the murine mhv coronavirus ns2-encoded phosphodiesterase that can degrade 2-5a, underscore the importance of rnase l pathway in antiviral immunity (zhao et al., 2012) . compared to its roles against rna viruses, evidence regarding the antiviral role of rnase l against dna viruses (e.g., herpesviruses and vaccinia virus) is limited (rasmussen et al., 2009; xiang et al., 2002; zheng et al., 2001) , although dna viruses can produce dsrna (weber et al., 2006) . hepatitis b virus (hbv) is a hepatotropic dna virus that can cause acute and chronic hepatitis in humans. more than 350 million people worldwide are chronic carriers of the virus and are at risk of progression of the infection to liver cirrhosis and hepatocarcinoma (lok and mcmahon, 2007) . ifn-a is currently the approved treatment for chronic hepatitis b (perrillo, 2009) . however, the mechanism by which ifn-a inhibits hbv replication is not completely understood. a recent etiological study that aimed to identify a potential link between naturally occurring rnase l gene variations (e.g., r462q) and prostate cancer suggested that this gene is correlated with chronic hepatitis b and hiv infections (arredondo et al., 2012) . however, there are few experimental data on the association between hbv and rnase l. in an early study, hbv replication was notably inhibited following poly(i:c) http://dx.doi.org/10.1016/j.antiviral.2014.01.021 0166-3542/ó 2014 elsevier b.v. all rights reserved. administration to hbv transgenic mice, but the extent of inhibition was not significantly different between groups of mice that were rnase l +/+ or rnase l à/à (guidotti et al., 2002) . while these data indicated that rnase l is not likely to mediate the antiviral activity of ifn against hbv, it was not determined whether the catalytic activity of rnase l was activated in these mice. in the present study, we adopted human hepatoma cells that permit hbv infection to address whether the oas/rnase l system plays a role in the inhibition of hbv, although hbv is not known to carry or produce dsrna. our data indicate that ligand-mediated activation of these enzymes results in a marked inhibition of hbv replication in both infected and transfected cells. we also provided evidence for differential roles of oas family members in this process. these results suggest that hbv replication can be regulated through the activation of rnase l-mediated rna decay pathways. hbv stock was prepared from the culture supernatant of hepg2.2.15 cells (sells et al., 1987) . the cells were maintained in dmem supplemented with 10% fbs (hyclone), 200 lg/ml g418 (a.g. scientific) and 50 lg/ml gentamicin (invitrogen). the cells were split 1:4 every 7 days, and the culture supernatant was concentrated as described (vincent et al., 2011) . to prepare viral dna the viral stock was treated with 200 lg/ml proteinase k for 1 h at 37°c in 10 mm tris (ph 7.5), 10 mm edta and 1% sds, followed by phenol/chloroform/isoamyl alcohol (pci) treatment. viral dna was measured by real-time qpcr with a sybr qpcr kit (kapa biosystems) and a myiq system (bio-rad laboratories) using hbv-specific primers (suppl. table 1 ). pcr was conducted by denaturation at 95°c for 30 s, followed by 40 cycles of denaturation at 95°c for 3 s and annealing/elongation at 60°c for 20 s. the viral genome copy number was calculated based on a standard curve generated with pgem-hbv1.2 and was indicated as genome equivalents (geq). for the infection assay, hepg2 cells (atcc hb-8065) were stably transfected with the ntcp gene (a generous gift from wenhui li of nibs, china) and maintained in dmem with 2 lg/ml blasticidin (invivogen). the ntcp-supplemented cells were incubated for 7 days before infection in primary hepatocyte maintenance media (pmm). pmm consists of williams e medium supplemented with 10% fbs, 10 ng/ml egf (invitrogen), 5 lg/ml transferrin, 3 lg/ml insulin, 2 mm l-glutamine, 18 lg/ml hydrocortisone, 40 ng/ml dexamethasone, 5 ng/ml sodium selenite, 2% dmso (all from sigma) and 50 lg/ml gentamicin. the cells were seeded in 24-well plates ($10 5 cells/well) and inoculated the following day with hbv at $2000 geq/cell in pmm (without dmso) containing 4% peg8000. the viral inoculum was removed 16 h later, and the cells were further incubated (up to 9 days in most experiments) in pmm; the media was changed every 3 days. for the transfection assay, huh-7 cells grown in dmem with 5% fbs were transfected with 1 lg/well of pgem-hbv1.2, a replicon containing the 1.2-mer hbv genome. viral polymerase gene expression was nullified in the hbv1.2(p-) construct due to a t to c mutation in the first atg codon and a deletion of t from the second atg codon; these changes did not alter the expression of the core gene that overlaps in the same region of the genome (ryu et al., 2008) . in hbv1.2(x-), the expression of the viral x gene was blocked with two stop codons introduced adjacent to the first and second atg codons of the x gene by substitution of t for c at nt positions 22 and 259 (cha et al., 2009 ). hbv1.2(c-42) expresses an assembly-defective core protein due to deletion of the 42nd codon (leu) of the capsid gene (koschel et al., 2000) . capsid-associated viral dna was extracted from infected cells that were lysed for 20 min on ice in buffer [50 mm tris-hcl (ph 7.5), 1 mm edta, 0.2% np-40 and 150 mm nacl]. the lysate was clarified by centrifugation at 15,000g, and the supernatant was transferred to a new tube and gently mixed for 12 h with an anti-hbc antibody (dako) and protein a/g plus-agarose (santa cruz biotechnology). the beads were collected by centrifugation at 1000g and washed 3 times in the lysis buffer. viral dna was hbcag merge extracted with 200 lg/ml of proteinase k for 1 h at 37°c in 10 mm tris (ph 7.5), 10 mm edta and 1% sds; it was then extracted by pci and measured by southern blot and real-time pcr as described above. viral cccdna was extracted by pci from infected cells that were lysed for 4 h at 65°c in lysis buffer [50 mm tris-hcl (ph 8.0), 50 mm edta, 100 mm nacl and 1% sds] supplemented with proteinase k (200 lg/ml). the extracted dna ($500 ng) was treated with 10 u of plasmid-safe dnase (epicentre technologies) for 8 h at 37°c followed by dnase inactivation at 70°c for 30 min. an aliquot ($20 ng) of extracted dna was denatured at 95°c for 5 min and amplified by qpcr using cccdna-specific primers (suppl. table 1 ). the amplification protocol included 45 cycles of denaturation at 95°c for 30 s, annealing at 62°c for 25 s and elongation at 72°c for 45 s. the hbv cccdna copy number was calculated based on a standard curve generated with pgem-hbv1.2. viral pgrna was measured by real-time qpcr. total rna was extracted from infected cells with rnaiso plus (takara), treated with rq1 dnase and reverse transcribed with the improm-ii rt system (promega). cdna derived from 50 ng of total rna was amplified by qpcr as described for the capsid dna assay. transcripts of rnase l, p53, oas1, oas2 and oas3 were measured by real-time pcr from 1 lg of total cellular rna under the same conditions described above. gapdh mrna was used as a normalization control. viral rna and capsid dna were measured by northern and southern blotting as previously described (park et al., 2011) . infected cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.5% triton x-100 and immunostained with anti-hbcag (dako) followed by alexa 568-conjugated goat anti-rabbit igg (invitrogen). rnase l was immunoblotted and detected with anti-rnase l (invitrogen) or anti-flag antibody (sigma). tubulin was probed with an antibody obtained from applied biological materials. the rnase l expression plasmid p3xflag-rnase l and a nuclease-defective construct, r667a, have been previously described (kwon et al., 2013) . poly(i:c) was obtained from sigma. trimeric 2 0 ,5 0 adenylates, either in 5 0 -monophosphorylated (p2-5a) or . intracellular pgrna at 9 days post-infection was measured by rt-qpcr ( ⁄ p < 0.05, ⁄⁄ p < 0.01). (b) aliquots of huh-7 or hepg2 cells either grown in dmem or further incubated in pmm for 5 days were immunoblotted for rnase l ($82 kda). human lung carcinoma (a549) cells grown in dmem were also probed. tubulin served as a loading control. (c) rnase l mrna levels in hepg2 cells incubated in pmm for the indicated periods were measured by rt-qpcr and are shown as fold-induction relative to the initial level. for comparative purposes, p53 mrna is also shown. unphosphorylated (2-5a) forms were provided by chemgenes corp. pcr primers and sirnas were purchased from integrated dna technologies and genepharma, respectively, and their nucleotide sequences are shown in suppl. table 1 . jetpei (polyplus) was used for dna transfection. lipofectamine 2000 (invitrogen) was used for transfection of poly(i:c), 2-5as and sirnas. ifn-a was obtained from r&d systems, inc. studies on hbv have been hampered by the lack of a robust cell culture system that permits the full life cycle of viral growth. a recent study identified a bile acid transporter protein named sodium taurocholate cotransporting polypeptide (ntcp) that binds the nterminal pre-s1 domain of hbv envelope protein l and mediates the entry of hbv and its surrogate virus hdv (yan et al., 2012) . it was shown that supplementation of human hepatoma cells such as huh-7 and hepg2 with ntcp supports hbv infection. to address the role of rnase l in this process, we prepared a pool of hepg2 cells stably transfected with the ntcp gene. while the ntcpexpressing cells were routinely maintained in dmem, for experiments involving hbv infection, the cells were incubated in pmm for several days before infection, which appeared to render the cells more susceptible to viral infection. infected cells were further incubated (up to 9 days in most experiments) in pmm, and the media was changed every 3 days. immunostaining indicated that the number of cells positive for hbcag, the viral capsid protein, increased with time, although it was less than 5% of the total cells at 9 days post-infection (fig. 1a) . to follow viral replication, we measured the viral pregenomic rna (pgrna) by rt-qpcr. at 9 days post-infection, pgrna reached up to $2 â 10 6 copies per lg of total cellular rna (fig. 1b) . in contrast to these results, expression of hbcag and pgrna was markedly reduced when infected cells were transfected with poly(i:c). viral dna in the capsids, measured by real-time pcr of intracellular viral capsids immunoprecipitated with anti-hbc antibody, was similarly decreased. intracellular viral cccdna was also significantly reduced at 9 days post-infection, while no inhibition was observed at 2 days post-infection (suppl. fig. s1 ). these results demonstrate the profound antiviral effect of poly(i:c) on hbv expression and replication in this cell-based infection system. the antiviral effect of poly(i:c) observed above was likely due to the type i ifn-inducing response mediated by dsrna-binding cellular receptor proteins such as tlr3 and rig-i. indeed, we observed strong induction of ifn-b and oas2 mrnas in hepg2-ntcp cells approximately 12 h after poly(i:c) transfection (suppl. fig. s2 ). among the ifn-related cellular factors, we focused on rnase l because while the induction of oas depends on the ifn response, the activation of its endonuclease function requires dsrna. to more specifically address the antiviral activity, if any, of rnase l against hbv, we transfected the infected cells with synthetic 5 0 -monophosphorylated trimeric 2 0 ,5 0 -adenylates (p2-5a), a specific activator of rnase l. viral pgrna expression was inhibited by up to $70% in a p2-5a dose-dependent manner, whereas no inhibition was observed in the cells either mock-transfected or transfected with unphosphorylated 2-5a ( fig. 2a) . because p2-5a is a unique and specific activator for rnase l, these results strongly indicated that the observed inhibition of hbv was the result of rnase l activation. however, unlike the situation in liver tissue, where abundant expression of rnase l was observed, huh-7 and hepg2 cells express only low levels of rnase l (kwon et al., 2013; malathi et al., 2007; zhou et al., 2005) . interestingly, we identified rnase l proteins in an amount that was detectable by immunoblotting of hepatoma cells that were incubated for 5 days in pmm, whereas it was not detected in the same type of cells grown in dmem (fig. 2b) . our analysis indicated a steady increase of rnase l mrna in the two hepatoma cell lines during incubation in pmm (up to $50-fold in 8 days), whereas the level of p53 mrna remained stable (fig. 2c) . although we could not explain this phenomenon, the elevated expression of rnase l provided an opportunity to address the role of rnase l through sirna-mediated suppression. inhibition of pgrna by p2-5a was almost fully reversed in the rnase l knockdown cells, demonstrating a specific role for rnase l in the inhibition of hbv (fig. 3) . compared with this result, poly(i:c)-mediated inhibition was rescued by $50%, suggesting that other effectors in addition to rnase l also contributed to the inhibition. the antiviral activity of rnase l was further characterized in another hepatoma cell line, huh-7, following transient transfection of the viral genome construct hbv1.2. including the 3.5-kb pgrna, all viral mrnas (2.4, 2.1 and 0.9 kb in length) were markedly reduced when the replicon cells were further transfected with rnase l (fig. 4a) . as in the infection experiments, poly(i:c) was required for the antiviral activity of rnase l, although a mild inhibitory effect was observed even without poly(i:c) (fig. 4b ). this mild inhibitory effect of rnase l (shown in the lane 2 vs. lane 1 of fig. 4a) suggested that some oas activation occurred under these conditions, most likely due to non-specific dsrnas (e.g., read-through transcripts) produced off the plasmid dna templates. in contrast, no inhibition was observed with r667a, which harbors a missense mutation in the ribonuclease domain of rnase l (dong et al., 2001) . the strong antiviral effect and concomitant occurrence of rrna cleavage (shown in the lane 5) confirmed the poly(i:c)-mediated activation of ectopically expressed rnase l. poly(i:c) alone (without ectopic rnase l expression) had no effect (lanes 1 vs. 4; lanes 3 vs. 6). this result is consistent with the low level of endogenous rnase l expression, as we reported previously (kwon et al., 2013) , and no induction of ifn-b or isg expression was observed in this type of hepatoma cell upon poly(i:c) transfection (described below) as reported (li et al., 2005) . therefore, the observed antiviral activity of transfected poly(i:c) was primarily contributed by the activated ribonuclease function of rnase l. the activation of rnase l was also confirmed with p2-5a transfected huh-7 cells (suppl. fig. s3 ). because the first step in the hbv replication cycle is the synthesis of viral mrna and pgrna off the viral genome, downregulation of viral transcripts by rnase l would subsequently affect viral dna replication as well. as expected, the capsid-associated viral dna was barely detectable in rnase l-activated cells, indicating that the downregulation of viral transcripts resulted in the severe inhibition of viral dna synthesis, including the new synthesis of viral relaxed-circular (rc) and duplex-linear (dl) dnas (fig. 4c, 2nd lane of the southern blot). to further substantiate this result, we examined hbv with mutations that have critical effects on viral dna replication. hbv1.2(p-) is incapable of dna replication because viral pol gene expression is nullified due to a t to c mutation in the first atg codon and a t deletion in the second atg codon; however, these mutations do not alter the expression of the core gene that overlaps the same region (ryu et al., 2008) . hbv1.2(c-42) is also replication-defective because the core gene lacks the 42nd codon (leu) and thus cannot form assembled capsids (koschel et al., 2000) . our analyses indicated that the transcripts of these mutants, which were not affected despite their replication defects, were degraded by poly(i:c) and rnase l similar to those of the wild-type genome. a similar result was obtained for the third mutant, hbv1.2(x-), in which the viral x gene was nullified due to stop codons introduced adjacent to the first and second atg codons (cha et al., 2009) . despite nullification of this critical protein, viral transcription and dna replication were not affected as reported previously for this type of hepatoma cell (melegari et al., 1998) . likewise, all viral transcripts of this mutant were degraded by poly(i:c) and rnase l similar to those of the wild-type genome. thus, rnase l-dependent downregulation of viral rnas (and dna) as observed for wild-type hbv was observed for all three mutants regardless of their replication capability. rnase l activation requires 2-5a, which is synthesized by oas. in humans, three closely related and linked genes, oas1, oas2 and oas3, code for oas proteins. oas expression is induced by type i ifns, but dsrna is required for the activation of their catalytic activity. our rt-pcr analysis showed that oas1 and oas3, but not oas2, were constitutively expressed in huh-7 and hepg2 cells before all three genes were further induced by ifn-a treatment (fig. 5) . as mentioned above, poly(i:c) had no effect on the expression pattern of the three oas genes in huh-7 cells, yet it induced all three oas genes in hepg2 cells. therefore, it is likely that in huh-7 cells, the constitutively expressed endogenous oas1 and/or oas3 proteins were catalytically activated by poly(i:c) and contributed to rnase l activation as observed above. to address this hypothesis, we attempted to knock down oas expression using a specific sirna. despite the notable suppression of oas1 (by up to 60%), inhibition mediated by rnase l and poly(i:c) was not affected (fig. 6) , suggesting that oas1 does not play a major role in this process. interestingly, a moderate rescue of viral rna was observed in the oas1 knockdown cells even prior to the transfection of rnase l and poly(i:c). most likely, oas1 was activated by non-specific dsr-nas produced off the plasmid dna templates, resulting in the activation of endogenous rnase l that was present at a low level. compared with oas1, viral transcripts were partially but significantly rescued by oas3 sirnas, although smearing of the signals might be still indicative of rna degradation, consistent with incomplete suppression of oas3 (fig. 7) . this result indicated that oas3 plays an important role in activating rnase l in the presence of poly(i:c). however, unlike oas1, oas3 knockdown had no effect without poly(i:c). because both oas1 and oas3 are constitutively expressed in these cells prior to poly(i:c) treatment, as shown in fig. 5 , these results suggest that the two enzymes differ in their requirement for dsrna; specifically, oas3 might be activated more efficiently with poly(i:c). in vitro data indicated that oas3 was more sensitive to dsrna ($100 times less dsrna is required for activation) (marie et al., 1997) . to examine the role of oas2, we stimulated its expression by ifn-a treatment following transfection with oas2 sirna. viral rna was partially rescued with one of the two sirnas, and this rescue was correlated with the extent of oas2 mrna suppression (fig. 8) . ifn-a treatment alone did not show apparent inhibition of hbv transcripts, as we previously reported for this type of hepatoma cell (park et al., 2011) . our results indicate that oas2 and oas3, but not oas1, are dependent on poly(i:c) under these conditions. thus, oas family members differ in their requirement for dsrna and contribute differentially to rnase l activation under different conditions. among ifn-related cellular factors, rnase l has been shown to inhibit a number of rna viruses and few dna viruses. in the typical antiviral pathway, viral dsrna activates oas, which in turn activates rnase l through the synthesis of 2-5a. it is thought that rnase l-mediated viral rna decay leads to the inhibition of these viruses. in this study, we attempted to address whether the oas/ rnase l system plays a role in cellular restriction or ifn-mediated inhibition of hbv, although hbv is not known to carry or produce dsrna. with the newly established hepg2-ntcp cell culture system, which permits hbv infection, we showed that viral gene expression and replication can be profoundly reduced by 2-5aor poly(i:c)-mediated activation of rnase l. in hbv1.2-transfected huh-7 cells, it was further confirmed that this type of inhibition occurred mostly through viral rna degradation via the ribonuclease function of rnase l. in contrast to the effect of the specific ligand 2-5a, the poly(i:c)-mediated antiviral effect was not fully rescued by rnase l knock down. while the latter result implies that other effectors also contribute to the inhibition observed, it might be worth considering the ways by which poly(i:c) might have functioned in our study and in previous studies performed with hbv-transgenic animals. intravenous administration of poly(i:c) resulted in a dramatic reduction of viral replication; however, no significant difference was observed between groups of rnase l +/+ or rnase l à/à animals with respect to the extent of inhibition (guidotti et al., 2002) . a subsequent study showed that poly(i:c) inhibited hbv through induction of type i ifn responses, as evidenced by intrahepatic isg expression and requirement for the cytokine receptors in this process (isogawa et al., 2005) . while these data indicate that rnase l is not likely to mediate the antiviral activity of ifn against hbv, it was not determined whether the catalytic activity of rnase l was activated in these mice. most likely, poly(i:c) that was injected intravenously into the animals triggered ifn induction via tlr signaling. however, the lack of apparent difference in viral replication between rnase l +/+ or rnase l à/à animals (despite intrahepatic induction of oas) suggested that rnase l was most likely not activated. consistent with this notion, the steady state level of viral rna was not affected in these animals, whereas hbv dna was profoundly inhibited. in contrast, all forms of hbv rnas were profoundly reduced in our cell-based assays, indicating the efficient activation of rnase l in poly(i:c)transfected cells. this direct rnase l-activating effect of poly(i:c) was more evident in the huh-7 cell, in which tlr3-mediated signaling is known to be inefficient (li et al., 2005) . our rnase l knockdown experiment performed in hepg2 cells, showed $50% rescue of poly(i:c)-mediated viral rna reduction, suggesting that other factors in addition to rnase l contributed to the inhibition. indeed, we observed strong induction of ifn-a and oas2 mrnas in hepg2 cells within 12 h of poly(i:c) transfection. studies exploring how the ifn-mediated antiviral responses (those triggered by the secreted ifns or by the direct activation of prr with pamp) inhibit hbv indicated that various steps of viral replication are affected (reviewed in chang et al., 2012) . although many of these studies highlight posttranscriptional inhibition mechanisms, cellular effectors responsible for this type of regulation have not been identified. recently, it was reported that zinc finger antiviral protein (zap), a cellular protein known initially as a retroviral restriction factor, inhibits hbv through the viral rna decay process (mao et al., 2013) . while zap itself retains no ribonuclease activity, it is known to bind viral (and cellular) rnas through its n-terminal domain, which contains four zinc finger motifs and stimulates rna degradation by recruiting various host rna processing machineries, including exosomes. according to these authors, expression of at least one isoform of zap was induced by ifn signaling, and ectopic expression of zap markedly reduced viral rna. while rnase l differs from zap in that it requires specific ligands to activate its ribonuclease function, it is also possible that rnase l acts in combination with other cellular factors or as part of the cellular machinery during viral rna decay. regarding the suggested relationship between naturally occurring rnase l gene variation (e.g., r462q) and chronic hepatitis b (arredondo et al., 2012) , we did not find any defect in hbv inhibition with this variant in our transfection assay (data not shown). this result was consistent with the reduced 2-5a-mediated oligomerization activity but intact ribonuclease function of this allele (xiang et al., 2003) . we showed that oas1 and oas3 were constitutively expressed in huh-7 cells before their expression was further induced, along with that of oas2, by ifn-a treatment. elevated oas activity has been found in the sera of hepatitis c patients following ifn therapy, and the level of oas activity was found to be correlated with their virological responses to this therapy (kim et al., 2006) . among chronic hepatitis b patients, the frequency of a nonsense mutation in the r567 codon of oas3 was reported to be higher among non-responders to ifn therapy compared with responders (ren et al., 2011) . although we did not test this allele of oas3, our knockdown data support the role of oas3, along with that of oas2, in the activation of rnase l. in summary, our data indicated that hbv replication can be regulated through the activation of rnase l-mediated rna decay pathways with exogenously provided ligands. specifically, small molecules that mimic natural ligands (such as 2-5a) would represent a novel therapeutic strategy. short communication: rnasel alleles and susceptibility to infection by human retroviruses and hepatitis viruses stimulation of hepatitis b virus genome replication by hbx is linked to both nuclear and cytoplasmic hbx expression new insights into the role of rnase l in innate immunity the innate immune response to hepatitis b virus infection: implication for pathogenesis and therapy basis for regulated rna cleavage by functional analysis of rnase l and ire1p interferon-regulated pathways that control hepatitis b virus replication in transgenic mice toll-like receptor signaling inhibits hepatitis b virus replication in vivo 0 -,5 0 -oligoadenylate synthetase response ratio predicting virological response to peg-interferon-alpha2b plus ribavirin therapy in patients with chronic hepatitis c hepatitis b virus core gene mutations which block nucleocapsid envelopment the oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities the ribonuclease l-dependent antiviral roles of human 2 0 ,5 0 -oligoadenylate synthetase family members against hepatitis c virus distinct poly(i-c) and virusactivated signaling pathways leading to interferon-beta production in hepatocytes chronic hepatitis b small self-rna generated by rnase l amplifies antiviral innate immunity inhibition of hepatitis b virus replication by the host zinc finger antiviral protein 69-kda and 100-kda isoforms of interferon-induced (2 0 -5 0 )oligoadenylate synthetase exhibit differential catalytic parameters cloning and characterization of a novel hepatitis b virus x binding protein that inhibits viral replication pkr-dependent mechanisms of interferon-alpha for inhibiting hepatitis b virus replication benefits and risks of interferon therapy for hepatitis b herpes simplex virus infection is sensed by both toll-like receptors and retinoic acid-inducible gene-like receptors, which synergize to induce type i interferon production polymorphisms of interferon-inducible genes oas associated with interferonalpha treatment response in chronic hbv infection hepatitis b virus polymerase suppresses translation of pregenomic rna via a mechanism involving its interaction with 5 0 stem-loop structure production of hepatitis b virus particles in hep g2 cells transfected with cloned hepatitis b virus dna viral encounters with 2 0 ,5 0 -oligoadenylate synthetase and rnase l during the interferon antiviral response hepatitis b virus impairs tlr9 expression and function in plasmacytoid dendritic cells doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses blockade of interferon induction and action by the e3l double-stranded rna binding proteins of vaccinia virus effects of rnase l mutations associated with prostate cancer on apoptosis induced by 2 0 ,5 0 -oligoadenylates sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology increased severity of hsv-1 keratitis and mortality in mice lacking the 2-5a-dependent rnase l gene mapping of the human rnasel promoter and expression in cancer and normal cells we are grateful to wenhui li for providing us with the ntcpexpressing plasmid. this work was supported by the basic science research program of the national research foundation (#2012-008126) of the republic of korea. some preliminary studies were supported by a grant from korea university in 2011-2012. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral.20 14.01.021. key: cord-325280-4whzcmqv authors: takizawa, naoki; yamasaki, manabu title: current landscape and future prospects of antiviral drugs derived from microbial products date: 2017-10-11 journal: j antibiot (tokyo) doi: 10.1038/ja.2017.115 sha: doc_id: 325280 cord_uid: 4whzcmqv viral infections are a major global health threat. over the last 50 years, significant efforts have been devoted to the development of antiviral drugs and great success has been achieved for some viruses. however, other virus infections, such as epidemic influenza, still spread globally and new threats continue to arise from emerging and re-emerging viruses and drug-resistant viruses. in this review, the contributions of microbial products isolated in institute of microbial chemistry for antiviral research are summarized. in addition, the current state of development of antiviral drugs that target influenza virus and hepatitis b virus, and the future prospects for antivirals from natural products are described and discussed. viral infectious diseases cause significant morbidity and mortality in humans. lower respiratory infections are the deadliest communicable disease, causing 3.2 million deaths globally in 2015. moreover, in 2015 acquired immunodeficiency syndrome (aids) caused by human immunodeficiency virus (hiv) infections killed 1.1 million people and 36.7 million people were infected with hiv globally. to combat viral diseases, both vaccines and antiviral drugs have been employed. as a result of global efforts, smallpox caused by variola virus infections was eradicated in 1979 because of smallpox vaccination (by vaccinia virus). however, the development of effective vaccines is difficult and time consuming for certain viruses that can escape from the host immune response. therefore, the continual development of antiviral drugs and vaccines is required to combat viral diseases. approximately 90 drugs have been approved to treat human infectious diseases caused by the following nine viruses: hiv, human cytomegalovirus, hepatitis b virus (hbv), hepatitis c virus (hcv), herpes simplex virus, influenza virus, respiratory syncytial virus, varicella zoster virus and human papillomavirus. in addition, many antiviral drugs are being evaluated in clinical trials. one of the threats of viral infections is emerging and re-emerging viruses, such as ebola virus, severe acute respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus and zika virus. effective antiviral drugs or vaccines against these emerging viruses are not currently available. drug-resistant viruses, which can emerge because of the high mutation rate of the viral genome exhibited by many viruses, particularly rna viruses, also represent a threat to global health. almost all currently approved antiviral drugs have been produced by chemical synthesis, but natural products have contributed to the development of antiviral drugs by providing insights into the synthesis of chemical compounds. viruses must utilize host cellular machineries to propagate and our knowledge of virus-host interactions has recently been advanced. natural products exhibit great structural diversity and complexity. thus, both viral and host proteins involved in viral propagation can be targeted to develop antiviral drugs from natural products. in this review, we summarize antiviral microbial products discovered by institute of microbial chemistry (imc) and discuss antiviral compounds against influenza virus and hbv, because these two viruses threaten global human health now and antiviral drug against these two viruses have been developed. antiviral compounds isolated in imc are summarized in table 1 . in addition, we provide a summary of natural compounds for broad-spectrum antivirals and discuss the future potential uses of microbial products as antivirals. natural compounds with antiviral activities focused in this review are summarized in table 2 . the contributions of imc to antiviral research nucleos(t)ide analogs are a major class of approved antiviral drugs that exert therapeutic effects through incorporation into viral dna and rna to inhibit virus replication. to date, they have been used as a versatile platform to develop both narrow and broad-spectrum antivirals. moreover, natural products have provided some motivation to develop new nucleos(t)ide analogs. 1,2 two arabinosyl nucleosides, spongouridine and spongothymidine, were isolated from marine sponges and subsequently contributed to the synthesis of vidarabine (arabinosyladenine), which is used for the treatment of herpes simplex virus infections. professor umezawa and colleagues identified nucleoside analogs from actinobacteria, such as formycin, 3,4 coformycin 5 and oxanosine, 6-8 some of which exhibit an antiviral activity. currently, several classes of anti-hiv drugs are available for hiv treatment and the combination therapies using them, termed highly active antiretroviral therapy that targets multiple steps of the virus life cycle, significantly reduce morbidity and mortality. zidovudine (azidothymidine) was first approved for hiv treatment by the us food and drug administration in 1987. this drug had initially been used for chemotherapeutic intervention against hiv infection, but unmet medical needs of increased efficacy, reduced adverse effects and a higher barrier to drug-resistant mutations remained, prompting a continuing effort to develop new anti-hiv drugs. in this context, imc had isolated microbial products with anti-hiv activity, such as benanomicins a and b, 9 kijimicin, 10 and bellenamine and its homologs. 11 among them, benanomicins also exhibited anti-fungal activity, which may be advantageous for the treatment of aids patients with fungal infections. in addition, 14-o-acyladriamycins 12 and aminoacridines 13 were synthesized as hiv reverse transcriptase inhibitors. meanwhile, other research groups found that several compounds discovered by imc exhibit inhibitory activities against other viruses: phleomycin (anti-poliovirus activity), 14 fusaric acid (broad-spectrum antiviral activity against herpes simplex virus, varicella-zoster virus, hbv, hcv and sindbis virus), 15 aclacinomycin a (anti-phage φx174 and λ activities), 16 clazamycin b (anti-herpes simplex virus activity) 17 and sarkomycin (anti-rna phage f2 activity). 18 among them, phleomycins and aclacinomycins were discovered as antibiotics that exhibit antitumor activity, presumably as a consequence of interactions with cellular dna. 19 currently, microbial secondary metabolites are well established to contain many compounds with structural and chemical diversity. professor umezawa et al. postulated that certain compounds among them may target enzyme reactions and started to search for enzyme inhibitors from a microbial fermentation broth in 1965. this pioneering work has contributed to various biochemical advances and to therapeutic approaches for certain diseases. for example, in the field of virus research, leupeptin, an inhibitor of serine and cysteine proteases from streptomyces roseus, 20 has been shown to prevent glycoprotein-mediated entry of marburg virus, suggesting the role of unknown host proteases in glycoprotein activation. 21 antipain and elastatinal, inhibitors of serine and cysteine proteases from actinomycetes, 22,23 were used to inhibit poliovirus 2a protease. 24, 25 notably, pepstatin, an aspartic proteinase inhibitor isolated from several species of streptomyces, 26 made a significant contribution to the development of a key class of anti-hiv drugs in highly active antiretroviral therapy. hiv proteinase processes viral polyproteins to generate mature proteins and is essential for the virus life cycle. the acetyl-derivative of pepstatin has been reported to be highly effective against hiv protease through a transition state mimic, which led to the rational design of peptide-based protease inhibitors. 27, 28 furthermore, siastatin b, an analog of sialic acid, was isolated from streptomyces verticillus var. quantum as a sialidase inhibitor. 29 although siastatin b showed no effect on the sialidase activity of influenza virus neuraminidase (na), its epimeric analog significantly inhibited this enzymatic activity and virus propagation in vitro, as described below. 30 together, imc has identified many microbial products with antiviral activities, some of which have played prominent roles as a unique pharmacophores for antiviral drugs. seasonal influenza is acute respiratory infections caused by influenza viruses. there are three types of seasonal influenza viruses-type a, b and c; influenza a and b viruses cause outbreaks and epidemics. the world health organization estimates that annual epidemics caused by influenza a(h1n1), a(h3n2) and b viruses result in~3 to 5 million cases of severe illness and 250 000 to 500 000 deaths, globally. 31 furthermore, an influenza pandemic, such as those of the 'spanish' influenza in 1918 (h1n1),' 'asian' influenza in 1957 (h2n2),' 'hong kong' influenza in 1968 (h3n2),' 'russian' influenza in 1977 (h1n1)' and 2009 h1n1 influenza (swine-origin h1n1), occurs when new influenza a virus against which the human population has no immunity emerges as a consequence of 'genetic reassortment'. 32 as these epidemics and pandemics still occur, the continued discovery and development of new antiviral drugs and vaccines against influenza infections are required. a total of eight drugs (matrix 2 (m2) inhibitors (amantadine and rimantadine), na inhibitors (zanamivir, oseltamivir, peramivir and laninamivir octanoate) and viral rnadependent rna polymerase inhibitors (ribavirin and favipiravir) have been approved for the treatment of influenza infections. 1-adamantanamine (amantadine) was the first synthetic compound shown to inhibit influenza virus propagation 33 and was approved in 1966 to treat influenza a virus infections. the target of amantadine is m2 integral membrane protein, which exhibits h + ion channel activity. acidification of virus-containing endosomes activates m2 ion channel activity. 34 acidification in the virion interior is necessary to allow the viral genome to diffuse away from the endosomal membrane. amantadine inhibits proton translocation by binding to the transmembrane domain of the m2 protein tetramer, thereby inducing the release of the viral genome into the cytoplasm 35 ( figure 1 ). however, amantadine and rimantadine, a derivative of amantadine, are not currently used to treat influenza virus infections because drug-resistant viruses can emerge rapidly (both the a/h1n1pdm and a/h3n2 seasonal epidemic strains have the amantadine-resistance s31n mutation) and the central nervous system side effects are particularly common in patients treated with amantadine. hemagglutinin (ha) and na are two major glycoproteins expressed by both influenza a and b viruses. ha mediates the binding of viruses to target cells via terminal sialic acid and na cleaves terminal sialic acids from host cells. na activity facilitates the release of progeny influenza virus by digesting sialic acids in the ha receptors ( figure 1 ). the sialidase activity of na has been targeted in the development of anti-influenza drugs. siastatin b is an analog of sialic acid and that inhibits the sialidase activity of clostridium perfrigens nas, but does not inhibit that of influenza virus (a/aichi/2/68 strain) na and newcastle disease virus (sato strain) hn. 29 a siastatin b epimeric analog, 3-episiastatin b, can inhibit the sialidase activity of influenza a and b viruses na and propagation of influenza a virus. 30 a synthetic analog of neuraminic acid, 2,3-didehydro-2-deoxy-nacetylneuraminic acid (neu5ac2en), was found to be an inhibitor of influenza virus na. 36 neu5ac2en is considered to be a transition state analog that can bind to the active site of na. [37] [38] [39] based on a structural analysis of the na-neu5ac2en complex, analogs of neu5ac2en were synthesized and the specificity for influenza virus na and activity of these analogs were tested. among these analogs, 4-guanidino-neu5ac2en (zanamivir) exhibited antiviral activity against various influenza a and b viruses. 40 the guanidine group in zanamivir is suggested to form salt bridges with glu119 in the na active site and strong charge-charge interactions with glu227 of na. because of poor oral bioavailability, zanamivir is administered by inhalation. to improve oral bioavailability, novel analogs were synthesized based on x-ray crystallographic analysis of na with key analogs. 41 one analog, gs4071, inhibited influenza a virus na activity. the na inhibition activity of gs4071 was comparable to that of zanamivir. oseltamivir is an ethylester prodrug of gs4071. the oral bioavailability of oseltamivir was improved compared with that of gs4071. 42 after zanamivir and oseltamivir, two na inhibitors, peramivir and laninamivir octanoate, were launched. 43, 44 peramivir is administered by intravenous administration and laninamivir octanoate is administered by inhalation. these two drugs are used as a single-dose treatment for influenza a and b viruses. viral polymerase complex is a promising target for the development of anti-influenza drugs, because transcription and replication are critical steps for virus propagation. moreover, the viral polymerase complex acts by a specific molecular mechanism for viral transcription and replication. the viral polymerase complex is a heterotrimer composed of three subunits: pa, pb1 and pb2. at the initiation of viral mrna transcription, influenza viral polymerase cannot synthesize the 5′-cap structure of mrna that is necessary for translation and uses a unique prospects for microbe-derived antiviral drugs n takizawa and m yamasaki 'cap-snatching' mechanism to 'steal' the cap structure of mrna from host mrnas. 45 host-cell pre-mrna is bound to the pb2 subunit via its 5′-cap structure and can be cleaved at nucleotide 10-13 by the endonuclease activity of the pa subunit. using this capped fragment as a primer, the viral polymerase complex synthesizes viral mrna. these processes have been targeted in the development of anti-influenza drugs and two inhibitors, vx-787 (pimodivir) and s-033188, are under phase 2 and 3 clinical trials, respectively. vx-787 is a pb2 inhibitor that binds to the cap-binding pocket in pb2. 46, 47 the molecule 3-pyrimidineazainodole, vrt-0761704, was identified as a lead compound for anti-influenza virus activities by phenotypic screening, and the target of this compound was determined to be the pb2 subunit. based on structural information for the pb2-m 7 gtp and pb2-vrt-0761704 complex, vx-787 was synthesized. vx-787 represents an orally bioavailable compound that is only effective for influenza a virus, not influenza b virus. s-03318 specifically inhibits the endonuclease activity of pa and thus prevents the cleavage of host pre-mrnas. s-03318 is effective for both influenza a and influenza b virus infections. two approved drugs, ribavirin and t-705 (favipiravir), inhibit influenza viral rna synthesis (figure 1 ). ribavirin is a synthetic nucleoside analog that is metabolized to the triphosphate form (ribavirin-tp). 48 ribavirin-tp inhibits the rna polymerase complex of influenza virus by competing with atp and gtp. 49 ribavirin has not been used to treat influenza virus infection, but has been used for hcv infections along with pegylated interferon. t-705 is a pyrazine derivative that is metabolized to t705-4-ribofuranosyl-5′-triphosphate in cells. 50 ,51 t705-4-ribofuranosyl-5′-triphosphate acts as a specific inhibitor of influenza virus rna synthesis. t705-4-ribofuranosyl-5′triphosphate directly inhibits viral rna synthesis and is incorporated into synthesized rna in the monophosphate form (t-705rmp) without chain termination. [52] [53] [54] the incorporated t-705rmp can induce the mis-incorporation of incorrect nucleotides, so a large fraction of progeny virions from t-705-treated cells do not exhibit infectivity. t-705 has been approved for use against pandemic influenza infections in japan. all of the approved and clinical trial drugs mentioned above are synthesized, but natural products for anti-influenza virus drugs have been screened and studied. na has been targeted for the anti-influenza virus activity of natural products because high-throughput assay systems for measurements of na activity have been constructed. reported natural products that show inhibition of na can be classified into five groups: flavonoids, oligostibenes, coumarins, diarylheptanoids and others. 55 ha is also targeted for the screening of antiinfluenza virus drugs. a novel compound with a pentacyclic structure, stachyflin isolated from stachybotrys, inhibits conformational changes of ha at low ph, thereby inhibiting membrane fusion between virus and host cells. [56] [57] [58] hepatitis b virus according to the world health organization estimates in 2016, the annual number of deaths resulting from viral hepatitis is~1.4 million, which is comparable to that of hiv and tuberculosis; 47% of those deaths can be attributed to hbv. 59 approximately 257 million individuals are chronically infected with hbv worldwide and have an elevated risk of developing liver cirrhosis and hepatocellular carcinoma. 60 current antiviral drugs approved by the us food and drug administration include immunomodulators (interferon-α and pegylated interferon-α) and nucleos(t)ide analogs (lamivudine, adefovir, telbivudine, entecavir and tenofovir), which are useful for therapeutic treatment to interrupt the disease progression. however, these drug classes have certain limitations; for immunomodulators, a partial efficacy and side effects exist, and for nucleos(t)ide analogs, the emergence of drug-resistant virus strains can occur during long-term use. thus, an unmet need remains for the development of novel anti-hbv drugs. although microbial products with anti-hbv activities, as compared with phytochemicals, 61,62 are limited to date, microbial metabolites may represent a fundamental source for compounds that interfere with the virus life cycle, because of structural diversity and various biological activities. hbv is a small enveloped hepatotropic dna virus with an~3.2 kb partially double-stranded, relaxed circular dna genome that encodes polymerase, precore (hbv e antigen), core (capsid), surface antigens (small, middle and large) and hbx protein (transcriptional activator). as shown in figure 2 , the virus enters hepatocytes by binding to the cellular receptor(s), which includes sodium taurocholate cotransporting polypeptide. 63 following release of the nucleocapsid, relaxed circular dna is transported into the nucleus and converted into covalently closed circular dna (cccdna). the cccdna is quite stable in the form of a minichromosome and serves as a template for the transcription of all viral mrnas. among these mrnas, the 3.5 kb pregenomic rna is translated into polymerase and core proteins, which is then encapsidated together with polymerase that serves as a rna replicative intermediate. the pregenomic rna is reverse transcribed into minus-strand dna and degraded by the rnase h activity associated with the polymerase during this process. hbv polymerase subsequently synthesizes plus-strand dna with various lengths, which generates relaxed circular dna. finally, the matured nucleocapsids are released from infected cells after the acquisition of an envelope that contains the surface antigens in the endoplasmic reticulum and golgi apparatus to produce progeny virions, or to be recycled to the nucleus to supply the cccdna pool. to identify promising lead compounds for the drug development, it is important to select potential therapeutic targets for chronic hbv infection from the virus life cycle. although various anti-hbv drugs, which is beyond the scope of this review, are the subject of preclinical and clinical trials, 64,65 their inhibitory mechanisms should provide helpful data to make reasonable selections of such key targets for the drug screening for microbial products. potential applications of microbial products in anti-hbv drug development approved nucleos(t)ide analogs competitively inhibit dna elongation of viral polymerase after the conversion to triphosphate form by cellular enzymes. although nucleos(t)ide analog inhibitors can be highly effective in preventing liver disease progression, these drugs are not curative and require long-term use, which is associated with a risk of drug-resistance and side-effect. to overcome these drawbacks, several nucleos(t)ide analogs are currently in preclinical and clinical trials, which indicates that active site of the dna polymerization remains an indispensable drug target for hbv treatment. indeed, a new tenofovir derivative, tenofovir alafenamide (vemlidy), was recently approved and its improved pharmacological properties contribute to the reduction of nephrotoxicity. to date, microbial metabolites have yielded various nucleos(t)ide analogs, as mentioned above, including oxetanocin a and its derivative that exhibits anti-hbv activity. 66, 67 prospects for microbe-derived antiviral drugs n takizawa and m yamasaki the ideal goal of anti-hbv treatments is the loss of viral surface antigens, hbsag, which is correlated with dramatic reduction of incidence of hepatocellular carcinoma. however, current nucleos(t)ide inhibitors are ineffective in this regard, because they have little effects on the cccdna pool that serves as the template for hbsag expression. to improve treatment outcomes, novel anti-hbv agents that target other steps of viral dna synthesis, in combination with current hbv drugs, may promote cccdna decay and provide a higher barrier to resistance. 65, 68 the core protein self-assembles to form a nucleocapsid in which viral dna synthesis is carried out, and several inhibitors of this process are in phase i/ii clinical trials. furthermore, such inhibitors may facilitate cccdna decay because the strong inhibition of viral dna synthesis is thought to completely block the recycling of nucleocapsids into the nucleus for cccdna replenishment. 69, 70 thus, perturbing the capsid assembly process would be a promising approach for novel hbv treatment. a derivative of leucamide a, a cyclic heptapeptide derived from sponge, was found to interfere with this process. 71, 72 furthermore, rnase h-mediated pregenomic rna degradation is another essential step for the viral dna synthesis and a novel inhibitor of this activity is undergoing preclinical trials. 64 as the nucleolytic cleavage is dependent on a metal ion coordinated by the active site of rnase h, a class of metal-chelating inhibitors might have a potential for the development of novel rnase h inhibitors. although β-thujaplicinol, which showed inhibitory activity toward hbv rnase h, is of plant origin, 73, 74 such hydroxylated tropolones are often found in microbial metabolites. 75 taken together, it appears likely that microbial metabolites represent an enthralling resource of new chemical entities that target hbv dna synthesis. in general, natural product screening is a time-consuming process that requires the fractionation and isolation of active compound(s) from complex extracts of an unknown composition. recently, we developed a system to facilitate the detection of the replicating hbv genome using adenovirus vectors, 76 which can be used to quantitatively evaluate the inhibitory potency of a test compound in a shorter period of time than other model systems for studying hbv replication using cell lines. similarly, new efficient detection methods for virus replication would allow for the rapid drug screening of natural resources. as hbv infection is significantly influenced by various of host factors, host factor-targeted antivirals are prospective therapeutic approaches. 77, 78 myrcludex-b, a peptide that mimics the n-terminus of the pres1 in the large surface antigen that is involved in binding to sodium taurocholate cotransporting polypeptide, can block virus entry to hepatocytes and is currently in phase ii clinical trials. 79, 80 recently, cyclosporine a, a fungal cyclic polypeptide, and its analogs have been reported to inhibit the virus entry by targeting sodium taurocholate cotransporting polypeptide. 81, 82 such macrocyclic peptide scaffolds often have the advantage of occupying an interface that is required for protein-protein interactions, such as receptor binding and capsid assembly, as described above. moreover, host enzymes that are essential for virus replication represent promising drug targets. deoxynojirimycin is an iminosugar α-glucosidase inhibitor and its analogs have shown to reduce the hbv secretion from infected cells. 83, 84 iminosugars have a potential for development as antiviral drugs that disrupt the folding of viral glycoproteins by inhibiting host glucosidase, which is localized to the endoplasmic reticulum. 85 deoxynojirimycin is of plant origin, but this class of compounds is also found in microbial metabolites, such as siastatin b as described above. 29 although the effects of siastatin b on hbv replication are figure 2 life cycle of hepatitis b virus (hbv). hbv enters hepatocytes following binding to the cellular receptor(s) including sodium taurocholate cotransporting polypeptide (ntcp). after uncoating, relaxed circular dna (rcdna) within a released nucleocapsid is transported into the nucleus due to nuclear localization signal on the capsid. in the nucleus, rcdna is converted into cccdna by viral and cellular enzymes. the cccdna is quite stable in the form of a minichromosome that is responsible for persistent hbv infection and transcribed by host rna polymerase ii to generate four mrnas as follows: 3.5 kb pregenomic rna (pgrna) and precore mrna, 2.4 and 2.1 kb surface antigens mrna, and 0.7 kb hbx mrna. pgrna is translated into the viral polymerase and core proteins, which is then encapsidated together with polymerase. within the nucleocapsid, the pgrna is reverse transcribed into minusstrand dna and degraded by the rnase h activity associated with the polymerase followed by plus-strand dna synthesis. the 2.4 and 2.1 kb mrna are translated into large (lhbs), and middle (mhbs) and small (shbs) surface antigens, respectively. finally, the matured nucleocapsids are enveloped by lipid bilayer containing these surface antigens at the endoplasmic reticulum (er) and golgi apparatus and progeny virions are released from infected cells. in parallel, a huge amounts of subviral hbsag particles are assembled in the absence of nucleocapsid and released. the other 3.5 kb mrna is translated into precore protein that is proteolytically processed to hbeag. hbx has a multifunctional role in activation of various viral and cellular promoters and enhancers. a full color version of this figure is available at journal of antibiotics online. prospects for microbe-derived antiviral drugs n takizawa and m yamasaki presently unknown, host glucosidase involved in the antigen and virion secretion might be a suitable target for anti-hbv drug screening for microbial metabolites. furthermore, several therapeutic vaccines for the treatment of chronic hbv infection are in preclinical and clinical trials. this approach aims to stimulate either specific or nonspecific immune responses against hbv to eliminate the cccdna pool, while adjuvants may work to enhance vaccination. previously, bestatin (ubenimex), an immunomodulator discovered by imc, 86 has been reported to enhance immune responses for hiv vaccination in a murine model, 87 although its effect on hbv vaccination is unknown. compounds with adjuvant activity on hbv vaccination might be present in microbial metabolites. recently, deadly disease outbreaks caused by emerging and re-emerging viruses have become increasingly threatening, including outbreaks of nipah virus in malaysia, hendra virus in australia, hantavirus in the united states, ebola virus in africa, severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses in china and mideast, zika virus in oceania and americas, and the influenza virus h5n1, h1n1 and h7n2 subtypes. no efficient vaccine or specific antiviral drug for these emerging and re-emerging virus diseases is yet available. not only antiviral drugs against specific virus infections, but also broad-spectrum antiviral drugs need to be developed. here we summarize candidates of broad-spectrum antiviral drugs, nucleoside analogs and drugs against host proteins and discuss the potential of natural compounds for broad-spectrum antiviral drugs. nucleoside analogs are candidates for use as broad-spectrum antiviral drugs. ribavirin is known to be a broad-spectrum rna virus inhibitor and an antiviral candidate for emerging infectious diseases, such as dengue virus, norovirus, and hendra and nipah viruses. [88] [89] [90] the potential activity of ribavirin against broad-spectrum rna viruses can be exerted by both the direct and indirect inhibition of viral polymerase activity. ribavirin monophosphate inhibits inosine-5′-monophosphate dehydrogenase, which catalyzes the reaction from inosine-5′-monophosphate to xanthosine monophosphate, thereby decreasing intracellular gtp levels. 91 nucleotide pool imbalances that result from ribavirin treatment could cause a reduction of viral polymerase activity and the incorporation of alternative nucleotides for gtp. recent reports suggest that t-705 is also a broadspectrum rna virus inhibitor for viruses such as ebola virus. 92, 93 the detailed mechanism of the anti-viral activity of t-705 against these rna viruses is not known and merits further study. both bcx4430 and gs-5734 are nucleoside analogs and are the subject of clinical trials for ebola virus disease. 94, 95 they also exhibit broad-spectrum antiviral effects against rna virus families, including filoviruses, arenaviruses, paramyxoviruses and flaviviruses. [96] [97] [98] [99] as mentioned above, natural products have contributed to the development of antiviral nucleotide analogs. new classes of nucleotide analogs derived from natural products will be promising lead compounds for emerging viruses. drugs against non-viral proteins (that is, host proteins) are also broad-spectrum antiviral drug candidates. viruses use common cellular machinery from entry into to exit from host cells. inhibitors against such cellular machinery could inhibit viral propagation. these host targeted drugs are not yet approved as antivirals, but research and development are ongoing. rna viruses are known to use host membrane compartments for replication and trafficking. thus, lipid metabolism is a candidate target of antiviral drugs. statins, which were first isolated from penicillium citrinum, are hydroxymethylglutaryl-coenzyme a reductase inhibitors. treatment with statins decreases levels of low-density lipoprotein cholesterol. 100 antiviral effects of stains have been reported for hbv, hiv, influenza virus, dengue virus, human cytomegalovirus and hcv. [101] [102] [103] [104] [105] [106] the antiviral activity of statins may be a consequence of disruption of the membrane components that viruses use, along with changes in membrane trafficking. serine palmitoyltransferase inhibitors, myriocin isolated from myriococcum albomyces 107 and na255 isolated from fusarium incarnatum, 108 can also inhibit the propagation of hcv, hbv and influenza virus. [108] [109] [110] serine palmitoyltransferase acts in the sphingomyelin synthesis pathway and sphingomyelin is an essential component of 'lipid rafts'. myriocin inhibits the intracellular transport of influenza virus glycoproteins and na255 disrupts the hcv replication complex. drug repositioning (also known as drug repurposing) is of growing interest to reduced time and cost associated with drug developments. cyclosporine a (csa) is an approved immunosuppressive drug and it has been revealed that csa is effective against viruses such as hiv, human papillomavirus, influenza virus, hcv, coronaviruses and human cytomegalovirus. [111] [112] [113] [114] [115] [116] csa can be isolated from hypocladium inflatum gams and inhibits prolyl isomerase cyclophilins, which are implicated in the regulation of protein conformation. 117 the antiviral mechanism of csa involves the inhibition of the correct folding of viral proteins by cyclophilins. csa exerts immunosuppressive effects as the csa-cyclophilin a complex is formed by the inhibition of calcium-dependent phosphatase activity by calcineurin, which leads to the inhibition of key activators of t cells. therefore, csa derivatives that lack the inhibitory activity of calcineurin, but maintain the inhibitory activity of cyclophilins have been semi-synthesized. 118 a csa derivative, alisporivir (deb025), with ribavirin has been studied as an anti-hcv drug and is the subject of a clinical trial. 119 today, viral proteins are the major target of antiviral drugs. antiviral drugs against specific target proteins have been successful against important infectious diseases, such as hiv and hcv. with rapidly increasing knowledge of protein structure, development of in silico tools, and methods for chemical and genetic modification, more antiviral drugs will be developed that will contribute to the future elimination of specific infectious diseases. concurrently, we now face the threat of emerging and re-emerging infectious diseases with no available specific antiviral drug. to prevent a pandemic of these infections, broadspectrum antiviral drugs also need to be developed. natural products are a rich source and promising starting points to identify lead compounds for antivirals that target host proteins. studies of virushost interactions and 'omics' data from virus infected cells will help us to accelerate the development of screening systems and the identification of potential drug targets. we anticipate that novel antiviral drugs derived from microbial products will contribute to both the elimination of specific virus infections and emerging virus infections in the future. we dedicate this review to prof hamao umezawa. role of marine natural products in the genesis of antiviral agents a new antibiotic, formycin antiviral effect of formycin and formycin b mode of inhibition of coformycin on adenosine deaminase synthesis and anti-hiv activity of unusual nucleoside oxanosine derivatives the x-ray structure determination of oxanosine new antifungal antibiotics, benanomicins a and b inhibit infection of t-cell with human immunodeficiency virus (hiv) and syncytium formation by hiv kijimicin: an inhibitor of human immunodeficiency virus in acutely and chronically infected cells new bellenamine homologs inhibiting human immunodeficiency virus type i infectivity inhibition of human immunodeficiency virus-associated reverse transcriptase by 14-o-acyladriamycins inhibition of human immunodeficiency virus-associated reverse transcriptase by aminoacridines differential effect of phleomycin on the infectivity of poliovirus and poliovirus-induced ribonucleic acids conservation of multifunctional ribosomal protein metallopanstimulin-1 (rps27) through complex evolution demonstrates its key role in growth regulation in archaea, eukaryotic cells, dna repair, translation and viral replication phage inactivation by aclacinomycin a and its analogues clazamycin b is antibiotic 354 an improved screening method for antiphage antibiotics and isolation of sarkomycin and its relatives modes of action of phleomycin, bleomycin and formycin on hela s3 cells in synchronized culture new protease inhibitors from actinomycetes cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss2 expression antipain, a new protease inhibitor isolated from actinomycetes elastatinal, a new elastase inhibitor produced by actinomycetes inhibition of proteolytic activity of poliovirus and rhinovirus 2a proteinases by elastase-specific inhibitors bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores pepstatin, a new pepsin inhibitor produced by actinomycetes effective blocking of hiv-1 proteinase activity by characteristic inhibitors of aspartic proteinases rational design of peptide-based hiv proteinase inhibitors purification and characterization of a sialidase inhibitor, siastatin, produced by streptomyces synthesis of 3-episiastatin b analogues having anti-influenza virus activity world health organization media centre. influenza (seasonal) fact sheet constraints, drivers, and implications of influenza a virus reassortment antiviral activity of 1-adamantanamine (amantadine) multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza a m2 proton channel structural basis for proton conduction and inhibition by the influenza m2 protein inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-n-acetylneuraminic acid structure of influenza virus neuraminidase b/lee/40 complexed with sialic acid and a dehydro analog at 1.8-a resolution: implications for the catalytic mechanism molecular modeling studies on ligand binding to sialidase from influenza virus and the mechanism of catalysis characterisation of an ionisable group involved in binding and catalysis by sialidase from influenza virus rational design of potent sialidase-based inhibitors of influenza virus replication influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity penetration of gs4071, a novel influenza neuraminidase inhibitor, into rat bronchoalveolar lining fluid following oral administration of the prodrug gs4104 cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities cs-8958, a prodrug of the new neuraminidase inhibitor r-125489, shows long-acting anti-influenza virus activity. antimicrob. agents chemother influenza virus rna polymerase: insights into the mechanisms of viral rna synthesis preclinical activity of vx-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase pb2 subunit. antimicrob. agents chemother discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 broad-spectrum antiviral activity of virazole: 1-f8-d-ribofuranosyl-1,2,4-triazole-3-carboxamide inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate in vitro and in vivo activities of anti-influenza virus compound t-705 mechanism of action of t-705 against influenza virus the ambiguous basepairing and high substrate efficiency of t-705 (favipiravir) ribofuranosyl 5'-triphosphate towards influenza a virus polymerase t-705 (favipiravir) induces lethal mutagenesis in influenza a h1n1 viruses in vitro mechanism of action of t-705 ribosyl triphosphate against influenza virus rna polymerase influenza neuraminidase: a druggable target for natural products stachyflin and acetylstachyflin, novel anti-influenza a virus substances, produced by stachybotrys sp. rf-7260. ii. synthesis and preliminary structure-activity relationships of stachyflin derivatives stachyflin and acetylstachyflin, novel anti-influenza a virus substances, produced by stachybotrys sp. rf-7260. i. isolation, structure elucidation and biological activities a new strategy toward the total synthesis of stachyflin, a potent anti-influenza a virus agent: concise route to the tetracyclic core structure world health organization. global health sector strategy on viral hepatitis world health organization media centre. fact sheets a review of non-nucleoside anti-hepatitis b virus agents naturally derived anti-hepatitis b virus agents and their mechanism of action sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus hepatitis b virus therapy: what's the future holding for us? effect of oxetanocin g, a novel nucleoside analog, on dna synthesis by hepatitis b virus virions anti-hepatitis b virus activities of purine derivatives of oxetanocin a emerging antivirals for the treatment of hepatitis b hepadnavirus envelope proteins regulate covalently closed circular dna amplification control of cccdna function in hepatitis b virus infection isothiafludine, a novel non-nucleoside compound, inhibits hepatitis b virus replication through blocking pregenomic rna encapsidation effect of a hepatitis b virus inhibitor, nz-4, on capsid formation β-thujaplicinol inhibits hepatitis b virus replication by blocking the viral ribonuclease h activity hydroxylated tropolones inhibit hepatitis b virus replication by blocking viral ribonuclease h activity studies on lipoxygenase inhibitors. i. my3-469 (3-methoxytropolone), a potent and selective inhibitor of 12-lipoxygenase, produced by streptoverticillium hadanonense ky11449 efficient genome replication of hepatitis b virus using adenovirus vector: a compact pregenomic rna-expression unit host factor-targeted hepatitis b virus therapies recent insights into hepatitis b virus-host interactions efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein the entry inhibitor myrcludex-b efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis b virus cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilin-independent interference with the ntcp receptor cyclosporin a and its analogs inhibit hepatitis b virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (ntcp) secretion of human hepatitis b virus is inhibited by the imino sugar n-butyldeoxynojirimycin treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking iminosugar antivirals: the therapeutic sweet spot bestatin, an inhibitor of aminopeptidase b, produced by actinomycetes adjuvant effect of ubenimex on a dna vaccine for hiv-1 rna synthesis during infection by hendra virus: an examination by quantitative real-time pcr of rna accumulation, the effect of ribavirin and the attenuation of transcription interferons and ribavirin effectively inhibit norwalk virus replication in replicon-bearing cells antiviral effects if ribavirin and 6-mercapto-9-tetrahydro-2-furylpurine against dengue viruses in vitro mechanism of action of 1--d-ribofuranosyl-1,2,4-triazole-3-carboxamide (virazole), a new broad-spectrum antiviral agent successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model post-exposure efficacy of oral t-705 (favipiravir) against inhalational ebola virus infection in a mouse model protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys antiviral activity of the adenosine analogue bcx4430 against west nile virus and tick-borne flaviviruses bcx4430, a novel nucleoside analog, effectively treats yellow fever in a hamster model efficacy of the broad-spectrum antiviral compound bcx4430 against zika virus in cell culture and in a mouse model gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ml-236a and ml-236b fungal metabolites, having hypocholesterolemic activity statins reduce dengue virus production via decreased virion assembly different anti-hcv profiles of statins and their potential for combination therapy with interferon protective effect of fluvastatin on influenza virus infection statin compounds reduce human immunodeficiency virus type 1 replication by preventing the interaction between virion-associated host intercellular adhesion molecule 1 and its natural cell statin compounds reduce human immunodeficiency virus type 1 replica simvastatin potentiates the anti-hepatitis b virus activity of fda-approved nucleoside analogue inhibitors in vitro inhibitory effects of statins on expression of immediate-early 1 protein of human cytomegalovirus in virus-infected cells myoriocin, a new antifungal antibiotic from myriococcum arbomyces host sphingolipid biosynthesis as a target for hepatitis c virus therapy host sphingolipid biosynthesis is a promising therapeutic target for the inhibition of hepatitis b virus replication intact sphingomyelin biosynthetic pathway is essential for intracellular transport of influenza virus glycoproteins cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein l1 from the l2/dna complex following virus entry inhibition of human immunodeficiency virus and growth of infected t cells by the immunosuppressive drugs cyclosporin a and fk 506 the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporin a inhibits the influenza virus replication through cyclophilin a-dependent and -independent pathways cyclophilin a is required for efficient human cytomegalovirus dna replication and reactivation cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes mechanisms of action of cyclosporine cyclophilin inhibitors as antiviral agents the combination of alisporivir plus an ns5a inhibitor provides additive to synergistic anti-hepatitis c virus activity without detectable crossresistance we thank all members and alumni of imc, particularly dr akio nomoto. the authors declare no conflict of interest. key: cord-265472-b1s4stvz authors: guimarães, luísa eça; baker, britain; perricone, carlo; shoenfeld, yehuda title: vaccines, adjuvants and autoimmunity date: 2015-10-31 journal: pharmacological research doi: 10.1016/j.phrs.2015.08.003 sha: doc_id: 265472 cord_uid: b1s4stvz abstract vaccines and autoimmunity are linked fields. vaccine efficacy is based on whether host immune response against an antigen can elicit a memory t-cell response over time. although the described side effects thus far have been mostly transient and acute, vaccines are able to elicit the immune system towards an autoimmune reaction. the diagnosis of a definite autoimmune disease and the occurrence of fatal outcome post-vaccination have been less frequently reported. since vaccines are given to previously healthy hosts, who may have never developed the disease had they not been immunized, adverse events should be carefully accessed and evaluated even if they represent a limited number of occurrences. in this review of the literature, there is evidence of vaccine-induced autoimmunity and adjuvant-induced autoimmunity in both experimental models as well as human patients. adjuvants and infectious agents may exert their immune-enhancing effects through various functional activities, encompassed by the adjuvant effect. these mechanisms are shared by different conditions triggered by adjuvants leading to the autoimmune/inflammatory syndrome induced by adjuvants (asia syndrome). in conclusion, there are several case reports of autoimmune diseases following vaccines, however, due to the limited number of cases, the different classifications of symptoms and the long latency period of the diseases, every attempt for an epidemiological study has so far failed to deliver a connection. despite this, efforts to unveil the connection between the triggering of the immune system by adjuvants and the development of autoimmune conditions should be undertaken. vaccinomics is a field that may bring to light novel customized, personalized treatment approaches in the future. vaccines have been a preventive treatment option available for over 200 years. they have been proven to be effective in preventing infections that previously had high morbidity and mortality. an example of this is the eradication of small pox, which was mainly attributed to successful vaccination programs. preventing a high burden disease has since proven to be a cost effective measure and, as such, vaccines have become a part of multiple national health programs. these promising results led to the development of more and more vaccines and to the study of its applicability in other fields such as cancer prevention and treatment. vaccines are drugs administered to healthy individuals, and much like other drugs, vaccines are associated with adverse events. usually the described adverse events are transient and acute, but may rarely present with hypersensitivity and induction of autoimmunity that may be severe and fatal. these adverse events play an important role in the life of the vaccinated patients. immune mediated diseases arise from various different sources; these include environmental, genetic, hormonal and immune defects. the combination of these defects can be described as the mosaic of autoimmunity [1] . patient background can be used as a clue to determine the response that may be elicited following drug administration. it has been proven that infectious agents may elicit an autoimmune disease in a prone subject through various mechanisms, including, but not limited to, molecular mimicry, epitope spreading and polyclonal activation [2] . scientific findings suggest that autoimmunity may be triggered by vaccine adjuvants, of which aluminum compounds (aluminum hydroxide and phosphate) have been the most studied and the most widely used. adjuvants are molecules, which, in combination with antigens, enhance immunological response. this enables an easier and more effective recognition of "non self", which in turn permits the triggering of adaptive and innate immune responses [3] . recently a new syndrome was described: "autoimmune/inflammatory syndrome induced by adjuvants" (asia). this embodies a spectrum of reactions, which are usually mild but may also be severe. these reactions are attributed to adjuvant stimulation, which can include chronic exposure to silicone, tetramethylpentadecane, pristane, aluminum, infectious components and other adjuvants. all of these environmental factors have been found to induce autoimmunity and inflammatory manifestations by themselves both in animal models and in humans. the mechanisms of this disease will be described in further detail [4] . this review will focus on general mechanism of vaccines, adjuvant-induced autoimmunity, and on vaccines and the specific autoimmune diseases that they may trigger. adjuvants approved to date for human vaccines are: aluminum, mf59 in some viral vaccines, mpl, as04, as01b and as02a against viral and parasitic infections, virosomes for hepatitis b virus (hbv), human papilloma virus (hpv), hepatitis a virus (hav), and cholera toxin for cholera. adjuvants may be composed of several different compounds. currently, oil based adjuvants, virosomes, toll-like receptors (tlrs) related adjuvants, mpl, adjuvants made of unmethylated cpg dinucleotides and tuftsin have all been described. it is of great interest the understanding of the mechanisms related to the adjuvant effect, as well as to aluminum salts. aluminum acts through multiple pathways, which do not depend solely on tlrs signaling. each of these pathways leads to an enhanced host immune response [5] . there are many oil based adjuvants. one is incomplete freund adjuvant (ifa), which contains water in oil emulsion. another is complete freund adjuvant (cfa), which is the same as ifa, except that it also contains killed mycobacteria in addition to water in oil emulsion. usually, cfa is used for primary vaccination and ifa for boosting. recent oil based adjuvants that have been developed are mf59 (novartis ® ), as03 (glaxosmithkline ® ), advax tm which are based on inulin compounds (vaxine tm pty) and qs-21/iscoms, which are immune stimulating complexes composed of cholesterol and phospholipid with or without antigen (table 1) . virosomes are adjuvants that contain a membrane-bound hemagglutinin and neuraminidase obtained from the influenza virus. both components facilitate the uptake into antigen presenting cells (apc) and mimic the natural immune response [6] . leucocyte membranes have membrane bound pattern recognition receptors (prrs) called tlrs, which are responsible for detecting most (although certainly not all) antigen-mediated infections. their activation leads to adaptive immune responses. for this reason, many adjuvants that are used today are directed to prrs. these adjuvants are called tlrs related adjuvants [7] . mpl is a series of 4'monophosphoryl lipid a obtained from the purification of a modified lipopolysaccharide (lps) of salmonella minnesota. bacterial deoxyribonucleic acid (dna) is immunostimulatory due to unmethylated cpg dinucleotides. vertebrate dna has relatively low amounts of unmethylated cpg compared to bacterial dna. the adjuvant effect of cpg is enhanced when conjugated to protein antigens. this adjuvant is being tested in vaccines directed at infectious agents, allergens and tumor cells [8] [9] [10] . another type of adjuvant is tuftsin. tuftsin is an auto adjuvant, which is a natural self-immunostimulating tetrapeptide (thr-lys-pro-arg). this tetrapeptide is a fraction of the igg heavy chain molecule produced by enzymatic cleavage in the spleen [11] . its functions include: binding to receptors on neutrophils and macrophages to stimulate their phagocytic activity, increasing tumor necrosis factor alpha (tnf␣) release from human kupffer cells enhancing secretion of il1 by activating macrophages, activation of macrophages expressing nitric oxide (no) synthase to produce no and enhancement of murine natural cell mediated cytotoxicity in vitro [11] [12] [13] . in summary, it is an adjuvant with minor side effects with a promising effect in restoring innate immune mediated response. adjuvants may exert their immune enhancing effects according to five immune functional activities: 1. translocation of antigens to the lymph nodes where they can be recognized by t cells. 2. antigen protection enabling longer exposure. 3. enhanced local reaction at the injection site. 4. induction of the release of inflammatory cytokines. 5. interaction with prrs, specifically tlrs [22] . a adjuvant effect the term "adjuvant effect" refers to the co-administration of an antigen with a microbial specific factor to enhance an antigenspecific immune response in vivo. the microbial components of adjuvants activate apcs to produce pro-inflammatory cytokines ("non-specific" signal 2) and to up-regulate molecules essential for antigen presentation. these molecules include major histocompatibility complex (mhc) class ii (antigen-specific signal 1) and b7-1/2. these innate immune events allow a more effective presentation to the adaptive immune system, resulting in an augmented activation and clonal expansion of t cells [23] . in accordance to this effect, if self-antigens are used, an autoimmune response can be elicited [24] . it has been shown that auto-reactive t-cells that surpass tolerance mechanisms can be triggered by exogenous adjuvants to become auto-aggressive [25] . infectious agents are able to naturally generate their adjuvant effect and can induce autoimmunity [26] . an example of this is the causality between viral infection and myocarditis. half the cases of myocarditis are preceded by an acute viral infection. infectious myocarditis in humans can be reproduced in experimental murine models of myocarditis [27] . it has also been shown that the autoimmune reaction elicited by an infectious agent can be effective in treating cancer. an example of this is that bladder administration of bcg (bacille calmette-guérin) has been shown to be effective against superficial bladder cancer development [28] . it can be inferred that the adjuvant effect can be used against specific tumor derived molecules, so that these molecules can be recognized as "non self". prr-pamp (pattern recognition receptor-pathogen-associated molecular patterns) interactions activate the apcs to promote antigen-specific lymphocytic responses [29] . the definition of pamps has now broadened, in that the recognized structures do not need to be pathogens. thus the concept of "microbe-associated molecular patterns" (mamps) and of "danger/damage-associated molecular patterns" (damps) were defined based on the notion that the endogenous host molecules signal danger or damage to the immune system [30] . tlrs are single-transmembrane prrs localized on cell surface and endosomal membranes. from all the prrs, these are the most studied. tlrs play a crucial role in innate immune response to "non self" and are biosensors of tissue damage. the interaction between the four known tlrs adapters: myd88, tirap/mal, tram and trif, in tlr signaling, shape the innate immune response. besides prrs the innate immune system also detects proteolytic enzymes generated during infection [31] . merging the response to different prrs signaling may be the pathway for developing customized responses to different aggressions [32] . b experimental models of adjuvants many animals have been used in experimental models of adjuvant-related autoimmune conditions [33] . these include primates, salmons, rabbits and swine; however, the most common are murine models. murine models include autoimmune prone strains, models of autoimmune disease and autoimmune resistant strains ( table 2 ). an interesting model is that described by lujan et al. the authors described that a commercial sheep, inoculated repetitively with aluminum-containing adjuvants vaccinations, developed an acute neurological episode with low response to external stimuli and acute meningoencephalitis few days after immunization. an excitatory phase, followed by weakness, extreme cachexia, tetraplegia and death appeared. this was suggested to be part of the spectrum of asia syndrome. moreover, the biopsy of the nervous tissue of experimental animals indicated the presence of alum [48] . c toxicity of aluminum adjuvants aluminum nanoparticles have both a unique capacity of surpassing the blood brain barrier (bbb) and of eliciting immune inflammatory responses. these are probably the reasons why aluminums' most sensitive target is the brain, and also why documented side effects are mostly neurologic or neuropsychiatric [49, 50] . aluminum is present in nature, not only as a vaccine adjuvant, but also in food, water and cosmetics. it has been described as a neurotoxin because even when a relatively small amount of aluminium reaches the brain [49] , is can act as a genotoxin [51] , a prooxidant [52] , it can be proinflammatory [51] , act as an immunotoxin [5] and also as an endocrine disruptor [53] . aluminum interferes with many essential cellular processes. memory, concentration, speech deficits, impaired psychomotor control, reduced seizure tolerance and altered behaviour are manifestations of aluminium neurotoxicity. moreover, alzheimer's [54] , amyotrophic lateral sclerosis, parkinsonism dementia [55] , multiple sclerosis [56] , and neurological impairments in children have been linked to aluminum neurotoxicity [57] . brain susceptibility to aluminum compounds is possibly due to the brain's high metabolic requirement, to the fact that it possesses a large area of biological membranes and to the relatively low concentration of antioxidants [54] . aluminum adjuvants exert their immunostimulatory effect through many different pathways that activate both the innate and adaptive immune systems. one of the most significant is the activation of the nlrp3 inflammasome pathway [58] . nlpr3 activation has been shown to trigger type 2 diabetes. by using nlpr3 knockout mice it has been demonstrated that the absence of inflammasome components leads to a better maintenance of glucose homeostasis and higher insulin sensitivity [59] . on the other hand, activation of the inflammasome and its downstream components: pro-inflammatory cytokines il-1␤ and il-18 are strongly implicated in the development of several central nervous system (cns) disorders [60] . the vast majority of people are consuming higher amounts of aluminum through dietary and parenteral intake than what expert authorities consider safe. upper limits set by us food and drug administrations (fda) for aluminum in vaccines are set at no more than 850 g/dose. these values were not based on toxicity studies, but on the minimum amount needed for aluminum to exert its effect as an adjuvant [51] . the quantities of aluminum to which infants, in their first year of age are exposed, have been considered safe by the fda. however the scientific basis for this recommendation does not take into account aluminum persistence in the body. the concern about aluminum in dietary intake has been reinforced by the food and agriculture (fao) who expert committee, which lowered the provisional tolerable weekly intake of aluminum from 7 mg/kg/bw (490 mg/week, for an average 70 kg human) to 1 mg/kg/bw (70 mg/week) [61] . the amount of dietary intake of aluminum has risen in urban societies to up to 100 mg/day considering the widespread use of processed convenience foods. however, only about 0.25% of dietary aluminum is absorbed into systemic circulation and most of it is thereafter eliminated through the kidneys [54] . absorption of aluminum by the skin from ointments and cosmetics containing aluminum has been shown. moreover, the presence of aluminum in breast tissue was associated with breast cancer [62] . aluminum compounds persist for up to 8-11 years post vaccination in human body. this fact, combined with repeated exposure, may account for a hyper activation of the immune system and subsequent chronic inflammation [63] . the clinical and experimental evidence collected so far identify at least three main risks associated with aluminum in vaccines: 1. it can persist in the body. 2. it can trigger pathological immunological responses. 3. it can pass through the bbb into the cns where it can trigger immuno-inflammatory processes, resulting in brain inflammation and long-term neural dysfunction. there is a link between allergies and autoimmunity since both are the result of an abnormal immune response [3, 4] . metals such as mercury, aluminum, nickel and gold are known to induce immunotoxic effects in humans. the immunologic effects of these metals include immunomodulation, allergies and autoimmunity. they may act either as immunosuppressants or as immune adjuvants. metals bind firmly to cells and proteins and thus have the ability to modify autologous epitopes (hapetenization). t-cells then recognize the proteins as foreign and trigger an autoimmune response [64] . hypersensitivity caused by metals may be referred to as type iv delayed hypersensitivity. the reaction is considered delayed because the first symptoms appear 24-48 h after exposure, because it is mostly t-cell mediated and the gold standard for diagnosis of delayed type hypersensitivity is patch testing [65] . in mercury-sensitized patients, even mercury concentrations within the normal range might provoke neuroallergic reactions in the brain [66] . identifying metal sensitivity and removal of the sensitizing metals, such as dental amalgam, have been proved successful by showing symptom improvement in patients with previous autoimmune diseases. these diseases included fibromyalgia, autoimmune thyroid diseases and orofacial granulomatosis [67] [68] [69] [70] (table 3 ). the timeline regarding the field of vaccinology has been divided in two generations, the first regarding the administration of inactivated pathogens in whole or live attenuated forms (e.g., bacillus calmette guerin (bcg), plague, pertussis, polio, rabies, and smallpox) and the second regarding vaccines assembled from purified microbial cell components, also referred as subunit vaccines (e.g., polysaccharides, or protein antigens) [78] . this latter approach there are obstacles to conventional vaccine development methods such as non-cultivable in vitro pathogens (e.g., hepatitis c, papilloma virus types 16 and 18, and mycobacterium leprae), antigen hypervariability (e.g., serogroup b meningococcus, gonococcus, malaria), opportunistic pathogens (e.g., staphylococcus aureus) and rapid evolving pathogens such as human immunodeficiency virus (hiv) [79] . vaccine research gained a new perspective as the genomics field emerged over the last decades. bacterial genomes have been sequenced and analyzed making it possible to choose the best candidate vaccine antigens by using the concept of reverse vaccinology [80] . the main known factors influencing the observed heterogeneity for immune responses induced by vaccines are gender, age, ethnicity, co-morbidity, immune system, and genetic background. the interaction between genetic and environmental components will dictate the response to vaccines. studying the vaccine and the host will enable the development of customized treatment options. the combination of genetics, epidemiology and genomics in vaccine design has been denominated "vaccinomics" [81] . the importance of genetic influence is supported by twins and siblings studies, which show familial aggregation. this suggests that genomics is crucial in inter-individual variations in vaccine immune responses [82] . both human leukocyte antigen (hla) and non-hla gene markers have been identified as markers for immune response to vaccines. multiple studies have shown connections between hla gene polymorphisms and non-responsiveness to the hbv vaccine [83] . hla region is divided in three sub regions: class i is associated with the induction and maintenance of cell-mediated immune response, class ii is associated with presentation of exogenous antigens to helper t cd4+ cells and class iii, where immune non hla related genes are located. normal human tissue has at least 12hla antigens, and although new recombinant haplotypes may occur, it is inherited mostly intact from progenitors [84] . hla allelic differences are associated with different responses to vaccines, either by hyper or hypo responsiveness. we can infer that a similar response may be associated with different safety in relation to the development of autoimmune reactions to vaccines, particularly in the patients with genetic predisposition to an enhanced response to vaccine inoculation [85] . furthermore, patients that share the same hla, for instance siblings, have been diagnosed with asia following similar environmental stimuli [86, 87] . autoantibodies help to diagnose certain autoimmune diseases, however, they can also be found in healthy individuals. thus, autoimmune diseases cannot be diagnosed based solely on antibody detection [88] . inoculation of vaccines triggers autoimmune responses that result in the development of autoantibodies. many studies have been carried out in animals, healthy subjects and patients with autoimmune diseases to understand if this development is of clinical significance [89] [90] [91] [92] . a difference in eliciting the production of autoantibodies in healthy humans has been observed between adjuvanted and non-adjuvanted influenza vaccines [93] . the annual influenza vaccine has been the most heavily researched vaccine, along with hpv and pneumococcal vaccines as far as their relationship with patients who have previously been diagnosed with an autoimmune disease [94] [95] [96] . autoantibody induction after hpv vaccination was also shown in adolescent girls with systemic lupus erythematosus (sle) [97] . although induction of autoantibodies was proven following vaccine administration, there have been no proven relation with disease diagnosis in either of the specific groups studied so far [92, 98] . it has been widely demonstrated that autoantibodies can develop years before the manifestation of a full-blown autoimmune disease [99] . moreover, the development of a specific autoantibody is also genetically determined, and the link between genetic, autoantibodies and vaccines may become an even more intriguing area of research [100] . silicones are synthetic polymers that can be used as fluids, emulsions, resins and elastomers making them useful in diverse fields. they were thought to be biologically inert substances and were incorporated in a multitude of medical devices such as joint implants, artificial heart valves, catheters, drains and shunts. of all the silicone-containing products, the most famous are most likely breast implants. silicon is one of the substances suspected to induce asia [5] . it is currently believed that exposure alone is not enough to trigger the disease but that it requires the presence of additional risk factors (e.g., genetic susceptibility, other environmental factors) [4] . silicone exerts local tissue reactions. some of these reactions are considered para-physiological, such as capsular tissue formation around an implant. other reactions are viewed as abnormal, like when capsular contractures and allergic reactions to silicone or platinum (catalyst used in silicone polymerization found in minute concentrations in implants) occur [101] . cutaneous exposure to silicone with cosmetics or baby bottles could potentially sensitize patients [102] . there is also a systemic component of silicone exposure related to diffusion of silicone through the elastomer envelope, commonly termed "bleeding". it may arouse systemic effects as it degrades and fragments in tissue, it can also spread throughout the body and lead to the development of cancer or autoimmune phenomena [103] . patients with ruptured implants complain more frequently of pain and chronic fatigue when compared to patients with intact implants [104] . anti-silicone antibodies were found to be present in human sera more frequently in patients who have undergone silicone breast implants, however, their pathological significance remains uncertain [105] . the same was seen for other antibodies such as autoantibodies directed against dsdna, ssdna, ssb/la, silicone and collagen ii, which were found to be present in increased levels in patients after exposure to silicone [106] . it has also been shown that the formation of autoantibodies is directly related to implant duration. several autoimmune diseases have been linked to silicone exposure including rheumatoid arthritis (ra), systemic lupus erythematosus (sle), polymyositis, systemic sclerosis (ssc) and fibromyalgia. although asia symptoms may arise 24 years after the onset of exposure to silicone implants [107] , most of the follow-up periods are short and concluding evidence is yet to come regarding this causality. there have been published case reports, epidemiologic and research studies that suggest a connection between several vaccines and certain autoimmune conditions, notwithstanding that, overall the benefits of vaccination outweigh the risks. thrombocytopenia has been reported as the main adverse event following mmr vaccine. after mmr vaccine the onset of immune thrombocytopenic purpura (itp) usually occurred within 6 weeks at a risk rate of 1:22,000-25,000 mmr vaccine doses, while the incidence of itp following infections is 1:6000 for measles and 1:3000 for rubella [108] . as the risk of thrombocytopenia is higher in patients who experience natural infection with measles, mumps or rubella than in those receiving the vaccine, vaccination is encouraged. arthralgia complaints have also been reported and they may present as transient arthralgia, acute arthritis and rarely chronic arthritis [109] . some risk factors have been found to be associated with the development of arthritis in vaccinated patients such as: female gender, older age, prior seronegativity and specific hla alleles [110] . yf vaccine is only advisable to people in, or going to endemic areas. the risk of developing yf vaccine-associated neurologic disease (yel-and) is inversely proportional to age [111] . this is why children aged <6 months cannot be vaccinated and <8 months, except during epidemics [112] . vaccination is not advisable to people >60 years because of possible higher risk of severe adverse effects (saes) even though the incidence remains low [113] . besides being a vaccine for mycobacterium tuberculosis (tb), the bcg has proved effective as immunotherapy for bladder cancer. although the mechanism is yet to be fully understood, it is thought that bcg binds to fibronectin forming complexes that enable the recognition as "non-self" by the innate immune response of th1 cells. ultimately the pathways result in the apoptosis of tumor cells [114] . because of its effect in treating non-muscle-invasive urothelial carcinoma, as well as superficial bladder tumors, it was expected that bcg could play a role in treating other types of cancer, despite data having not corroborated this hypothesis so far. adverse events vary according to the site and method of administration. intradermal administration of bcg has been reported to elicit arthritis [115] , dermatomyositis [116] and takayasu's arteritis (ta) [117] among others. intravesical treatment for bladder cancer can cause reactive arthritis (rea) [118] . the risk relies on a systemic reaction composed of an early infective phase (pcr positive and response to anti-tb treatment) and a late hypersensitivity reaction [119] . hbv is a dna virus of the hepadnaviridae family, responsible for acute and chronic liver disease. hbv vaccines are considered the first efficient vaccines against a major human cancer. hbv vaccines have reduced the risk of developing chronic infection and they also have proved to reduce the incidence of liver cancer in children [120] . the vaccine has been associated mainly with autoimmune neuromuscular disorders. they include, but are not limited to: optic neuritis, guillain-barre syndrome (gbs), myelitis and multiple sclerosis (ms), systemic lupus erythematosus (sle), arthritis, vasculitis, antiphospholipid syndrome (aps) and myopathy [121] . hbv vaccine is the most common immunization associated with acute myelitis. there are studies that indicate that the pathogenicity behind such vaccine and autoimmunity might be based on cross-reactivity between hbv antigen (hbsag) epitopes, yeast antigens, as well as other adjuvants contained in the vaccine itself [122] . up to 90% of cervical cancer deaths, occur in developing countries that lack the ability to fully implement the papanicolau (pap) screening programs. hpv poses a special challenge in vaccine safety. hpv is necessary for the development of cervical cancer. however, most women infected with hpv will not develop the disease since 70% of infections will resolve within a year and up to 90% within 2 years without specific treatment. over the course of decades, cancer may result in a small proportion of the remaining infected women. death rate from cervical cancer in 9-20 year old girls is zero and long-term benefits are yet to be proven. in this specific case, short term risks to healthy subjects can prove to pose a heavier burden than cervical cancer [123] . there are at least 100 types of hpv strains, 15 of which have been pathologically associated with cancer. two vaccines, gardasil tm and cervarix tm , are commercially available against hpv. both contain the l1 capsid proteins of several hpv strains as antigens. gardasil tm contains serotypes 16, 18, 6, 11. these antigens are combined with aluminum (al) hydroxyphosphate sulphate as an adjuvant. cervarix tm contains a combination of the oil-based adjuvant monophosphoryl lipid a (mpl) and al hydroxide (aso4) as adjuvant and is directed at strains 16 and 18 [124] . there have been several reports of post-licensure adverse events, some of which have even been fatal [125] . compared to other vaccines, an unusually high proportion of adverse drug reactions has been reported associated with hpv vaccines [126] . in 2008, australia reported an annual adr rate of 7.3/100,000, the highest since 2003. this increase was almost entirely due to adrs reported following the commencement of the national hpv vaccination program for females aged 12-26 years in april 2007 (705 out of a total of 1538 adrs records). the numbers only decreased after the cessation of the catch-up schedule. although the percentage of convulsions attributable to the hpv vaccine decreased, the overall report remained comparable between 2007 and 2009 (51% and 40% respectively). these reports do not prove the association, but show that there is a higher frequency of adrs related to hpv vaccines reported worldwide, and that they fit a consistent pattern (i.e., nervous system-related disorders rank the highest in frequency) that deserves further investigation [126] [127] [128] . indeed, several autoimmune diseases have been linked to hpv immunization. examples include gbs, ms, acute disseminated encephalomyelitis (adem), transverse myelitis (tm), postural orthostatic tachycardia syndrome (pots), sle, primary ovarian failure (pof), pancreatitis, vasculitis, immune thrombocytopenic purpura (itp) and autoimmune hepatitis (ah) [123] . influenza is an acute viral infection that affects the respiratory tract and is caused by influenza type a-c viruses of the orthomyxoviridae family [129] . h1n1 mortality rates in the 2009 outbreak showed high risk in those aged 70 years and older, presence of chronic diseases and delayed admission. risk of infection was lower in those who had been vaccinated for seasonal influenza with 2008/9 trivalent inactivated vaccine [130] . studies have demonstrated that influenza vaccine is safe and immunogenic in patients with sle or rheumatoid arthritis (ra), diminishing the risk of respiratory infections [129] . it has been shown that adjuvanted vaccine had more local reactions but did not increase systemic adverse reactions [131] . molecular mimicry has been suggested as a mechanism to explain an autoimmune response following influenza vaccination. however, a causal relationship between influenza vaccines and induction of autoimmune diseases remains unproved [129] . diseases or symptoms reported after influenza vaccination include mostly neurological syndromes such as gbs [ref] . nonetheless, influenza vaccines should be recommended for patients with ms, because influenza infection is associated with increased risk of exacerbations. that being said, influenza vaccinations showed increased risk of autoimmune responses suggestive of asia [132] , vasculitis [133] and aps [134] among others. meningococcal disease is caused by neisseria meningitidis. one of the following five serogroups causes almost every invasive disease: a-c, y, and w-135. vaccines available so far for its prevention encompass either pure polysaccharide vaccines that use purified bacterial capsular polysaccharides as antigens, or protein/polysaccharide conjugate vaccines, which use the polysaccharide molecule plus diphtheria or tetanus toxoid as tcell-stimulating antigens. n. meningitidis serogroup b (menb) menb glycoconjugate vaccines are not immunogenic and hence, vaccine design has focused on sub-capsular antigens [135] . menb capsular polysaccharide is composed of a linear homopolymer of ␣(2 → 8) n-acetyl-neuroaminic acid (polysialic acid; psa). menb psa and psa found on neural cell adhesion molecules are structurally identical. as a result of this, it has been proposed that infection with menb or vaccination with psa may be associated with subsequent autoimmune or neurological disease [136] . no evidence of increased autoimmunity was found to be associated with meningococcal serogroup b infection [136] . regarding vaccination, the inoculation does not cause autoimmune diseases but may unmask autoimmune phenomena in genetically predisposed individuals. local reactions are more frequent in individuals vaccinated with quadrivalent meningococcal conjugate vaccines compared to plain polysaccharide vaccines. the intramuscular administration of the conjugate vaccine (versus subcutaneous for that of polysaccharide) may, in part, explain the higher reactivity [137] . diseases previously associated with meningococcal vaccines are gbs [138] , henoch-schönlein purpura (hsp) [139] and bullous pemphigoid (bp) [140] . streptococcus pneumoniae (pneumococcus) is the main cause of bacterial community-acquired pneumonia and meningitis in western countries, as well as the cause of more than 800,000 children deaths in developing countries [141, 142] . there are three anti-pneumococcal vaccines commercially available. two of these are conjugated to a protein carrier (pcv7 and pcv13) and one is not conjugated (ppv23). ppv23 was licensed in 1983 and consists of the capsular polysaccharides of twentythree different streptococcus pneumoniae serotypes (1-5, 6b, 7f, 8, 9n, 9 v, 10a, 11a, 12f, 14, 15b, 17f, 18c, 19f, 19a, 20, 22f, 23f, and 33f). it does not elicit immunological memory because the immune response it triggers is t-cell independent. it is usually administered to the elderly (above 65 years), as it is believed to be less effective in children. pcv7 is composed of the most frequent serotypes 4, 6b, 9 v, 14,18c, 19f, and 23f. pcv13 is directed at serotypes 1, 3-5, 6a, 6b, 7f, 9 v, 14, 18c, 19a, 19f, and 23f. contrary to ppv23 both pcv7 and pcv13 have an aluminum adjuvant in their composition that elicits a t-cell mediated response [143] . ever since vaccines were introduced in the healthcare system, prevalence, fatality and admissions for invasive pneumococcal disease have decreased significantly [144] . vaccine adverse events vary depending on whether the vaccine is adjuvanted or not. in a non adjuvanted vaccine, local reactions are present in 9% of people vaccinated intra muscularly and in 24% of those immunized sub-cutaneously [145] . in conjugated vaccines, this percentage rises to 50% [146] . systemic reactions such as fever, irritability, decreased appetite and sleep disturbances occur in 80-85% of recipients of pcv or ppv. symptoms like arthralgia, arthritis, myalgia, paresthesia and fatigue are more frequent in patients post ppv. this may be related to the fact that the vaccines are administered to different age groups. autoimmune risk following ppv vaccine is very low. only 14 case reports were found after ppv vaccine. six of these referred to reactivation of a previous autoimmune disorder. studies directed to access vaccine safety in subjects with autoimmune diseases showed immunization was safe [147, 148] . tetanus toxoid (tt) is a potent exotoxin produced by the bacteria clostridium tetani. the toxin has a predominant effect on inhibitory neurons, inhibiting release of ␥-aminobutiric acid (gaba). when spinal inhibitory interneurons are affected the symptoms appear [149] . the vaccine against c. tetani contains deactivated tetanus toxoid plus an adjuvant (usually aluminium hydroxide). the most studied and prevalent disease associated with tt is antiphospholipid syndrome (aps), but cns complications have also been reported such as optic neuritis, acute myelitis and encephalomyelitis [150] . in mice, the immune response to tt depends on genetic background and to the specific adjuvant used for immunization. naive balb/c mice, immunized with tt, developed antibodies directed to tt, dsdna and ␤2gpi and were extremely sick [90] . aps is an autoimmune disease characterized by the occurrence of thrombotic events. patients suffering from this condition have recurrent fetal loss, thromboembolic phenomena, thrombocytopenia as well as neurological, cardiac and dermatological involvement [151] . the serological marker of aps is the presence of antiphospholipid antibodies (apl), which bind negatively charged phospholipids, platelets and endothelial cells mainly through the plasma protein beta-2-glycoprotein-i (b2gpi). the presence of igg and igm anti-cardiolipin antibodies (acl) and lupus anticoagulant is associated with thrombosis in patients with aps [151] . ␤2gpi was identified as the most important antigen in aps. ␤2gpi has several properties in vitro which define it as an anticoagulant (e.g., inhibition of prothrombinase activity, adenosine diphosphate-induced platelet aggregation, platelet factor ix production) [152] . passive transfer of anti-␤2gpi antibodies induce experimental aps in naïve mice and thrombus formation in ex vivo model [153] . evidence suggests that the molecular mimicry mechanism between ␤2gpi and tt is one of the possible causes for aps. besides tt, aps has also been reported following hbv and influenza virus vaccination, although data are scarce [154, 155] . sle is a multisystem autoimmune disease characterized by the production of a variety of autoantibodies. igg isotype antibodies to double-stranded dna (dsdna) are thought to be diagnostic markers and their presence correlates with disease pathogenesis. several factors including genetic, hormonal, environmental and immune defects are involved in the induction of autoantibodies in this disease [156] . post vaccination manifestations of sle or lupus like syndrome have been reported and range from autoantibody induction to full blown clinical disease. reports have been published associating sle to hbv, mmr, dtp, hpv, influenza, bcg, pneumococcal and small pox vaccinations [157] . vaccination in sle diagnosed patients is associated with disease exacerbation and decreased antibody response, which may be due to the underlying disease and the frequent use of immunosuppressive drugs [158] . a temporal link between sle and hbv vaccination is the only relation that has been demonstrated [159] . several studies have demonstrated an increased prevalence of hpv in individuals with lupus compared to the general population, which has increased awareness for the need to vaccinate this high-risk population [160] . to do so, the association between immunization with hpv vaccines and sle like symptoms, as well as the higher incidence of flares in known lupus patients must be taken into account. vasculitis is the name given to a group of autoimmune mediated diseases, which involve blood vessels of different types and sizes. they can be categorized according to several disease features indluding: the type of vessel affected, organ distribution, genetic predisposition and clinical manifestation [161] . so far, 18 cases of large vessel vasculitis have been detected. this includes 15 cases of giant cell arteritis (gca) following influenza vaccination, 2 cases of takayasu disease (td), and one case of large cell arteritis involving subclavian and renal arteries following hbv vaccines. two of these patients had previous received the diagnosis of ankylosing spondylitis and polymyalgia rheumatica (pmr)-like illness [162] . one case of polyarteritis nodosa (pan) following the administration of tetanus and bcg vaccine is described. all other cases of pan in adults follow the administration of hbv vaccine [163] [164] [165] . case reports of medium vessels vasculitis -both polyarteritis nodosa and kawasaki disease (kd) -have also been published in pediatric patients. kd has been described one day after the second dose of hbv vaccine and following yellow fever vaccine [166, 167] . two cases of pediatric patients with pan have been reported two months after receiving the hbv vaccine [164, 165] . eosinophilic granulomatosis with polyangiitis (egpa) after tetanus vaccination [163] and following hbv vaccine [168] have been reported. there are also 3 cases of microscopic polyangiitis (mpa) and 6 cases of granulomatosis with polyangiitis (gpa) following influenza vaccines in the literature [169, 170] . henoch schönlein purpura (hsp) is the most common vasculitis of childhood. it is generally benign and self-limited. it is mediated by iga immune complex deposition in various tissues as well as in small-sized blood vessels. genetic risk factors play an important role in the pathogenesis of the disease: it is associated with hla-drb*01, 07 and 11. hsp was associated with seasonal influenza, influenza a (h1n1), pneumococcal and meningococcal disease, hepatitis a virus (hav), hbv, anti-human papilloma virus (hpv) vaccines, and following multiple combinations of vaccines, such as typhoid, cholera and yellow fever [139, [171] [172] [173] . leukocytoclastic vasculitis has been associated with several vaccines, including influenza vaccine [174] , hav vaccine [175] , hbv vaccine [176] , pneumococcal vaccine [177] , varicella [178] , rubella, smallpox [179] and the anthrax vaccine [180] . dermal vasculitis with pan uveitis has also been described following mmr vaccine [181] . ra is the most prevalent chronic inflammatory arthritis affecting the synovial membrane of multiple diarthrodial joints. although its etiology has not been completely clarified, deregulation of the immune system is evident with a preponderance of inflammatory cytokines and immune cells within the joints. ra has an estimated heritability of 60%, leaving a substantial proportion of risk to environmental factors. immunizations have previously been proposed as potential environmental triggers for ra. in the norfolk arthritis register database, 19 of the first 588 patients reported receiving a tetanus vaccination within 6 weeks prior to the onset of arthritis. similarly, a transient rise in rf titer was recorded in 10 out of 245 military recruits 2-3 weeks after receiving concomitant immunization against tetanus, typhoid, paratyphoid, mumps, diphtheria, polio and smallpox. however, only 2 showed a persistent elevation in titer and none developed arthritis [182] . several mechanisms have been proposed to explain the putative association between vaccination and the initiation of ra, the most prominent of which are molecular mimicry and non-specific immune system activation [182] . vaccines who have been associated with ra include rubella vaccine in which reactive arthritis occurs in 5% of recipients. controlled studies failed to show persistent arthritis or arthralgia in these patients [110] . patients following hbv vaccine showed an increase of arthritis in a vaers study, but this was not seen in a large retrospective epidemiological study [183] . data so far suggest that vaccines carry an insignificant role in the pathogenesis of ra. several mechanisms are being studied to produce vaccines mainly targeting inflammatory cytokines as "antigens" such as tnf, aiming to induce high titers of endogenous neutralizing anticytokine antibodies with the goal of breaking natural th tolerance to auto antigens. other cytokines, namely il-1 il-6, mif, rantes, il-18, mcp-1 are also being tested [184] . another vaccine related therapy uses autologous t cell lines to induce a specific immune response by the host's t cells directed against the autoimmune (vaccine) t cells [185] . this strategy has been successful in mouse models and has shown encouraging results in a small pilot study of 15 ra patients, where 10 patients showed a clinical response, defined by acr 50 improvement criteria [186] . uctd is a clinical condition characterized by signs, symptoms and laboratory tests suggestive of a systemic autoimmune disease but that does not fulfill the criteria for any defined connective tissue disease (ctd). such patients with clinical manifestations suggestive of systemic connective tissue disease but not fulfilling any existing criteria are quite frequent: 12-20% of the patients initially asking for a rheumatologic evaluation may at least temporarily be diagnosed as affected by 'undefined' or 'undifferentiated' connective tissue disease. comparing studies on these diseases is unfeasible because of the inexistence of defined criteria for diagnosis [187] . within 5 years of follow-up, patients usually evolve to defined ctds, which include sle, systemic sclerosis (ssc), primary sjögren's syndrome (pss), mixed connective tissue disease (mctd), systemic vasculitis, poly-dermatomyositis (pm/dm) and ra. maintaining an undefined profile for 5 years makes evolving into ctds less probable and the diagnosis of "stable uctd" reliable [188] . disease etiology is a concern and it has been associated with vitamin d deficiency and silicone implants, both of which lead to an imbalance in proinflammatory and anti-inflammatory cytokines [189] . vaccines have also been associated with this disease, namely the hbv vaccine [190] . etiopathogenesis of uctd is unknown and it has been suggested it might fall on asia spectrum since symptomatic similarities are striking and uctd etiopathogenesis has been associated with adjuvants [122] . aa is an autoimmune disease, characterized by one or more well demarcated oval and round non-cicatricial patches of hair loss. the disease may affect any hair bearing part of the body and has a great impact on a patient's self-esteem and quality of life. depending on ethnicity and location, aa is the most prevalent skin disease. aa prevalence varies and is estimated to be between 0.1-0.2% in the united states and 3.8% in singapore [191, 192] . as with any other autoimmune disease, the development of aa encompasses genetic and environmental factors. environmental factors associated with aa development are emotional and/or physical stress, infections and vaccines [193] . secondary syphilis is one of the most well studied examples, however epstein barr virus [194] and herpes zoster [195] infections have also been related to the development of the disease. as far as vaccines go, hbv vaccine has been associated with aa development. in one study of 60 patients, 48 developed aa after vaccination with hbv vaccine. of those 48 patients, 16 were rechallenged, and the reappearance of disease was witnessed [196] . in mice this association failed to be established [197] . one case of aa was witnessed following tetanus toxoid, as well as two case reports following hpv and mmr vaccine [198] [199] [200] . itp is an autoimmune disease defined by a platelet count of less than 105 platelets/l without overlapping diseases. it can present with or without anti-platelet-antibodies. thrombocytopenia is relatively common and the overall probability of developing itp was 6,9% in a cohort of 260 patients. it was also found that 12% of patients developed an overlapping aid other than itp [201] . the etiology of the disease is yet to be fully understood but it has been detected following infectious diseases, such as helicobacter pylori, hepatitis c virus (hcv), novel influenza a infection, rotavirus infection and human immunodeficiency virus (hiv) [202] . itp onset has also been reported, although rarely, as a severe adverse event following vaccine administration. this was more often observed after measles-mumps-rubella (mmr), hepatitis a and b, diphtheria-tetanus-acellular pertussis (dtap), and varicella vaccinations [203] . molecular mimicry has been suggested as a possible mechanism for the development of itp, namely following helicobacter pylori infection. its eradication has been shown to increase platelet count and diminish the levels of anti-caga antibody in a subset of h. pylori infected subjects with itp [204] . these data point towards a beneficial role of h. pylori eradication in chronic itp. two cases of itp following anti-rabies vaccine have been reported and one after hpv vaccine. reactivation of itp was reported two weeks after a tick-borne encephalitis vaccination [202] . the most consistent association with itp is with the mmr vaccine [205] . however, it should be emphasized that the number of cases are fewer than expected without vaccination. t1d is due to antigen specific reactions against insulin producing beta cells of the pancreas. much like other autoimmune diseases, t1d results from a combination of genetic, environmental, hormonal and immunological factors. environmental factors such as pathogens, diet, toxins, stress and vaccines are believed to be involved in the beginning of the autoimmune process [206] . although the mechanisms by which viral infections cause autoimmune diabetes have not been fully clarified, there is some evidence to suggest a role for natural infections in the pathogenesis of t1d mellitus in susceptible individuals [207] . it has been hypothesized that vaccination could trigger t1d in susceptible individuals. although post-vaccination t1d may be biologically plausible, cumulative evidence has not supported an increased risk of t1d following any vaccine [208] . several experimental data have suggested that, depending on the timing, vaccination might exert a protecting or aggravating effect on the occurrence of diabetes [209] . a study suggests that haemophillus influenza type b vaccine might be a risk factor in the induction of islet cell and anti-gad antibodies measured at one year of age [210] but there are previous studies that show no association between hib and t1d [211] . in a cohort of american military officers diagnosed with t1d, there was no association found between vaccination and t1d diagnosis [212] . available data about a relation between the mumps vaccine and t1d are still incomplete and their interpretation is difficult because of miscellaneous confounding factors associated with the development of t1d [213] . association between hemagglutinin 1 neuraminidase 1 (h1n1) vaccines and t1d is so far unproven [214] . in humans, it has been hypothesized that early-age bcg vaccination is associated with the risk of t1d. the few studies conducted to date provided no consistent evidence of an association. there are, however, studies showing a possible temporary boost of the immune function after vaccination [215] . studies also show that among bcg-vaccinated children who test positive for islet autoantibodies, there is a higher cumulative risk of t1d [216] . in animal experiments it has been observed that bcg seems to have a protective effect against diabetes, however researchers have yet to translate this benefit to humans [217] . in all, studies results do not support any strong association between vaccination and t1d. narcolepsy is a sleep disorder described as excessive sleepiness with abnormal sleep pattern characterized by uncontrollable rapid eye movement (rem) events which occur at any time during the day. these event and may or may not be accompanied by a loss of muscle tone (cataplexy) [218] . a plethora of data indicates that narcolepsy is caused by the lack of orexin (also known as hypocretin), an important neurotransmitter, which is involved in the regulation of the sleep cycle. in narcolepsy patients, a loss of orexin producing neurons in the hypothalamus and low levels of orexin in the cerebrospinal fluid (csf) has been reported [218] . narcolepsy has been shown to have an autoimmune background. antibodies against tribbles 2 (trib2) have been found in these patients, which may be related to the pathogenesis of disease. an experimental model of narcolepsy in mice has been made by passive transfer of total igg from narcolepsy patients into the animal's brains through intra ventricular injection [219] . environmental factors like influenza a virus and streptococcal infections have been associated with disease onset. interestingly, fever by itself without the diagnosis of an infectious etiology was found to be a risk factor for narcolepsy [220] . several groups have studied and found an increase in the incidence of narcolepsy diagnosis following the introduction of influenza vaccination, specifically, aso3-adjuvanted pandemrix tm vaccine. this association was shown in finland especially in [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] year-olds, but also in case reports from other countries [221] . other studies failed to find an association. the actual infection with h1n1 has been associated with disease development in china, however no such relationship has been noted in europe [220] . the above-mentioned associations are specifically related to the aso3-adjuvanted pandemrix tm vaccine. the same association has not been reported for other h1n1 adjuvanted or non-adjuvanted vaccines. the major difference between the aso3 and the mf59 adjuvants is the presence of the ␣-tocopherol. ␣-tocopherol is unique in that it can achieve the highest and longest antibody response by producing an enhanced antigenspecific adaptive immune response. in vitro it was shown that ␣-tocopherol could increase the production of orexin as well as increase the proteosome activity. this increased production of orexin fragments may facilitate antigen presentation to mhc class ii, thus triggering an autoimmune process [220] . all these data together support the relationship between the h1n1 pandemrix tm vaccine and the development of narcolepsy. gluten induced enteropathy, gluten sensitive enteropathy, or more commonly called celiac disease (cd) is a life-long autoimmune condition mainly of the gastrointestinal tract, specifically affecting the small intestine. the abnormal immune response crates autoantigens which are directed towards tissue transglutaminase (ttg). the two main autoantibodies and the most widespread serological markers to screen for the disease are anti ttg and anti endomysium. two additional auto-antibodies, namely: anti deaminated gliadin peptide and anti-neoepitope ttg were found recently to be reliable for cd screening as well [222] . cd is an autoimmune disease induced by well-known nutritional environmental factors. the non-dietary ones are less studied and established. several infectious disease have been linked to its development, the so-called infectome [193] . a clear cause-effect relation is yet to be established for most of the pathogens associated with cd. what has been shown, however, is that in countries with low economic status, inferior hygiene conditions and higher infectious load, cd prevalence is lower [223] . an epidemiologic relationship was established in 2006 between rotavirus infection and cd. data showed that in genetically predisposed individuals, rotavirus infection was related to childhood cd development [224] . in subsequent research studies, a celiac peptide was recognized and proved to share homology with rotavirus major neutralizing protein vp7 and with the cd autoantigen ttg. the antibodies directed against the viral protein vp7 were shown to predict the onset of cd and induce typical features of cd in the intestinal epithelial cell-line t84 [225] . it has also been suggested that rotavirus vaccine alters b and t behavior, as the percentage of b-cells was higher in the vaccinated infants [226] . rotavirus vaccine as an inducer of cd is still in discussion and warrants further study. pmr is an autoimmune inflammatory rheumatic disease characterized by raised inflammatory markers with pain and morning stiffness of shoulders and pelvic girdles and synovitis of proximal joints and extra-articular synovial structures. its diagnosis is clinical and it is typically a disease of the elderly occurring mainly in subjects above 70. etiopathogenesis of pmr remains unknown, but genetic and environmental factors play a role [227] . a close temporal relationship has been ascertained concerning epidemics of mycoplasma pneumoniae, chlamydia pneumonia, parvovirus b19 and peaks of cases of pmr and giant cell arteritis, however this is not clearly proven [228] . cases of pmr following vaccination have rarely been reported. however, it is believed that post vaccination pmr may be underreported due to its symptomatic similarities with the transient effects of vaccines, namely: arthralgia, myalgia and low-grade fever. this leads to failure in establishing a chronological relationship when the disease is diagnosed. most of the reported cases are associated with seasonal influenza vaccine (inf-v). often, the time interval between vaccine administration and symptoms onset varies from one day, to three months. three cases were reported with associated giant cell arthritis. a case report of relapsing pmr after four years of remission following tetanus vaccination has also been reported [229, 230] . acute disseminated encephalomyelitis (adem) is an inflammatory demyelinating disease of the central nervous system (cns). adem is usually poly-symptomatic with encephalopathy (i.e., behavioral change or altered level of consciousness). it affects mostly children and young adults and has higher prevalence in males. its incidence is 0.6-0.8 per 100 000 per year [231] . although there is no concrete evidence of a clear pathogenic association, adem has been associated with immunization or previous viral infection. post-vaccination adem accounts for only 5-10 percent of all cases, while post-infectious adem accounts for 66 percent of all cases of adem [232] . the hypothesis that better describes these associations is molecular mimicry. t-cells targeting human herpesvirus-6 (hhv-6), coronavirus, influenza virus and epstein-barr virus (ebv) have been shown to cross-react with myelin basic protein (mbp) antigens. anti-mbp t-cells were detected in patients following vaccination with simple rabies vaccine [233] [234] [235] . in a post experimental therapy for alzheimer's disease with a vaccine that contained aggregates of synthetic a␤42 fragments of amyloid precursor protein, adem was shown to develop in mice [236] . the experimental model of ms, eae mice, may be induced with injection of a␤42, but only when the latter is administered together with the complete freund's adjuvant [237] . this observation points to the importance and central role of the adjuvants in induction of adem and autoimmunity in general [238] . the overall incidence of post vaccination adem is estimated to be 0.1-0.2 per 100 000 and a higher risk has been reported following immunization against measles. other vaccines accountable for post-vaccination adem include vaccines against the varicella zoster, the rubella, the smallpox and the influenza viruses [239] . surprisingly, certain vaccines such as anti-tetanus vaccine were shown to have a negative correlation with adem (statistically significant decreased risk) [240] . hbv immunization has been studied as a possible cause for adem but was later associated with clinically isolated syndrome (cis) (a first time occurring demyelinating episode that may, or not develop to ms) and complete conversion to ms [241] . as far as case reports are concerned, adem was associated with vaccination with influenza, hepatitis a and b, mmr, hpv and tetanus [121, 242, 243] . bullous dermatoses are characterized by the presence of blisters and autoantibodies against structural components of the skin: desmosomal proteins (in pemphigus), adhesion molecules of the dermal-epidermal junction (in pemphigoid diseases), and epidermal/ tissue transglutaminase (in dermatitis herpetiformis). the most frequent autoimmune bullous diseases are bullous pemphigoid (bp) and pemphigus vulgaris (pv). bp is more frequently observed in the elderly, while the age of onset of pv is between 40 and 60 years. neither of the diseases have any gender preference [244] . bp and pv etiology is, so far, poorly understood. both diseases have been associated with various environmental factors, which include emotional and/or physical stress, infections and vaccinations [244] . genetic predisposition has also been studied with overexpression of certain hla class ii alleles. these include hla-dqb1*0301, drb1*04, drb1*1101, and dqb1*0302. these alleles have been found to be more prevalent in bp patients than in the general population [245] . pv is associated with certain hla class ii loci such as hla-dr4 and hladr14 alleles (drb1*0401 and drb1*0402, which is prevalent in ashkenazi jews, iranian and sardinian patients). other loci include drb1*1401 (common among japanese and italian patients) and two dqb1 alleles (dqb1*0302 and dqb1*0503), which are strongly associated with pv. bp and pv patients' sera were found to have significantly higher prevalence of antibodies to hepatitis b virus, hepatitis c virus, helicobacter pylori, toxoplasma gondii and cytomegalovirus [244] . as far as vaccination is concerned, bp developed in patients following influenza, diphtheria, tetanus, pertussis, hepatitis b, bcg, polio and herpes zoster vaccines [140, 246, 247] furthermore, reactivation of bp following influenza vaccination was reported in one case report [248] . new onset pv was associated with: influenza vaccine, hepatitis b vaccine, anthrax vaccine, typhoid booster and rabies vaccination. in addition, exacerbation of pv after vaccination was also reported following influenza vaccine and tetanus vaccine [121] . iim compose a group of skeletal muscles diseases in which myositis without a recognized cause occurs. iim is usually subdivided in 4 entities: dermatomyositis (dm), polymyositis (pm), inclusion body myositis (sibm) non-specific myositis (nsm) and immune mediated necrotizing myopathy (iam) [249] . iim prevalence is around 1.1 × 10 −6 cases, with a bimodal age of distribution that peaks in childhood and again between 45 and 55 years. dm is the most common inflammatory myopathy while pm is the least frequent. despite exhibiting similar clinical symptoms, the subsets of iim exhibit significant immunopathological variation. dm begins with the activation of the complement and formation of membrane attack complexes (mac). in pm and sibm the fundamental process is related to cd8+ t cells mediated cytotoxicity [249] . it is unclear what breaks the tolerance and drives the immune response to induce iim. so far, dm, pm and sibm have been linked to vaccination. several cases have been reported in the literature associating different vaccines with the development of idiopathic inflammatory myopathies. 119 cases of iim had been reported to vaers database up to june 2013. out of these 119 cases, 33 were classified as pm, 85 as dm and an only one as a sibm. dm has been reported after almost any vaccine, however only a few studies have attempted to clarify the possible relationship between dm and vaccination. pm is a frequent misdiagnosed disorder. some reports have associated previous immunization, especially hepatitis b vaccine with pm [250] . despite being recently differentiated from other iim, sibm has already been related to hbv vaccine [250] . some vaccines associated with myositis are mmr vaccine, smallpox vac-cine, poliomyelitis (ipv), diphtheria and tetanus toxoid, influenza, hpv and bcg [250] . fms is an entity that is related to the inability of the cns to modulate pain. the conditioned pain modulation process in the cns appears to be compromised among many fms patients, which might explain the enhanced pain sensation experienced by these patients [251] . the etiology of fms is yet to be unveiled. genetic predisposition, physical trauma (particularly to the cervical spine), emotional stress (to various stressors) as well as a variety of infections have been linked with fms. vaccines have been associated with the triggering of fms namely rubella and lyme disease vaccines [252] . there are several reports of fibromyalgia-like disease after vaccination, specifically hpv (martinez-lavin journal of clinical rheumatology 2014). the medical community and regulatory agencies should be aware of these possible adverse effects aiming at defining their magnitude. chronic fatigue syndrome (cfs) is a disease characterized by disabling fatigue, headaches, concentration difficulties and memory deficits (90%). other symptoms such as sore throat (85%), tender lymph nodes (80%), skeletal muscle pain and feverishness (75%), sleep disruption (70%), psychiatric problems (65%) and rapid pulse (10%) are often observed. it more frequently affects women and has a prevalence of 0.2-2.6% [253] . although disease etiology is still unknown, there are several pathogens, such as epstein-barr virus (ebv), which have been associated with cfs. patients often have higher titers of igm to the ebv viral capsid antigen. cytomegalovirus and human herpes virus 6 antibodies were also detected more often in cfs patients, although other reports failed to replicate these results. parvovirus b19 infection has also been suggested as a trigger to cfs [253] [254] [255] . vaccine inoculation has also been appointed as a probable cause. vaccinations against rubella, q fever and hepatitis b were found to be associated with higher risk of developing cfs while meningococcal vaccine, poliovirus and influenza vaccine were not. surprisingly, staphylococcus toxoid vaccine appeared to have a protective effect [121, 256, 257] . defined in 2011 by shoenfeld and agmon-levin asia syndrome is characterized by hyperactive immune response to adjuvants [4] . as previously stated, asia incorporates four known medical conditions: siliconosis, gws, mmf, and post-vaccination phenomena [4] . recently, the sick building syndrome (sbs) was proposed as a candidate for the asia spectrum [258] . all of these diseases satisfy several criteria for fms and seid [252] . a macrophagic myofasciitis (mmf) mmf has been described as an emerging condition of unknown cause characterized by a pathognomonic lesion in muscle biopsy mixing large macrophages with submicron to micron-sized agglomerates of nanocrystals in their cytoplasm and lymphocytic infiltrates. these lesions were related to aluminum deposits in muscle following immunization with aluminum containing vaccines [63] . mmf lesion is now universally recognized as indicative of a long-lasting persistence of aluminum adjuvant at the site of prior intramuscular immunization. the long-lasting mmf lesion should be considered as a biomarker of aluminum bio persistence in a given individual. patients with mmf have higher reported myalgia with incidence being up to 90%. its etiology is not clear but genuine muscle weakness is rare and the diagnosis of fibromyalgia is also rare. higher prevalence of chronic fatigue syndrome (cfs) in patients with mmf has been reported as well. cognitive impairment has been associated with mmf: in one series of 105 mmf patients, up to 97% had attention and memory complaints and neuropsychological tests were abnormal in 89% [259] . b gulf war syndrome (gws) gws is a clinical entity specifically related to a certain time and place in history. it was described among veterans of the military conflict occurring in 1990-1991 in the persian gulf. the syndrome is characterized by chronic fatigue, musculoskeletal symptoms, malaise and cognitive impairment. it clinically overlaps with post traumatic stress disorder (ptsd), fms, cfs and other functional disorders [260] . the unique conditions that have been associated so far with disease development are the exposure to extreme climate in the persian gulf, exposure to various chemicals (pesticides, depleted uranium), stress provoked by prolonged waiting without actual combat and the intense exposure to vaccinations of the soldiers for fear of biological weaponry [260] . comparing gulf war veterans and veterans of the bosnian conflict, multiple vaccinations administered to servicemen in the gulf war was identified as a unique exposure [261] . the mechanism through which vaccination exposure may lead to the development of functional symptoms is not completely understood. the possibility that a shift from th1 to th2 type reactions could be of pathogenic significance was raised and is supported by an increased frequency of allergic reactions, low natural killer cell activity and low levels of interferon ␥ and il-2 in these patients [262] . one study with gws patients showed a connection between anti-squalene antibodies and symptoms development. this was refuted by a larger study that found no association between antisqualene antibodies and chronic multi-symptom illness [263] . c asia registry a registry is a collection of data related to patients with the same specific characteristic. it is often the first approach in the study of an area of inquiry. in rare diseases, registries are often the way to get a sufficiently sized sample of patients which can be used either for epidemiological or research purposes. asia syndrome may be underreported because of unawareness and failure to connect the syndrome with the exposure. this registry was created to fully understand the clinical aspects of disease and compare patients from all over the world in order to have fully validated criteria for disease diagnosis and also to define demographic and environmental history of disease. the asia syndrome registry website can be found on the following link: https://ontocrf.costisa.com/en/web/asia. only cases reported by physicians are accepted. to make an informed decision in medicine, there is always a need to weigh the pros and cons. ards may play an important role in deciding whether vaccination is or is not appropriate to a patient. in these cases, patients are immunosuppressed on account of their diagnosis and even more so if they are under specific immunomodelatory medication [4] . if the efficacy of vaccination is reduced, there is a potential for development of disease flares following vaccination. in the case of live vaccines, its inoculation may even be enough to trigger disease in the host. for these specific reasons, live vaccines are generally contraindicated in patients receiving immunosuppressant medication. there is a need for screening and treatment of latent tuberculosis infection (ltbi) before starting anti-tnf-alpha therapy. the same is true for vaccination. preferably, even recommended vaccination (see table 5 ) should be administered before the initiation of disease-modifying anti-rheumatic drugs (dmards) because these may reduce vaccine efficacy [264] . immunosuppression equals high risk of infection and lower vaccine efficacy. taking into account safety concerns and efficacy, the eular recommendations for immunizations in aiird patients are: • assess vaccination status in initial investigation. • administer vaccines in a stable disease phase. • live attenuated vaccines are to be avoided especially if immunosuppressive agents are being administered. bcg is not recommended. • administer vaccines ideally before starting dmards and anti-tnf␣ agents. • influenza and 23-valent polysaccharide pneumococcal vaccination is recommended. • tetanus toxoid vaccination is recommended following recommendations of general population, in case of major and/or contaminated wounds in patients receiving rituximab in the previous 24 weeks tetanus ig is indicated. • hpv and herpes zoster should be considered. • in hyposplenic/asplenic patients, influenza, pneumococcal, haemophilus influenza b and meningococcal c are advisable. • hepatitis a and b is recommended in patients at risk. • travel patients should be immunized according to general population guidelines except for live attenuated vaccines, which are to be avoided [148] . vaccines have many beneficial effects in combating infectious diseases and preventing mortality and morbidity. they have also proved to be effective cancer treatments by immunomodulation, as demonstrated by the intravesical administration of bcg to treat superficial bladder cancer [28] . vaccines are however, linked to autoimmunity. beneficial outcomes, like the adjuvant effect are based on immunity triggering and enhanced immunity mechanisms. these same responses account for autoimmunity exertion. vaccines induce the production of autoantibodies, but their pathologic effect is yet to be unveiled. although vaccines are widely considered safe, there are subjects with predispositions to whom vaccines pose a bigger threat. an example is the fact that animal models with autoimmune predispositions develop autoimmune disease following adjuvant exposure. as many as 1% of recipients of aluminum containing adjuvants may be sensitized to future exposure [269] . silicon-induced inflammatory fibro proliferative response is irrefutable and well documented. the presence of anti-silicone antibodies and silicone-associated autoimmune phenomena seems very plausible. asia syndrome and aluminum safety studies show that the use of aluminum containing "placebo" in control groups in vaccine safety studies should be carefully evaluated. new studies must be performed using a proper placebo to adequately test vaccine safety. another evident failure in vaccine safety studies are the short-term periods which are evaluated. continued immune system activation has been observed to be a potential mechanism of disease. a disease which is poorly understood so far. vaccine recommendations should be reassessed frequently in different subsets of the population. this does not invalidate the need for vaccines, however, the lower the possibility of exerting adverse events, the easier it will be for the potential benefits to outweigh the risks. vaccinomics represents a major breakthrough in vaccine development and can lead to the development of targeted vaccines to peptides most likely to be immunogenic [81] . a predictable response to vaccine can be achieved by differentiating the host variability. this can be achieved namely in genetics and pathogen variability. developing a vaccine accordingly will lead to increased specificity in treatment and leave less room for adverse events. by using immunomodulation, vaccinomics can also give rise to novel therapies for autoimmune diseases. there are several reports of cases of autoimmunity diseases following vaccines but despite in vitro positive results and due to both the limited number of cases and the long latency period of the diseases, every attempt for an epidemiological study has failed to deliver a connection. classification as asia syndrome, in detriment of classic specific autoimmune diseases, could be the key to finding effective preventative therapeutic strategies. it will enable the study of bigger patient clusters with earlier diagnoses. future studies that could help clarify the association between vaccinations, adjuvants and autoimmunity should ideally have a different design, more long-term data and should include autoimmune phenomena as well as large-scale epidemiological studies of autoimmune diseases. the mosaic of autoimmunity infections and autoimmunity-friends or foes? autoimmune/inflammatory syndrome induced by adjuvants (asia) 2013: unveiling the pathogenic, clinical and diagnostic aspects asia-autoimmune/inflammatory syndrome induced by adjuvants adjuvants and autoimmunity adjuvant activity of immunopotentiating reconstituted influenza virosomes (irivs) interfaces questions and possibilities in toll-like receptor signalling immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis immunotherapeutic strategies for the treatment of malignant melanoma does pegylated interferon alpha-2b confer additional benefit in the adjuvant treatment of high-risk melanoma? tuftsin on the 30-year anniversary of victor najjar's discovery tuftsin a naturally occurring immunopotentiating factor. i. in vitro enhancement of murine natural cell-mediated cytotoxicity tuftsin and tuftsin conjugates potentiate immunogenic processes: effects and possible mechanisms the novel adjuvant ic31 strongly improves influenza vaccine-specific cellular and humoral immune responses in young adult and aged mice improving vaccine delivery using novel adjuvant systems prototype alzheimer's disease epitope vaccine induced strong th2-type anti-abeta antibody response with alum to quil a adjuvant switch cpg oligodeoxynucleotides trigger protective and curative th1 responses in lethal murine leishmaniasis tuftsin-azt conjugate: potential macrophage targeting for aids therapy enhanced immune response induced by a potential influenza a vaccine based on branched m2e polypeptides linked to tuftsin construction of a synthetic immunogen: use of the natural immunomodulator polytuftsin in malaria vaccines against resa antigen of plasmodium falciparum stimulating effect of tuftsin and its analogues on the defective monocyte chemotaxis in systemic lupus erythematosus immunological concepts of vaccine adjuvant activity recent advances in the discovery and delivery of vaccine adjuvants using eae to better understand principles of immune function and autoimmune pathology toll pathway-dependent blockade of cd4 + cd25 + t cell-mediated suppression by dendritic cells viruses as adjuvants for autoimmunity: evidence from coxsackievirus-induced myocarditis pathogenesis of myocarditis and dilated cardiomyopathy bacillus calmette-guerin immunotherapy of superficial bladder cancer pattern recognition receptors and control of adaptive immunity how dying cells alert the immune system to danger role of protease-activated receptors in inflammatory responses, innate and adaptive immunity novel signaling interactions between proteinase-activated receptor 2 and toll-like receptors in vitro and in vivo autoimmune (auto-inflammatory) syndrome induced by adjuvants (asia)-animal models as a proof of concept the endogenous adjuvant squalene can induce a chronic t-cell-mediated arthritis in rats percutaneous exposure of adjuvant oil causes arthritis in da rats exacerbation of collagen-induced arthritis in rats by rat cytomegalovirus is antigen-specific comparative study of adjuvant induced arthritis in susceptible and resistant strains of rats. i. effect of cyclophosphamide aloh3-adjuvanted vaccine-induced macrophagic myofasciitis in rats is influenced by the genetic background induction of plasma cell tumours in balb-c mice with 2,6,10,14-tetramethylpentadecane (pristane) paraffin oil injection in the body: an obsolete and destructive procedure induction of lupus autoantibodies by adjuvants adjuvant immunization induces high levels of pathogenic antiphospholipid antibodies in genetically prone mice: another facet of the asia syndrome induction of the asia syndrome in nzb/nzwf1 mice after injection of complete freund's adjuvant (cfa) manifestations of systemic autoimmunity in vaccinated salmon comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs comparison of tissue reactions produced by haemophilus pleuropneumoniae vaccines made with six different adjuvants in swine delayed acquisition of neonatal reflexes in newborn primates receiving a thimerosal-containing hepatitis b vaccine: influence of gestational age and birth weight autoimmune/autoinflammatory syndrome induced by adjuvants (asia syndrome) in commercial sheep slow ccl2-dependent translocation of biopersistent particles from muscle to brain aluminum hydroxide injections lead to motor deficits and motor neuron degeneration nanomolar aluminum induces pro-inflammatory and pro-apoptotic gene expression in human brain cells in primary culture the pro-oxidant activity of aluminum regulatory role of zinc during aluminium-induced altered carbohydrate metabolism in rat brain aluminum and alzheimer's disease: after a century of controversy, is there a plausible link? exposure to aluminium and the subsequent development of a disorder with features of alzheimer's disease elevated urinary excretion of aluminium and iron in multiple sclerosis aluminum-induced entropy in biological systems: implications for neurological disease the immunobiology of aluminium adjuvants: how do they really work? fatty acid-induced nlrp3-asc inflammasome activation interferes with insulin signaling inflammasome signaling at the heart of central nervous system pathology summary and conclusions of the sixty-seventh meeting of the joint fao/who expert committee on food additives if exposure to aluminium in antiperspirants presents health risks, its content should be reduced macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle structural basis of metal hypersensitivity diagnosis and treatment of metal-induced side-effects mercury and multiple sclerosis the beneficial effect of amalgam replacement on health in patients with autoimmunity metal-induced inflammation triggers fibromyalgia in metal-allergic patients the role of environmental factors in autoimmune thyroiditis orofacial granulomatosis associated with hypersensitivity to dental amalgam, oral surg nephrotic syndrome due to ammoniated mercury endemic clustering of multiple sclerosis in time and place mercury exposure and antinuclear antibodies among females of reproductive age in the united states: nhanes, environ. health perspect gold nephropathy: serologic data suggesting an immune complex disease nickel-induced allergy and contact dermatitis: does it induce autoimmunity and cutaneous sclerosis? an experimental study in brown norway rats aluminum in the central nervous system (cns): toxicity in humans and animals, vaccine adjuvants, and autoimmunity six revolutions in vaccinology from pasteur to genomics: progress and challenges in infectious diseases systems vaccinology vaccinomics, adversomics, and the immune response network theory: individualized vaccinology in the 21st century studies of twins in vaccinology vaccinomics current findings, challenges and novel approaches for vaccine development the hla genomic loci map: expression, interaction, diversity and disease the link between genetic variation and variability in vaccine responses: systematic review and meta-analyses development of polyarthritis after insertion of silicone breast implants followed by remission after implant removal in 2hla-identical sisters bearing rheumatoid arthritis susceptibility genes autoimmune/auto-inflammatory syndrome induced by adjuvants (asia) after quadrivalent human papillomavirus vaccination in colombians: a call for personalised medicine anticardiolipin and anti-beta(2) glycoprotein i antibodies in sera of 61 apparently healthy children at regular preventive visits autoimmunity and hepatitis a vaccine in children bacterial induction of autoantibodies to beta2-glycoprotein-i accounts for the infectious etiology of antiphospholipid syndrome vaccination-induced systemic autoimmunity in farmed atlantic salmon autoimmune response following annual influenza vaccination in 92 apparently healthy adults high non-specific t lymphocyte response to the adjuvanted h1n1 vaccine in comparison with the h1n1/h3n2/b-brisbane vaccine without adjuvant short and long-term effects of pandemic unadjuvanted influenza a(h1n1) pdm09 vaccine on clinical manifestations and autoantibody profile in primary sjögren's syndrome pneumococcal vaccination of patients with systemic lupus erythematosus: effects on generation of autoantibodies immunogenicity and safety of a quadrivalent human papillomavirus vaccine in patients with systemic lupus erythematosus: a case-control study safety and immunogenicity of the quadrivalent hpv vaccine in female systemic lupus erythematosus patients aged 12 to 26 years anti-phospholipid antibodies following vaccination with recombinant hepatitis b vaccine development of autoantibodies before the clinical onset of systemic lupus erythematosus genetics and autoantibodies silicone and autoimmunity platinum in the environment: frequency of reactions to platinum-group elements in patients with dermatitis and urticaria silicone and scleroderma revisited rupture of silicone gel breast implants and symptoms of pain and fatigue the association between silicone implants and both antibodies and autoimmune diseases a comparison of autoantibody production in asymptomatic and symptomatic women with silicone breast implants silicone implant incompatibility syndrome (siis): a frequent cause of asia (shoenfeld's syndrome) management of chronic childhood immune thrombocytopenic purpura: aieop consensus guidelines rubella-associated arthritis. i. comparative study of joint manifestations associated with natural rubella infection and ra 27/3 rubella immunisation risk of chronic arthropathy among women after rubella vaccination. vaccine safety datalink team efficacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every 10 years risk of yellow fever vaccine-associated viscerotropic disease among the elderly: a systematic review bacillus calmette-guérin immunotherapy for genitourinary cancer arthritis after bcg vaccine in a healthy woman dermatomyositis after b.c.g. vaccination aetiopathogenesis of takayas's arteritis and bcg vaccination: the missing link? reactive arthritis induced by intravesical bcg therapy for bladder cancer: our clinical experience and systematic review of the literature systemic granulomatosis and hypercalcaemia following intravesical bacillus calmette-guérin immunotherapy universal hepatitis b vaccination in taiwan and the incidence of hepatocellular carcinoma in children. taiwan childhood hepatoma study group vaccines and autoimmunity hepatitis b vaccination and undifferentiated connective tissue disease: another brick in the wall of the autoimmune/inflammatory syndrome induced by adjuvants (asia) adverse reactions to human papillomavirus (hpv) vaccines sustained efficacy and immunogenicity of the hpv-16/18 as04-adjuvanted vaccine up to 7.3 years in young adult women detection of human papillomavirus l1 gene dna fragments in postmortem blood and spleen after gardasilreg. vaccination-a case report human papillomavirus (hpv) vaccine policy and evidence-based medicine: are they at odds? annual report: surveillance of adverse events following immunisation in australia annual report: surveillance of adverse events following immunisation in australia influenza vaccine and autoimmunity infection and death from influenza a h1n1 virus in mexico: a retrospective analysis altered response to a(h1n1) pnd09 vaccination in pregnant women: a single blinded randomized controlled trial adverse events following immunization with vaccines containing adjuvants antineutrophil cytoplasmic antibody vasculitis associated with influenza vaccination hughes syndrome) met the autoimmune/inflammatory syndrome induced by adjuvants (asia) meningococcal group b vaccines comparative long-term adverse effects elicited by invasive group b and c meningococcal infections immune response, antibody persistence, and safety of a single dose of the quadrivalent meningococcal serogroups a, c, w-135, and y tetanus toxoid conjugate vaccine in adolescents and adults: results of an open, randomised, controlled study vaccines and guillain-barré syndrome henoch-schölein purpura and polysaccharide meningococcal vaccine infantile bullous pemphigoid developing after hexavalent, meningococcal and pneumococcal vaccinations the risk of sequelae due to pneumococcal meningitis in high-income countries: a systematic review and meta-analysis burden of disease caused by streptococcus pneumoniae in children younger than 5 years: global estimates fda. pneumococcal 13-valent conjugate vaccine (diphtheria crm197 protein) prevnar 13 ® decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine comparative reactogenicity and immunogenicity of 23 valent pneumococcal vaccine administered by intramuscular or subcutaneous injection in elderly adults safety and immunogenicity of a 13-valent pneumococcal conjugate vaccine pneumococcal polysaccharide vaccination in rheumatoid arthritis patients receiving tocilizumab therapy eular recommendations for vaccination in adult patients with autoimmune inflammatory rheumatic diseases tetanus a review of the literature optic neuritis and myelitis after booster tetanus toxoid vaccination antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1000 patients crystal structure of human beta2-glycoprotein i: implications for phospholipid binding and the antiphospholipid syndrome induction of anti-phospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anti-cardiolipin antibodies characterization of antiphospholipid antibodies in chronic hepatitis b infection influenza vaccination and the production of anti-phospholipid antibodies in patients with systemic lupus erythematosus unraveling the soul of autoimmune diseases: pathogenesis, diagnosis and treatment adding dowels to the puzzle vaccines and autoimmune diseases of the adult effect of belimumab on vaccine antigen antibodies to influenza, pneumococcal, and tetanus vaccines in patients with systemic lupus erythematosus in the bliss-76 trial ten cases of systemic lupus erythematosus related to hepatitis b vaccine prevalence of cervical human papillomavirus infection in women with systemic lupus erythematosus revised international chapel hill consensus conference nomenclature of vasculitides large artery vasculitis following recombinant hepatitis b vaccination: 2 cases etiology and precipitating factors of necrotizing angiitis with respiratory manifestations. 5 case reports cutaneous polyarteritis nodosa in a child following hepatitis b vaccination systemic polyarteritis nodosa following hepatitis b vaccination kawasaki disease in an infant following immunisation with hepatitis b vaccine yellow fever vaccination and kawasaki disease churg-strauss vasculitis with brain involvement following hepatitis b vaccination anca-associated vasculitis following influenza vaccination: causal association or mere coincidence? influenza vaccination in anca-associated vasculitis henoch-schönlein purpura after hepatitis a vaccination henoch-schönlein purpura following influenza a h1n1 vaccination henoch-schönlein purpura following influenza vaccinations during the pandemic of influenza a (h1n1) leukocytoclastic vasculitis after influenza vaccination vasculitis related to hepatitis a vaccination hypersensitivity vasculitis after hepatitis b vaccination leukocytoclastic vasculitis after pneumococcal vaccination allergic reaction to varicella vaccine vasculitides associated with infections, immunization, and antimicrobial drugs lymphocytic vasculitis associated with the anthrax vaccine: case report and review of anthrax vaccination panuveitis and dermal vasculitis following mmr vaccination can immunisation trigger rheumatoid arthritis? risk of rheumatoid arthritis following vaccination with tetanus, influenza and hepatitis b vaccines among persons 15-59 years of age anti-cytokine vaccination in autoimmune diseases exploiting t. cell crosstalk as a vaccination strategy for rheumatoid arthritis vaccination with selected synovial t cells in rheumatoid arthritis undifferentiated ctd a wide spectrum of autoimmune diseases analysis of the evolution of uctd to defined ctd after a long term follow-up undifferentiated connective tissue disease after silicone-gel testicular implantation connective tissue disease following hepatitis b vaccination prevalence of alopecia areata in the first national health and nutrition examination survey tracing environmental markers of autoimmunity: introducing the infectome onset of alopecia areata after epstein-barr virus infectious mononucleosis hair loss after varicella zoster virus infection hair loss after routine immunizations recombinant human hepatitis b vaccine initiating alopecia areata: testing the hypothesis using the c3h/hej mouse model alopecia universalis in an adult after routine tetanus toxoid vaccine telogen effluvium following bivalent human papillomavirus vaccine administration: a report of two cases acute-onset madarosis following mmr vaccination long-term outcome of otherwise healthy individuals with incidentally discovered borderline thrombocytopenia immune thrombocytopenic purpura (itp) associated with vaccinations: a review of reported cases epidemiology of paediatric immune thrombocytopenia in the general practice research database effect of eradication of helicobacter pylori in children with chronic immune thrombocytopenia: a prospective, controlled, multicenter study, pediatr immune thrombocytopaenic purpura: an autoimmune cross-link between infections and vaccines vaccination may be associated with autoimmune diseases viral infections and molecular mimicry in type 1 diabetes childhood immunizations and type 1 diabetes: summary of an institute for vaccine safety workshop. the institute for vaccine safety diabetes workshop panel stimulation of the developing immune system can prevent autoimmunity vaccinations may induce diabetes-related autoantibodies in one-year-old children lack of association between receipt of conjugate haemophilus influenzae type b vaccine (hboc) in infancy and risk of type 1 (juvenile onset) diabetes: long term follow-up of the hboc efficacy trial cohort vaccination and risk of type 1 diabetes mellitus in active component u. s. military difficulties in assessing the relationship, if any, between mumps vaccination and diabetes mellitus in childhood, pubmed,ncbi neurological and autoimmune disorders after vaccination against pandemic influenza a (h1n1) with a monovalent adjuvanted vaccine: population based cohort study in bacille calmette-guérin vaccination and incidence of iddm in montreal, canada neonatal bacille calmette-guerin vaccination and type 1 diabetes bacille calmette-guérin/dnahsp65 prime-boost is protective against diabetes in non-obese diabetic mice but not in the streptozotocin model of type 1 diabetes narcolepsy: autoimmunity, effector t cell activation due to infection, or t cell independent, major histocompatibility complex class ii induced neuronal loss? passive transfer of narcolepsy: anti-trib2 autoantibody positive patient igg causes hypothalamic orexin neuron loss and sleep attacks in mice is narcolepsy a classical autoimmune disease? the pandemrix-narcolepsy tragedy: how it started and what we know today a novel algorithm for the diagnosis of celiac disease and a comprehensive review of celiac disease diagnostics infections may have a protective role in the etiopathogenesis of celiac disease rotavirus infection frequency and risk of celiac disease autoimmunity in early childhood: a longitudinal study a subset of anti-rotavirus antibodies directed against the viral protein vp7 predicts the onset of celiac disease and induces typical features of the disease in the intestinal epithelial cell line t84 influence of early environmental factors on lymphocyte subsets and gut microbiota in infants at risk of celiac disease; the proficel study cervical human papillomavirus infection in mexican women with systemic lupus erythematosus or rheumatoid arthritis giant cell arteritis and polymyalgia rheumatica after influenza vaccination: report of 10 cases and review of the literature postvaccine vasculitis: a report of three cases clinical study of childhood acute disseminated encephalomyelitis, multiple sclerosis, and acute transverse myelitis in fukuoka prefecture inflammatory/post-infectious encephalomyelitis high level of cross-reactivity in influenza virus hemagglutinin-specific cd4+ t-cell response: implications for the initiation of autoimmune response in multiple sclerosis cross-reactivity with myelin basic protein and human herpesvirus-6 in multiple sclerosis myelin basic protein-reactive autoantibodies in the serum and cerebrospinal fluid of multiple sclerosis patients are characterized by low-affinity interactions tauopathy-like abnormalities and neurologic deficits in mice immunized with neuronal tau protein acute/relapsing experimental autoimmune encephalomyelitis: induction of long lasting, antigen-specific tolerance by syngeneic bone marrow transplantation autoimmune/inflammatory syndrome induced by adjuvants (shoenfeld's syndrome): clinical and immunological spectrum post-vaccination encephalomyelitis: literature review and illustrative case vaccinations and risk of central nervous system demyelinating diseases in adults acute disseminated encephalomyelitis cohort study: prognostic factors for relapse neuromyelitis optica following human papillomavirus vaccination hbv vaccine and dermatomyositis: is there an association? autoimmune blistering diseases of the skin pemphigus: etiology, pathogenesis, and inducing or triggering factors: facts and controversies does influenza vaccination induce bullous pemphigoid? a report of four cases bullous pemphigoid after herpes zoster vaccine administration: association or coincidence? reactivation of bullous pemphigoid after influenza vaccination pathophysiology of autoimmune polyneuropathies a review on the association between inflammatory myopathies and vaccination fibromyalgia and overlapping disorders: the unifying concept of central sensitivity syndromes etiology of fibromyalgia: the possible role of infection and vaccination infection and vaccination in chronic fatigue syndrome: myth or reality? review part 2: human herpesvirus-6 in central nervous system diseases pathogenesis of parvovirus b19 infection: host gene variability, and possible means and effects of virus persistence effects of staphylococcus toxoid vaccine on pain and fatigue in patients with fibromyalgia/chronic fatigue syndrome vaccination as teenagers against meningococcal disease and the risk of the chronic fatigue syndrome the sick building syndrome as a part of the autoimmune (auto-inflammatory) syndrome induced by adjuvants selective elevation of circulating ccl2/mcp1 levels in patients with longstanding post-vaccinal macrophagic myofasciitis and asia cognitive functioning in gulf war illness health of uk servicemen who served in persian gulf war gulf war syndrome: is it due to a systemic shift in cytokine balance towards a th2 profile antibodies to squalene in us navy persian gulf war veterans with chronic multisymptom illness vaccination in adult patients with auto-immune inflammatory rheumatic diseases: a systematic literature review for the european league against rheumatism evidence-based recommendations for vaccination in adult patients with auto-immune inflammatory rheuma eular recommendations for vaccination in paediatric patients with rheumatic diseases vaccination against yellow fever among patients on immunosuppressors with diagnoses of rheumatic diseases updated consensus statement on the use of rituximab in patients with rheumatoid arthritis safety and efficacy of meningococcal c vaccination in juvenile idiopathic arthritis unexpectedly high incidence of persistent itching nodules and delayed hypersensitivity to aluminium in children after the use of adsorbed vaccines from a single manufacturer key: cord-285505-8norumv6 authors: vere hodge, r. anthony title: meeting report: 27th international conference on antiviral research, in raleigh, nc, usa date: 2014-09-16 journal: antiviral res doi: 10.1016/j.antiviral.2014.08.009 sha: doc_id: 285505 cord_uid: 8norumv6 the 27th international conference on antiviral research (icar) was held in raleigh, north carolina, usa from may 12 to 16, 2014. this article summarizes the principal invited lectures. john drach (elion award) described the early days of antiviral drugs and their novel modes of action. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept nucleoside analogs. replacing thymine by 5-chlorouracil led to the generation of a new form of escherichia coli. adrian ray (prusoff award) demonstrated how prodrugs can markedly improve both the efficacy and safety of potential drugs. the keynote addresses, by david margolis and myron cohen, tackled two emerging areas of hiv research, to find an hiv “cure” and to prevent hiv transmission, respectively. these topics were discussed further in other presentations – a cure seems to be a distant prospect but there are exciting developments for reducing hiv transmission. tdf-containing vaginal rings and gsk-744, as a long-lasting injection, offer great hope. there were three mini-symposia. although therapy with tdf/ftc gives excellent control of hbv replication, there are only a few patients who achieve a functional cure. myrcludex, an entry inhibitor, is active against both hbv and hdv. the recent progress with hbv replication in cell cultures has transformed the search for new antiviral compounds. the hbv capsid protein has been recognized as key player in hbv dna synthesis. unexpectedly, compounds which enhance capsid formation, markedly reduce hbv dna synthesis. the development of bcx4430, which is active against marburg and ebola viruses, is of great current interest. this article provides an overview of the invited lectures at the 27th international conference on antiviral research, sponsored by the international society for antiviral research (isar), which was held in raleigh, north carolina, usa from may 12 to 16, 2014 . it begins with reports of lectures by the recipients of isar's three major awards, held in memory of gertrude elion, antonín holý and william prusoff. these are followed by brief summaries of the keynote addresses and the three mini-symposia on ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. because this review article simply provides short accounts of oral presentations, it is not generally accompanied by references to the scientific literature. any descriptions of favorable treatment outcomes should not be taken as recommendations for clinical use. 2. gertrude elion memorial award lecture: collaborative antiviral studies for the discovery of drugs to treat cytomegalovirus infections john c. drach, ph.d., university of michigan, ann arbor, michigan, usa (fig. 1) . gertrude b. (trudy) elion was born in new york city and was pleased to work for the burroughs wellcome co. when based in new york but was concerned when it transferred to research triangle park, north carolina, not many miles from this year's meeting site. however, within just a few months she declared that she was ''at home'' in north carolina. she was awarded the nobel prize in physiology or medicine in 1988 for her pioneering work in purine biosynthesis which paved the way for the discovery of drugs to treat organ rejection, cancer and viral diseases. the focus of john's presentation was on the research conducted in his own and his collaborators' laboratories that ultimately led to the invention of three compounds which were discovered to have antiviral activity against human cytomegalovirus (hcmv) and which later entered clinical trials: bdcrb pyranoside (gw275175x) (phase i), maribavir (phases i, ii and iii) and cyclopropavir (phase i). his major collaborators included karen biron, charles shipman, leroy townsend, and jiri zemlicka. to date, there are only five fdaapproved drugs for treatment of hcmv infections: cidofovir, fomivirsen, foscarnet, ganciclovir and valganciclovir. being inspired by the presence of a naturally-occurring 5,6dimethylbenzimidazole nucleotide in vitamin b12, research on benzimidazole nucleosides was initiated by medicinal chemists in the 1950s and '60s. this led to the synthesis of a trichloro analog in townsend's laboratory at the university of utah and later the discovery of its activity against hcmv in john's laboratory. much work, in both their laboratories at the university of michigan, established that it and its 2-bromo analog (bdcrb) have excellent activity against hcmv with very low cytotoxicity. surprisingly, it was found to be inactive against other herpes viruses and it did not need conversion to a triphosphate to be active against hcmv. collaborative studies with karen biron at burroughs wellcome established that, unlike many other anti-virals that inhibit viral dna synthesis such as ganciclovir (gcv), these compounds acted by a novel mechanism, inhibition of viral dna processing. it was the viral resistance studies which revealed the viral targets, pul89 and pul56. these two proteins, with pul104, form a complex known as the terminase which cuts newly synthesised hcmv dna into unit lengths for packaging into virions. although bdcrb had many desirable properties in vitro, it had poor pharmacokinetics in mice and monkeys due to hydrolysis of its glycosidic bond; therefore it was not developed for human use. much additional work in drach's and townsend's laboratories at michigan and by biron's group at burroughs wellcome ultimately led to two potential drug candidates, bdcrb pyranoside and maribavir (fig. 2) . both compounds have excellent activity against hcmv, low toxicity, and excellent pharmacokinetics. clearly, their modes of action differed markedly from that of gcv. quite unexpectedly, they have different mechanisms of action. bdcrb pyranoside has a mechanism of action very similar to its parent compound bdcrb, inhibition of dna processing. in contrast, maribavir inhibits dna synthesis, albeit indirectly. it is a 2isopropylamine derivative of bdcrb except that it has the unnatural l-sugar configuration. its mechanism of action involves inhibition of the viral kinase (pul97), which phosphorylates another viral protein, pul44. phosphorylated pul44 is necessary for viral dna synthesis. thus inhibition of pul97 by maribavir inhibits viral dna synthesis. interestingly, pul97 is also the kinase that activates (phosphorylates) gcv. resistance studies confirmed that a single mutation in ul97, resulting in a mutation in the kinase (leu397arg), was necessary and sufficient for resistance to maribavir. in a further study of resistance, virus already resistant to bdcrb was passaged in increasing concentrations of maribavir and resistant virus was isolated. this strain grew at the same rate as the wild-type virus and was resistant to both bdcrb and maribavir. as expected, resistance to bdcrb was due to known mutations in ul56 and ul89. however, no mutations were found in ul97. further investigation showed that a single base change in ul27 (t1004c) was necessary and sufficient for resistance to maribavir. the role of the encoded protein was then unknown but the amino acid mutation (leu335pro) is in the middle of the protein. similarly, biron's group detected resistance due to mutations in the ul27 gene. further research studies on maribavir have been summarized in previous icar scientific reports. cyclopropavir (cpv, fig. 3 ) was synthesized in the laboratory of jiri zemlicka, karmanos cancer institute, detroit, michigan. it is a guanosine nucleoside analog which is very active against hcmv. unlike the benzimidazole nucleosides, it also inhibits epstein-barr virus (ebv) and human herpesvirus 8 (hhv-8). like gcv, it is phosphorylated by the kinase encoded by ul97. it is more potent in vitro and in vivo than ganciclovir but has a somewhat different pattern of resistance. in one resistant strain, the key mutation formed a stop codon resulting in a truncated pul97 kinase protein. the phosphorylation of cpv by pul97 is more efficient than that of gcv, with a considerably lower k m and higher v max . interestingly, the phosphorylation of cpv to its monophosphate (cpv-mp) by pul97 is stereoselective; only the (+) isomer of cpv-mp is formed. a single enzyme, gmp kinase, phosphorylates cpm-mp to both its di-and triphosphates. in contrast, acyclovir and gcv require additional cellular enzymes to convert their diphosphates to active triphosphates. cyclopropavir is currently in phase i clinical trials for the treatment of hcmv infections. 3. the antonín holý memorial award lecture: from modified nucleoside to a chemically modified genome piet herdewijn, rega institute for medical research, ku leuven, belgium (fig. 4) . the 2013 icar began with a symposium, on the legacy of the late antonín (tony) holý , at which the establishment of a new isar award in medicinal chemistry was announced. the awardee is to be a senior scientist of international stature in medicinal chemistry and who has made innovative contributions impacting antiviral drug discovery or development. piet is, therefore, the first to receive this award. in the late 1970s, the potent activities of bvdu and bvarau against herpes simplex virus type 1 (hsv-1) and varicella zoster virus (vzv) were discovered; this work motivated piet to start antiviral research with the synthesis of carbocyclic bvdu. through to the early 1990s, he synthesized several other nucleoside analogs with bicyclic bases having good activity against hsv-1 and vzv. during the 1990s, emphasis switched to investigating the effect of modifying the sugar ring, in particular the synthesis of six-membered rings containing an oxygen or a double bond. piet showed examples of compounds with activity against hsv-1, hsv-2, vzv and hcmv. back in 1984, erik de clercq showed piet a paper on aids, one of the authors being phil furman. this publication stimulated the search for anti-hiv compounds. many compounds were discovered with potent activities (and good selectivity indices) against hiv. piet worked out the first structure-activity relationships of anti-hiv dideoxy nucleosides. starting in the late 1980s, tony holý synthesised a series of phosphonates. at the 2013 icar, erik de clercq recalled how this work led, ultimately, to tenofovir, which was to become a major success for treating hiv-infected patients. from its first introduction in 2001, its market share has increased to well over 40%. in 2002, having a single-pill regimen was agreed as a way forward to simplify, and thereby enhance, hiv therapy. this led to atripla being approved in 2006, complera in 2011 and stribild in 2012. tenofovir, in its various prodrug forms, is now available in over 130 countries and is distributed widely to the known hivinfected population. in line with this research, piet synthesized phosphonate nucleosides, with a threose sugar moiety, which showed anti-hiv activity in the same range as 9-(2-phosphonylmethoxyethyl) adenine (pmea). piet's work had taken a different pathway. it is possible to link several nucleotides together to form aptamers. for example (fig. 5) , the above antiviral nucleosides, which have a 6-membered ring in place of the natural furanose, could be incorporated into hexitol nucleic acid (hna) aptamers. x-ray studies revealed the structures of hna-rna duplexes and hna-hna duplexes, the latter having a similar overall form to that of an rna-rna duplex with the same base sequence. hna-containing aptamers were shown to be potent and specific inhibitors of trans-activating region (tar)mediated transcription. normally, an hiv encoded protein, transactivator of transcription (tat), binds to cellular factors and to the viral tar rna regulatory element, resulting in a vastly increased rate of transcription of all hiv genes. hna-containing aptamers prevents this interaction and so inhibit hiv replication. it took four years to engineer a polymerase that would utilise hnas to assemble a strand complementary to a dna template. in line with this research, hexitol-modified sirna has shown good activity in an in vivo anti-hbv model. this success stimulated the concept that it may be possible to generate new forms of biologically active dna. in order to pursue this idea, a culture system with twin growth chambers was devised. alternative nutrient media could be fed into the chambers and the culture from one chamber could be used to seed the second chamber, the former culture being removed. in this example, the aim was to replace thymine with 5-chlorouracil ( fig. 6) using escherichia coli. initially, the nutrient contained 10% 5-chlorouracil and 90% thymine. with each cycle, seeding one chamber from the previous one, the proportion of 5-chlorouracil was increased. after 180 days, in which there had been about 4000 generations of e. coli, thymine had been replaced totally by 5-chlorouracil. an interesting outcome was that the alternative base led to a change not only in the genotype but also in the phenotype; the ''new'' e. coli cells were much longer than the original. this is the first example of a dna polymerase being adapted through evolutionary pressure to accept a nucleotide analog, resulting in the generation of a new living organism. prusoff young investigator award lecture: use of nucleotide prodrugs to enhance selectivity of anti-hiv and -hcv agents adrian s. ray, gilead sciences inc., foster city, ca, usa (fig. 7) . adrian started his lecture with photos of william (bill) prusoff and reminisced of his days with bill, raymond schinazi and yung-chi (tommy) cheng. adrian presented examples to illustrate two models of how a prodrug strategy can transform a potential drug into a much improved clinical candidate. in the first, the prodrug alters the distribution of the pharmacologically active nucleotide analog to tissues where viral infection is taking place (on-target) and away from tissues resulting in adverse events (off-target). in the second, the prodrug enables one to select a drug candidate based more directly on the intrinsic properties of the active nucleotide-triphosphate analog via by-passing an inefficient activation (phosphorylation) of the corresponding nucleoside analog. sofosbuvir (sovaldi ò ), a prodrug of 2 0 -f-2 0 -c-meump, was approved in the usa on 6th december, 2013 for treatment of patients with hepatitis c. this is a fine example of a prodrug enhancing the activity of the parent compound. the nucleoside analogue, 2 0 -f-2 0 -c-meu, is poorly active due to restricted phosphorylation to the monophosphate. sofosbuvir, a nucleotide analogue prodrug of 2 0 -f-2 0 -c-meu, delivers the monophosphate into the cell and this is then further phosphorylated efficiently to give high levels of the triphosphate which inhibits hcv rna polymerase. adrian recalled being much impressed by a result reported at the meeting in 2007 of the american association for the study of liver diseases (aasld). in a phase ii monotherapy trial in patients with hcv, at day 3, the viral loads were reduced by log 10 3.2 and log 10 1.1 for vx-950 (1250 mg bid, n=10) and rg-7128 (1500 mg bid, n=8), respectively. however, from day 4 to 13, the polymerase inhibitor (rg-7128) had continued to reduce the viral load, reaching a reduction of log 10 2.7. on the other hand, the protease inhibitor (vx-950) did not give a sustained reduction, with the viral load starting to increase from day 6. at day 13, the viral load was only log 10 2.2 less than baseline. nucleotide analogues have two advantages over other classes of inhibitors. there is a high genetic barrier to resistance selection, due to the hcv rna polymerase being highly specific for its natural substrates and template. this specificity can be altered but only under extreme evolutionary pressure (see section 3). also, nucleotide analogs often have pan-genotype activity because the active site of the hcv ns5b polymerase is so highly conserved. as an example of how prodrugs can impact a discovery program, allowing for more targeted delivery and for the optimization of the intrinsic properties of the triphosphate, adrian presented the history of the gs-6620 program. the c-adenine analogue (2 0 -c-me-4-aza-7,9-dideazaa, c-nuc1) was compared to the corresponding n-nucleoside, mk608. in a genotype1b replicon assay, the ec 50 values were 2.5 lm and 0.08 lm respectively. however, their triphosphates were equally effective against hcv ns5b polymerase (ic 50 values both 0.3 lm). in the replicon system, the triphosphate of the n-nuc (mk608) was formed more efficiently than that of the c-nuc1, thus explaining the lower activity of the c-nuc1. however, in primary human hepatocytes, c-nuc1 was phosphorylated to the triphosphate more efficiently than the n-nuc (mk608). this illustrates the importance of using primary human cells. c-nuc1 seemed to have a benign in vitro toxicity profile, including not inhibiting the mitochondrial dna polymerase-gamma, but it had very significant toxicity in animals. in a collaboration between gilead and craig cameron at pennsylvania state university, the researchers sought to identify the toxicity target(s) for ribonucleotide analogues, including c-nuc1 and others that had been stopped in phase ii trials. these studies showed a correlation between c-nuc1 and the phase ii candidates, r1626, nm283 and bms986094/idx184. all the latter were efficiently incorporated into rna by the mitochondrial rna polymerase (>70% of the corresponding natural nucleotide). the triphosphate of c-nuc1 was also an efficient substrate (22% the rate of atp). in contrast, the active nucleotide analogs, formed by drugs approved for the treatment of hcv, were poor substrates. ribavirin was poorly incorporated (about 5%) and sofosbuvir was below the limit of detection (= 0.02%). more extensive in vitro and cell culture evaluation of the compounds could have saved the expense of taking them into clinical trials. understanding that the mitochondrial rna polymerase is an important target for ribonucleotide toxicity, the gilead team sought analogs that were not incorporated by this polymerase. adding a cn group to the 1 0 position of c-nuc1 did not change its activity as an hcv ns5b polymerase inhibitor (ic 50 0.3 lm) but it did reduce incorporation in the mitochondrial rna assay (<0.02%). however, in the absence of a nucleotide prodrug to bypass the first phosphorylation step, the resulting di-substituted nucleoside analog would not be a drug candidate because it was not efficiently activated in cells. application of a nucleotide prodrug strategy allowed this nucleotide to be pursued further. oral absorption, delivery of the monophosphate into hepatocytes and high hepatic extraction were criteria used as part of the prodrug optimization process. a nucleotide prodrug, gs-464335 (a mixture of diastereoisomers at phosphorous) was well absorbed in dogs (>80%). comparing the pre-hepatic and post-hepatic plasma drug levels, about 80% of the absorbed drug was taken up by the liver. inside cells, gs-464335 was converted to the corresponding monophosphate which was efficiently converted to the triphosphate. at 24 h, the triphosphate levels remained about 2-fold above the ic 90 value. a pure stereoisomer was selected and later named gs-6620. in a phase ii trial (900 mg, bid 5 days), the mean reduction in hcv load was about log 10 1.5. two subjects achieved hcv rna <25 iu/ ml. however, the pharmacokinetics and antiviral responses were highly variable. whereas the activity results were disappointing, clinical proof of concept was observed in terms of safety. gs-6620 did have a markedly improved safety profile relative to c-nuc1, progressing through chronic toxicology studies in rats and dogs at relatively high doses. the story of gs-6620 illustrates both how nucleotide prodrugs enable further progression of candidates and also the complexity of predicting the behavior of nucleotide prodrugs across species. one wonders what cell culture test or animal model may have predicted such variability. when selecting famciclovir as the prodrug for penciclovir, one potential prodrug was rejected because the pharmacokinetics in rats varied widely between individual animals (vere hodge et al., 1989) . a recent publication by adrian and his team highlights the metabolism of gs-6620 by carboxylesterase 2, an enzyme highly expressed in the human small intestine but not uniformly expressed in different animal species, as a possible reason for the highly variable and suboptimal intestinal absorption of gs-6620 in humans (murakami et al., 2014) . the focus of adrian's talk then switched to hiv. over the last 15 or 20 years in north america, the hiv-infected population has been changing, becoming older (now 33% over 50 years old vs <10% in 1995) and more likely to be obese (in every usa state, >20% adults with bmip30). this has led to a shift in the focus of antiretroviral therapy (art), from solely control of hiv replication to now include tolerability in older, possibly obese, patients. the first example given for hiv was how application of a different prodrug strategy can markedly change the distribution even when delivering the same pharmacologically active nucleotide analog. the first approved prodrug of tenofovir (tfv) was tfv disoproxil fumarate (tdf). more recently, tfv alafenamide (taf) has been progressed into clinical development. a key difference in the properties of the two prodrugs is their stability in plasma, with half-lives of 0.4 and 90 min, respectively. even with a short halflife, tdf gave better delivery of tfv into cells, as indicated by the hiv ec 50 values in cell culture assays but there clearly was room for improvement; the ec 50 values for tfv, tdf and taf are 1.2, 0.015 and 0.003 lm respectively. whereas the gain in cell culture ec 50 value may be modest, this is not the only gain. the increased stability of taf allows it to load on-target cells and tissues (e.g., lymph nodes) for a longer period of time resulting in increased lymphoid cell and tissue levels at greatly reduced circulating tfv levels, leading to less exposure to off-target tissues (e.g., kidney). in monotherapy studies after oral dosing with tdf (300 mg) and taf (25 mg), the plasma tfv auc is reduced from 1920 to 268 ng.h/ml respectively whereas the reduction in hiv load from baseline is improved, from log 10 0.97 to log 10 1.46 copies/ml, respectively, reflecting the more efficient delivery of taf to target cells and tissues. clearly the lower dose of taf (25 mg) relative to tdf (300 mg) will give taf a marked advantage when considering combination pill therapy. understanding how marked a difference a prodrug can make from the taf example, adrian went on to describe how a prodrug approach transformed a new nucleotide project in which intrinsic properties of the pharmacologically-active nucleotide analog were optimized. their starting point was gs-2128 (d4api), which had good activity against both wild-type and resistant hiv strains but was an active inhibitor of mitochondrial polymerase-gamma. on comparing the known structures of hiv rt and mitochondrial polymerase-gamma, differences in the 2 0 -binding pocket were noted. this led to gs-9148 in which 2 0 -f was added to gs-2128 (fig. 8) . compared to tfv, gs-9148 was about 3-fold less active against wild-type hiv but maintained better activity against resistant strains (k65r and multiple thymidine analog resistance mutations). most importantly, it was inactive (ic 50 >300 lm) against mitochondrial polymerase gamma. more than 50 prodrugs were synthesized and evaluated in metabolism studies and in dogs (intravenous and oral administration). then the enantiomers were tested separately in dogs. this led to the selection of gs-9131. whereas tfv is efficiently utilised by renal uptake transporters, gs-9148 was poorly taken into the kidney. no adverse renal findings were observed with the prodrug (gs-9131) in 28-day studies in rats, dogs and monkeys at the highest doses tested (300 mg, 20 mg and 30 mg/kg daily, respectively). in summary, this work has given examples of the prodrug approach being used successfully both to increase selectivity (by loading on-target tissues vs off-target tissues) and to increase activity (via by-passing metabolic constraints). adrian presented cases in which a prodrug strategy was able to fulfil the full potential of a selective, active triphosphate analog and enable its further progression as a clinical candidate. the keynote speakers were david margolis and myron cohen (fig. 9) . david margolis, university of north carolina, nc, usa in hiv-infected patients, there is a long-lasting reservoir of hiv in the form of integrated viral dna in resting cd4+ memory cells of the host immune system. therefore, even if it were possible to eliminate 100% of viral replication, a reservoir of hiv would remain. there may be reservoirs in other long-lived cells. to date, there is only one known hiv patient who has been cured of his infection, the ''berlin patient''. he was treated for cancer by chemotherapy followed by a bone-marrow transplant. being ccr5 +/à, the chemotherapy had a greater chance to remove all the ccr5+ve cells. the bone marrow donor was ccr5àve. although this patient continues to have no sign of hiv infection, this is hardly a viable treatment option for most hiv-infected patients. even in subjects with hiv replication well controlled by therapy, 70% have detectable plasma viremia which does not appear to decay over time (at least two years). to improve the sensitivity of the assay for hiv, 4 billion lymphocytes are mixed with antibody attached to magnetic beads. this selects for the cd4+ t cells, about 0.2-1 billion cells. the limit of detection is 1 copy of hiv rna/million cells, limit of quantitation is 10 copies/million cells. to reduce the reservoir of hiv, it was suggested that activation of integrated hiv in resting cd4+ t cells would give renewed hiv rna synthesis and possibly result in cell death either due to viral cytopathic effects or resulting from hiv-specific immune responses. a small clinical trial was set up to test this hypothesis. vorinostat (vor), a clinically approved drug for treating certain cancers, has been shown to bind to the active site of histone deacetylases. after a single dose, there was an increase in hiv rna (1.5 to 5-fold, mean 2.6-fold). of these subjects, 5 elected to continue with multiple doses. from the 11th to 22nd vor dose, acetylation of histones and activation of hiv rna synthesis became refractory to therapy. also, it is not known what proportion of cells, with latent hiv, can be activated. whereas a single vor dose did increase the expression of hiv rna, this is not an effective therapy for removing the hiv reservoir. myron cohen, university of north carolina, nc, usa myron noted that there are 2.5 million new hiv infections each year. in this context, anal sex may be an important factor because just one or a few virions of hiv can be infective; within 3 weeks, there is rapid virus replication throughout the body and latent hiv reservoirs of ''founder virus'' are already formed. although anal sex has been associated with homosexual couples, myron pointed out that it is not uncommon amongst heterosexual couples. although behavioral education should be encouraged, it can never be the whole answer. various approaches to the prevention of hiv transmission are being evaluated. monoclonal antibodies, broad neutralising antibody (bnab) and vaccines may have potential for prevention of transmission, but most progress is being made with dapivirine rings containing tdf. these are designed to stay in the vagina for a month. phase iii trials are ongoing. a long-acting hiv integrase inhibitor, gsk 1265744 (generally known as gsk 744), is administered i.m. once every 3 months; a two-year safety trial will be required. phase i trial has been completed and phase ii trial is being planned. by analogy with tuberculosis therapy, in which the infectious state is disabled prior to a complete cure, one wonders if hiv transmission rates may decrease with effective art use. in 2005, the hiv prevention trials network (hptn) initiated a study (hptn 052) which enrolled 1,763 hiv sero-discordant couples (couples that have one member who is hiv-infected and the other who is hiv-uninfected), mostly (97%) heterosexual couples. the infected partner had to be well enough not to require immediate art. the couples were randomised to have either immediate or delayed art. both groups received the same care including counselling on safe sex practices, free condoms, treatment for sexually transmitted infections and regular hiv testing. in may 2011, it had been announced that there had been 27 hiv transmissions in the delayed art group (877 couples) compared to only 1 in the immediate art group (886 couples), a 96% reduction. in these 28 cases, the hiv strain was linked to the partner. this is the first randomised clinical trial to show that treating an hivinfected individual with art can reduce the risk of hiv transmission to an uninfected partner. even with ''safer-sex'' counselling, there were 60 pregnancies in the delayed art group, despite that group having more incentive for safer-sex. following the announcement of this result, all infected participants were offered art. myron reported the 10th annual review of this study. in the delayed art group, there had been a total of 28 cases of hiv transmission with the hiv strain linked to the partner and 11 cases of unlinked transmission. in the one case of hiv transmission in the immediate art group, infection had been detected at day 85 of the study and further investigation suggested that the infection event was on day 1. clearly, early art is highly beneficial. cdc guidelines now recommend that all hiv infected patients should have art. 6. mini-symposium: hepatitis b virus anna lok, university of michigan, mi, usa the number of people infected with hbv world-wide, as estimated by the who and cdc in 2007, was between 223 and 240 million, but was declining due to vaccination. in the usa, vaccine use has led to a steady decline in the rate of new infections, decreasing from about 10/100,000 residents in the 1980s to about 1/100,00 today. in contrast, the prevalence of chronic hepatitis b among immigrants remains high, with no decreasing trend. when infection is acquired early in life, chronic infection is the norm. high viral load is associated with progression to liver cancer. there are 7 fda-approved drugs to treat chronic hbv infection, including entecavir (etv), emtricitabine (ftc) and tdf. with several years of continuous therapy, hbeag loss is achieved in about 40% of patients but hbsag loss (the ultimate goal, seen as a ''cure'') is still a distant prospect for most patients. however, cirrhosis can be reduced by long-term antiviral treatment. in one tdf trial at 5 years, 344/348 patients had a liver biopsy which showed that 73% of patients had improved fibrosis scores (p2 units) and that most other patients had no worsening. tdf has now been used for 6 years without detecting hbv resistance, making it one of the first line drugs. tdf is generally well tolerated but its rare side effects include nephrotoxicity (see above for a possible switch to taf when it is approved), reduction in bone mineral density and very rarely lactic acidosis. despite the major progress made in hbv therapy, there remain various challenges. one is cost, about $60,000-$72,000 for 5-year tdf therapy. pharmacy claims show that adherence is a problem; doses used are less than doses prescribed. there is a lack of accurate prediction of how hbv disease will progress in individuals. hbv dna can be integrated into the human genome at an early stage of infection. fortunately, the integrated viral dna is usually not the complete viral genome and patients, who achieve hbsag loss, rarely relapse. stefan mehrle, university of heidelberg, germany (stephan urban, head of hepatitis b research group, university of heidelberg, was originally scheduled to give this presentation). some chronic hbv-infected subjects are co-infected with hepatitis delta virus (hdv). this is a defective virus that replicates only in the presence of hbv. current antiviral drugs do not inhibit hdv. recently, heparan sulphate proteoglycan (hspg) has been shown to be essential for binding both hbv and hdv to primary hepatocytes. in 2012, human sodium taurocholate co-transporting polypeptide (hntcp) was identified as a functional receptor for hbv and hdv. hntcp is also designated as a solute carrier protein 10a1 (slc10a1). hntcp was shown to be a binding factor for the pres1 domain of the hbv l envelope protein. this interaction was found to be essential for hbv and hdv infection. whereas hbv replication is poor in cell lines derived from hepatocytes (e.g. hepg2 and huh-7) in which hntcp is usually weakly expressed, hbv replication is possible in primary human hepatocytes. the critical discovery was that over-expression of hntcp in hepg2 or huh-7 cells conferred susceptibility to hbv and hdv infection. myrcludex-b is a lipopeptide derived from amino acid residues 2-48 of the pres1 region of the hbv l protein. because it quickly (within 5 min) targets the liver, it is being developed for liver imaging and for drug targeting. it also acts as an entry inhibitor for hbv and hdv by interrupting binding between the hbv l protein and hntcp. it specifically inhibits hntcp-mediated taurocholate transport but the effect on hbv replication is much greater. myrcludex-b activity has been investigated in vivo using scid mice reconstituted with human hepatocytes. with prophylactic treatment, not one infected hepatocyte was seen. following therapeutic treatment, at week 6 post-infection, there were a few isolated infected cells. after the end of therapy, the infection seems to spread but only to neighboring cells. myrcludex-b has been synthesised on a 100 g scale. toxicology evaluation in 3 chimpanzees has been completed and clinical trials have been initiated. in a phase i trial using a 20 mg dose, myrcludex-b was well tolerated. results of a further phase i trial are due to be reported later this year (2014). a dose-ranging phase ii trial has been started. kyong-mi chang, university of pennsylvania, pa, usa anti-hbs antibodies clearly play a critical role in controlling hbv disease. their presence has been accepted as an indication of an ''effective cure''. however, these antibodies appear late in the disease course and so they must have a limited role in the early stages of the disease. what is the role of t-cell responses? in contrast to other viruses, there is a delayed onset, about 4-8 weeks rather than days. cd4+ t cells regulate the adaptive response, cd8+ t cells attack hbv-infected cells. the national institute of diabetes and digestive and kidney diseases (niddk), part of the usa national institutes of health (nih), is supporting a prospective clinical trial to investigate hbv-specific t cell responses during the course of hbv disease. there are no clear t cell differences relative to hbv genotype. the t cell responses are highest during acute hbv infection. during the chronic phase of hbv disease, t cell responses remain suppressed. in conclusion, there are a lot of players in the immune control of hbv infection but their relative contributions and how they adapt to control hbv replication are still largely unknown. 6.4. diversifying the hepatitis b pipeline: current efforts to explore novel mechanisms andrea (andy) cuconati, institute for hepatitis & virus research, pennsylvania commonwealth institute, pa, usa current hbv therapy using nucleotide anti-virals has been highly effective in controlling the infection but a ''cure'', as defined by hbsag seroconversion, has remained elusive. at best after 5 years, the rate is about 25%. other approaches are needed. myrcludex-b (see section 6.2) is the lead entry inhibitor. nvr-1221, an encapsidation inhibitor, is entering clinical trials. in addition. studies with novel nucleotide analogues are ongoing. the hbv field has been transformed recently by the introduction of cell-based antiviral assays. stefan mehrle (see section 6.2) has been leading the way. the assay read-out will need to be optimized for high-throughput screening (hts) but, already, the assay has shown some ''hits''. a few compounds inhibited encapsidation of viral rna. (the hbv virion contains partly double stranded (ds) dna but the reverse-transcription from rna to dna occurs within the capsid.) within the cell, hbv dna is transported into the nucleus where the viral dna forms covalently closed circles (cccdna). two specific inhibitors of cccdna formation have been found. current nucleotide anti-hbv compounds do give large reductions of hbv dna in plasma but only a minimal reduction in levels of the hbs antigen (about log 10 0.1). in contrast, one ''hit'', hbf-0259 inhibited surface antigen production but not genomic replication. structure-activity-relationship (sar) studies have given the current lead compound, hbv-0215. in conclusion, the cell-based assay, with complete replication of hbv, has markedly improved the screening for anti-hbv compounds although further optimization is still needed to give hts capability. adam zlotnick, university of indiana, in, usa. over the last few years, there has been much progress towards understanding the critical role of the hbv core protein -it is much more than just a protective coat for the genome because it plays a major role in the hbv life cycle. the core protein, being 183 amino acids long, is known as cp 183 . the first 149 amino acids are involved in core assembly whereas the last 34 residues, rich in serines and arginines, bind to rna. phosphorylation of the serines, particularly s155, s162 and s172, is required for specific packaging of full length hbv rna complexed to the polymerase (reverse transcriptase -pregenomic rna; rt-pgrna). this rt-pgrna complex initiates encapsidation. the core consists mainly of cp 183 but also includes other proteins (about 0.5%). adam showed us a computer model of the core, using different colours to highlight the various critical components. inside the core, the area of highest density (highlighted in red) represented the polymerase which was attached to the inner surface of the core. the ''other proteins'' in the core were shown in blue. the current thinking is that the polymerase, initially acting as a reverse transcriptase, is attached to, and guided by, an ''inside railway track''. this enables the polymerase to jump to the other end of the rna to start the reverse transcription into dna and then jump again to the other end to start, but never complete, the replication of the complementary dna strand. the self-assembly of the core is an energetically ''downhill'' process. somewhat surprisingly, it is possible to get mutations in which the core is even more stable but the rt activity is reduced. the phenylpropenamide derivative, at-130, fills a pocket in the core and so stabilizes it, similar to the change in amino acids in the mutants. in the presence of at-130, core assembly occurs faster; hence it is known as a core assembly enhancer (as adam mentioned, not a term much loved by industry, their preference is for core assembly inhibitors). regardless, the whole capsid structure changes. the binding of only a few drug molecules is required to make the core non-functional. it seems that it is easier to find compounds to enhance core assembly than inhibitors. 6.6. targeting cccdna to cure chronic hepatitis b massimo levrero, sapeinza universita' di roma, italy. the current hbv therapies of choice are tdf alone or with etv. these drugs have an extensive safety record with use up to 7 years. however, as for other nucleoside/nucleotide analogs, there is only a limited (about 1 log 10 ) reduction in the levels of hbv cccdna. the half-life of hbv cccdna seems to be long, but is still unknown. hbv replication parallels host gene expression, in that they involve the acetylation of histones, for example h 3 and h 4 . both host transcription factors and viral proteins bind to the cccdna. massimo summarized various assays to study different stages of cccdna during the replication cycle. potentially, these assays would allow the study of various approaches: to reduce or clear cccdna, to silence cccdna or to prevent the formation of new cccdna so that it would eventually be removed by dilution and cell death. for proof-of-concept, known ''epigenetic'' compounds, which act as transcription inhibitors, have shown that cccdna can be silenced. by reducing histone acetylation, the cccdna becomes too compact to allow transcription. this approach mimics, partly, therapy with interferon. this research is still at an early stage. due to time constraints, the next two speakers were asked to present brief summaries. john morrey (utah state university, ut, usa) described four mouse models but all stages of the life cycle of hbv can be studied only in the chimeric mouse model, in which human hepatocytes are used. however, this model lacks the potential to study the immune system and it is very expensive. stephan menne (georgetown university, dc, usa) described the woodchuck model. woodchuck hepatitis virus (whv) resembles the human virus and the disease in animals has many similarities to that in humans. neonatal infection becomes chronic in about 60-75% of cases. these chronic cases have virtually a 100% life-time risk of developing cancer, the time scale being about 1 year of chronic infection, followed by cancer at years 3 to 4. the use of microbicides is an active area of research for the prevention of transmission of hiv. david katz (duke university, nc, usa) described how mathematical models may aid drug product design. for example, if it is assumed that the microbicide gel is 400 microns thick, the epithelium is 200 microns and the stroma (connective tissue) is 3000 microns and if the partition coefficient between gel and epithelium in known, then it is possible to model drug transfer and suggest how various other parameters, for example the size of the subject, may modify drug delivery. it is important that different disciplines work together, for example biophysicists with behavioral scientists. biophysics can help an understanding of complex physical phenomena but human behavior can be both complex and highly variable. ralph baric (university of north carolina, nc, usa) noted that a particular infective agent, for example norovirus (nov), may cause subclinical or serious disease in different individuals. in general, animal models are designed to give consistent outcomes rather than aiming to mimic the genetic diversity found in human subjects. in a collaborative effort, mice from 8 ''founder'' strains, including 3 wild-derived strains, were selected. the 5 founder laboratory strains were all derived ultimately from a single female mouse ca 1900. the susceptibility of the 8 founder strains to severe acute respiratory syndrome coronavirus (sars-cov) differed widely (ld 50 p10 6 -10 2 ). the founder strains were cross-bred. although ca 90% of the genes was equally distributed among the new mouse lines, there were gene combinations not seen previously. after infecting mice from the different founder strains with a constant sars-cov inoculum and measuring virus load at a set time after infection, there was a correlation between virus load and disease (as measured by vascular cuffing). it was possible to relate the effect to chromosomes 3 (27%) and 13 (20%). hopefully, identification of the important genes may be achieved. by keeping the virus inoculum constant, this system better represents the clinical spectrum of disease. when using this system to evaluate a potential vaccine, it was found that mice, under the age of one year, could be protected. however, there was a range of effectiveness, from good protection to inactive. these variations may give a representation of human diversity. angela kashuba, university of north carolina at chapel hill, nc, usa in four clinical studies, truvada [a combination pill containing tdf and emtricitabine (ftc)] was taken once daily to prevent hiv transmission, known as pre-exposure prophylaxis (prep). the adherence rates were unexpectedly poor in all four studies, particularly low in the study including at risk women. for example in one study, ''high adherence'' was defined as subjects taking at least 80% of drug doses and was achieved by only 54% of subjects. possible reasons may have been the apparent risk of side-effects (the long consent form included 7 pages of side-effects) and the perception that the subjects, as individuals, were not particularly at risk of infection by hiv. importantly, the trial did confirm the concept that prep could be effective. there was >90% protection in those subjects generally taking 7 doses/week and there was some protection, albeit much less, in subjects taking 2 doses/week. adherence rates, reported by subjects, were appreciably higher than the rates evidenced by drug blood level measurements taken just before the next dose (i.e. 24 h after previous dose). in an attempt to better understand and model these data, the drug concentrations (tdf/tfv and ftc) in various tissues were measured. the ratio between drug concentrations in blood and tissue samples differed greatly for tdf/tfv, with less variations for ftc. concentration ratios of tdf/tfv were about 50 in rectal tissue but only 0.2 in vaginal tissue. for ftc, the ratios were 2.6 and 1.3, respectively. when considering the possible consequences of missed doses, the time scale for hiv infection is an important factor. it is thought that hiv takes about 1-3 h to reach the epithelial cells. clearly, adherence is a critical factor for efficacy and so a real-time objective method for measuring adherence is urgently needed before further clinical studies are initiated. 7.4. the novel nucleoside analog bcx4430 exhibits broad-spectrum antiviral activity and confers post-exposure protection against ebola and marburg viruses travis k. warren, usamriid, fort detrick, md, usa ebola and marburg viruses are members of the filovirus family. even in recent outbreaks of these diseases, including the current ebola epidemic in west africa, care workers are becoming infected and dying. drugs, which are being investigated for treating these diseases, are progressed under the fda ''animal rule''. bcx4430 is a c-nucleoside adenine analog (fig. 10 ) which is being progressed by biocryst pharmaceuticals inc. (warren et al., 2014) . in cell culture assays, bcx4430 is active against ebola and marburg viruses, (ec 50 ca 1 lm). with bcx4430 at 30 lm, there was no detectable incorporation into host dna or rna. in rats, bcx4430 is efficiently activated (phosphorylated) to the triphosphate. in a primer-extension assay, there is some read-through beyond a single residue of bcx4430, but there is effective chain termination after the first bcx4430 residue where the template has two consecutive uridine residues. bcx4430 has been tested in rodent and nonhuman primate models of marburg hemorrhagic fever. in mice, there was a dose response (30, 20, 3.3 and 1.1 mg/dose, bid) with full protection at the two higher doses (survivors, 100%, 100%, 95% and 83% respectively). in an experiment with dosing starting at different times (4 h pre-infection, 24, 48, 72, 96 and 120 h post-infection vs placebo), the placebo-treated mice died on days 6, 7 and 8 with one survivor (10%). in the treated groups, the percent survival was 80, 100, 80, 100, 100 and 30, respectively. in guinea pigs, bcx4430 (bid) with treatment starting at different times (1 h pre-infection, 24, 48 and 72 h post-infection) there was full protection (100% survival) for the pre-infection and 24 h groups, with reduced efficacy at the later start times. in cynomolgus monkeys, bcx4300 treatment was started at 1, 24 and 48 h post-infection. in the placebo group, all 6 animals died within days 9 to 12. in all the treated groups, virus loads were reduced by more than log 10 3. there was one late death in the 1 h group but the other 17 monkeys survived. various markers of potential organ damage were reduced in all treated groups. encouraged by these results, 14-day toxicology trials have recently been completed without any serious concerns. biocryst is developing bcx4430 under the fda animal rule and indenabling work is ongoing. when asked about viral resistance, travis explained that it is not ethically permissible to create resistant strains of marburg virus, but samples collected from the monkeys are being sequenced to look for mutations indicative of drug resistance. as yet, mitochondrial toxicity has not been examined. had similar bone marrow transplants, initially seemed to have been ''cured'' but hiv was detected after 70 and 200 days, respectively. latent hiv can survive in various long-lived cells for decades, especially in memory t cells. when these cells proliferate, the integrated hiv genome is duplicated as the cell divides and the cells survive so long as hiv remains silent. compounds known to activate all t-cells are too toxic to become a clinical therapy. however, latency-reversing agents (lra) have greater specificity, ideally activating only the integrated hiv, leading to the death of hiv-containing t-cells. there remains another possibility (perhaps a less popular view) that there is continued low rate of hiv replication. two clinical studies have been initiated in subjects with undetectable plasma hiv levels. raltegravir, an hiv integrase inhibitor, was added to the background therapy. latent hiv is mostly integrated into host dna but hiv may also form episomal circular dna. the proportion of the circular form increases with raltegravir treatment. in the two clinical studies, 13/45 and 9/15 subjects, respectively, had detectable hiv circles which then decayed. this implies that some de novo infection of cells is ongoing. on the other hand, art works well, with no evidence of sequence evolution in the hiv circles at 48 weeks. is it possible that raltegravir is inducing a single round of hiv replication, to give an increase in hiv circles? derek sloan, gilead sciences, foster city, ca, usa like vorinostat, (vor), romidepsin (rmd) is a histone deacetylase inhibitor which is used clinically to treat cancer. memory cd4+ t cells were taken from hiv subjects on suppressive art; ex-vivo treatment with rmd (40 nm) induced a 6-fold increase in intracellular hiv rna which persisted for 48 h. in contrast, a much higher concentration of vor (1 lm) gave a 2 to 3-fold lower response which was only transient. rmd also increased levels of extracellular hiv rna and virions. encouragingly, this ex-vivo induction of latent virus was seen at rmd concentrations that are below the levels of drug achieved in humans by clinical doses of rmd. accordingly, in a phase i/ii trial in hiv-infected subjects on art, rmd gave a better and more sustained response than vor. about 1.5% of cells containing hiv provirus were activated. although this is far too low a percentage to eliminate the latent hiv reservoir, it is hoped that combination of such lra, which give improved results in ex-vivo cell assays, may give better clinical efficacy. gilead scientists have started screening for novel lras. ''gs-1'' has been identified as a hit by hts. research on this lead is at a very early stage. gilead workers are also investigating other approaches. for example, gs-9620 is a toll-like receptor 7 (tlr7) agonist and it acts as an immune stimulator. although it is being evaluated in phase ii studies for the treatment of chronic hbv infections, the potential effect on hiv reservoirs is being investigated. in siv-infected monkeys, oral dosing of tlr7 agonist induced the activation of immune effector cells such as cd8+ t cells and nk cells. based on these data, tlr7 agonists are being further investigated for their effect on latent siv reservoirs in monkeys which have good virological suppression. another approach is to use anti-envelope antibodies. broadly neutralising antibodies (bnabs) are very effective in preventing siv infection when the viral load is low but less effective against a high-load virus challenge. in addition, a prophylactic cmv-vector-based siv vaccine was effective in preventing siv infection in rhesus monkeys. this and similar vaccines are being tested in vivo for their effects on the latent siv reservoirs. in summary, lras are able to activate hiv provirus in memory cd4+ t cells and thereby may enhance the recruitment of immune effector cells to destroy provirus-containing cells. however, a ''cure'' for hiv infection is still a distant prospect. furthermore, latent hiv reservoirs are heterogeneous and so a combination of approaches will likely be required. gerardo garcia-lerma, centers for disease control and prevention, atlanta, ga, usa proof-of-concept studies for prep, are mostly conducted in nonhuman primates. these can be used either to model a single highdose infective challenge or repeated low inoculations, about 10-50 tissue culture infective doses (tcid 50 ). since 2005, rhesus macaque models have been used in a long series of investigations. in a study, in which the monkeys were treated daily with either oral tdf or tdf/ftc and given a weekly siv inoculum rectally, tdf/ftc gave a longer delay in infection than did tdf alone. when using the vaginal infection route, tdf/ ftc gave 100% protection. in contrast, there was far less protection in clinical trials -why? one possible reason may have been that women were having the contraceptive injection, depot medroxyprogesterone acetate (dmpa). a study, in macaque monkeys given dmpa, confirmed that dosing with tdv/ftc gave good drug levels in plasma and in vaginal secretions. therefore, this did not explain the poor protection in the clinical trial. the macaque model has been used successfully to investigate various situations that are presented in the clinic. when macaques were co-infected with siv and a bacteria and treated with tdf/ftc for 12 weeks, there was good, but not complete, protection (80%). with ftc-resistant virus, tdf/ftc remained protective. in this case, ftc-resistant virus has increased susceptibility to tdf. with the k65r mutant hiv, there was protection against a low inoculum but only partial protection (ca 50%) against a high inoculum. whereas daily dosing seems to be acceptable for patients living with hiv, another option for prep is desirable. gsk-1265744 (generally known as gsk-744) is an hiv integrase inhibitor. it can be formulated with nano-particles to provide an injectable drug depot. in the macaque model, gsk-744, injected once monthly, gave full protection against repeated rectal and vaginal exposures. because metabolism of gsk-744 is much slower in humans than macaques, it was expected to remain effective in humans for up to three months. a phase i study confirmed that drug levels remained above the predicted effective level with a 20-week dosing interval. a phase ii trial is planned. another approach is to use vaginal rings, which have been in clinical use as contraceptive devices for years. in the macaque model, tdf-containing rings, replaced every 4 weeks, gave full protection. a phase iii trial has just been initiated. another option, elvitegravir (evg) and taf are being evaluated in a biodegradable polymer. although daily dosing with tdf/ftc has not proved sufficiently successful as prep in clinical use, it has proved that prep is an achievable aim and this has encouraged the progression of other options. courtney fletcher, university of nebraska, omaha, ne, usa atripla was the first triple combination pill taken once daily for hiv therapy. it contained tdf, ftc and efavirenz (efv). the macaque model has been used to investigate the differing tissue distributions of these drugs and how viral replication may be continuing wherever the drug concentrations are lowest. there are two approaches: tissue homogenates and tissue cells. tissue homogenates give both the intracellular and extracellular drug amounts. from tissues, mononuclear cells (mncs) are collected and the intracellular drug concentration measured. this approach is preferred by courtney but this option may be constrained by sample size and the drug concentration may be underestimated. for exam-ple, with raltegravir, after the mncs have been washed 3 times, the drug concentration is very low. much higher raltegravir concentrations are found when the mncs are cleaned by a rapid spin through oil. comparing an oil spin and repeated washes, the oil process gives higher drug levels, typically about 50% higher. following initial studies in macaques, a clinical study, in 32 subjects, investigated distribution of the drugs from atripla in peripheral blood mononuclear cells (pbmc) and various tissues (see above). in 12/32 subjects, there are data on the time to reduce hiv load to <48 copies/ml. in plasma, the time was 3-4 months. in lymphoid tissues, there was a much slower rate of hiv decline. also, patient variability was noted, with the faster responders having the higher drug levels. a drug may be absorbed from the gastrointestinal tract either going via the portal vein to the liver and then into blood circulation or via the lymphoid system. blood flow is about 200 times faster than lymphoid flow. when the water/1-octanol partition-coefficient (logp) of a drug is <5, absorption tends to be via the blood route. the prodrug approach can be used to alter absorption or, as for tfv, stability of the prodrugs (tdf and taf) can influence the relative concentration in lymphoid tissues (see above). this year, the three major award lectures exemplified the strength of icar, covering very different areas of research. john drach (elion award) described his journey through the early days of antiviral research, which led to the identification of novel modes of antiviral action that had not been envisaged previously. piet herdewijn (holý award) used evolutionary pressure to select dna polymerases that accept novel nucleoside analogs. the replacement of thymine by 5-chlorouracil led to the generation of a new form of e. coli. i suggest that this work has important implications in conventional antiviral research. with hiv and hcv protease inhibitors, the genetic barrier is limited by the ability of the viral protease and its substrate (the viral polyprotein cleavage sites) to co-mutate so that the virus can become resistant to the antiviral drug. so far, polymerase inhibitors have not suffered the same fate but this work shows that a poor choice of nucleotide analog could result in a resistant virus with a new type of rna in which the drug replaces a natural nucleoside. adrian ray (prusoff award), describing work at gilead, demonstrated how the prodrug concept can markedly improve both the efficacy and safety of potential drugs. their progress with hiv and hcv therapies has been remarkable. the keynote addresses tackled two emerging areas of hiv research. david margolis summarized work aiming to eradicate hiv from infected subjects and myron cohen described current progress with approaches to prevent hiv transmission. i found both these presentations to be informative and stimulating. hiv ''cure'' still seems to be a distant prospect. in contrast, prior to exposure prophylaxis (prep) has been shown to be an achievable aim although the need for daily dosing is a barrier to success. gerardo garcia-lerma described recent progress which is likely to radically change the prospects for therapeutic convenience and success. tdf-containing vaginal rings, which need replacing only once a month, are being evaluated. another exciting prospect is gsk-744 which has been formulated as a long-lasting injection. a phase i trial confirmed that the drug may be administered at 3month intervals. in the absence of a proven hiv vaccine, prep with drugs has become the most promising strategy to reduce hiv infection rates among high-risk populations. this conference also included three interesting mini-symposia: ''hepatitis b virus'', ''research triangle park'' and ''challenges in hiv infection, treatment and prevention''. an innovation this year was a session devoted to the european training network, euvirna and introduced by frank van kuppeveld. all the 18 euvirna fellows, who attended icar, gave short presentations at this session. for further information, please see the isar news (24.1) in the september issue of antiviral research for an account by frank van kuppeveld. for many years, the clinical symposium was, for me, a major highlight of icar. in my report for the 2013 icar, i expressed a hope regarding hcv therapy: ''there is the prospect that the first nucleotide analogue will be licensed by the time of our next icar meeting. the combination of a nucleotide analogue and a ns5a inhibitor looks set to transform hcv therapy across all genotypes. as for hiv, single-pill, once-daily regimens are following on quickly''. on 6th december 2013, sofosbuvir (sovaldi ò ) was the first nucleotide analog to be approved in the usa by the food and drug administration (fda) for treatment of patients with hcv. approval by the european union followed soon afterwards, in january 2014. a ns5a inhibitor, ledipasvir, formulated as a single fixed-dose combination pill with sofosbuvir, is progressing quickly through clinical trials. with such remarkable progress being achieved since the 2013 icar, i was disappointed to discover that there was no presentation on this topic at this year's icar. a paper (sofia, 2014) , which was part of a symposium in antiviral research on ''hepatitis c: next steps toward global eradication'', emphasizes recent successes. after completing therapy, a sustained virological response for 12 weeks (svr12) is regarded as a cure for hcv-infected patients. the combination of sofosbuvir/ledipasvir has shown remarkable results in clinical trials, with svr12 in the range 95-100% across genotypes. this combination was well tolerated. a nda for the sofosbuvir/ledipasvir combination pill was submitted recently. i do not recall any previous antiviral trials in which the ''intention-to-treat'' analyses showed 100% success rates. perhaps similar to the hcv symposium in antiviral research, i hope that the 2015 icar, which will be held in rome, will have a mini-symposium which will include an account of this remarkable progress. it would be interesting to have an update on the clinical impact of this combination therapy for hcv and to have an assessment on the prospects for global eradication of hcv. beside this one disappointment, there were many excellent presentations and i would like to add my thanks to the isar officers and conference committee for organizing another interesting and successful icar. metabolism and pharmacokinetics of anti-hepatitis c virus nucleotide prodrug gs-6620 beyond sofosbuvir: what opportunity exists for a better nucleoside/nucleotide to treat hepatitis c? selection of an oral prodrug (brl 42810; famciclovir) for the antiherpesvirus agent brl 39123 protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 acknowledgements i wish to thank all those authors who have kindly provided me with copies of their presentations and for giving me valuable comments. also, i thank the president of isar for asking me to prepare this meeting report. key: cord-312965-5hcb15xc authors: qi, yan-fei; zhang, hong; wang, juan; jiang, yanfang; li, jinhua; yuan, ye; zhang, shiyao; xu, kun; li, yangguang; li, juan; niu, junqi; wang, enbo title: in vitro anti-hepatitis b and sars virus activities of a titanium-substituted-heteropolytungstate date: 2011-11-23 journal: antiviral res doi: 10.1016/j.antiviral.2011.11.003 sha: doc_id: 312965 cord_uid: 5hcb15xc a structural determined heteropolytungstate, [k(4)(h(2)o)(8)cl][k(4)(h(2)o)(4)pti(2)w(10)o(40)]·nh(2)oh 1, has been synthesized and evaluated for in vitro antiviral activities against hepatitis b (hbv) and sars virus. the identity and high purity of compound 1 were confirmed by elemental analysis, nmr, ir analysis and single-crystal x-ray diffraction. the compound 1, evaluated in hepg 2.2.15 cells expressing permanently hbv, significantly reduced the levels of hbv antigens and hbv dna in a dose-dependent and time-dependent manner. ec(50) values were determined to be 54 μm for hbeag, 61 μm for hbsag and 2.66 μm for supernatant hbv dna, as compared to 1671, 1570, 169 μm, respectively, for the commercially-available hepatitis b drug adefovir dipivoxil (adv). intracellular cccdna, pgrna and hbcag were also found to be decreased by compound 1 in a concentration-dependent manner. cytotoxicity results showed that compound 1 has low toxicity in hepg 2 cells with cc(50) value of 515.20 μm. the results indicate that compound 1 can efficiently inhibit hbv replication in hepg 2.2.15 cells line in vitro. additionally, compound 1 also shows high anti-sars activity at an ec(50) of 7.08 μm and toxicity with a cc(50) of 118.6 μm against mdck cells. hepatitis b virus (hbv) infections continue to be a major public health problem worldwide (barraud et al., 1999) . more than 400 million people worldwide are currently infected with hepatitis b virus. approximately 20% of hbv patients will develop chronic hepatitis, and are at significant risk of developing cirrhosis or liver hepatocarcinoma. hbv is the prototype of hepadnaviridae, a family of small enveloped hepatotropic dna viruses that can infect the liver of human (marion and robinson, 1983) . chronic hepatitis b patients are commonly treated with either interferon alpha (inf-a), or the nucleoside analog lamivudine (3tc), adefovir, entecavir or telbivudine which are the synthetic reverse transcrip-tase inhibitors (de clercq, 1999; delmas et al., 2002; buster and janssen, 2006) . however, none of these therapies are completely safe and effective. although direct antiviral therapy with amivudine or adefovir could efficiently control chronic active hepatitis b, drug resistance or renal toxicity could develop progressively several months after the initiation of therapy. it is thus still urgently required to identify effective anti-hbv agents. polyoxometalates (poms) are inorganic cluster-like complexes and constituted from oxide anion and transition metal cations. these complexes have shown potential applications in multitudinal fields such as catalysis, medicine and functional materials müller, 1991, 1994) . especially, the medicinal properties of poms have been a subject of interest (witvrouw et al., 2000; judd et al., 2001; shigeta et al., 2003; yamase, 2005) . these compounds have low toxicity for cultured cells, and relatively less expensive than the ''chemical'' antiviral drugs. recently, poms have been reported to inhibit the replication of rna viruses and dna viruses in vitro and in vivo, such as the human immunodeficiency virus, severe acute respiratory syndrome (sars) virus, influenza virus and herpes simplex virus (rhule et al., 1998; dan et al., 2002) . the activity of poms against hepatitis b virus was also suggested by zoulim (1999) . the mechanism of action of poms remains to be fully elucidated, but may occur at any of the life cycle stages, including viral adsorption, penetration, or reverse-transcription (dan et al., 2002; shigeta et al., (domaille and knoth, 1983; ozeki and yamase, 1991) shows high antiviral activity. the interesting biological results of [pw 10 ti 2 o 40 ] 7à prompted us to explore the antiviral activity of compounds containing [pw 10 ti 2 o 40 ] 7à anions as potential anti-hbv agents. it was also reported that the heteropolyoxotungstates [pw 10 ti 2 o 40 ] 7à have broad-spectrum anti-rna virus activity (dan and yamase, 2006) . therefore, it is necessary to further explore the antiviral activity of compounds containing [pw 10 ti 2 o 40 ] 7à . severe acute respiratory syndrome (sars), a disease seriously threatening human health caused by the single-stranded rna coronavirus, spread in 29 countries in early 2003, presenting a worldwide public health concern drosten et al., 2003; ksiazek et al., 2003) . the research attention in exploitation of anti-sars treatments has been mainly focused on vaccines, antiviral drugs, and their integration of traditional chinese medicine and western therapy. however, no effective therapeutic drug is available to date (stadler et al., 2003) . as a part of our ongoing antiviral drug discovery program, a number of pom analogs have been synthesized and evaluated for their potential antiviral activity (li et al., 2004a,b the results indicate that compound 1 exhibits strong antiviral activities against the hbv and sars viruses with low cytotoxicity, indicating that it is a potential medicinal candidate against hbv and sars viruses. all the chemicals were of analytic grade and used without further purification. compound 1, was freshly prepared and characterized. w and ti in 1 were determined by a leaman inductively coupled plasma (icp) spectrometer. infrared spectrum was recorded in the range 400-4000 cm à1 on an alpha centaurt ft/ir spectrophotometer using kbr pellets. the 183 w nmr spectrum was obtained on a bruker am-500 spectrometer operated at 500 mhz with d 2 o as the solvent. quantitative rt-pcr was performed with abi 7300 sequence detection system (roche, germany). (domaille et al., 1983) was added into 50 ml distilled water with stirring. to this solution, 1.03 g (15.0 mmol) nh 2 oháhcl and 1.04 g (2.8 mmol) lacl 3 á7h 2 o was added in sequence. the solution was heated to 70°c for more than 2 h in a water bath, then filtered and kept slow evaporation in an undisturbed place at room temperature. colorless crystals of compound 1 were isolated in 1 week with the yield of 60% based on k 7 pti 2 w 10 the measurement for compound 1 was collected on a rigaku r-axis rapid ip diffractometer with mo-ka monochromated radiation (k = 0.71073 å a 0 ) at 150 k. empirical absorption correction was applied. the structure was solved by the direct method and refined by the full-matrix least-squares on f 2 using the shelxl-97 software (sheldrick, 1997) . all of the non-hydrogen atoms except the disordered atoms o(1), ow(2), ow(3) and cl(1) were refined anisotropically. all the crystallographic parameters are tabulated in table 1 . images were created with the diamond program. hepg 2.2.15 cells (provided by the department of infectious diseases of the 1st hospital, jilin university, pr china) were cultured in dulbecco's modified eagle's medium (dmem; gibco) containing a 10% fetal bovine serum (fbs; gibco), 100 u/ml penicillin, 100 u/ml streptomycin, and 200 lg/ml g418 (growth medium). during the experiments, the cells were grown in the media as described above without g418. the cell lines were incubated at 37°c in 5% carbon dioxide atmosphere. prior to exposures to drugs, the cell viability was verified to be >85% according to the standard trypan blue exclusion test. hepg 2 (provided by the department of infectious diseases of the 1st hospital, jilin university, pr china) were cultured in dulbecco's modified eagle's medium (dmem; gibco) containing a 10% fetal bovine serum (fbs; gibco), 100 u/ml penicillin, and 100 u/ml streptomycin. the cell lines were incubated at 37°c in 5% carbon dioxide atmosphere. sars virus (provided by academy of military medical sciences, beijing, pr china) was propagated in african green monkey kidney cells (vero-e 6 ). vero cells were propagated in dmem supplemented with 10% fbs, 2 mm l-glutamine, 50 u/ml penicillin, 50 lg/ml streptomycin and bicabouate. the cell lines were incubated at 37°c in 5% carbon dioxide atmosphere. drugs were sterilized by filtration prior to use. each agent was dissolved in dmem to generate the appropriate doses for experimentation. non-treated cells (dmem alone) were used as negative controls. 2.5. anti-hbv activity of compound 1 the cytotoxicity of compound 1 was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide table 1 crystal data and structure refinements for 1. formula h 27 clk 8 no 53 pti 2 w 10 fw 3202.75 (mtt) assay as previously described (kodama et al., 1996) . the optical absorbance was read on a plate reader (bio-rad co.) at a wavelength of 490 nm. briefly, hepg 2 cells were plated on 96-well plates at a density of 5.0 â 10 4 cells/ml. after a 24 h in period of incubation, the dilutions of 1, and adv at different doses were added. hepg 2 cells in the negative control group were treated with the same volume of medium. after a period of incubation, mtt solution (0.15 ml, 5 mg/ml in 0.01 m pbs) was added to each well. the cells were incubated for another 4 h at 37°c, and then dmso (0.15 ml) was added. the cytotoxicity was measured by the reduction of mtt observed in mitochondria at 24, 48, and 72 h after the initial treatment. the cc 50 was defined as the concentration of drug that achieved 50% cytotoxicity against cultured cells, as calculated by the bliss method (han et al., 2008) . supernatants of cells treated with compound 1 or adv and nontreated cells were collected. the semi-quantitative detection of hbsag and hbeag were estimated using elisa assay kits (shanghai kehua co., ltd., china). the supernatants of treated and non-treated cells were collected. the hbv dna from the medium was extracted using hbv real quant pcr kit (qiagen kits, china). the abi 7300 sequence detection system (applied biosystems) was used to quantify the purified dna. pcr was performed under the following conditions: 37°c for 5 min and 94°c for 1 min, followed by 42 amplification cycles at 95°c for 5 s and 60°c for 30 s. hepg 2.2.15 cells were harvested by trypsin digestion and washed three times with phosphate buffered saline (pbs, ph 7.3). cells were counted and used to evaluate the presence of hbv replicative intermediates: 1 â 10 6 cells for cccdna, 3 â 10 6 cells for pgrna. total rna was isolated from cells using the trizol reagent (invitrogen). for rt-pcr analysis, rna was reverse-transcribed at 42°c for 90 min using a commercially available cdna synthesis kit (invitrogen). pcr was performed with gene specific primers for hbv and b-actin: hbv nt2429-2451 (forward), 5 0 -ctcaatctcgggaatctcaatgt-3 0 ; hbv nt2659-2636 (reverse), 5 0 -tggataaaacctagcaggcataat-3 0 ; b-actin nt2396-2415 (forward), 5 0 -acggccaggtcatcaccat-3 0 , b-actin nt2438-2457 (reverse), 5 0 -aggctggaagagtgcctcag-3 0 . qpcr was performed on the abi 7300 sequence detection system using the sybr green kit (invitrogen). a standard curve was constructed by the simultaneous amplification of serial dilutions of the expression plasmid encoding hbv. target cdnas were normalized to the endogenous rna levels of bactin. gene expression was determined using the relative quantifiwhere ct was the fractional cycle number that reached a fixed threshold, ctest was the test of hbv in compound 1 and adv exposed groups, and ccontrol was the reference control (rna from non-treated cells). the fold increase was calculated using 2 àddct (livak and schmittgen, 2001) . since hbv cccdna is structurally similar to plasmid, intracellular cccdna was extracted from cells by an alkali lysis procedure with a qiagen plasmid mini kit (ca) in order to remove most of cellular chromosomal dna and non-supercoiled relaxed circular (rcdna). purified cccdna was dissolved in 50 ll te buffer (10 mm, ph 8.0), and 10 ll of the product was further treated with plasmid-safe™ atp-dependent dnase (psad; epicentre technologies, wi) to remove any remaining single-stranded virus dna, rcdna or cellular chromosomal dna. intracellular cccdna was quantified by selective fluorescent pcr with taqman ò mgb probe capable of amplifying and checking cccdna more efficiently than rcdna. the hbv primers were: nt1562-1579 (forward), 5 0 -ttctcatctgccggaccg-3 0 ; nt1883-18 64 (reverse), 5 0 -cacagcttggaggcttgaac-3 0 . the taqman mg b probes were: nt1836-1855 (reverse), 5 0 fam-cctaat catctc ttgttcat-mgb 3 0 . qpcr was performed to detect intracellular cccdna by using the realquant pcr kit (invitrogen). cell lysates were prepared by using ripa (radio immunoprecipitation assay) lysis buffer supplemented with 1% (v/v) phenylmethanesulfonylfluoride (pmsf; biyuntian co., ltd., china). proteins were separated by electrophoresis on an 8% sodium dodecyl sulfate-polyacrylamide gel, transferred onto immobilon-p membranes (millipore), and analyzed by standard western blot technique using anti-hbcag antibodies (santa cruz biotechnologies). b-actin (santa cruz biotechnologies) was used as the normalization control. the anti-sars virus activity was estimated by mtt assay (sigma). the optical absorbance was read on a plate reader (bio-rad co.) at a wavelength of 570 nm for mtt. vero-e 6 cell cultures (2 â 10 5 cells/ml) were prepared in a 96-well plate. after a 24 h period of incubation, sars virus stock (0.1 ml per well) and different doses of compound 1 (0.1 ml per well) were added. in the control group, 0.1 ml medium was added. the plates were incubated at 37°c in a humidified 5% co 2 atmosphere for 2 days until maximum cytopathic effects were observed in the untreated, negative control cultures. the cytopathic effects were quantitated by the mtt assay. briefly, 30 ll of mtt solution prepared in dmem was added to each well and plates were incubated at 37°c for 4 h. the mtt solution was removed without disturbing the cells and 60 ll of dmso was added to each well to dissolve formazan crystals. after gently shaking the plates for 5 min, the absorbance from each well was measured at 540 nm. the percentages of protection were calculated as [(a à b)/(c à b) â 100], where a, b, and c stand for the absorbances of wells containing compound 1 and virus (a), virus (b) and cell (c) only, respectively. data were expressed as mean ± sd (standard deviation). all experiments were performed in triplicate. statistical significance was evaluated by the one-way analysis of variance (anova) and student's t-test. multiple comparisons were statistically analyzed using sas software version 8.0 (significance was established at p < 0.05). x-ray crystallography shows that compound 1 consists of a 3-d polyoxoanion framework [k 4 (h 2 o) 4 pti 2 w 10 o 40 ] 3à , one-dimensional (1-d) chainlike [k 4 (h 2 o) 8 cl] 3+ cations and the isolated hydroxylamine nh 2 oh. in compound 1, the polyoxoanion [pti 2 w 10 o 40 ] 7à exhibits a well-known keggin structure (fig. 1a) . the central p atom is surrounded by a cube of eight oxygen atoms with each oxygen site half-occupied. the p-o distance is 1.51(2) å. in the two crystallographically unique surrounding metal sites of the polyoxoanion, there exists a site occupancy disorder in the w2 center, that is, both ti and w2 atom share the same site with 25% occupancy, respectively, forming the polyoxoanion [pti 2 w 10 o 40 ] 7à . (fig. 1a) . in asymmetric unit of compound 1, the k1 center (see fig. 1b ) is octa-coordinated with four l 2 -o atoms of one polyoxoanion (o2 and o5), two terminal oxygen atoms of other two polyoxoanions (o6), and two coordination water molecules (ow1 and ow3a). the k-o distances range from 2.640(11) to 2.900(15) å. thus, the k1 centers can be regarded as a joint to connect all the polyoxoanions together. the k2 center (fig. 1c ) also exhibits the eight coordination environment with two terminal o atoms (o4) of two polyoxoanions, four coordination water molecules (ow2 and ow3) and two cl anions. the k-o distances are in the range of 2.39(2)-2.71(3) å, while the k-cl distance is 2.80(4) å. it is interesting that all coordination atoms are shared by two k2 centers. based on this connection mode, the k centers form a 1-d chain via these double-bridging atoms ( fig. 2a) . in the asymmetric unit of 1, ow2, ow3 and cl possess the 50%, 50% and 12.5% occupancies, respectively, thus, the 1-d chainlike cations can be described as [k 4 the growth of the hepg 2 cells in the presence of various concentrations of compound 1 was examined. the results of the cytotoxicity experiments are given in fig. 3 . the cytotoxicity of fig. 1. (a) the structure of polyoxoanion of compound 1 showing the coordination sites with k centers. coordination environments of (b) k1 center and (c) k2 center. the levels of hbsag, hbeag and extracellular hbv dna in the medium were measured at different time points in the control group, adv group, and compound 1 group, respectively, as shown in fig. 4 . after treatment, the levels of hbeag, hbsag and extracellular hbv dna in the drug-treated groups were decreased significantly compared to that in the control group in a concentrationdependent manner (p < 0.05). the levels of hbeag, hbsag and extracellular hbv dna decreased with time in hepg 2.2.15 cells, indicating that the anti-hbv activity of compound 1 is time-dependent (p < 0.01). as shown in fig. 4a , the inhibition ratio of hbeag in compound 1-treated cultures reached the peak value 65.89% at 93.38 lm at day 9 and still kept about 40.43% at day 11. at the same time, the inhibition ratio of hbsag (shown in fig. 4b ) reached the peak value of 72.10% at 93.38 lm at day 9 and was still about 37.66% at day 11. the inhibition ratios of hbeag and hbsag in the adv group were 57.03% and 56.22% at 2000 lm, respectively. the inhibitory values of hbeag and hbsag in adv group decreased at day 11. interestingly, the inhibition rates of compound 1 on extracellular hbv dna at day 11 (4 days after the end of exposure to the drug) were lower than those observed on day 9. however, they were higher than those on day 7. at the same time, the inhibition values of adv for extracellular hbv dna declined and they were lower than the values at day 7, as shown in fig. 4c . these result indicate that compound 1 may have a persistent effect on suppressing hbv. the 50% effective concentration (ec 50 ) values of hbeag, hbsag and extracellular hbv dna for compound 1 and adv are shown in table 2 . the therapeutic index (ti) of compound 1 was higher than that of adv. to characterize the anti-hbv mechanism of compound 1, the amounts of intracellular viral pgrna and dna were measured in the control group, adv group, and compound 1 group at different concentrations, respectively, at day 5, as shown in fig. 5 . the results revealed that the levels of intracellular hbv pgrna and cccdna were decreased with elevation of the compound 1 concentration, as compared to those detected from the control group (p < 0.05). the maximum inhibition ratios of pgrna and dna were 36.50% and 92.57% at 100 lm in the compound 1 group, which are higher than the ratios in the positive control group (avd group, p < 0.05). these results suggested that compound 1 apparently impacting viral cccdna replication and viral pgrna transcription in hepg 2.2.15 cells, and that the anti-hbv mechanism of compound 1 seems to be similar to that of adv. the inhibitory effects of adv and compound 1 on hbs(c)ag protein levels were determined with western blot. hepg 2.2.15 cells were treated with compound 1 and adv, respectively. the cell extracts were prepared and analyzed at day 5. as shown in fig. 6 , the levels of intracellular protein levels of hbcag were reduced with elevation of the compound 1 concentration in comparison with that in the control group (p < 0.05), indicating that the anti-hbv hbcag activity of compound 1 is concentration-dependent. in addition, the inhibitory effect of compound 1 was higher than that of adv at the same dose. aiming at evaluating the antiviral activity against sars virus of compound 1, the cytotoxicity of compound 1 in vero-e 6 cells was first measured. the results showed that the maximal noncytotoxic concentration (cc 0 ) and the 50% cytotoxic concentration (cc 50 ) of compound 1 were 31.2 lm and 118.6 lm, respectively. according fig. 3 . cytotoxicity of compound 1 and adv in hepg 2 cells. a p < 0.05 for the drug groups vs. control group. for compound 1: b p < 0.05 for the drug group vs. drug at 90 lm group. c p < 0.05 for the drug groups vs. drug at 180 lm group. d p < 0.05 for the drug group vs. drug at 360 lm group. e p < 0.05 vs. drug at 720 lm group. for adv: b p < 0.05 for the drug group vs. drug at 250 lm group. c p < 0.05 for the drug groups vs. drug at 500 lm group. d p < 0.05 for the drug group vs. drug at 1000 lm group. e p < 0.05 vs. drug at 2000 lm group. to the cytotoxic results, compound 1 was diluted at five non-toxic concentrations (31.2, 15.6, 7.8, 3.9 , and 1.95 lm) and the anti-sars virus activity was checked with mtt assay. as shown in fig. 7 , the inhibition ratio of compound 1 increased with concentration, indicating that the anti-sars virus activity of compound 1 is concentration-dependent. it is noteworthy that the sars virus was completely inhibited at 31.2 lm. the 50% effective concentration (ec 50 ) and the 90% effective concentration (ec 90 ) were 7.08 and 21.0 lm, respectively. ti of compound 1 was 16.75. currently, nucleoside analogs play an important role in the therapy of hbv, however, some disadvantages of these agents include side effects, drug resistance and costs which limit their clinical use in hbv-infected patients. polyoxometalates, as nonnucleoside analogs, have been proven to exhibit broad inhibitory activity against human immunodeficiency viruses (hiv-1 and hiv-2), herpes simplex virus, and influenza virus. especially, the titanium-containing polyoxotungstates have shown antiviral activity against a variety of enveloped rna or dna viruses. herein, we synthesized a titanium-substituted-heteropolytungstate [k 4 (h 2 o) 8 cl][k 4 (h 2 o) 4 pti 2 w 10 o 40 ]ánh 2 oh, and examined the anti-hbv activities in vitro. hepg 2.2.15 cells as a useful ''in vitro'' model are widely used for the evaluation of novel anti-hbv drugs and chosen in our experiments. the cell line contains multiple copies of hbv genome that can stably integrate into the host cell genome. the results indicate that compound 1 inhibited hbv dna, hbsag and hbeag antigens in the culture medium in a concentrationand-time dependent manner. a lower concentration of the compound 1 was required to effectively inhibit secreted hbv dna than to inhibit secreted antigens. it is possible that the compound acts on the exported virions outer protein coats. although the mechanisms mediating the antiviral effects by compound 1 remain unfig. 4 . the inhibitor effect of control group, adv groups, and compound 1 groups on secretion of hbeag (a), hbsag (b) and extracellular viral dna (c) from the medium of hepg 2.2.15 cells. a p < 0.05 vs. the corresponding negative control. b p < 0.05 vs. the corresponding 7 days outcome of the same dose. c p < 0.05 vs. the corresponding 11 days outcome of the same dose. table 2 anti-hbv activity, cytotoxicity and the selective index (ti) of compound 1 and adv in vitro. hbsag hbv dna clear, we have deduced that compound 1 might block the secretion of hbv containing nucleocapsids or destabilize hbv dna-containing nucleocapsids. interestingly, we observed that the in vitro anti-hbv properties of compound 1 against rebound of serum hbv dna, hbsag and hbeag were more robust than those of positive group, adv, as indicated by evaluation of treated cells 4 days after termination of treatment. this result shows that compound 1 has a sustained anti-hbv activity. the levels of intracellular hbv dna, rna and hbcag protein were also reduced by compound 1 in a concentration-dependent manner. the adv drug competitively inhibits hbv polymerase, which is structurally similar to datp. it is known to reduce the levels of hbv dna and hbsag both in vitro and in vivo by a phosphorylation event that facilitates its physical incorporation into nascent fig. 5 . the inhibition effect on the levels of intracellular pgrna (a) and cccdna (b) in control group, adv groups, and compound 1 groups at different concentrations. a p < 0.05 vs. the corresponding negative control. b p < 0.05 vs. the corresponding adv groups at the same concentration. viral dna by the activity of hbv polymerase during replication. in our study, we observed an apparent reduction in the levels of intracellular hbv-specific rna and dna following compound 1 treatment. these data also reveal that the anti-hbv mechanism of compound 1 may be similar to that of the nucleoside analog. since the hbv pgrnas were transcribed from cccdna, we presume that hbv-specific transcripts may be affected by compound 1. it is also important to consider that the hbv pgrna was inhibited in drugtreated cells (he et al., 2008) . hbcag, hbsag and hbv polymerase are translated from pregenome mrna, and the minus strand hbv dna are transcribed from the pregenome mrna template. in this study, it was found that compound 1 could inhibit the levels of hbcag, hbsag and hbv dna protein expressed in a concentration-dependent manner in vitro. these results suggested that compound 1 apparently interfered with viral pgrna transcription in hepg 2.2.15 cells. moreover, compound 1 shows broad-spectrum antiviral activity. it can efficiently inhibit sars virus in vero-e 6 cells in vitro with low toxicity against mdck cells. in summary, a heteropolytungstate has been prepared and characterized. the in vitro experimental results show that compound 1 has potential anti-hbv and anti-sars virus activities. further experiments are underway now, which include evaluation of in vivo activities and determination of the mechanism of anti-hbv activity of compound 1. enhanced duck hepatitis b virus gene expression following aflatoxin b1 exposure antiviral treatment for chronic hepatitis b virus infection-immune modulation or viral suppression? the memory effect of heteropolyoxotungstate (pm-19) pretreatment on infection by herpes simplex virus at the penetration stage prevention of the interaction between hvem, herpes virus entry mediator, and gd, hsv envelope protein, by a keggin polyoxotungstate perspectives for the treatment of hepatitis b virus infections inhibitory effect of adefovir on viral dna synthesis and covalently closed circular dna formation in duck hepatitis b virus-infected hepatocytes in vivo and in vitro ti 2 w 10 po 40 7à and preparation, properties, and structure determination by tungsten-183 nmr identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus in vivo and in vitro antihepatitis b virus activity of total phenolics from oenanthe javanica inhibition of hepatitis b virus replication by pokeweed antiviral protein in vitro polyoxometalate hiv-1 protease inhibitors. a new mode of protease inhibition application of a gastric cancer cell line (mkn-28) for anti-adenovirus screening using the mtt method a novel coronavirus associated with severe acute respiratory syndrome heteropolymolybdate-amino acid complexes: synthesis, characterization and biological activity synthesis, structural characterization and biological activity of polyoxometalate-containing protonated amantadine as a cation analysis of relative gene expression data using real-time quantitative pcr and the 2 àddct method hepadna viruses: hepatitis b and related viruses structure of a dititanodecatungstophosphate polyoxometalate chemistry: an old field with new dimensions in several disciplines polyoxometalates: from platonic solids to antiretroviral activity polyoxometalates in medicine broad spectrum anti-rna virus activities of titanium and vanadium substituted polyoxotungstates sars -beginning to understand a new virus potent anti-hiv (type 1 and type 2) activity of polyoxometalates: structure-activity relationship and mechanism of action anti-tumor, -viral, and -bacterial activities of polyoxometalates for realizing an inorganic drug therapy of chronic hepatitis b virus infection: inhibition of the viral polymerase and other antiviral strategies the inhibition of 1 to sars virus in different concentrations supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.antiviral.2011.11.003. key: cord-350964-0jtfc271 authors: van nguyen, dung; van nguyen, cuong; bonsall, david; ngo, tue tri; carrique-mas, juan; pham, anh hong; bryant, juliet e.; thwaites, guy; baker, stephen; woolhouse, mark; simmonds, peter title: detection and characterization of homologues of human hepatitis viruses and pegiviruses in rodents and bats in vietnam date: 2018-02-28 journal: viruses doi: 10.3390/v10030102 sha: doc_id: 350964 cord_uid: 0jtfc271 rodents and bats are now widely recognised as important sources of zoonotic virus infections in other mammals, including humans. numerous surveys have expanded our knowledge of diverse viruses in a range of rodent and bat species, including their origins, evolution, and range of hosts. in this study of pegivirus and human hepatitis-related viruses, liver and serum samples from vietnamese rodents and bats were examined by pcr and sequencing. nucleic acids homologous to human hepatitis b, c, e viruses were detected in liver samples of 2 (1.3%) of 157 bats, 38 (8.1%), and 14 (3%) of 470 rodents, respectively. hepacivirus-like viruses were frequently detected (42.7%) in the bamboo rat, rhizomys pruinosus, while pegivirus rna was only evident in 2 (0.3%) of 638 rodent serum samples. complete or near-complete genome sequences of hbv, hev and pegivirus homologues closely resembled those previously reported from rodents and bats. however, complete coding region sequences of the rodent hepacivirus-like viruses substantially diverged from all of the currently classified variants and potentially represent a new species in the hepacivirus genus. of the viruses identified, their routes of transmission and potential to establish zoonoses remain to be determined. unlike many other communicable diseases, the burden of viral hepatitis has substantially increased over the last two decades to recently become the seventh leading cause of mortality worldwide. viral hepatitis now causes more deaths than tuberculosis, aids or malaria each year. hepatitis c virus (hcv) and hepatitis b virus (hbv) are responsible for >90% (96% in 2013) of viral viruses 2018, 10, 102 2 of 12 hepatitis-related mortality and disability. as such, these hepatitis viruses are the targets of efforts to combat viral hepatitis [1] , including hbv vaccination, development of hcv vaccines, and highly effective drugs. in contrast, hepatitis e virus (hev) is endemic in many low-income countries [2] but usually causes self-limiting hepatitis. infection with hev occasionally results in liver failure [1] . hbv, hcv, and hev are members of virus families hepadnaviridae, flaviviridae, and hepeviridae, respectively. hbv has a partially double-stranded dna genome with 4 overlapping open reading frames (orfs) [3] , whereas hcv and hev have a single-stranded rna genome [4, 5] . while the origins of these human viruses are unknown, rodents and bats are putative reservoir hosts because they host a diverse array of hepadnaviridae [6] [7] [8] , hepeviridae [7, [9] [10] [11] [12] [13] [14] [15] [16] , and genera pegivirus [17] [18] [19] and hepacivirus [17, 19, 20] of the flaviviridae family including homologues of the human hepatitis viruses under question. among these, it is of concern that a bat hepadnavirus may possess the ability to infect human liver cells [6] . several factors may contribute to the risk of zoonotic rodent or bat virus transmission. rodent meat is a popular source of protein for human consumption in vietnam, particularly in the mekong delta, where rats (rattus spp. and bandicota indica) are commonly trapped and sold live in wet markets [21] . the total annual consumption of rat meat in vietnam is estimated at 3300-3600 tonnes [22] . hoary bamboo rats (rhizomys pruinosus) are additionally farmed for human consumption. moreover, bat faeces collected from bat caves or farms is used as natural fertilizer ("guano") in vietnam. as rodents and bats are reservoirs or carriers of a significant number of zoonotic pathogens [23] and viruses with unknown zoonotic potential, there are health risks that are associated with exposure to these animals. however, a previous study [22] showed none of the surveyed rat catchers or processors were aware of infection risks from contact with live rats. consequently, no precautions were taken for the handling of rodents. in the search for viral diversity and zoonotic viruses, novel paramyxovirus and coronavirus in vietnamese bats and rats were detected in a previous study [24] . here, we report the detection pegivirus and human hepatitis-related viruses in these mammals. rodent and bat samples were collected within the vizions (vietnam initiative on zoonotic infections) framework [25] for the screening of zoonotic microorganisms [21, [26] [27] [28] . rodent samples. as it is important and essential to understand the risk associated with rodents, including those sold in the markets, a total of 435 rats purchased from markets in five of twelve provinces in the mekong delta during 2012-2015 and 82 farmed bamboo rats purchased from a market in dak lak in 2014-2015 were enrolled. in addition, 226 trapped rats were also included. rat trapping was carried out in different locations (pig and poultry farms, rice fields, fruit groves, tropical forests, markets, slaughter-house) in the provinces of dong thap during march 2013 and dak lak in april 2014, as previously described [27] . serum and liver samples were collected post-mortem. species identification of rats was carried out on the basis of morphological characteristics and sequencing of a conserved housekeeping gene [27] . all of the samples were stored in sterile tubes with rna later at −20 • c until nucleic acid extraction. special precautions were taken to avoid cross-contamination. bat samples. a total of 157 bats were trapped at six sites in the provinces of dong nai (in cat tien national park) and quang ngai in may 2013 using mist nets and harp traps as described [26] . trapped bats were speciated according to morphology [29] , and liver samples were collected and stored as described above for rats. rna was manually extracted from 638 rodent serum samples using qiaamp viral rna mini kit (qiagen, manchester, uk) and following instructions from the manufacturer. liver samples from 157 bats and 470 rodents were subjected to nucleic acid extraction using allprep dna/rna mini kit (qiagen, manchester, uk). briefly, about 30 mg of liver per sample was first lysed and homogenised using tissuelyser (qiagen, manchester, uk). the lysate was applied to an allprep dna spin column for dna to bind onto the column. ethanol was added to the flow-through and rna and bound to the membrane when the sample was passed through an rneasy spin column. after washing steps, dna and rna was eluted separately in 50 µl of nuclease-free water. extracted nucleic acid was used in screening for the targeted hepatitis viruses. in order to minimize contamination, pcr mastermix preparation, and the addition of templates were carried under separated laminar flow hoods and lab spaces. all of the surfaces, tubes, and equipment were uv irradiated between each pcr. reverse transcription using superscript iii reverse transcriptase (invitrogen, paisley, uk) was performed according to the manufacturer's instruction. synthesized cdna was screened for hepaciviruses and pegiviruses using a semi-nested pcr protocol with broad spectrum degenerate primers, which can detect all known hepaciviruses and pegiviruses. amplification conditions (using gotaq from promega, southampton, uk) included 95 • c for 3 min, and 30 cycles of denaturation (94 • c, 30 s), annealing (55 • c, 30 s) and elongation (72 • c, 30 s). similarly, hev was screened using broadly reactive primers targeting viral rna-dependent rna polymerase as described in drexler et al., 2012 [11] . dna extracted from liver samples was used for screening of hbv. degenerate primers targeting a highly conserved region of the polymerase gene of sequences from all known hbv hosts were designed for a nested pcr protocol using the above amplification conditions. primers for screening are listed in table s3 . the length of the sequenced screening fragments (excluding primer sequences) of homologues of hbv, hev, hcv, and pegivirus was 257, 284 and 360 nucleotides, respectively. for rodent hepacivirus, hev and pegivirus genomes, extracted rna from representative positive samples was subjected to deep sequencing using an illumina platform. libraries were prepared from total extracted rna using the nebnext ultra directional sequencing kit (new england biolabs, hitchin, uk) with omission of actinomycin d, then pooled and sequenced on a hiseq 4000 instrument (illumina, nr saffron walden, uk). quality control and trimming of reads were performed before genome mapping using clc genomics workbench (qiagen bioinformatics, redwood city, ca, usa) with the default affine gap cost parameters. the closest related virus genomes (genbank numbers kc815310, jx120573 and kj950934 for hepacivirus, hev and pegivirus, respectively) were used as templates for genome mapping. additional primers were designed using the obtained contigs for gap fillings. for bat hbv, primers were designed using sequences available from genbank and the obtained sequences from screening. these primers amplified amplicons, with overlapping regions as presented in table s4 . all of these nested or hemi-nested pcr protocols used superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, paisley, uk) for rna viruses or q5 high-fidelity dna polymerase (new england biolabs, hitchin, uk) for hbv in the first round pcr, according to instructions from the manufacturers. q5 high-fidelity dna polymerase was also used in the second round pcr. two real-time pcr primer sets (table s5 ) in the 5 untranslated region of bamboo rat hepaciviruses and the screening fragment of other rat hepaciviruses were designed using sequences available from this study. the targeted regions were amplified from positive samples and cloned into pgem-t easy vector (promega, southampton, uk) for in vitro transcription, as previously described [30] . the obtained rna transcripts were used to generate standard curves of the real-time pcr assays for measurement of rodent hepacivirus rna titers using superscript iii reverse transcriptase (invitrogen, paisley, uk) and powerup sybr green master mix (thermo fisher scientific, northumberland, uk). sequences were imported into sse (simmonic sequence editor) [31] for the alignment and calculation of sequence distance values from reference sequences of known viruses from which sequence identities were inferred. sequence distances instead of sequence identities in the regions used for classification of hepaciviruses and pegiviruses were presented to easily compare with the species p-distance thresholds set in the proposed update to the taxonomy of the genera hepacivirus and pegivirus [32] . maximum-likelihood phylogenetic trees were reconstructed using the mega 7.0 software package [33] with 1000 bootstrap resamples. the best-fitting model for each sequence dataset (as shown in figure captions) was first determined and used for phylogenetic reconstruction. sequences obtained in this study have been assigned the following genbank accession numbers mg600410-mg600465. nucleic acid that was extracted from liver samples of 157 bats (29 species; table s1 ) and 470 rodents (six species) was screened for pegivirus and human hepatitis b, c, e viruses and their homologues ( table 1 ) by nested and semi-nested pcr assays with degenerate primers. hepaciviruses were the most commonly detected (8.1% of rodent samples, from three species), followed by hepatitis e related virus (3% of rodent samples, from four species) while hepatitis b related viral dna was only detectable in three bats (2 species). most of the hepacivirus positive samples were from farmed hoary bamboo rats in dak lak province although the predominantly sampled rat species was rattus argentiventer. coinfection with hepacivirus and hev was observed in a sample from rattus losea. all liver samples from bats were negative for hepacivirus and hepatitis e related virus and no sample was positive for pegivirus. and 335 other rats whose liver samples were screened for hepacivirus as above. hepacivirus rna was only detected in serum samples of 10 bamboo rats with positive liver samples. pegivirus rna was detected in two samples from rattus tanezumi. two real-time pcr assays specific for bamboo rat hepaciviruses, and other hepaciviruses were used to determine viral rna concentration in the positive samples. the concentration of rna ( figure 1 ) was high in both liver tissue (median, 3.35 × 10 7 copies/gram; range, 0.9 × 10 5 -1.16 × 10 9 ) and sera (median, 5.7 × 10 6 copies/ml; range, 2.3 × 10 6 -2 × 10 7 ). viruses 2018, 10, x 5 of 12 ( figure 1 ) was high in both liver tissue (median, 3.35 × 10 7 copies/gram; range, 0.9 × 10 5 -1.16 × 10 9 ) and sera (median, 5.7 × 10 6 copies/ml; range, 2.3 × 10 6 -2 × 10 7 ). amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [17, 18] , china [7] and vietnam [16] . rodent hepacivirus sequences (360 nucleotides) formed two well supported clades (figure 2a) . clade 1 included all of 35 sequences from rhizomys pruinosus which shared 84.5-100% pairwise nucleotide identity while three sequences (nucleotide identity of 89-99%) from rattus losea and rattus argentiventer grouped in clade 2. these clades differed from each other by mean distances of 39.6% and 33.2% at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (<12% nucleotide and <3% amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc815310) with the corresponding distances of 40% and 36%, respectively ( table 2 ). the amino acid p-distances of the obtained hepacivirus sequences and kc815310 in the regions 1123-1566 and 2536-2959 (numbered relative to m62321) were 30% and 32%, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure 2b ) [32] . the other bamboo rat hepaciviruses in clade 1 may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade 2, they likely belong to species hepacivirus g due to their low amino acid p-distances (7.6-8.4%) in the screening region to kj950938. the 5' untranslated region sequences of these hepaciviruses contain two mir-122 seed sites (cacucc), which were located 51 nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the 15 hev sequences (284 nucleotides) from four rodent species were 84.4-99.3% identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure 3a) . the obtained complete genome of rat hev comprised of 6960 nucleotides excluding the poly a tail. its concatenated orf1 and orf2 differed by 6.8% (table 2 ) at amino acid level to the closest match (jx120573) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure 3b ), according to the latest proposal for classification of hepeviruses [34] . amplicons derived from pcr screening experiments were all successfully sequenced and complete or near complete genome sequences were determined for representatives of the viruses by pcr or deep sequencing. after exclusion of primers, the obtained screening sequences from each targeted virus clustered in one or two clades of those with high identity and were most closely related to sequences previously reported from rodents or bats from the us [17, 18] , china [7] and vietnam [16] . rodent hepacivirus sequences (360 nucleotides) formed two well supported clades (figure 2a) . clade 1 included all of 35 sequences from rhizomys pruinosus which shared 84.5-100% pairwise nucleotide identity while three sequences (nucleotide identity of 89-99%) from rattus losea and rattus argentiventer grouped in clade 2. these clades differed from each other by mean distances of 39.6% and 33.2% at nucleotide and amino acid levels, respectively. while the four vietnamese bamboo rat hepacivirus genomes were highly similar to each other (<12% nucleotide and <3% amino acid distances in the complete coding region (cds)), they were remarkably different from the closest sequence (kc815310) with the corresponding distances of 40% and 36%, respectively ( table 2 ). the amino acid p-distances of the obtained hepacivirus sequences and kc815310 in the regions 1123-1566 and 2536-2959 (numbered relative to m62321) were 30% and 32%, respectively. the newly identified hepaciviruses therefore could be provisionally classified as members of a new hepacivirus species (figure 2b ) [32] . the other bamboo rat hepaciviruses in clade 1 may also belong to this new virus species on the basis of the high sequence identity in this clade. similarly, although complete genome sequences were not determined for hepaciviruses in clade 2, they likely belong to species hepacivirus g due to their low amino acid p-distances (7.6-8.4%) in the screening region to kj950938. the 5' untranslated region sequences of these hepaciviruses contain two mir-122 seed sites (cacucc), which were located 51 nucleotides from each other, as consistent with the suspected hepatotropism of the viruses. in contrast to host specificity of rodent hepaciviruses, the 15 hev sequences (284 nucleotides) from four rodent species were 84.4-99.3% identical to each other and phylogenetically interspersed with each other and with reference sequences from other rat species (figure 3a) . the obtained complete genome of rat hev comprised of 6960 nucleotides excluding the poly a tail. its concatenated orf1 and orf2 differed by 6.8% (table 2 ) at amino acid level to the closest match (jx120573) and these two sequences therefore belong to a same genotype in the species orthohepevirus c (figure 3b) , according to the latest proposal for classification of hepeviruses [34] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure 4 ). the two bat hbv complete genome sequences comprised 3275 and 3302 nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table 2 . bat031 consistently showed greatest sequence identity to ky905324 in all 4 orfs. in contrast, bat033 shared highest identity to ky905328 in the p and s orfs, but was more similar to ky905324 and ky905327 in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure 5a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj950934 in the region 888-1635 (numbered relative to u22303) was 17.8% and the two viruses could be classified as members of species pegivirus j (figure 5b) , according in the update to the taxonomy of the pegivirus genus [32] . the three hbv variants (from bat species hipposideros pomona and hipposideros larvatus) clustered with known bat hbv sequences (figure 4) . the two bat hbv complete genome sequences comprised 3275 and 3302 nucleotides. as with other hepadnaviruses, the bat hbv genomes have four open reading frames (orfs) encoding the surface (s), polymerase (p), core (c), and x proteins. a detailed comparison of the orfs of these viruses with their closest sequences is shown in table 2 . bat031 consistently showed greatest sequence identity to ky905324 in all 4 orfs. in contrast, bat033 shared highest identity to ky905328 in the p and s orfs, but was more similar to ky905324 and ky905327 in the orfs encoding for x and c, respectively. the two vietnamese pegivirus sequences from rattus tanezumi grouped with a sequence previously reported from rattus norvegicus (figure 5a ). the amino acid p-distance between the obtained rodent pegivirus sequence and kj950934 in the region 888-1635 (numbered relative to u22303) was 17.8% and the two viruses could be classified as members of species pegivirus j (figure 5b) , according in the update to the taxonomy of the pegivirus genus [32] . the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence (0-5%) reported in previous studies [6, 7, 11, 19] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high (42.7%). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [35, 36] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [37] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [38, 39] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [40] . equine hepacivirus has been shown to be transmittable via direct inoculation [41] and via vertical transmission [42] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [43] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus (23.3% in firth et al. 2014 [18] ) indicates other more efficient transmission routes may exist such as via saliva the present study reports the findings of pegivirus and human hepatitis-related viruses in vietnamese rodents and bats. the detection of hepacivirus, hev homologue and pegivirus in a number of rodent species, and hbv homologue in hipposideros larvatus indicates wider host ranges of these viruses. whereas, the identified rat hev and bat hbv showed relatively high sequence identity to previously characterized viruses infecting rattus rattus, rattus tanezumi and hipposideros pomona, the rodent pegivirus and hepacivirus showed substantial sequence distances to their closest sequences and represent new pegivirus variants and a new hepacivirus species. this highlights the incomplete genetic characterization of these viruses. thus far, only one complete genome and two complete coding sequences (including the one from this study) of rodent pegivirus are available on genbank. more highly divergent hepacivirus and pegivirus sequences are expected to be discovered from rodents in future studies. the absence of bat hev, hepacivirus, pegivirus, and low detection frequency of bat hbv are consistent with their low prevalence (0-5%) reported in previous studies [6, 7, 11, 19] . contrastingly, the prevalence of hepacivirus in the farmed bamboo rats was unprecedentedly high (42.7%). the inbred nature of farmed bamboo rats that were investigated in this study may have contributed to susceptibility to infection and likelihood of persistence [35, 36] . the existence of relatively homozygous individuals may play a key role in the maintenance of pathogens in a population [37] . the high prevalence of hepacivirus in bamboo rats also indicates the need to reconsider transmission routes of hepaciviruses. among hepaciviruses, the transmission route of hcv has been relatively well studied, while those of other hepaciviruses are mostly speculative [38, 39] . as a bloodborne virus, hcv is thought to be mainly transmitted through injections or blood transfusion. however, this does not explain how a range of divergent hcv strains have been maintained for centuries in some rural populations in central africa and southeast asia [40] . equine hepacivirus has been shown to be transmittable via direct inoculation [41] and via vertical transmission [42] . rodent hepacivirus may utilize a similar transmission route as experimental intravenous injection of the supernatants of homogenised liver tissues from infected rats lead to viraemia [43] . the high prevalence of hepacivirus in bamboo rats (in this study) and commensal rattus norvegicus (23.3% in firth et al. 2014 [18] ) indicates other more efficient transmission routes may exist such as via saliva and bites, the zoonotic potential of the detected viruses is unknown and requires further investigation. while the identified rodent hepaciviruses appear host species specific, four rodent species were infected with highly similar hev homologues, which were phylogenetically interspersed, indicative of low host species specificity. this is a characteristic that may lead to their establishment and emergence in new hosts. understanding the receptor usage for cell entry of hev in rodents and other host species would potentially help predict the host range of the virus. furthermore, a surrogate assay with pseudotyped viruses carrying surface/envelope proteins of the identified viruses may be useful in assessing their potential to infect human liver cells. such an assay was used to show that a bat hbv could infect primary human hepatocytes [6] . in summary, this study demonstrated the wide circulation of diverse pegivirus and human hepatitis-related viruses in new rodent and bat species. the presented findings form a framework for future investigations of human transmission risk, now that the rodent and bat species infected with these viruses have been identified and the human contact groups are better defined (e.g., bamboo rat farmers, rat catchers, rat sellers, and bat farmers). the transmission routes of the identified viruses are to be determined. the following are available online at http://www.mdpi.com/1999-4915/10/3/102/ s1. the global burden of viral hepatitis from 1990 to 2013: findings from the global burden of disease study the global burden of hepatitis e virus genotypes 1 and 2 in 2005 hepatitis b: the virus and disease genetic organization and diversity of the hepatitis c virus hepatitis e virus: molecular virology, clinical features, diagnosis, transmission, epidemiology, and prevention bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in china hepatitis virus in long-fingered bats hepatitis e virus in rats hepatitis e virus genotype 3 in wild rats, united states bats worldwide carry hepatitis e virus-related viruses that form a putative novel genus within the family hepeviridae complete genome sequence of a rat hepatitis e virus strain isolated in the united states hepatitis e virus in norway rats (rattus norvegicus) captured around a pig farm novel hepatitis e virus genotype in norway rats detection of a novel hepatitis e-like virus in faeces of wild rats using a nested broad-spectrum rt-pcr characterization of full genome of rat hepatitis e virus strain from vietnam identification of rodent homologs of hepatitis c virus and pegiviruses detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus bats are a major natural reservoir for hepaciviruses and pegiviruses evidence for novel hepaciviruses in rodents rodents and risk in the mekong delta of vietnam: seroprevalence of selected zoonotic viruses in rodents and humans. vector-borne zoonotic dis market study of meat from field rats in the mekong delta aciar monograph rodent-borne diseases and their risks for public health detection of potentially novel paramyxovirus and coronavirus viral rna in bats and rats in the mekong delta region of southern viet nam the vietnam initiative on zoonotic infections (vizions): a strategic approach to studying emerging zoonotic infectious diseases bartonella species and trombiculid mites of rats from the mekong delta of vietnam. vector borne zoonotic dis how important are rats as vectors of leptospirosis in the mekong delta of vietnam? vector borne zoonotic dis a guide to the mammals of southeast asia large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in vietnam sse: a nucleotide and amino acid sequence analysis platform proposed update to the taxonomy of the genera hepacivirus and pegivirus within the flaviviridae family mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets consensus proposals for classification of the family hepeviridae parasite-mediated selection against inbred soay sheep in a free-living, island population disease susceptibility in california sea lions consanguinity and susceptibility to infectious diseases in humans hepacivirus cross-species transmission and the origins of the hepatitis c virus viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species the virus whose family expanded experimental transmission of equine hepacivirus in horses as a model for hepatitis c virus vertical transmission of hepatitis c virus-like non-primate hepacivirus in horses mouse models of acute and chronic hepacivirus infection key: cord-320106-thre6r63 authors: xu, zhihui; ren, xiaoqiang; liu, yan; li, xiaodong; bai, siyu; zhong, yanwei; wang, lin; mao, panyong; wang, huifen; xin, shaojie; wong, vincent wai-sun; chan, henry lik-yuen; zoulim, fabien; xu, dongping title: association of hepatitis b virus mutations in basal core promoter and precore regions with severity of liver disease: an investigation of 793 chinese patients with mild and severe chronic hepatitis b and acute-on-chronic liver failure date: 2010-09-17 journal: j gastroenterol doi: 10.1007/s00535-010-0315-4 sha: doc_id: 320106 cord_uid: thre6r63 objective: to investigate the features of hepatitis b virus (hbv) basal core promoter/precore (bcp/pc) mutations and genotypes in a large number of mild/severe chronic hepatitis b (chb-m/chb-s), and acute-on-chronic liver failure (aclf) patients and analyze the clinical implications of the virologic features. patients and methods: sera of 793 (325 chb-m, 170 chb-s, and 298 aclf) patients admitted to or who had visited beijing 302 hospital from january 2005 to december 2008 were collected and successfully amplified for the hbv bcp/pc and a 1225-bp-long s/pol (nt 54–1278) gene regions. biochemical and serological parameters and hbv dna level were routinely performed. viral dna was extracted and subjected to a nested pcr. genotypes/subgenotypes were determined based on complete genomic sequence or on analysis of the 1225-bp-long s/pol-gene sequence. hbv genotyping was performed by direct pcr sequencing followed by molecular evolutionary analysis of the viral sequences. a p value of <0.05 (two-sided) was considered to be statistically significant. conclusions: our findings suggest that chb patients infected with bcp/pc mutant viruses are more susceptible to severe hepatitis and aclf than those with the bcp/pc wild-type virus and that aclf patients with pc mutant viruses have an increased risk of death. as such, the hbv pc mutation is a potential predictive indicator of aclf outcome. hepatitis b virus (hbv) chronically infects about 350 million people worldwide and 93 million people in china, with high risk of evolution to liver cirrhosis (lc) and hepatocellular carcinoma (hcc) [1, 2] . chronic hbv infection leads to a wide spectrum of clinical presentations, including the asymptomatic carrier state, mild and severe chronic hepatitis b (chb-m, chb-s), and acute-onchronic liver failure (aclf). in china, patients with hepatitis b-related aclf account for more than 80% of total aclf cases due to the high prevalence of chronic hbv infection. without transplantation, such patients have a high mortality rate (60-80%), resulting in 22,600 deaths annually [3] . the pathogenesis of the severity of chronic hbv infection remains largely unclear. both viral and host factors may play a role. hbv is a highly variable virus and is classified into at least eight genotypes (a-h) that may vary in geographical distribution, viral characteristics, and relationship to clinical outcomes [4] . hbv mutations in basal core promoter (bcp) and precore (pc) regions have attracted special attention because the bcp mutation may enhance hbv replication in vitro and the pc mutation abrogates translation of the hbe antigen (hbeag) which is considered to be a tolerant protein to buffer immune attack of the infected hepatocytes [5] [6] [7] . studies have been performed to clarify these virologic features in patients with acute liver failure (alf) who developed fulminant hepatitis from acute hbv infection. the occurrence of the bcp double mutation a1762t/g1764a and the pc mutation g1896a has been documented to be higher in alf patients than in those with acute hepatitis b [8] [9] [10] [11] [12] [13] [14] . a few other bcp/pc mutations have also been reported to be associated with increased hbv replication capacity and/or reduced hbeag expression in vitro and, in some cases, also associated with alf occurrence [15] [16] [17] [18] . however, the findings have been inconsistent, and to date no obvious link has been identified between hbv bcp/pc mutations and alf or fulminant hepatitis development [19] [20] [21] [22] . such clinical results may be partly due to inadequate sample sizes and the interference of viral genotypes. there is still a paucity of data on the association of hbv bcp/pc mutations and genotypes with aclf occurrence. liu et al. [23] reported that these virologic features seemed not to be associated with the fulminant and subfulminant exacerbation of chb, but their sample size was small. the severity of chronic hbv infection may exhibit a progressive process, i.e., from chb-m to chb-s and then to aclf in many cases. in the study reported here, we investigated the features of hbv bcp/pc mutations and genotypes in a large number of chb-m, chb-s, and aclf patients and analyzed the clinical implications of the virologic features. sera of 793 patients who were admitted to or visited beijing 302 hospital from january 2005 to december 2008 were collected and successfully amplified for the hbv bcp/pc and a 1,225-bp-long s/pol (nt 54-1278) gene regions. the patient cohort comprised 325 chb-m, 170 chb-s, and 298 aclf patients. the patients came from various areas of china, but mainly from the north of the country. the diagnostic criteria were based on the management scheme of diagnostic and therapy criteria of viral hepatitis [24] and diagnostic and treatment guidelines for liver failure [25] , issued by the chinese society of infectious diseases and parasitology and the chinese society of hepatology, respectively, and have been described in our previous studies [26, 27] . briefly, all patients had persistent seropositivity of hbsag for at least 6 months before enrollment. chb-m patients met the following criteria: a history of chronic hepatitis based on a histopathological diagnosis and/or compatible laboratory data and ultrasonographic findings, with mild to moderate liver disease activities that did not reach the criteria of chb-s. chb-s patients had severe liver disease symptoms, including obvious clinical manifestations and significant alterations in their biochemical parameters, such as significant serum alanine aminotransferase (alt) elevation. based on the biochemical parameters, the diagnosis of chb-s had to meet at least one of the following criteria: (1) serum albumin level b32 g/l; (2) serum total bilirubin (tbil) [85.5 lmol/l; (3) plasma prothrombin activity (pta) was 60-40%; (4) serum cholinesterase \4,500 iu/l. aclf patients met the following criteria: recent development of increasing jaundice (tbil [171.0 lmol/l or rapid increase to [17.1 lmol/l/day) and decreasing pta (\40%), with a recent development of complications, such as hepatic encephalopathy (cgrade 2), or an abrupt and obvious increase of ascites or spontaneous bacterial peritonitis or hepatorenal syndrome. the criteria for aclf have been widely used in china and are similar (but not exactly identical) with the newly issued asian pacific association for the study of the liver (apasl) criteria [28] . for all patients, there was no evidence of hcc or other metastatic liver disease; no evidence for concomitant hepatitis c/d virus (hcv/hdv) or human immunodeficiency virus (hiv) infection or autoimmune liver disease. the study was approved by the ethics committee of beijing 302 hospital. the detection of biochemical and serological parameters and hbv dna level were routinely performed in the central clinical laboratory of beijing 302 hospital. the lower limit of hbv dna detection is 500 copies/ml (equivalent to 100 iu/ml). detection of the bcp/pc mutations viral dna was extracted and subjected to a nested pcr as described elsewhere [29] . the primers were 5 0 -gac gtc ctt tgt yta cgt cc-3 0 (sense, nt 1413-1432) and 5 0 -tct gcg acg cgg cga ttg ag-3 0 (antisense, nt 2403-2422) for the first-round pcr and 5 0 -act tcg ctt cac ctc tgc ac-3 0 (sense, nt 1583-1602) and 5 0 -atc cac act cca aaa gay acc-3 0 (antisense, nt 2257-2277) for the second-round pcr. the first-round pcr consisted of equilibrating at 94°c for 3 min, followed by 10 the genotypes/subgenotypes were determined based on complete genomic sequence or on analysis of the 1225-bplong s/pol-gene sequence, which was amplified by an inhouse nested pcr assay [29] . a total of 380 hbv complete genomic sequences from individual patients were used for hbv genotyping, including 115 sequences from chb-m patients (genbank accession no.: fj386574-386689 except for fj386590), 120 sequences from chb-s patients (fj562218-562340 except for fj562263, 562306, and 562338), and 145 sequences from aclf patients (eu939536-939681 except for eu939680). hbv genotyping was performed by direct pcr sequencing followed by molecular evolutionary analysis of the viral sequences using mega 4 software. standard reference sequences were acquired from the online hepatitis virus database (http://www.ncbi.nlm.nih. gov/projects/genotyping/formpage.cgi). continuous variables were expressed as mean ± standard deviation (sd) or median. differences in continuous data were evaluated using student's t test, analysis of variance (anova), or nonparametric wilcoxon signed-ranked test, where appropriate, and categorical data were analyzed using the chi-square test and fisher's exact test. multivariate analyses with logistic regression were used to determine independent factors. statistical analysis was carried out in spss ver. 16 .0 software (spss, chicago, il). a p value of .05 (two-sided) was considered to be statistically significant. clinical background, hbv genotype, and bcp/pc mutation profiles of the patients table 1 summarizes the clinical background, hbv genotypes, and bcp/pc mutations in the three groups of patients. hbv genotypes c and b were detected in the samples of 648 and 145 patients, respectively. hbv/c2 and hbv/b2 were the dominant subgenotypes and found in 96% and 92% individual genotypes, respectively. the hbv genotyping based on the 1225-bp-long gene fragment had 100% (380/380) concordance for the classification of genotypes b and c and 98.7% concordance for the classification of subgenotypes b2 and c2 (b2 100%, c2 98.3%) in comparison with the genotyping based on complete hbv genomes. a slightly higher ratio of genotype b to c was found in aclf patients than in chb patients. compared to chb patients, aclf patients had significantly higher mutation incidences at eight of the ten sites of interest, including t1753v (c/a/g), a1762t, g1764a, c1766t, and t1768a in the bcp region and g1862t, g1896a, and g1899a in the pc region. notably, the frequencies of a1762t, g1764a, and g1896a hotspot mutations and the average substitution number at the ten sites of interest of the viral sequences increased in a stepwise manner in the three groups of patients, namely, chb-m \ chb-s \ aclf patients. correspondingly, the incidence of the bcp/pc wild-type sequence decreased in the same order. in addition, two interesting triple bcp mutations, t1753v/a1762t/g1764a and a1762t/ g1764a/c1766t/t1768a-triplet (with any three substitutions at the four sites), were more frequently detected in aclf patients than in chb patients. the frequencies of former triple mutations were 15.7, 15.3, and 29.2% for chb-m, chb-s, and aclf patients, respectively (p \ 0.01). the frequencies of latter triple mutations were 3.4, 4.7, and 12.8% for chb-m, chb-s, and aclf patients, respectively (p \ 0.01). the quadruple mutation a1762t/g1764a/c1766t/t1768a was detected in four aclf cases only. to exclude the potential influence on the results brought by age differences among patient groups, patients aged from 25 to 38 and 39 to 52 years were analyzed as independent two subsets. as shown in table 2 , patients in different illness categories had a similar age within each subset; the frequencies of a1762t, g1764a, g1896a and the average substitution number remained significantly different among the three illness groups as did the stepwise increase in the order of chb-m[chb-s[aclf patients. in addition to the ten interested sites, significant difference in variant frequencies among the three illness categories was also found at a1846t (chb-m 8.3%, chb-s 36.5%, aclf 37.5%, p \ 0.01). because hbv genotype influences bcp/pc mutational rates, we analyzed the bcp and pc mutations in genotype b and c viruses individually. table 3 summarizes the profiles of the bcp/pc mutations in patients infected with genotypes b or c virus, respectively. in patients infected with genotype b virus, a statistical difference in the occurrence of mutations among the three groups of patients was only observed at a1762t and g1764a, whereas in those infected with genotype c virus, there was a significant difference for four mutations among the groups, i.e., t1753v, g1862t, g1896a, and g1899a. the average substitution number/ sample clearly increased in a stepwise manner in the order of chb-s\chb-m\aclf patients for both genotypes b and c virus infection. the triple mutational pattern table 1 clinical features, hbv genotype, and bcp/pc mutation profile of the 793 patients enrolled in the study the clinical and virologic characteristics in relation to mortality of aclf patients of the 298 patients with aclf, 184 patients had a fatal outcome and 90 patients survived[6 months after the onset of liver failure. older age (c40 years), higher tbil level (c408 lmol/l), lower pta (b24%), higher alt level (c311 iu/ml), and the hbv pc mutation were detected as independent risk factors of death ( table 5 ). the cutoff values were the median for biochemical parameters. a age of 40 years, 10 5 copies/ml for hbv dna, and 3 for the substitution number/sample were selected as cutoff values because they were the integral values closest to the median. the outcome of hbv infection depends on the interplay between the virus, the hepatocytes, and the host's immune response. hbv bcp/pc mutations are considered to be likely involved in the driving factors of disease progression of hbv infection because bcp/pc mutations affect viral replication and/or hbeag expression, which may in turn impact on immune responses to the virus. for example, it has been proposed that expression of hbeag during perinatal infection, the major mode of hbv transmission in asia, induces immune tolerance. another potential role of hbeag in promoting persistent infection is to mimic cp so as to buffer the immune attack of the infected hepatocytes by the anti-hbc antibodies [5] . when the balance is disrupted by the emergence of hbv mutants with different phenotypes, the altered virus-cell relationship might trigger robust immune responses of the host in some instances, causing extensive hepatocyte necrosis [30] . thus, the investigation of virologic features may shed light on the pathogenesis and identify predictors of aclf development. however, the development of aclf is related to multiple factors, all of which need to be studied extensively. clinical manifestations of aclf are different from those of fulminant hepatitis on the basis of acute hbv infection. aclf manifestation also differs from the acute exacerbation of chb, which is defined as clinical symptoms along with an abrupt rise in serum alt ([200 iu/l [31] or [500 iu/l [32] ). aclf patients are relatively rare compared to the large population of chb patients. the beijing 302 hospital is one of the largest hospitals for infectious and liver diseases in china and is well known for its management of hepatitis b. patients from various areas of china come to the hospital seeking treatment, and this has allowed us to collect a larger sample of aclf patients. the efficiency of pcr amplification of hbv bcp/pc regions is greatly reduced by the nick-gap structure encompassed in the regions. a sensitive nested pcr assay with a lower limit of detection of 2000 copies/ml was developed to overcome this obstacle. the technique allowed us to analyze samples of quite low viral load, as in our previous severe acute respiratory syndrome (sars)coronavirus studies [33, 34] . a significantly higher incidence of the bcp/pc mutations at eight of the ten sites of interest was found in aclf patients than in chb patients. in particular, the occurrence of three hotspot bcp/pc mutations and average substitution number in the ten sites of interest increased with the severity of illness in a stepwise fashion ( table 1 ), suggesting that the accumulation of the bcp/pc mutations could be one of the driving factors of illness severity. taking into account that a longer duration of infection may increase the incidence of hbv bcp/pc mutation, we particularly compared different illness groups of comparable ages to minimize this factor. the results showed that the increasing trend of the three hotspot mutations (a1762t, g1764a, and g1896a) with disease severity remained unchanged in age-matched patients ( table 2 ). in addition to variants at the ten sites of interest, the frequency of a1846t was found to increase in chb-s and aclf patients compared to chb-m patients, suggesting that this variant may also be associated with disease severity. a1846t has been reported to be more frequently detected in hbeag-negative hbv infection than in hbeag-positive hbv infection [35] , but the mechanism needs further clarification. more frequent bcp mutations and less frequent pc mutations were detected in genotype c virus than in genotype b virus which confirmed previous results [36, 37] . interestingly, t1754g was an exceptional case which was more often seen in genotype b virus infection. the influence of hbv genotypes on the severity of hbv infection is still uncertain. as genotype b and c viruses have different bcp/pc mutational features, their influence on clinical outcome may be associated with the bcp/pc mutation. nevertheless, the average substitution number per sample in bcp/pc regions clearly increased with the severity of the illness categories in both genotype b and c, suggesting that the accumulation of the bcp/pc mutations increased the risk of aclf occurrence regardless of the hbv genotypes. it has been reported that the co-existence of a1762t/ g1764a with t1753v, c1766t, and t1768a may enhance viral replication in vitro and is associated with alf and advanced liver disease [38] [39] [40] . we found that t1753v-, c1766t-, and/or t1768a-containing triple mutations were more frequent in aclf patients than in chb patients, suggesting highly replicative strains with t1753c, c1766t, and t1768a bcp mutations were also most likely to be associated with aclf development. among the 57 cases with triplet b patterns, 30 (52.6%) were a1762t/ g1764a/c1766t, nine (15.8%) were a1762t/g1764a/ t1768a, and 18 (31.6%) were g1764a/c1766t/t1768a. noticeably, the last pattern has not been documented earlier and mainly occurred in aclf patients. in the luciferase reporter system, the natural g1764a/c1766t/t1768a mutant sequences have been proven to have a 1.8-fold higher transcriptional regulation activity on average than the wild-type sequences generated by reverse site-directed mutagenesis (data not shown). it has been suggested that the bcp mutations frequently emerge at the late hbeag phase of infection, whereas the pc mutations usually emerge later, at the height of the anti-hbe immune response [15] . therefore, we classified three bcp/pc mutation statuses as bcp-/pc-, bcp?/pcand bcp±/pc? and analyzed their implications. the results showed that chb-m patients infected with bcp/pc mutants exhibited more enhanced inflammatory responses (significant increase in tbil and alt levels and a slight decrease of viral load) than those with the bcp/pc wildtype virus, whereas chb-s and aclf patients did not. in contrast, chb-s patients infected with bcp/pc mutants had lower tbil and hbv dna levels than those with the wild-type virus. to date, few studies have been able to show an association between these clinical parameters and hbv bcp/pc mutations. bcp mutants have been shown to be positively correlated with increased alt levels in chronic hbv carriers and chb patients [41] , and reduced viremia associated with bcp/pc mutants was explained by the fact that enhanced hbv replication of the mutants would efficiently stimulate immune reactions, leading to the enhanced destruction of viral particles [13, 15] . the alternation of alt and hbv dna levels may be influenced by the use of antiviral treatment; furthermore, a blood test at a single time point may bias the evaluation of disease activity for some chb patients with fluctuating alt and hbv dna levels, although these influences were relatively proportionate in large-size samples in this study. further genotypic and phenotypic studies need to be performed to make a clearer outline. the rates of hbeag loss and anti-hbe positivity increased in a stepwise manner along with the emergence of bcp mutation and pc mutation in chb-m patients. in chb-s and aclf patients, bcp mutation alone did not significantly increase hbeag-negative/anti-hbe-positive rates, suggesting that bcp mutations may have diverse influence on hbeag seroconversion in different illness categories. there have been reports of the bcp double mutations being associated with lower viral loads in hbeag-positive individuals [42] and the pc mutation being correlated with high levels of hbv dna in hbeagnegative chb patients [43] . however, these associations were not observed in our study (data not shown). mutant viruses frequently coexist with wild-type viruses, and subpopulations comprising \20% of the total hbv population may be missed by direct sequencing techniques [44] . this may account for the detection of g1896a mutants in some hbeag-positive patients. interestingly, we found that aclf patients infected with pc mutants had a significantly higher mortality than those with pc wild-type hbv, indicating that the hbv pc mutation could serve as a predictive indicator for aclf outcome. in summary, our findings suggest that chb patients infected with bcp/pc mutant virus are more susceptible to severe hepatitis and aclf than those with the bcp/pc wild-type virus and that aclf patients with pc mutant virus have an increased risk of death. the results of this study further our knowledge of the virologic factors that are associated with the severity of chronic hbv infection. european association for the study of the liver. easl clinical practice guidelines: management of chronic hepatitis b hepatitis b virus in tenofovir-naive chinese patients with chronic hepatitis b contains no mutation of rta194t conferring a reduced tenofovir susceptibility characteristics of acute and sub-acute liver failure in china: nomination, classification and interval influence of hepatitis b virus genotype on the long-term outcome of chronic hepatitis b in western patients hepatitis b virus e antigen variants hepatitis b virus genetic variability and evolution possible association of vigorous hepatitis b virus replication with the development of fulminant hepatitis fulminant hepatitis b: induction by hepatitis b virus mutants defective in the precore region and incapable of encoding e antigen hepatitis b virus strains with mutations in the core promoter in patients with fulminant hepatitis two core promoter mutation identified in a hepatitis b virus strain associated with fulminant hepatitis result in enhanced viral replication clinical and molecular virological difference between fulminant hepatic failures following acute and chronic infection with hepatitis b virus mutations in the basic core promotor and the precore region of hepatitis b virus and their selection in children with fulminant and chronic hepatitis b influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis b virus infection association of hepatitis b virus subgenotypes and basal core promoter/precore region variants with the clinical features of patients with acute hepatitis genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis b virus core promoter mutants detection and significance of a g1862t variant of hepatitis b virus in chinese patients with fulminant hepatitis initial load of hepatitis b virus (hbv), its changing profile, and precore/core promoter mutations correlate with the severity and outcome of acute hbv infection clinical outcome and virological characteristics of hepatitis b-related acute liver failure in the united states hepatitis b virus genomes of patients with fulminant hepatitis do not share a specific mutation properties of hepatitis b virus genome recovered from vietnamese patients with fulminant hepatitis in comparison with those of acute hepatitis hepatitis b virus: prevalence of precore/core promoter mutants in different clinical categories of indian patients no significant correlation exists between core promoter mutations, viral replication and liver damage in chronic hepatitis b infection precore/core promoter mutations and genotypes of hepatitis b virus in chronic hepatitis b patients with fulminant or subfulminant hepatitis chinese society of infectious diseases and parasitology; and chinese society of hepatology. management scheme of diagnostic and therapy criteria of viral hepatitis chinese society of infectious diseases and parasitology, severe liver diseases and artificial liver group, chinese society of hepatology. diagnostic and treatment guidelines for liver failure imbalanced intrahepatic cytokine expression of interferon-c, tumor necrosis factor-a, and interleukin-10 in patients with acute-on-chronic liver failure associated with hepatitis b virus infection compartmentalization and its implication for peripheral immunologically-competent cells to the liver in patients with hbv-related acute-on-chronic liver failure acute-on-chronic liver failure; consensus recommendations of the asian pacific association for the study of the liver (apasl) features and clinical implications of hepatitis b virus genotypes and mutations in basal core promoter/precore region in 507 chinese patients with acute and chronic hepatitis b hepatitis b virus mutants and fulminant hepatitis b: fitness plus phenotype role of genotype and precore/basal core promoter mutations of hepatitis b virus in patients with chronic hepatitis with acute exacerbation detection of hbv core promoter and precore mutations helps distinguish flares of chronic hepatitis from acute hepatitis b sars-associated coronavirus quasispecies in individual patients genetic variation of sars coronavirus in beijing hospital clinical significance of hepatitis b virus (hbv) genotypes and precore and core promoter mutations affecting hbv e antigen expression in taiwan epidemiological study of hepatitis b virus genotypes, core promoter and precore mutations of chronic hepatitis b infection in hong kong relationship of genotypes of hepatitis b virus to mutations, disease progression and response to antiviral therapy basal core promoter, precore region mutations of hbv and their association with e antigen, genotype, and severity of liver disease in patients with chronic hepatitis b in india effect of basal core promoter and pre-core mutations on hepatitis b virus replication sequential accumulation of the mutations in core promoter of hepatitis b virus is associated with the development of hepatocellular carcinoma in qidong the precore/core promoter mutant (t1762a1764) of hepatitis b virus: clinical significance and an easy method for detection the association of hbv core promoter double mutations (a1762t and g1764a) with viral load differs between hbeag positive and anti-hbe positive individuals: a longitudinal analysis hepatitis b virus genotypes and g1896a precore mutation in 486 spanish patients with acute and chronic hbv infection selection of a secretion-incompetent mutant in the serum of a patient with severe hepatitis b key: cord-351295-4toxlskr authors: lanave, gianvito; capozza, paolo; diakoudi, georgia; catella, cristiana; catucci, leonardo; ghergo, paola; stasi, fabio; barrs, vanessa; beatty, julia; decaro, nicola; buonavoglia, canio; martella, vito; camero, michele title: identification of hepadnavirus in the sera of cats date: 2019-07-23 journal: sci rep doi: 10.1038/s41598-019-47175-8 sha: doc_id: 351295 cord_uid: 4toxlskr hepadnaviruses infect several animal species. the prototype species, human hepatitis b virus (hbv), increases the risk of liver diseases and may cause cirrhosis and hepatocellular carcinoma. recently a novel hepadnavirus, similar to hbv, has been identified through transcriptomics studies in a domestic cat with large cell lymphoma in australia. herewith, a collection of 390 feline serum samples was screened for hepadnavirus. overall, the virus was identified in 10.8% of the sera with a significantly higher prevalence (17.8%) in the sera of animals with a clinical suspect of infectious disease. upon genome sequencing, the virus was closely related (97.0% nt identity) to the prototype australian feline virus sydney 2016. the mean and median values of hepadnavirus in the feline sera were 1.3 × 10(6) and 2.1 × 10(4) genome copies per ml (range 3.3 × 10(0)–2.5 × 10(7) genome copies per ml). for a subset of hepadnavirus-positive samples, information on the hemato-chemical parameters was available and in 10/20 animals a profile suggestive of liver damage was present. also, in 7/10 animals with suspected hepatic disease, virus load was >10(4) genome copies per ml, i.e. above the threshold considered at risk of active hepatitis and liver damage for hbv. overall, we detected hepadnavirus dna in 42/390 (10.8%) sera. dch dna was detected in 31 (17.8%) out of 174 sera collected with a request for diagnosis of infectious diseases (collection a) and in 11 (5.1%) out of 216 sera submitted to the laboratory for pre-surgical evaluation or for suspected metabolic or neoplastic disease (collection b) that were used to generate a baseline. the difference for dch prevalence was statistically extremely significant (p-value = 0.00006, or = 4.04005, ci95% = [1.96601; 8.30211]) when compared animals from collection a and collection b (fig. 1a) . moreover, there was no significant difference in terms of prevalence (p = 0.64125) between male (11.4%, 25/219) and female (9.9%, 17/171) individuals (fig. 1b) . we also analysed the distribution of hepadnavirus across various age groups (fig. 1c) . the prevalence of dch was higher in cats aged 4 to 7 months (20.5%, 8/39) although without statistically significant difference (p = 0.08061) compared to other age groups. we reconstructed the complete genome of the dch strain ita/2018/165-83, of approximately 3.2 kb (fig. 2) , which had 97.0% nucleotide sequence identity to the australian reference strain aus/2016/sydney across the entire genome. some differences were observed in the tree topologies between our analysis and previous studies 2 , likely accounted for by the alignment complexity. in the consensus phylogenetic tree (fig. 3) , a large group included bat and rodent viruses. the two feline hepadnaviruses segregated together in a defined branch within www.nature.com/scientificreports www.nature.com/scientificreports/ this large group. all the primate viruses were basal to this bat/rodent/feline group. also, there were two additional well-defined groups, encompassing avian and amphibian viruses, respectively. the findings of this study confirm that the novel hepadnavirus is a common component of the feline virome. the pathogenic role of this hepadnavirus, if any, is not yet known, but thus far, all hepadnaviruses have been reported to replicate preferentially in hepatocytes 1, 4 . in this study we observed a marked and significantly higher prevalence (17.8%, 31/174) in the cohort of cats with suspected infectious diseases (collection a) with respect to a group of animals (collection b) used as baseline. almost half of the sera positive for dch (14/31, 45 .2%) of this cohort were collected from cats with retroviral infection (fiv and/or feline leukemia virus, felv). in turn, the overall prevalence of retroviral infection in collection a was 24.1% (42/174), with 33.3% (14/42) being co-infections with dch with a statistically significant (p = 0.0048) correlation between dch and retrovirus positive cats. these findings echo what has been documented for hbv, which is more frequently observed in immunocompromised individuals 5 . reactivation of hbv is common in patients with immunosuppression 5 . even more interestingly, hepatopathy has been described in felv-infected cats, with icterus and various inflammatory and degenerative liver diseases 6 . feline retroviruses have been demonstrated to impair severely the immune system in infected cats and diseases associated with immune-suppression account for a large portion of the morbidity and mortality observed in felv-infected cats [7] [8] [9] . transmission of hbv in humans occurs through blood and other body fluids and contagion can also occur during sexual contact and by maternal/fetal route 1, 4 . transmission of fiv/felv in cats occurs with similar modalities, as the virus is present in blood and body fluids 9 . similar modalities of transmission might also be hypothesized for dch, since we found viremia in 10.8% of the cats. importantly, the presence of dch in the sera may pose unexpected risks in transfusion medicine. dch, along with feline retroviruses, bartonella spp. and feline hemoplasma, should be considered in the screening of donor subjects 10 . in cats there are not known viral agents strictly associated with hepatic disease, unlike what observed in human medicine where there are five major types of viral hepatitis (a to e) 11 . for diagnosis of hbv infection and for predicting the stage of infection in human patients, antigens, antibodies and viral genome are profiled/ quantified and this information is coupled with haematological and blood chemistry tests. in the case of dch, we can only use the molecular diagnostics and hematological and blood chemistry data, since there are no immunological reagents available for the diagnostics. out of 42 dch-infected cats, we could retrieve information on hematologic and serum biochemical parameters for 20 animals (table 1 ). in 10 of these, increased levels of markers indicative of structural or functional liver damage (i.e. ast, alt, alp, ggt and total bilirubin) were present. hbv load is considered relevant in infected human patients and varies markedly across the phases of hbv infection 1 . the lower threshold for risk of active hepatitis and liver damage is 10 4 viral genome equivalents of hbv per ml, equivalent to about 2000 iu (international unit)/ml 1 . usually, high hbv dna load in blood is found in acute infection, or in active chronic stages of disease, but the virus can reactivate after long periods of apparent remission, chiefly in immunosuppressed patients 1 . the mean and median values of dch viremia in feline sera figure 1 . results of the screening for dch in the feline sera. the prevalence of dch was evaluated in sera collected for the diagnosis of infectious diseases (collection a) and sera submitted to the laboratory for presurgical evaluation or for suspected metabolic or neoplastic disease (collection b) and used for comparison (panel a). the prevalence of dch was also evaluated in relation to sex (panel b) and age (panel c) of the animals. www.nature.com/scientificreports www.nature.com/scientificreports/ were 1.3 × 10 6 and 2.1 × 10 4 dna copies per ml (range 3.3 × 10 0 -2.5 × 10 7 dna copies per ml). in 7 out of 10 animals with suspected hepatic disease, dch load was >10 4 genome copies per ml. although this parallelism between hbv and dch is intriguing, whether a correlation also exists between dch replication and liver damage should be assessed in structured, larger observational studies. for instance, we also found the virus in cats with unaltered hepatic markers. this condition occurs in human patients with chronic hbv infection during the inactive "immune-control" phase, defined as the presence of hbv antigens in serum without alt elevation and with low viremic hbv-dna levels 12 . however, only in 3/10 cats with non-altered hepatic markers the virus titer was lower than 10 4 . the actual patho-biology of dch in cats should be determined in order to understand better the patterns of dch infection. several novel viruses have been discovered in cats in recent years 13, 14 . optimizing the diagnostic algorithms and gathering epidemiological data will help assessing the possible pathogenic role of these viruses in cats and eventually conceive strategies to protect their health. a total of 390 sera were collected from two different veterinary clinic laboratories located in apulia region, southern italy and tested upon request of the veterinarian practitioners after anamnesis, medical history and clinical examination. one-hundred seventy-four sera (collection a) had been collected for diagnosis of infectious diseases (fiv, felv, feline coronavirus (fcov), toxoplasmosis, hemoplasmosis, bacterial and fungal infections). a total of 147/174 (84.5%) sera were submitted with a suspect/request of diagnosis for fiv/felv, with 42 sera being positive for retrovirus. fisher's exact test was performed to the collection a to evaluate the correlation between dch positive cats and retrovirus positive cats. the significance level of the test was set at 0.05. of the other 27 sera (15.5%), 14 were sent for a suspect/request of diagnosis for coronavirus (with 7/14 being positive), 5 for toxoplasmosis (with 2/5 being positive), 2 for giardia (with 1/2 being positive). five sera were collected from animals with suspected bacterial/fungal infections and 1 serum (negative) was from a cat with suspected hemoplasmosis. a total of 216 sera (collection b) submitted to the laboratory for pre-surgical evaluation (n = 85) or for suspected metabolic (n = 127) or neoplastic (n = 4) disease was used for comparison to generate a baseline. information on the sera analyzed in the study is included in fig. 1 . the study was approved by the ethics committee of the department of veterinary medicine, university of bari (authorization 23/2018). all experiments were performed in accordance with relevant guidelines and regulations. www.nature.com/scientificreports www.nature.com/scientificreports/ total dna was extracted from collected sera by using qiaamp cador pathogen mini kit (qiagen, hilden, germany), according to the manufacturer's instructions. we performed sample screening using a pcr with consensus pan-hepadnavirus primers 2 and a pcr with primers specific for dch 3 . also, we screened sera using a quantitative pcr (qpcr) designed based on the sequence of the australian reference strain aus/2016/sydney (genbank accession nr. mh307930) ( table 2) . for qpcr, we calculated dch dna copy numbers on the basis of standard curves generated by 10-fold dilutions of a plasmid standard topo xl pcr containing a 1.4 kb long fragment of the polymerase region of the australian reference strain aus/2016/sydney (iq supermix; bio-rad laboratories srl, segrate, italy). we added 10 μl of sample dna or plasmid standard to the 15-μl reaction www.nature.com/scientificreports www.nature.com/scientificreports/ master mix (iq supermix; bio-rad laboratories srl, segrate, italy) containing 0.6 μmol/l of each primer and 0.1 μmol/l of probe. thermal cycling consisted of activation of itaq dna polymerase at 95 °c for 3 min and 42 cycles of denaturation at 95 °c for 10 s and annealing-extension at 60 °c for 30 s. we evaluated the specificity of the assay using a panel of feline dna viruses (parvovirus, herpesvirus and poxvirus). the qpcr assay was able to detect as few as 10 1 dna copies per ml of standard dna and 3.3 × 10 0 dna copies per ml of dna template extracted from clinical samples. dch quantification displayed acceptable levels of repeatability over a range of target dna concentrations, when calculating the intra-and inter-assay coefficients of variation within and between runs, respectively 15, 16 . we carried out inferential statistical analyses using the chi-squared test with yates' correction, the evaluation of the odds ratio (or) and 95% confidence interval (ci95%) with the online software medcalc easy-to-use statistical software (https://www.medcalc.org/calc/odds_ratio.php). the significance level of the test was set at 0.05. full genome sequences of hepadnaviruses were retrieved from the genbank database and aligned using geneious version 9.1.8 (biomatters ltd, auckland, new zealand) and the mafft algorithm 17 . a set of genome sequences used in a previous study 2 was integrated with additional genome sequences of hepadnaviruses of recent identification in mammalian, avian, amphibian species and in fish. the final dataset included 53 hepadnavirus genomes. phylogenetic analysis was performed using jmodel test (http://evomics.org/resources/software/ molecular-evolution-software/modeltest/) to evaluate the correct best-fit model of evolution for the entire dataset. bayesian analysis 18, 19 was therefore applied using four mcmc chains well-sampled and converging over www.nature.com/scientificreports www.nature.com/scientificreports/ one million generations (with the first 2000 trees discarded as "burn-in") and supplying statistical support with subsampling over 1000 replicates. the identified program settings for all partitions, under the akaike information criteria, included six-character states (general time-reversible model), a proportion of invariable sites and a gamma distribution of rate variation across sites (gtr + i + g). we also tried to perform phylogenetic analyses using other evolutionary models (maximum likelihood, neighbor joining) to compare the topology of phylogenetic trees. we could observe similar topologies with slight difference in bootstrap values at the nodes of the tree. accordingly, we did prefer to retain the bayesian tree. we deposited the nucleotide genome sequence of strain ita/2018/165-83 (mk117078) in genbank. all data generated or analyzed in this study are included in this published article. fields virology detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in china a novel hepadnavirus identified in an immunocompromised domestic cat in australia hepatitis b virus: virology, molecular biology, life cycle and intrahepatic spread chronic hepatitis b: update 2009 diseases associated with spontaneous feline leukemia virus (felv) infection in cats isolation of a t-lymphotropic virus from domestic cats with an immunodeficiency-like syndrome clinical and immunologic aspects of felv-induced immunosuppression clinical aspects of feline retroviruses: a review blood transfusion in cats: abcd guidelines for minimising risk of infectious iatrogenic complications viral hepatitis in principles and practice of infectious diseases update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld 2018 hepatitis b guidance seroprevalence for 2117-like vesiviruses in italian household dogs identification of a novel parvovirus in domestic cats novel parvovirus related to primate bufaviruses in dogs development and validation of a real-time pcr assay for specific and sensitive detection of canid herpesvirus 1 mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform mrbayes 3: bayesian phylogenetic inference under mixed models mrbayes: bayesian inference of phylogenetic trees this study was supported by the grant ricerca corrente 2017 "nuovi flussi diagnostici in sanità animale: dalla ngs alla banca antigeni". reference grant number b45e17000060005 -izs am 06/17rc (italian ministry of health). in addition, the authors would like to thank professor donatella ferraro, university of palermo for providing hbv positive controls. the study was conceived by v.m. experiments were performed by g.l., p.c., g.d. and c.c. data analysis was performed by g.l., m.c. and p.c. sample collection was accomplished by l.c., p.g., f.s. support for experiments and scientific feedback were provided by v.b., j.b., n.d., c.b., v.m. the manuscript was written by v.m., g.l., p.c. and m.c. and reviewed and approved by all authors. competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-333220-tcvs4beg authors: lee, szu-yuan; huang, jhen-gang; chuang, tsung-liang; sheu, jin-chuan; chuang, yi-kuang; holl, mark; meldrum, deirdre r.; lee, chun-nan; lin, chii-wann title: compact optical diagnostic device for isothermal nucleic acids amplification date: 2008-08-12 journal: sens actuators b chem doi: 10.1016/j.snb.2008.03.008 sha: doc_id: 333220 cord_uid: tcvs4beg we recently reported the successful use of the loop-mediated isothermal amplification (lamp) reaction for hepatitis b virus (hbv) dna amplification and its optimal primer design method. in this study, we report the development of an integrated isothermal device for both amplification and detection of targeted hbv dna. it has two major components, a disposable polymethyl methacrylate (pmma) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of dna amplification by-product, magnesium pyrophosphate. we have established a correlation curve (r(2) = 0.99) between the concentration of pyrophosphate ions and the level of turbidity by using a simulated chemical reaction to evaluate the characteristics of our device. for the applications of rapid pathogens detection, we also have established a standard curve (r(2) = 0.96) by using lamp reaction with a standard template in our device. moreover, we also have successfully used the device on seven clinical serum specimens where hbv dna levels have been confirmed by real-time pcr. the result indicates that different amounts of hbv dna can be successfully detected by using this device within 1 h. around the world, the hepatitis b virus is one of the most common viral pathogens, having infected more than 370 million people [1] . at present, serologic diagnosis of hbv infections is based on the viral antigens and antibodies. however, the reveal-hbv (risk evaluation of viral load elevation and associated liver disease/cancer-hepatitis b virus) study indicates that the serum level of hbv dna ( 10,000 copies/ml) is a strong risk predica-tor of hepatocellular carcinoma or cirrhosis [2, 3] . it would thus be clinically valuable to have a molecular diagnostic method for screening and monitoring the progress of hepatitis. however, the long reaction time of traditional pcr amplification and the high cost of thermalcycler are still major issues for such a screening application. therefore, it is important to develop a rapid and accurate diagnostic device for field applications as soon as possible [4] . even though many nucleic acid amplification methods are currently available, a low cost, yet rapid method would be extremely useful, especially in developing countries. there are several pcr-based amplification methods [5] , such as nucleic acid sequence-based amplification [6] and self-sustained sequence replication, for nucleic acid amplification. however, these methods require a precise instrument to provide the efficient thermal cycles and to shorten the total amplification time for a test run. in general, it takes up to 2.5 h for a pcr test with the present state of art equipment. alternatively, isothermal amplification methods, e.g., strand displacement amplification [7] , branched dna amplification, invader, rolling circle amplification [8] and loop-mediated amplification method (lamp) [9] , have been proposed for amplifying the targeted nucleic acid sequence under a single working temperature condition with special designs for the buffer system and primers to prevent non-specific amplification. it would thus allow for the development of a low-cost device for rapid pathogen detection. loop-mediated isothermal amplification (lamp), originally developed by notomi et al. [9] , utilizes a designed set of six primers, termed inner and outer primers, to recognize specific gene sequences, and a polymerase with strand displacement activity to generate large amounts of amplified product (>10 9 copies) within 1 h [10] . moreover, it has been shown that the well-known pcr inhibitors in the blood (e.g., heme) have little impact on the lamp reactions [11] . it is believed that the bacillus stearothermophilus (bst) dna polymerase used in the lamp reactions is more resistant to these inhibitors. therefore, the lamp reaction has been successfully applied for fast genetic screening tests in many acute infectious diseases, including mycobacterium tuberculosis [12] , severe acute respiratory syndrome virus [13] , human influenza viruses [14, 15] , avian influenza viruses [16] and herpes viruses [17, 18] , with special primer design and buffer adjustment. among these, the lamp method has been shown to have great promise for the amplification of hbv dna. in addition, the dna yield of the lamp reaction (10 g/25 l) is much higher than that of the traditional pcr (0.2 g/25 l) [19] . recently, we have successfully demonstrated that the level of turbidity, which is due to the by-product (magnesium pyrophosphate) of dna polymerization, has a high correlation to the amounts of amplified dna. then, we decided to work with a total of 25 l of lamp reaction volume because of the practical limitations of turbidity detection. in addition to the use of the lamp protocol, we have adapted a simulated chemical reaction to mimic the by-product production without using expensive polymerase and primers [20] . it can serve as an internal quality check for the system validation. therefore, it is our intention to design and implement a compact integrated device with a disposable chip and simple quantitative optical read-out for low-cost applications. in this study, the goal is to develop an integrated isothermal device for real-time detection of hbv viral dna via the lamp amplification method. it has two major components, a disposable pmma micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to precipitation of dna amplification by-product, magnesium pyrophosphate. we decided to work with a total of 25 l of lamp reaction volume after adding 2 l of dna sample because of the practical limitations of turbidity detection. the performance of this integrated isothermal device has been tested by using within and between runs. for the applications of rapid pathogens detection, we have successfully used the device on seven clinical serum specimens and confirmed hbv dna levels with real-time pcr. the results of using this device indicate that different amounts of hbv dna can be successfully detected within 1 h with a threshold level of 10,000 copies/ml, which is the recommendatory quantity of reveal-hbv study. this integrated isothermal device can be advantageous in a wide spectrum of field applications, including pathogen detection and gene testing. the design methodology of primers used has been discussed in our previous study [21] . in brief, the target dna sequences and the partial hbv polymerase gene sequences are collected from a public data base and then aligned to find highly conserved fragments. then, the primer design can be executed by using the available software, gene runner (hastings software, inc., hudson, ny, usa), to check design parameters. the melting temperature (t m ) of each primer is calculated by the nearest-neighbor t m theory. finally, the designed primers are synthesized by the contract services (quality systems, inc., taipei city, taiwan, roc) followed by having its concentration optimized with a reaction buffer in the amplified test. the partial hbv polymerase gene was directly cloned into pgem-t easy vector (promega, madison, wi, usa) as standard template dna. the lamp assay was performed in a total of 25 l of the mixtures, which contain 20 pmol each of ib-fip (5 -tggaattagaggacaaacgggtgctgctatgcctcatctt-3 ) and ib-bip (5 -gctcaaggaacctctatgtttcgatgatgggatgggaataca-3 ), 5 pmol each of ib-f3 (5 -ggcgttttatcatcttcct-3 ) and ib-b3 (5 -aggttacttgcgaaagcc-3 ), 10 pmol each of ib-loopf (5 -taccttgatagtccagaagaacc-3 ) and ib-loopb (5 -ctacggacggaaactgcac-3 ), 0.4 mm dntps, 1 m betaine, 20 mm tris-hcl (ph 8.8), 10 mm kcl, 10 mm (nh4) 2 so 4 , 6 mm mgso 4 , 0.1% triton x-100, 5 units of the bst dna polymerase large fragment (new england biolabs, ipswich, ma, usa), and 2 l of dna standard template-a partial hbv polymerase gene cloned into a pgem-t easy vector or purified dna. we utilized a set of six primers to recognize specific hbv gene sequences. this mixture was incubated in a mastercycler ® gradient pcr machine (eppendorf, hamburg, germany) or our miniaturized device at 65 • c for 1 h. the white precipitate of reaction by-product can be observed by naked eye. aliquots of 2.5 l of lamp products were electrophoresed in 2% agarose gels (1× tbe) and then stained with sybr green i dye for verification by fluorescent imager (geldoc-it imaging system, uvp, upland, ca, usa). in addition, the sequences of lamp product were confirmed by the abi 3100-avant dna sequencer (applied biosystems, foster city, ca, usa). our integrated isothermal device has two major components, a disposable polymethyl methacrylate (pmma) micro-reactor and a temperature-regulated optical detection unit (base apparatus) for real-time monitoring of the turbidity changes due to the precipitation of dna amplification by-product, magnesium pyrophosphate. the disposable micro-reactor is constructed from two pmma parts to create a reaction chamber of 5-mm optical path length by using uv-light curing adhesives (ultrawide gn150, everwide chemical company, yunlin county, taiwan, roc). for the lamp reaction, the reaction chamber is filled with 25 l reagent manually via micropipette and then is sealed with gluey aluminum foil. the base apparatus consists of an optical detection unit, a thin-film heater, a temperature controller, and a power supply. the optical detection unit (fs-v21g, keyence corporation, osaka, japan) employs a light emitting diode (led) light source at 533 nm and a phototransistor detector with an extension of collimated optical fibers for the collection of forward scattering light. the temperature controller has a 60 w power supply and two 2 in. × 2 in. thin-film kapton tm heaters (minco, minneapolis, mn, usa), which is controlled by a proportional-integral-derivative (pid) controller (anly electronics, taipei county, taiwan, roc) with one thermal coupler feedback. after the insertion of assembled micro-reactor chips into the base apparatus, we can initiate the hbv lamp reaction at 65 • c through out the whole experimental time course. at the same time, the scattering light intensity is measured by the optical detection unit. in general, turbidity refers to the scattering of light by particles and has to be measured and calculated indirectly from eq. (1) [22] assuming no absorption in the path length: where i 0 is the intensity of incident light and i 1 is the intensity of transmitted light. during the dna polymerization process, white precipitation of magnesium pyrophosphate will be produced in the presence of magnesium ions and pyrophosphate ions as shown in the following equations: instead of using dntp and dna polymerases to initiate the precipitation, we have adapted a simulated reaction, as shown in eq. (4), to mimic the production of magnesium pyrophosphate which is used for evaluating the performance of our device: this reaction is performed in a total of 1 ml solution containing the following reagents, 1× thermolpol buffer (20 mm tris-hcl (ph 8.8), 10 mm kcl, 10 mm (nh4) 2 so 4 , 2 mm mgso 4 , 0.1% triton x-100), 2 mm mgso 4 , gradient concentrations of k 4 p 2 o 7 or k 3 po 4 from 1.2 to 0.2 mm and double-distilled water. the mixture is incubated in the disposable micro-reactor at 65 • c for 1 h to measure the end-point turbidity by our system. in addition, this experimental result was confirmed by a uv-visible spectrometer (varain, palo alto, ca, usa) at 533 nm. it also can be used to establish the correlation curve between the concentration of pyrophosphate ions and the level of turbidity. a series of seven serum specimens were obtained from patients at national taiwan university hospital (ntuh). the serum hbv viral dna was extracted by using the qiaamp viral dna mini kit (qiagen, valencia, ca, usa). briefly, 800 l of viral particles lysis buffer (avl buffer) was mixed with 200 l of serum. the avl buffer will lyse viral particles under highly denaturing conditions to inactivate dnases and to provide optimum binding buffering conditions. the mixture was incubated at room temperature for 10 min and then mixed with 800 l of ethanol. then, 630 l of the mixture was transferred to the qiaamp spin column and was centrifuged at 6000 × g for 1 min. five-hundred microliters of washing buffer 1 (aw1 buffer) was added to the spin column and centrifuged at 6000 × g for 1 min. then, 500 l of washing buffer 2 (aw2 buffer) was added to the spin column and centrifuged at 20,000 × g for 1 min. the dna was eluted from the column by adding 40 l of rnase-and dnase-free water. after centrifuging at 6000 × g for 1 min, the supernatant contained the dna and was ready for use. in addition, the hbv dna viral load was determined by real-time pcr which used the reagent of quantiplex tm hbv dna assay (chiron corporation, emeryville, ca, usa) and the detection system of abi prism 7000 sequence detection system (applied biosystems, foster city, ca, usa). the hbv viral load of these seven samples ranged from an undetectable level to more than 5 × 10 7 copies/ml for the testing of our device. following the principle of lamp reaction, the amplified products elongated to a length of several kbp and when generated they showed complex cauliflower-like structures [9] . we demonstrated that the positive sample reveals many bands of different sizes after agarose gel electrophoresis (fig. 1a) . with an increase of lamp the structure of the disposable micro-reactor. the disposable micro-reactor has a reaction chamber and 5-mm optical pathway. the reaction chamber is 5 mm-long, 2 mm-wide and 5 mm in height. (b) the structure of the base apparatus. the base apparatus is made with pmma to create a detection pathway and sliding track. it has two slices of flexible heaters on the top and on the bottom sides. the light source fibers and the optical sensor are lined on the detection pathway and put close (about 10 mm) to each other. the reaction box is 60 mm-long, 50 mm-wide and 14 mm high. the detection pathway is 60 mm-long, 10 mm-wide, 5 mm-height and the sliding track is 35 mm-long, 6 mm-wide, 5 mm-height. (c) picture of the disposable micro-reactor. this micro-reactor is just a home-made product. the disposable micro-reactor is constructed from two components of pmma and glass slide cover. it has a reaction chamber and a 5-mm optical pathway without heaters or sensors. (d) picture of the base apparatus. for the lamp reaction, the reaction chamber is filled with 25-l reagent and then sealed by gluey aluminum foil. the disposable micro-reactor component can be inserted into the base apparatus and the hbv lamp reaction can be started under isothermal condition. this system can provide appropriate reaction conditions for hbv lamp dna amplification. products, a large amount of by-product, magnesium pyrophosphate (mg 2 p 2 o 7 ), is produced and precipitated in the reaction mixture. the white precipitate of lamp reaction by-product can be observed by naked eye (fig. 1b) . other than the optimization of primers for the lamp reaction, the reaction volume is a critical issue in the design of such a miniaturized device for amplification and detection. we decided to work with a total of 25 l of lamp reaction volume because of the practical limitations of turbidity detection. the prototype of our integrated isothermal device has two components: a disposable micro-reactor ( fig. 2a) and a temperature-regulated optical detection unit (base apparatus) (fig. 2b) . for the lamp reaction, the disposable pmma micro-reactor is filled with 25 l reagent (fig. 2c ) and then sealed with gluey aluminum foil. it can be inserted into the base apparatus and the hbv lamp reaction can be started under isothermal conditions. simultaneously, we can detect the turbidity of the lamp by-product in the base apparatus (fig. 2d ). to evaluate the performance of the base apparatus for dna amplification and detection, we have adapted a simulated chemical reaction to produce magnesium pyrophosphate. the simulated reaction utilizes potassium pyrophosphate and magnesium sulfate to synthesize magnesium pyrophosphate, as established in our previous study [20] . white precipitates can be observed when the concentration of pyrophosphate ions exceeds 0.4 mm. fig. 3a shows the results of a 1 h end point turbidity measurement by using a spectrometer (r 2 = 0.9955) or our own system (r 2 = 0.9989) at 65 • c. fig. 3b shows the reproducibility results of within run for turbidity detection in our device. it shows that the coefficient of variation (cv%) within run is very low (<6%). however, our system shows larger fluctuations between runs than does the spectrometer. this might be due to minor uncertainties in our fabricated devices, which will be able to control within acceptable limits with mass production of disposable chip. with a total of 25 l of reagents and sample dna in sealed reaction chamber, we can start the reaction under isothermal condition (65 • c) and monitor the intensity of scattering light by the optical unit. the real-time data on turbidity, decreases during the first 2 min, and then increases until reaching a plateau at 60 min. fig. 4a shows the superimposed plots of turbidity data from different initial concentrations of dna template. all of these samples show obvious changes in 30 min. from these results, it would be reasonable to take turbidity value of 30 min as a critical point for determining the end point of nucleic acid amplification. a linear relationship with a good correlation coefficient (r 2 = 0.9605) between measured values of turbidity and the initial concentration of dna template is shown in fig. 4b . the hbv lamp reaction is also performed in the thermalcycler at 65 • c and then the amplified products are analyzed by electrophoretic analysis to confirm the consistency of the experimental results between our new system and the traditional system (fig. 5) . in our previous study, we demonstrated that our hbv lamp reaction has great specificity and sensitivity (50 copies/25 l) [20] . to confirm the results of the lamp reaction for hbv dna template amplification and detection in the integrated isothermal device, seven clinical serum specimens were obtained from patients with chronic hepatitis b at national taiwan university hospital. all of the serum specimens have been quantified by realtime pcr analysis as well. table 1 shows the quantitative results of turbidity measurements when using the integrated isothermal device in the lamp reagents containing the different amounts of dna template from the clinical specimens. following the results of the reveal-hbv study [2] , the serum level of hbv dna ( 10,000 copies/ml) is a strong risk predicator of hepatocellular carcinoma or cirrhosis. our quantitative results are good indicators for distinguishing the reported hbv dna threshold level in serum in 1 h. in our previous study [20] , we have demonstrated that the hbv lamp reaction can amplify specific dna sequences in less than 1 h with high specificity and sensitivity (50 copies/25 l). unlike traditional pcr results, lamp products consist of several inverted-repeat structures. after agarose gel electrophoresis, the positive lamp reaction reveals many bands of different sizes. the sequences of lamp products were confirmed by abi 3100-avant dna sequencer. moreover, in a comparison of the lamp reaction with pcr, the most significant advantage of the lamp reaction is its ability to amplify specific dna sequences under isothermal conditions (65 • c) without thermo-cycling. it thus has great potential for the development of an integrated isothermal device with disposable micro-reactor for low-cost applications. following the principle of lamp reaction, both its yield of dna and magnesium pyrophosphate precipitate are greater than pcr reaction [19] . in our system, an oven-like space has been designed and implemented to provide appropriate temperature conditions. an optical detection unit with a green led light source is used to detect the lamp reaction by turbidity derived from the mag-nesium pyrophosphate formation. in general, turbidity refers to the scattering of light by particles. usually, turbidity is not measured directly, but it is estimated from the optical density (od). it is assumed that the intensity of the input light is constant and that the samples have no absorbing constituents (dna absorption peak: 260 nm) and that there are no changes in other loss mechanisms for the transmission of light, such as surface coatings, alignment, etc. then the changes in detected light intensity can be ascribed to changes in total scattering. thus, we interpret changes in detected light intensity as having resulted from changes in turbidity. after the integrated isothermal device is constructed, the performance of the base apparatus should be confirmed. first, we check the efficiency of the heating and optical detection unit. we have adapted a simulated chemical reaction to mimic the by-product (magnesium pyrophosphate) production without using expensive polymerase and primers. then, the results of the turbidity measurement from using a spectrometer are compared with the base apparatus. the reaction curves also have a similar trend between turbidity and initial amounts of potassium pyrophosphate. it shows that our system can provide appropriate reaction conditions for dna amplification and detection. second, we demonstrate that the results of within runs exhibit greater reproducibility of turbidity detection through the use of this base apparatus. this shows that our system has good stability. however, the results of between runs from our system show greater fluctuations than the results from the spectrometer, because of the disposable micro-reactor being a home-made product. in the future, we can use plastic molding for mass production to improve the quality and consistency of the micro-reactor and develop multi-channel micro-reactor type to achieve large-scale quantification test. the stability of light source and the precision of light-alignment are also important for the improvement of overall system performance. after the performance of micro-reactor system is confirmed, hbv lamp reactions can be transplanted into this system. the reaction curves decrease rapidly and achieve the lowest level of turbidity in 2 min. then, the reaction curves increase quickly and achieve the highest level of turbidity in 8 min. we think that this phenomenon is related to the principle of brownian motion [23] . when the reaction mixture is initially raised from room temperature to 65 • c, some microscopic clusters or particles may dissolve, causing an increase in the intensity of transmitted light. in 30 min, the turbidity of hbv lamp reaction obviously changes. the standard curve shows that the numeric of the turbidity are highly correlated to the initial amounts of hbv template dna (r 2 = 0.9605). after 30 min, gravity causes the numeric of turbidity to continually decrease because of the magnesium pyrophosphate precipitation. therefore, the numeric of turbidity in 30 min could be a critical point for judging the performance of the hbv lamp reaction. in addition, the hbv lamp reaction is also performed in the thermalcycler. the amplified products can be analyzed by electrophoresis to confirm the consistency of the lamp reaction between our device and thermalcycler. in fig. 5 , we show that the electrophoretic result of the hbv lamp reaction is performed in the thermalcycler (lane 1) or in our device (lane 2). it seems that there are many small fragments (<200 bp) of hbv lamp reaction as shown in lane 2. we presume that this phenomenon is related to the slow heat transfer in our device, which causes bst dna polymerase cannot elongate the lamp product under stable condition, initially. the reveal-hbv study indicates that serum level of hbv dna ( 10,000 copies/ml) is a strong risk predicator of hepatocellular carcinoma or cirrhosis [2] . seven serum specimens with chronic hepatitis b were collected from national taiwan university hos-pital. these clinical specimens had triplicate tests by hbv lamp reaction with this integrated isothermal device. the hbv viral load of these seven samples ranged from an undetectable level to more than 5 × 10 7 copies/ml for the testing of our device. the frequency of positive results in triplicate test represents by fractional number. in addition, our hbv lamp reaction has good sensitivity (50 copies/25 l). even though the quantitative results from our device are higher than the data from real-time pcr, these results (table 1 ) are valid to effectively distinguish the hbv dna threshold level in the serum samples according to current protocol. thus, using the integrated isothermal device provides great assistance for early diagnosing and monitoring the progress of chronic hepatitis b. however, there are several inhibitors from the blood samples that might potentially interfere with the amplification processes require further elucidations. in this study, the serum hbv viral dna was extracted by using the qiaamp viral dna mini kit. the purified dna is free of protein, nucleases, and other contaminants and inhibitors. in 2006, a study demonstrated that the lamp assay does not require purified dna for efficient dna amplification [11] . these pcr inhibitors have little impact on the lamp reactions even though there are inhibitors (e.g., heme) in blood could severely affect the amplification of dna in pcr assays. as dna polymerase have different susceptibilities to pcr inhibitors, they suspect that the bacillus stearothermophilus (bst) dna polymerase used in the lamp reactions is more resistant. in the future, the influence of various inhibitors, e.g., elevated levels of triglycerides, bilirubin, hemoglobin, and non-specific human dna, will be addressed when we directly detect dna from serum or heat-treated blood by lamp. it would also be necessary for us to show that systemic lupus erythematosus (sle) and rheumatoid arthritis have no impact on the method during large-scale clinical experiment. we will also try to improve the sensitivity and further reduce the reaction volume to 6 l by using surface mode of optical detection [24] [25] [26] . in conclusion, we have successfully demonstrated the feasibility of the lamp reaction for hbv dna amplification and detection within 1 h in this novel integrated isothermal device. using the lamp reaction in the disposable micro-reactor component described here can amplify hbv dna with high specificity and efficiency under isothermal conditions. the base apparatus component can also provide appropriate reaction conditions as well as a steady optical detection system for detecting turbidity derived from magnesium pyrophosphate formation without fluorescence labeling. thus, using this integrated isothermal device greatly assists early diagnosing and monitoring the progress of chronic hepatitis b. it can improve the prognosis of patients and enormously save medical expenses. in the future, we hope to provide a multichannel, portable, label-free, real-time monitoring medical device for rapid identification and quantification of pathogenic organisms and point-of-care applications. epidemiology and natural history of hepatitis b, semin. liver dis predicting cirrhosis risk based on the level of circulating hepatitis b viral load correlation of hbv dna levels in serum and liver of chronic hepatitis b patients with cirrhosis micro total analysis system (micro-tas) in biotechnology enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia nucleic acid sequence-based amplification strand displacement amplification-an isothermal, in vitro dna amplification technique mutation detection and single-molecule counting using isothermal rolling-circle amplification loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. intracellulare in sputum samples development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation detection of human influenza a viruses by loop-mediated isothermal amplification development of h5-rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h5 avian influenza virus infection rapid detection of epstein-barr virus dna by loop-mediated isothermal amplification method sensitive and rapid detection of herpes simplex virus and varicella-zoster virus dna by loop-mediated isothermal amplification detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation efficient, specific, compact hepatitis b diagnostic device: optical detection of the hepatitis b virus by isothermal amplification optimal hepatitis b virus primer sequence design for isothermal amplification turbidity measurements of bacterial cultures in some available commercial instruments brownian motion, fluctuation and life admittance loci design method for multilayer surface plasmon resonance devices design and fabrication of an alternating dielectric multi-layer device for surface plasmon resonance sensor a miniaturized germanium-doped silicon dioxide-based surface plasmon resonance waveguide sensor for immunoassay detection she made a dive to 2200 m below sea level on the the study was supported by grants from national science and technology program (nsc 94-2614-b-002-001) for biotechnology and pharmaceuticals of national science council. the authors thank the national science council for its assistance in facilitating international cooperation with the university of washington, seattle, usa. key: cord-005953-5z89yeb6 authors: nan title: abstracts des 114. internistenkongresses 2008 date: 2008 journal: med klin (munich) doi: 10.1007/s00063-008-1026-y sha: doc_id: 5953 cord_uid: 5z89yeb6 nan so far, platelets were regarded static cells without further movement once they were adherent to fibrinogen or other extracellular matrices. migration of adherent platelets has not been discussed by the scientific audience so far. methods and results: 2-dimensional gel-electrophoresis of platelets revealed that fibrinogen-adherent platelets synthesize or modify multiple proteins. most of these proteins include cytoskeletal proteins and proteins of the migration apparatus. we concluded that adherent platelets might be able to migrate on the surface of an extracellular matrix or endothelium. to demonstrate platelet migration we exposed fibrinogen-coated coverslips with adherent platelets to continuous flow under high shear rates as in the arterial vascular system. all non-adherent platelets were removed from the experiment. under continuous flow we observed shear stress induced mechanotaxis of human platelets some of which started to actively migrate along the direction of flow. migrating platelets were still tightly adherent to the extracellular matrix and would have otherwise been swept away by the continuous flow. we found, that these migrating platelets underwent cytoskeletal rearrangement and formed pseudopodia. to support that our observation is an active cellular process, we blocked cellular signal transduction on pi3-kinase level with ly294002/wortmannin. after exposure to ly294002/wortmannin, we could no longer observe platelet migration under flow conditions. we subsequently examined chemokine-directed migration of platelets inlcuding sdf1, rantes and ena-78. we created a chemokine source constantly releasing chemokines and thereby generating a gradient. as for the sdf1 (stromal cell derived factor-1; 10μg/ml) source we observed that about 30% of platelets adherent around this artificial source started to move towards the sdf-1 source. platelets were actively migrating for several hours in this in-vitro setting. during migration platelets polarized and formed pseudopodia with modification of several proteins of the migratory aparatus under sdf1 exposure. when the sdf-1 source was removed, platelets migrated without a specific direction. also with blockade of the cxcr4 receptor, the specific receptor for sdf-1, directed platelet migration could no longer be observed. we are currently performing inhibitory experiments on different levels of cell signalling to characterize the migratory apparatus of platelets. the role of other cytokines associated with cell migration including rantes and ena-78 will be investigated. we apply immunofluorescence, western-blot, 2-dimensional gel electrophoresis and mass spectrometry to differentiate the migratory apparatus of migrating cells. conclusion: platelets that adhere to fibrinogen are not irreversibly fixed on matrix surface. they are able to move into a specific direction guided by shear stress induced mechanotaxis or directed along a chemokine gradient . by these mechanisms, platelets might be able to migrate into a platelet clot to further stabilize the hemostatic plug. moreover, platelet migration may be a physiological mechanism to precisely cover denuded vascular lesions and further play a role in the recruitment of platelets to the site of vascular lesions or inflammation. influence of genetic variation in four obesity candidate genes on body mass index and metabolism j. aberle 1 , p. peitsmeyer 1 , n. a. beck 1 , f. u. beil 1 1 3. medizinische klinik, hamburg objective: it is believed that is in most cases obesity derives from an obesogenic environment on the basis of a genetic predisposition. a large number of genes and genetic variations have been associated with obesity. research methods and procedures: in a cohort of 1134 caucasion men and women with a body mass index (bmi) of 25 kg/m² or more we investigated four candidate genes at baseline and following a 3 months low fat caloric restriction diet by polymerase chain restriction and restriction digestion: the 1359 g/a variant of the cannabinoid receptor 1 (cb1), the p129t polymorphism in fatty acid amide hydrolase (faah), the l7p variation in neuropeptide y (npy) and the -420c>g polymorphism in resistin. results: comparing groups according to genotype for each gene separately revealed a significantly greater reduction of body weight and bmi in carriers of at least on mutant allele in cb1 as well as a greater reduction in triglycerides in resistin mutant allele carriers. we furthermore investigated gene-to-gene interaction: adding faah mutant alleles to carriers of at least one mutant allele in cb1, or resistin significantly influenced triglycerides, total-or ldl-cholesterol levels respectively. combination of npy n-allele with either faah t-allele or cb1 a-allele as well resulted in an increase of triglyceride levels. discussion: our results support the hypothesis of obesity as a complex genetic disorder with several genes involved in the development of an obese phenotype and obesity related comorbidities. rt-pcr, die konzentrationen sezernierter proteine mittels immunoassay quantifiziert. ergebnisse: die adiponektinstimulation von rasf resultierte in veränderungen der genexpression sowohl auf mrna-als auch proteinebene. unter diesen regulierten genen waren proinflammatorische zytokine ( schlussfolgerung: adiponektin nimmt einfluss auf rasf, indem u.a. die expression und sekretion von proinflammatorischen und prodestruktiven/matrixabbauenden mediatoren modifiziert werden. diese erkenntnisse könnten dabei helfen, die pathophysiologischen mechanismen in der ra besser zu verstehen und potentielle zielmoleküle zur therapeutischen intervention zu identifizieren. in dieser globalen studie wurde der nutzen einer verlängerten thromboseprophylaxe nach totalem hüftgelenkersatz untersucht. es wurde die kurzzeit-thromboseprophylaxe mit enoxaparin mit einer verlängerten thromboseprophylaxe von bis zu 5 wochen mit einem neuen, oralen, direkten faktor-xa. bei studienbeginn und nach 5 tagen wurde die wundfläche vermessen und eine 3 mm große biopsie aus der wunde entnommen, aus der mit hilfe der real-time pcr (taqman ® ) die mmp-mrna bestimmt wurde. die wundflüssigkeit wurde mit hilfe der gaze täglich gesammelt und mittels zymographie aufgearbeitet. ergebnisse: die expression von mmp-9mrna zeigte keine veränderungen in beiden gruppen. die mmp-2 und -9-konzentrationen in der wundflüssigkeit unterschieden sich nicht signifikant in beiden gruppen zu beginn der studie. allerdings wurde eine signifikante reduktion der aktiven form von mmp-2 zwischen dem 1. und 5. behandlungstag in der pi-gruppe verzeichnet (p=0,043). die ergebnisse von mmp-2pro zeigten in der therapiegruppe ebenfalls eine sinkende tendenz (25% reduktion zu 27% anstieg in der kontrollgruppe) erreichten aber nicht das signifikanzniveau. außerdem zeigte sich eine reduktion der wundgröße in der pi-gruppe von 24% im vergleich zur kontrollgruppe (p=0,006). schlussfolgerung: es traten keine veränderung der mmp-mrna-expression unter pi-therapie auf. allerdings fanden wir eine signifikante reduktion der biologisch aktiven form von mmp-2 in der wundflüssigkeit. die lokale behandlung mit einem pi beeinflusst also primär nicht die expression von proteasen, sondern scheint die wundflüssigkeit so zu modulieren, dass eine geringere aktivität von mmps auf der wundoberfläche zu finden ist und durch eine geringere proteolytische aktivität eine verbesserte wundheilung eintritt. einleitung einer antiretroviralen therapie in zwei ländlichen regionen in rwanda (n=11), fibrose in mehreren regionen (n=9). nur die gruppe, die baseline keine fibrose aufwies zeigte unter dreijähriger ert eine normalisierung der wanddicke (von 13,1± 0,9 auf 11,8 ± 1,0 mm; p<0,032) und eine weitgehende funktionelle normalisierung der regionalen herzfunktion (strain rate radial: von 2,35 ± 0,16 s-1 auf 2,95 ± 0,39 s-1, p=0,017; vergleichskollektiv 2,7 ± 0,3 s-1). die anderen beiden gruppen zeigten zwar in hinblick auf die wandstärken einen positiven effekt, hinsichtlich der herzfunktion konnten sie jedoch bei bereits deutlich erniedrigten funktionswerten zum baseline-zeitpunkt lediglich stabilisiert werden (reduktion der wandstärke nach 3 jahren ert: gruppe wenig fibrose= 10 mm; gruppe viel fibrose= 12 mm) schlussfolgerung: die enzymersatztherapie ist eine effektive langzeitbehandlung bei patienten mit fabry kardiomyopathie. hierbei fällt insbesondere auf, dass eine rechtzeitige therapie, bevor sich eine myokardiale fibrose entwickelt hat, indiziert ist. klinikgestützte poststationäre mobile gesundheitsservices; "hospital-to home®" -im poststationären versorgungsmanagement chirurgischer patienten adult-onset still´s disease (aosd) represents a rare systemic inflammatory disorder with an estimated incidence between 1 and 2 cases per million inhabitants in western europe. due to this small incidence only few data on diagnosis and therapy are available. the etiology of aosd remains elusive. currently the hypothesis of an exacerbated immune response based on dysregulation of cytokine mediated signalling cascades is favoured among scientists. according to resent results the proinflammatoy cytokine interleukin 1 (il-1) seems to play a central role in pathogenesis of the more common juvenile as well as adult-onset still´s disease. in view of that, we report about a fast and sustained response of a 44year old woman with aosd under first-line treatment with the il-1 receptor antagonist anakinra. the fast mode of action compared to conventional dmards and tnf inhibitors paired with an acceptable safety profile had prompted us to initiate a primary il-1 receptor blocking therapy. the observed rapid clinical response (cessation of fever within 12 hours) and normalization of highly elevated acute phase laboratory parameters within a few days encourage a first line use of anakinra in aosd-patients. longer observation periods and larger series of patients will be needed to answer open questions like optimal length of anakinra-treatment, long-term outcome and complications. cardiovascular complications in patients with diabetes mellitus are associated with increased oxidative stress in vascular tissue. a key event for no synthase (nos) uncoupling involves downregulation of bh4 synthesizing enzyme gtpcyclohydrolase i (gch-i). we here investigated the effects of at1-receptor block ade with chronic telmisartan therapy on gch-i expression, nos uncoupling and endothelial function in streptozotocin (stz)-induced diabetes mellitus (type i). diabetes was induced by single i.v. injection of stz (60mg/kg, 7 weeks) in male wistar rats (250g). telmisartan (25mg/kg/d) therapy did not improve blood glucose and body weight. aorta from diabetic animals had vascular dysfunction as revealed by isometric tension studies. ros produced by nadph oxidase, mitochondria and xanthine oxidase were increased in the diabetic group. nadph oxidase subunits mrna and protein expression was increased in response to stz. importantly, the expression of the gch-i and enos activating ser1177 phosphorylation was decreased by stz. therapy with telmisartan normalized all these parameters almost completely. the results of the present studies demonstrate for the first time that at1 receptor treatment of diabetic animals with telmisartan is able to prevent downregulation of the important bh4 synthesizing enzyme gch-i, which prevents enos uncoupling and an activation of cardiovascular superoxide sources. der einfluss der sauren sphingomyelinase auf die expression von matrix-metalloproteinase-1 in intestinalen epithelzellen und fibroblasten background: the calcineurin (cn)/nf-at signaling cascade takes a crucial role during t-cell activation and the development of myocardial hypertrophy. in addition to nfat, the phosphatase cn is also translocated into the nucleus. for nuclear import, specific carrier proteins, so-called importins, recognize nuclear localization sequences (nlss) in the sequence of the respective target proteins. we developed a synthetic import blocking peptide (ibp) which is identical to the nls sequence of cn. ibp prevented the interaction between cn and its importin and subsequently inhibited nuclear import of cn. methods and results: according to immunohistochemical studies both the subcellular localization of cn, the inhibitory effect of ibp and also the specificity of the blocking peptide were demonstrated. while the inhibitory effect within the t-cell activation is analyzed by [3h]-thymidine incorporation (7135 ± 2,503 vs. 2957 ± 1161 [%]), the impact on angiotensin ii stimulated cardiomyocytes was examined on the transcriptional (nfat reporter assay; 145 ± 10 vs. 108 ± 5 [%]) and on the translational level ([3h]-leucine incorporation; 254 ± 27 vs. 88 ± 49 [%]). by using ibp, the expression of hypertrophy markers, e.g. myosin heavy chain β, was suppressed, the cell size was not increased. interestingly, phosphatase activity was not effected (cna assay, biomol). the translocation of diverse transcription factors, like nfat, nfκb or erk2, was not effected, which illustrated the specifity of ibp. the results could be confirmed in a pilot animal study for heart failure. discussion: ibp prevents the nuclear translocation of calcineurin both in t-cells and in cardiomyocytes. the inhibitory activity is specific for calcineurin und does not affect the cn phosphatase activity. ibp has also an effect in vivo, where it has been demonstrated to suppress the development of myocardial hypertrophy in an animal model. in summary, ibp suppresses t-cell activation und prevents the development of myocardial hypertrophy. proteasome inhibitors suppress angiogenesis by altering endothelial vegfr-2 expression the ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells that controls a wide range of cellular regulatory proteins, including transcription factors and cell cycle regulatory proteins. recent evidence established the importance of the proteasome also in tumor development, showing anti-tumor and anti-angiogenic actions by selective inhibitors in vivo. as signaling via the vegfr2 pathway is critical for angiogenic responses to occur, we explored whether anti-angiogenic effects via proteasome inhibition were mediated in part through diminished endothelial vegfr2 expression. our studies show that different proteasome inhibitors (mg132, alln and lactacystin) all blocked vegfr2 expression in a time-and concentration-dependent manner, which was paralleled by respective inhibition of capillary like structure formation and endothelial cell migration. in contrast, tie-2 or vegfr-1 expression was not significantly affected by proteasome inhibitor treatment. the suppressive effects on vegfr2 expression were neither conveyed by increased shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms accounted for the observed effects. in line with this conclusion, proteasome inhibition significantly suppressed vegfr2 mrna accumulation. in addition, inhibitor treatment considerably decreased transcriptional activity of 5'-deletional vegfr2 promoter gene constructs. proteasome inhibition-mediated repression was conveyed by a gc-rich region, harboring one consensus sp1 binding site. subsequent emsa analyses demonstrated diminished constitutive sp1-dependent dna binding in response to proteasome inhibition. hence, vegfr-2 expression may constitute a critical molecular target of proteasome inhibitors that mediates their anti-angiogenic effects in vivo. loss of ifn-γ inducibility of the mhc class ii antigen processing pathway in head and neck cancer: evidence for posttranscriptional as well as epigenetic regulation abnormalities of the major histocompatibility complex (mhc) antigens by tumor cells impair the cellular immune response and promote tumor evasion from immune surveillance. so far, studies analyzing the mhc class ii expression levels in hnc are limited. therefore, we investigated the constitutive and interferon (ifn)-γ-regulated expression profiles of mhc class ii apm in various hnc cell lines and also analyzed the mhc class ii expression in hnc lesions. hnc cell lines analyzed in vitro lacked constitutive mhc class ii surface expression. despite the ifn-γ-mediated induction at the mrna level, six out of ten cell lines did not show any relevant mhc class ii surface expression. this phenomenon might be attributed to a posttranscriptional dysregulation of specific mhc class ii apm components. one cell line displayed a loss of ifn-γ induced ciita-expression that corresponded to impaired mhc class ii surface expression, which could be linked to hypermethylation of the ifn-γ-responsive ciita-promoter iv. in vivo, immunohistochemistry analyses of 35 patients revealed that about 86% of hnc tissues exhibit a negative or only marginally positive staining, whereas 14% displayed a heterogenous or highly positive mhc class ii surface expression. there was no statistical correlation between tumor differentiation and the mhc class ii expression in hnc lesions. taken together these results suggest a high frequency of mhc class ii abnormalities in hnc in vitro and in vivo, which could occur at different steps of the antigen processing pathway. this information may have a significant impact on practical and clinical aspects of tumor immunotherapeutic strategies. ciita versus ifn-γ induced mhc class ii expression in head and neck cancer cells effective and safe reduction of blood pressue by the combination of amlodipine 5/valsartan 160 mg in patients with hypertension and metabolic risk factors not controlled by amlodipine 5 mg or felodipine 5 mga subanalysis of the express-m trial introduction: atrial fibrillation (af) is the most common cardiac arrhythmia and frequently occurs in patients with coronary heart disease. prophylactic oral anticoagulation (oac) is indicated in patients with af and at intermediate or high risk for thrombembolic events. however, an anticoagulant regimen has not been standardized for patients with af after coronary stent implantation, which may require additional dual antiplatelet therapy. methods: we identified retrospectively 159 patients with af who underwent pci in our department from 1999-2004. we compared baseline variables and incidence of a combined endpoint (stroke, mi, death, bleeding) in patients receiving clopidogrel and aspirin (n=103, group 1), versus patients receiving the combination of clopidogrel and aspirin with low molecular weight heparin (lmwh) (n=42, group 2), versus patients receiving the combination of clopidogrel and aspirin with oac (n=14, group 3) at discharge. results: the groups were not relevantly different regarding classical risk factors, however, patients discharged with triple therapy including oac seemed to be at (30) higher risk: patients in group 3 had decreased left ventricular ejection fraction and increased inflammatory state as measured by plasma fibrinogen. in a median follow up of 1,4 years (range from 2 months to 5.7 years) 2 severe bleeding events, 4 myocardial infarctions, 13 strokes, and 9 cardiovascular deaths occurred. kaplan meyer analyses showed that the occurrence of severe events was not significantly different (p=0.433). conclusion: in our analyses no treatment regimen seemed to be superior. however, in the follow-up period incidence of myocardial infarctions and strokes were high in patients with solely clopidogrel and aspirin treatment, and incidence of cardiovascular deaths was high in patients receiving the combination of clopidogrel and aspirin with lmwh. the number of patients receiving the combination of clopidogrel and aspirin with oac (triple therapy) was low, however, only one severe event was found in the follow-up period in this group. the current data underline the importance for prospective trials investigating the optimal anticoagulant-/antiplatelet treatment in patients with af after coronary stent implantation. valvuloplastie der kalzifizierten aortenstenose: neue erfolge durch weiterentwicklung der herkömmlichen methode background: we have shown that assessment of cardiac output (co) by the inert gas rebreathing method provides reliable measurements as compared to magnetic resonance tomography (mrt) in patients with sinus rhythm. during atrial fibrillation, however, co measurement based on the determination of stroke volume may be impaired by heart rate variation, potentially leading to divergent results. the present study therefore aimed to compare co measurements by inert gas rebreathing to mrt in patients with atrial fibrillation. methods: in a prospective case-control study, we included 30 consecutive patients with atrial fibrillation undergoing cardiac mrt, and 30 control patients pair-matched for gender, co by mrt, height and weight from a collective of 250 patients with sinus rhythm undergoing mrt. the co of reclining patients was determined by inert gas rebreathing with dinitric oxide and sulfur hexafluoride (innocor, innovision, odense dk) immediately before or after the mrt. the data determined by mrt for co served as reference value comparing the methods by means of bland-altman analysis. the study collective covered a wide range of patients (18 to 79 years, median 67, 21 men in the atrial fibrillation group vs. 23 to 78, median 66 in the sinus rhythm group). bland-altman analysis showed a good correspondence of the two methods for co with an average deviation of 0.2±1.2 l/min in the atrial fibrillation group vs. 0.3±1.1 l/min in the sinus rhythm group. no statistically significant difference could be found between the two groups (p = 0.48, mann-whitney-test). conclusion: co measurements by mrt and by the rebreathing method with dinitric oxide and sulfur hexafluoride provide closely related results even in the presence of atrial fibrillation. the rebreathing method therefore allows an easy and reliable non-invasive determination of the co in this patient collective. the importance of the method for diagnosing and treating cardiac diseases remains to be assessed by further studies. when analyzing the plasma lipid profile for each polymorphism, we found the following differences in subjects with gallstones: significantly higher triglyceride and cholesterol levels in carriers of the common abcb11 allele and significantly lower hdl-cholesterol concentrations in carriers of the common rs31653 allele. however, differences were also detected in controls with regard to cholesterol and total hdl-cholesterol concentrations. our results do not support a link between common abcb4 and abcb11 polymorphisms, lithogenic dyslipidemia and gallstone risk. evaluation der prävalenz schlafbezogener atemstörungen bei patienten mit vorhofflimmern und unauffälliger linksventrikulärer pumpfunktion mittels kardiorespiratorischer polygraphie hintergrund: in erregbaren herzzellen sind spannungsgesteuerte natriumkanäle für die auslösung und weiterleitung von aktionspotenzialen verantwortlich. natriumkanäle bestehen aus einer die pore bildenden α-untereinheit und zusätzlichen β-untereinheiten. verschiedene isoformen der α-untereinheiten haben ganz bestimmte entwicklungs-und lokalisationsmuster im herzmuskel, und sie besitzen spezifische pharmakologische und funktionelle eigenschaften. das kugelfischgift, tetrodotoxin (ttx), ein spezifischer natriumkanalblocker, inhibiert konzentrationsabhängig verschiedene α-isoformen. die kardiale isoform nav1.5 wird durch mikromolekulare konzentrationen blockiert (ttxresistent), während neuronale ttx-sensitive isoformen bereits durch konzentrationen im nanomolaren bereich inhibiert werden. die α-untereinheiten sind mit zusätzlichen β-untereinheiten assoziiert. methoden und resultate: humanes vorhofmyokard wurde mittels der patchclamp-technik, kontraktilitätsmessungen, immunhistochemie und western blot untersucht. wir konnten zeigen, dass nicht nur kardiale ttx-resistente (ttxr) natriumkanäle am menschlichen herzen vorkommen, sondern ebenfalls neuronale ttx-sensitive (ttxs) isoformen exprimiert werden. ttxs-natriumkanäle tragen zu 15% zum gesamtnatriumstrom bei, während die restlichen 85% des stroms von ttxr-kanälen übernommen werden. funktionelle einschränkungen durch die blockade von neuronalen natriumkanälen konnten in kontraktilitätsmessungen ebenfalls nachgewiesen werden. immunhistochemische experimente bestätigten isoformenspezifische lokalisation der α-untereinheiten nav1.1 bis 1.6 sowie aller bekannten β-untereinheiten (1-4). schlussfolgerung: im humanen herzen kommen neben kardialen auch neuronale natriumkanäle vor. sie tragen funktionell 15% zum gesamtnatriumstrom im menschlichen herzen bei und haben ebenfalls einen einfluss auf die kontraktilität. p44/p42-mapk-aktivierung in hypokontraktilen gefäßen zirrhotischer ratten: geänderte mechanismen der aktivierung jedoch gleichzeitig die g-protein abhängige aktivierung der p44/p42 mitogenactivated protein kinase (mapk). die g-protein abhängige p44/p42 aktivierung durch den angiotensin-ii typ1 rezeptor (at1r) erfolgt dagegen über protein-kinase c. methoden: leberzirrhose bei ratten wurde durch gallengangsligatur (bdl) induziert. isolierte aorten wurden in vitro mit bzw. ohne angiotensin-ii und verschiedenen inhibitoren inkubiert. protein-expressionen und -phosphorylierungen wurden über western-blot analyse untersucht. die interaktion zwischen dem at1r und b-arrestin 2 wurde in co-immunopräzipitations-experimenten untersucht. ergebnisse: trotz ausbleibender angiotensin-ii induzierter aktivierung der g-protein abhängigen kontraktilen signalwege in aorten von bdl ratten kommt es in aorten zirrhotischer und nicht-zirrhotischer ratten zu einer angiotensin-ii stimulierten aktivierung der p44/p42 mapk. diese angiotensin-ii stimulierte erk-aktivierung ist in aorten von bdl ratten, nicht jedoch der nicht-zirrhotischen kontrollen, resistent gegenüber hemmung der protein-kinase c und a. gleichzeitig führte die stimulation mit angiotensin-ii zur bindung von b-arrestin 2 an den at1r, die bei bdl ratten verstärkt ist. diskussion: bei leberzirrhose kommt es in extrahepatischen gefäßen zu änderungen in der kopplung von vasokonstriktor-rezeptoren an ihre signalwege. anstatt g-protein abhängiger signalwege unter normalen umständen (z.b. kontraktions-kaskaden) werden bei leberzirrhose b-arrestin 2-abhängige signalwege aktiviert. the prediabetic and diabetic in vivo modification of circulating low density lipoproteins decreases their potential to stimulate adrenal steroidogenesis context: biochemical modification of low density lipoprotein (ldl) has been implicated in the pathogenesis of impaired glucose tolerance (igt) and type 2 diabetes mellitus (dm2) and its related micro-and macrovascular disease. objective: since ldl serves as a major cholesterol source for adrenal steroidogenesis we investigated whether oxidative and glycoxidative modifications of ldl isolated from igt and dm2 subjects influence aldosterone and cortisol release from human adrenocortical cells. in a cross-sectional study ldl were isolated from 30 subjects with normal glucose tolerance (ngt), 30 subjects with igt, and 26 patients with dm2. individual oxidation and glycoxidation characteristics of ldl apolipoprotein b-100 were assessed by gas chromatography-mass spectrometry (gc-ms) analysis. human adrenocortical cells (nci-h295r) were incubated for 24h with 100 μg/ml of isolated ldl and after removal of supernatants the cells were further stimulated for next 24h with angiotensin ii. aldosterone and cortisol release were then quantified in the supernatants after 24h and 48h, respectively. results: circulating ldl from igt and dm2 subjects were substantially more oxidized and glycoxidized than the ldl samples from ngt subjects. each of the five measured oxidation/glycoxidation markers was significantly positively associated with glycemic control, measured as hba1c. ldl stimulated aldosterone and cortisol release from adrenocortical cells. however, hormone secretion was inversely related to the degree of ldl modification. conclusion: prediabetic and diabetic biochemical ldl modifications may promote impaired adrenocortical aldosterone and cortisol synthesis. die modulation der interaktion von nephrin und β-arrestin2 durch protein-kinase c alpha steuert die nephrin-vermittelte endozytose und signaltransduktion zielsetzung: hepatische sternzellen (hscs) sind die primären fibrogenen zellen in der fibrotischen leber. selektive apoptose von hscs stellt einen neuen antifibrotischen therapieansatz dar. wir haben kürzlich gezeigt, dass das endocannabinoid 2-arachidonoylglycerol (2-ag) die proliferation von hscs blockieren sowie apoptose induzieren kann. hepatozyten sind jedoch gegenüber diesen mechanismen resistent. der exakte molekulare mechanismus dieser selektiven zelltod-induktion in hscs ist noch nicht geklärt. ziel dieser arbeit war es, eine mögliche rolle von cox-2 im differentiellen zelltod-verhalten primärer hscs und hepatozyten durch 2-ag-metabolisierung in pro-apoptotischen prostaglandin-d2-glycerolester (pgd2-ge) zu überprüfen. methoden: primäre hepatozyten und hscs wurden durch collagenaseperfusion aus lebern gesunder ratten isoliert. 2-ag-oder pgd2-ge-induzierte apoptose wurde mittels ldh-assay und western blot für caspase 3-und parp-cleavage analysiert. bildung von reaktiven sauerstoffspezies (ros) wurde durch dcfda-fluoreszenz ermittelt. cox-2-expression wurde mittels western blot bestimmt. ergebnisse: hscs zeigten eine signifikante cox-2-proteinexpression, wohingegen hepatocyten keine wesentliche cox-2-expression aufwiesen. behandlung von primären in kultur aktivierten hscs mit 2-ag induzierte einen dosisabhängigen zelltod (70% nach 24h bei 10μm), begleitet von parp-und caspase 3-cleavage. pgd2-ge induzierte in primären hscs ebenfalls dosisabhän-gig apoptose. dagegen verursachte 2-ag in primären hepatozyten sogar bis zu konzentrationen von 100μm keinen zelltod, pgd2-ge jedoch führte ab konzentrationen von 25μm zum zelltod. 2-ag und pgd2-ge führten zur bildung von ros in hscs, was auf gemeinsame ros-abhängige apoptose-signalwege hinweist. schlussfolgerung: hscs und hepatozyten weisen einen signifikanten unterschied in der expression von cox-2 auf, was eine unterschiedliche metabolisierung des endocannabinoids 2-ag bedingt. die produktion von pgd2-ge durch cox-2 in hscs trägt möglicherweise zur disparaten empfindlichkeit von hepatozyten und hscs gegenüber 2-ag-induzierter apoptose bei. die rolle des hypoxie-induzierbaren-faktors 1a in der strahlentherapieresistenz von humanen adenokarzinomzellen der lunge (a549) zielsetzung: hypoxie hat einen entscheidenden einfluss auf die chemo-und strahlentherapieresistenz von soliden tumoren und somit auf die prognose von tumorerkrankungen. die hypoxie-induzierbaren-faktoren (hif-1a und hif-2a) sind transkriptionsfaktoren, die über spezifische zielgenaktivierung adaptive prozesse in den hypoxischen tumorarealen steuern. in dieser studie wurde die strahlensensibilität der adenokarzinomzelllinie der lunge (a549) unter dem einfluss von hif-1 und hif-2 untersucht. methoden: im ersten schritt wurde die auswirkung von bestrahlung 6 gy (1x, 4x, 10x) auf die a549 zellen untersucht (zellzählung, kolonie-assay, immunzytofluoreszenz: caspase-3, kerngrößen, multinuklei). unter normoxischen und hypoxischen bedingungen wurde die hif-1a und hif-2a expression mittels western blot und pcr mit und ohne radiatio untersucht. die strahlentherapieresistenz unter inhibition von hif (sirna) wurde in vitro (kolonie-assay, zellzählung) und in vivo (tumor growth delay assay) untersucht. ergebnisse: bestrahlung mit 6 gy führte zu einer reduktion der proliferation von a549 zellen ohne beeinflussung der apoptose. gleichzeitig änderte sich die kernmorphologie mit einem anstieg der kerngröße und vermehrten multinuklei unter fraktionierter bestrahlung. dies konnte auf einen anstieg der "seneszenten" zellen zurückgeführt werden. ein direkter einfluss einmaliger radiatio auf die expression von hif-1 oder hif-2 wurde nicht beobachtet. allerdings konnte über die inhibition von hif-1a mittels sirna eine erhöhung der strahlensensibilität in den a549 zellen in vitro erreicht werden. dies wurde in vivo mittels tumor growth delay assay bestätigt. schlussfolgerung: die aktivierung von hif-1a in hypoxischen tumorarealen hat einen entscheidenden einfluss auf die sensibilität von a549 zellen gegenüber bestrahlung. conditional overexpression of neuronal nitric oxide synthase is cardioprotective in ischemia-reperfusion introduction: we previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nnos, nos1) inhibited l-type ca 2+ -channels. we now hypothesize that nnos overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion. we assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nnos overexpression. nos-activity (22 ± 1.5 vs. 29 ± 1μm/sec, n=18, p<0.05) was significantly enhanced after nnos overexpression. co-immunoprecipitation experiments indicated interaction of nnos with sr ca 2+ atpase and additionally with l-type ca 2+ -channels in nnos overexpressing animals. ica,l (reduction of 40±6 rel.%, n=12, p<0.05) as well as intracellular ca 2+ -transients and fractional shortening in cardiomyocytes were clearly impaired in nnos overexpressing mice (3.0 ± 0.4f/f0 vs. 2.2 ± 0.2f/f0, n=13, p<0.005 and 7.7 ± 1.3% vs. 3.8 ± 0.5%, n=13, p<0.05). in vivo examinations of the nnos overexpressing mice showed a decrease of +dp/dtmax (reduction for 52 ± 17%, n=12, p<0.05) as well as a reduced ejection fraction (43±5% vs. 63±9%, n=12, p<0.05). ischemia-reperfusion experiments showed a cardioprotective effect of nnos overexpression (30 min post-ischemia, lvdp 20±6 in non-induced animals vs. 60±11 mmhg in nnos overexpressing animals, n=6, p<0.05). discussion: in summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nnos resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the l-type ca 2+ -channel. in contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nnos overexpression acts cardioprotective. these effects might be caused by a reduction of myocardial ca 2+ -overload after reperfusion. hintergrund und ziele: die pathogenese der erhöhten apoptose in der hepatitis c virus (hcv)-infizierten leber ist weitestgehend unbekannt. für zahlreiche hcv proteine ist eine modulation von zellulären apoptosemechanismen beschrieben worden. trail, der tnf-related apoptosis-inducing ligand, wurde kürzlich als zytotoxisch für hcv-infizierte hepatozyten beschrieben. um die zugrunde liegenden molekularen mechanismen einer potentiellen rolle von trail bei der hcv-induzierten hepatitis und der viralen clearance zu studieren, untersuchten wir die effekte der hcv replikation auf die trail-induzierte apoptose in humanen hepatomazellen. methoden: huh7.5 zellen wurden mit dem in zellkultur infektiösen hcv stamm jfh-1 sowie den mutanten jfh-1/δe1e2, jfh-1/gnd und sgr-jfh-1 hcv rna transfiziert bzw. infiziert. apoptose wurde durch bestimmung von parp, caspase-8 und -9 spaltprodukten sowie mittels tunel-reaktion untersucht. ergebnisse: transfektion von huh7.5 zellen mit replikations-kompetenter jfh-1, nicht jedoch mit replikations-defizienter jfh-1/gnd rna, führte zu einer apoptose von huh7.5 zellen, nachgewiesen mittels parp spaltung und positiver tunel reaktion. die verstärkte trail apoptose war in mit jfh-1/δe1e2 und sgr-jfh-1 subgenomischer hcv rna transfizierten huh7.5 zellen ähnlich stark wie beim vollständigen jfh-1 virus, was auf eine unabhängigkeit der apoptose-sensibilisierung von strukturproteinen sowie die notwendigkeit der viralen replikation hindeutet. untersuchungen zur signaltransduktion der jfh-1-bedingten apoptose-sensibilisierung zeigen eine deutliche abhängigkeit von caspase-9 aktivierung sowie eine jfh-1-induzierte suppression von bcl-2 und bcl-xl. dies deutet auf eine zentrale bedeutung des mitochondrialen signaltransduktionsweges bei der jfh-1-induzierten apoptose hin. schlussfolgerung: unsere daten zeigen eine apoptosesensibilisierung von huh7.5 hepatomazellen durch hcv replikation, welche unabhängig von hcv strukturproteinen und wahrscheinlich mitochondrial beding ist. diese ergebnisse geben wichtige hinweise für die pathogenese der hepatitis c und die mechanismen der viralen clearance. bier, jedoch nicht ethanol stimuliert in vitro die enzymsekretion der pankreasazinuszelllinie ar4-2j und frisch isolierter pankreasazinuszellen der ratte a. gerloff 1 , m. v. singer 1 , p. feick 1 1 ii. medizinische klinik, universitätsklinikum mannheim, mannheim hintergrund: da der pankreasazinuszelle in der frühen phase der entwicklung der alkoholischen pankreatitis eine wichtige rolle zugesprochen wird, wurde die wirkung von alkohol (ethanol) auf die funktion dieser zelle bereits seit jahren (44) intensiv untersucht. allerdings wird alkohol meist in form von schmackhaften getränken konsumiert, die viele nichtalkoholische inhaltsstoffe enthalten, welche ebenfalls einen pathophysiologischen einfluss auf die funktion des pankreas haben können. ziel: vergleich der wirkung von bier und adäquaten ethanollösungen auf die proteinsekretion und signaltransduktion der pankreasazinuszelllinie ar4-2j (ratte) und frisch isolierter ratten-azinuszellen. methoden: ar4-2j-zellen wurden für 72h mit dexamethason differenziert und frisch isolierte azinuszellen durch collagenase-verdau des pankreas von spraque-dawley-ratten gewonnen. nach inkubation der zellen für 60 min. wurde die amylasefreisetzung als maß der proteinsekretion mit hilfe eines kommerziellen testkits bestimmt. ergebnisse: die inkubation von ar4-2j-zellen mit bier (1-10% (v/v)) bewirkte eine dosisabhängige stimulation der basalen amylasesekretion. reiner ethanol in vergleichbarer konzentration wie im bier hatte keinen effekt auf die amylasefreisetzung. die bestimmung von ldh nach 24h inkubation der ar4-2j-zellen zeigte, dass die bierinduzierte amylasefreisetzung nicht auf einer membranschädigung beruhte. die verwendung selektiver inhibitoren und des ca-indikators fura-2/am ergab, dass die bierinduzierte amylasesekretion vorwiegend durch die aktivierung von phospholipase c und der anschließenden kalzium-freisetzung aus intrazellulären speichern vermittelt wird. der stimulatorische effekt von bier auf die proteinsekretion war in frisch isolierten azinuszellen reproduzierbar. schlussfolgerung: unsere daten zeigen, dass der effekt von bier auf die sekretion von pankreasazinuszellen auf nichtalkoholische inhaltsstoffe zurückzuführen ist. diese inhaltsstoffe müssen bei der untersuchung von alkoholinduzierten pathologischen und funktionellen veränderungen berücksichtigt werden. pharmakokinetik und pharmakodynamik einer fckw-freien, fixen kombination aus beclometason-dipropionat und formoterol (-1,46, 0,59 und -2,45 mm hg) erreichte keine statistische signifikanz. bei den mit liraglutid behandelten patienten zeigte sich im vergleich zu placebo eine senkung der triglyceride (adjustiert) von -19% (0,65 mg, p=0,03), -15% (1,25 mg, p=0,09) und -22% (1,90 mg, p=0,01) . es gab keine klinisch relevanten und konsistenten unterschiede bezüglich der gesamtcholesterin-, hdl-, ldl-, apob-, il-6-, tnf-α-, leptin-und adiponectin-konzentrationen zwischen den liraglutid-behandlungsgruppen und placebo, eine signifikante reduktion des ldl-cholesterins wurde unter placebo im vergleich zu den aktiven behandlungs-gruppen beobachtet, das galt aber nicht für apob, bei dem lediglich in der 1,25 mg behandlungsgruppe ein unterschied zu verzeichnen war. es konnte ein ausgeprägter effekt von liraglutid auf die pai-1-konzentrationen mit reduktionen von -14% (0,65 mg, p=0,29), -29% (1,25 mg, p=0,02) bzw. -25% (1,90 mg, p=0,05) gegenüber placebo beobachtet werden. das galt auch für die bnp-konzentrationen mit dosisabhängigen senkungen von -26% (p=0,1), -30% (p=0,05) und -38% (p=0,01) gegenüber placebo für die entsprechenden liraglutid-behandlungsgruppen. die beobachteten dosisabhängigen senkungen des crp gegenüber placebo (-3%, -12% und -20%) erreichten keine statistische signifikanz. schlussfolgerung: die behandlung mit liraglutid war mit einer blutdrucksenkung und einer abnahme der triglycerid-, pai-1-und bnp-konzentrationen verbunden, die beobachteten effekte müssen in langzeitstudien bestätigt werden und bedürfen weiterer untersuchungen. objectives: the majority of treated hypertensive patients do not achieve target blood pressure levels <140/90 mmhg. one key reason is inadequate adherence with the prescribed drug regimen. dosing regimens are either not executed as prescribed (non compliance) or patients stop taking the medication (non persistence). it has been demonstrated that drug adherence with angiotensin receptor antagonists like valsartan is superior in comparison to other drug classes. the present study was designed to evaluate whether drug adherence could be further improved by the use of supportive tools. design and methods: 28 centers were randomized to provide pharmacological treatment with or without a set of supportive measures (e.g. structured physicianpatient interaction, printed information about hypertension, reminder stickers, pill box with alarm, home blood pressure measurement). 202 newly diagnosed patients or patients with stage 1 hypertension (blood pressure at baseline 149.8±6.2/93.9±4.4 mmhg) who had not been treated for at least 1 year were included in this trial. all patients entered the 34-week treatment phase with valsartan 160 mg. titration to valsartan 160 mg/hctz 12.5 mg was allowed if necessary. drug adherence was assessed by electronic monitors (medication event monitoring system -mems). results: patients treated with a valsartan-based therapy in combination with supportive measures demonstrated lower rates of treatment discontinuation. per-sistence at the end of the 8 months observation period was 88% in the control patients and 96% in patients receiving supportive measures. in addition, better execution of the dosing regimen (compliance) could be observed in patients receiving supportive measures but this effect tended to fade with time. bp control improved more in patients with the supportive measures. conclusions: drug adherence can be improved with the use of supportive measures. in this study, while the effect on compliance decreased over time, the effect of chosen supportive measures on persistence seems to be long lasting. randomized, double blind parallel group study to evaluate the reduction of the urinary albumin/ creatinine ratio by valsartan plus lisinopril versus lisinopril or valsartan alone in hypertensive patients with microalbuminuria -the valeria trial objectives: microalbuminuria is known as an independent predictor for stroke, myocardial infarction and death and has a higher prevalence in hypertensive subjects than in the general population. intensified inhibition of the renin-angiotensin-aldosterone system seems to be an efficient treatment option and might be achieved by dual blockage with arbs and aceis. it was the aim of the study to compare the efficiency and safety of a combination therapy comprising valsartan and lisinopril with valsartan and lisinopril monotherapy in patients with hypertension and microalbuminuria. methods: this was a randomized, double blind parallel group study. patients with hypertension (mean sitting diastolic blood pressure = 85 mmhg and < 110 mmhg) and microalbumiuria (urinary albumin/creatinine ratio (uacr) = 2.5 mg/mmol and = 25.0 mg/mmol for men and uacr = 3.5 mg/mmol and = 35.0 mg/mmol for women in the first morning urine sample on at least two of three visits) were eligible for participation. after a wash-out/placebo-run-in phase of max. 3 weeks, patient were randomized to treatment (1:1:1) with either lisinopril 10-40 mg (lis), valsartan 80-320 mg (val) or the combination of valsartan/linisopril 80/10-320/20 mg (com) for 30 weeks. results: median uacr at baseline in the com, val, and lis group was 11.6 mg/mmol, 7.5 mg/mmol, and 8.8 mg/mmol, respectively. at baseline, systolic/ diastolic bp for com, val, and lis was 150.4/90.1 mmhg, 153.1/91.9 mmhg, and 153.0/90.6 mmhg. treatment with com, val, and lis resulted in an uacr reduction of 64%, 53%, and 41% (median) after 30 weeks of treatment. the reduction achieved with com was significantly greater than with lis (p=0.036). normalization of microalbuminuria (uacr < 2.5 mg/mmol for men and < 3.5 mg for women) with com, val, and lis was achieved in 38%, 31% and 17% of the patients (p=0.0344 for com vs. lis). blood pressure differences between the groups were not statistically significant. all treatments were well tolerated. conclusion: in patients with hypertension and microalbuminuria the combination of valsartan and lisinopril provided a significantly better reduction of uacr and doubled the rate of patients with normalized uacr compared to lisinopril alone. analyse der mikrozirkulation der oberen extremitäten unter ldl-apherese the baseline ppg of the rebleeder (group b) was 24.43 +-2.63 mmhg before tips and 12,57 +-2.82 mmhg after tips insertion, giving an overall ppg reduction of 48,52%. 28 patients who underwent tips for variceal bleeding never had a bleeding episode again after tips (group a) . the baseline ppg of these patients was 21.8 +-3.79 mmhg before tips and 10,0 +-2,71 mmhg after tips, giving an overall ppg reduction of 54,15% statistical analysis showed that the ppg difference between 12,57 mmhg in rebleeder (group b) and 10mmhg in non-rebleeder (group a) was statistically significant (p-value < 0.05). patients with refractory ascites never suffered from an episode of bleeding after tips independently from their initial ppg reduction. conclusion: patients who underwent tips for recurrent variceal bleeding do have a significant higher risk for rebleeding with an initial ppg reduction to 12,57 mmhg whereas those patients in which the ppg reduction to 10 mmhg can be achieved a rebleeding episode is unlikely. we conclude that a ppg reduction to at least 10mmhg should be aimed to prevent further bleeding episodes. these findings differ from previous reports in which ppg of less than 12 mmhg is thought to prevent rebleeding. ergebnisse: tgr männchen jedoch nicht tgr-weibchen entwickeln eine albuminurie (tgr-c: 129 mg/24h) und glomerulosklerose, die durch flutamid zu >90% unterdrückt wird (tgr-fl: 6 mg/24h). testosterongabe induziert in tgr-weibchen jedoch nicht in wt-weibchen albuminurie (tgr-ovt: 87 mg/24h, wt-ovt: 1.7 mg/24h) und glomerulosklerose (schadensindex-tgr-ovt: 152, wt-ovt: 5,3). ovariektomie induziert keine albuminurie. testosteron stimuliert in tgr-und wt-weibchen das renale wachstum und die glomeruläre angiotensinogenexpression, das glomeruläre wachstum wird jedoch nur in tgr-weibchen erhöht. androgenrezeptoren werden im glomerulus geschlechtsunabhängig exprimiert. schlussfolgerung: at1 rezeptorüberexpression in podozyten transgener ratten induziert albuminurie und glomerulosklerose. testosteron ist ein entscheidender kofaktor, der möglicherweise über eine direkte stimulation der glomerulären angii bildung wirkt. hohe prävalenz der ass-non-responder bei acvb-patienten in den multiplen testanalysen hintergrund und zielsetzung: in der therapie der hiv-infektion sind wechselwirkungen und unverträglichkeit der mittlerweile zahlreich zur verfügung stehenden antiretoviralen medikamente ein grosses problem. mehr als 50% der antiretroviralen medikamente und begleitenden antiinfektiva gegen opportunistische infektionen weisen positiv geladene reste (kationen) auf. die ausscheidung organischer kationen über die leber und niere erfolgt u.a. über die organischen kationentransporter (oct) 1 und 2, die eine breite substratspezifität aufweisen. ziel der studie war die charakterisierung kationischer antiretroviraler medikamente und antiinfektiva als inhibitoren und substrate von oct 1/ 2. methoden: hek-293 zellen wurden stabil transfiziert mit humanem oct 1 und 2 kloniert im eukaryotischen expressionsvektor pcdna3. die ic50-werte wurden durch messungen der spezifischen aufnahme von 3h-gelabeltem 1-methyl-4-phenylpyridinium (mpp+) bestimmt. die quantitative bestimmung der spezifischen substrate erfolgte mittels liquid chromatography electrospray-ionizationtandem mass spectrometry (lc-esi-ms/ms). km und vmax wurden mit der michaelis menten gleichung bestimmt. ergebnisse: pentamidine (ic50(hoct1) = 0.4 (1) μmol/l (mittelwert (standardfehler); (oct2) 3.8(1)), nelfinavir (7(1); 33(11)), ritonavir (14(2); 51(22)), lamivudine (17(3); 13(3)), trimethoprim (20 (3); 131(12)), zalcitabine (24(5); 25 (7)), saquinavir (37 (7); 105(29)) und indinavir (37(6); 275(57)) zeigten eine signifikante hemmung von oct1/2. lamivudine und zalcitabine sind substrate von oct1/2 (lamivudine: oct1(vmax=2.0(0.2) nmol/mg/min; km=249(51) μmol/l) oct2 (vmax=1.1(0.2); km=248(86)) zalcitabine: oct1(vmax=1.0(0.1); km= 242(56); oct2(vmax=0.6(0.1); km=232 (13) ergebnisse: der endogene ligand s1p, der selektive s1p1-rezeptor agonist sew 2871 und der unselektive s1p-rezeptor agonist fty720 transaktivieren den ebenfalls bereits als vermittler protektiver intrazellulärer signalwege beschriebenen egf-rezeptor. damit assoziert ist die aktivierung von akt in ratten-kardiomyozyten. im rattenmyokard verbessert bei applikation beginnend mit reperfusion fty720, nicht jedoch der selektive s1p1 rezeptor agonist sew2871 die erholung der mechanischen funktion signifikant. in humanen myokardpräparaten zeigt fty720 ex vivo einen analogen effekt bei applikation unter reperfusion. zusammenfassung: stimulation von s1p1 und s1p3-rezeptoren induziert die aktivierung von akt in kardiomyozyten. eine verbesserung der erholung der mechanischen funktion lässt sich nur über stimulation des s1p3 rezeptors unter reperfusion erzielen. s1p-rezeptoren scheinen daher ein klinisch interessanter angriffspunkt für eine behandlung im sinne einer pharmakologischen postkonditionierung. systemic effects of glucose content in pd fluids on life span and neuronal function in c. elegans background: glucose content in peritoneal dialysis fluids has a decisive role in the formation of glucose degradation products (gdps) during sterilization and is thus associated with the degradation of peritoneal membrane integrity. absorption of gdps from the peritoneal cavity in peritoneal dialysis patients results in rising plasma age levels. one precursor in age formation is methylglyoxal, which is metabolized by glyoxalase-1. to further investigate systemic effects of glucose content in pd fluids on life span and neuronal function, wild type and glyoxalase-1 overexpressing c. elegans were used as model system. homologies to 70% of all human genes are preserved in c. elegans. thus, this model system allows not only performance of life span assays, but also analysis of neuronal function. methods: c. elegans were cultivated under normal conditions and in the presence of 1.5% and 4% glucose derived from peritoneal dialysis fluids to perform life span assays. simultaneously locomotive patterns as parameter of neuronal function and neuronal gfp expression were evaluated for each group. results: wild type c. elegans showed a significant reduction of life span of 65% under the influence of glucose 1,5% and of 77% using 4% glucose. in contrast, glyoxalase-1 overexpression led to an increased resistance towards glucose expo-sure with no significant reduction of life span under 1.5% or 4% glucose. additionally, with glucose exposure an impairment of neuronal function was observed in wild type c. elegans displaying severe changes in locomotive patterns, which were not present in transgenic c. elegans. neuronal gfp expression in wild type c. elegans declined significantly with glucose exposure without a dose dependant effect. conclusion:. glucose derived from pd fluids has a significant impact on life span and neuronal impairment in c. elegans. whether potential systemic toxicity and neurotoxicity in pd patients can be attributed to pd fluids has to be further evaluated. common pathways in endogenous major depression and depression during ifn-α therapy for chronic hepatitis c background: a major complication of combination therapy with pegylated interferon-alpha (ifn-α) and ribavirin in patients with chronic hepatitis c is the induction of depressive side effects. methods: to elucidate the underlying pathophysiological mechanisms, a total of 50 caucasian patients with histologically proven chronic hepatitis c were treated with standard combination therapy consisting of pegylated ifn-α2a and ribavirin. the transcriptional profile 12h before and 12h after the first injection of ifn-α was analysed using human genomic microarrays (affymetrix, hg u133a) and quantitative real-time rt-pcr. class prediction analysis was performed to identify genes which are differentially regulated in patients with or without ifninduced depression. furthermore, pbmc from 21 patients hospitalized for se vere major depression and 11 controls were cultivated in vitro with pegylated ifn-α2a to validate the data in this cohort. results: 11/50 hcv patients (22%) developed clinically relevant ifn-induced depression. using class prediction analysis, the development of depression could be predicted with 91% accuracy by 16 genes including dynlt1, gch1, tor1b, disc1, mef2a and st3gal5 that were previously described to be relevant for recurrent major depression or neuronal development in the brain. in accordance with this data, increased endogenous ifn-production and selective hyper-responsiveness of these genes to ifn stimulation were observed in hcvnegative patients with severe major depression. conclusions: these data suggest that selective hyper-responsiveness to exogenous (ifn therapy) or endogenous (endogenous depression) type i ifns may lead to the development of depressive symptoms. these data could lead to the discovery of novel therapeutic approaches to treat ifn-induced and major endogenous depression. renal toxicity of glucose degradation products in peritoneal dialysis purpose (zielsetzung): in peritoneal dialysis (pd) residual renal function contributes to improved patient survival and quality of life. glucose degradation products (gdp) impair not only the peritoneal membrane, but also appear in the systemic circulation with the potential for organ toxicity. here we show that in a model of advanced renal failure, gdp affect the structure and function of the remnant kidney. sprague-dawley rats were randomly assigned to a twostage subtotal nephrectomized (snx) or sham operation and were left untreated for 3 weeks. the snx+gdp group received chemically defined gdp intravenously for 4 weeks; the snx and the sham-operated rats remained without gdp. the complete follow-up for all groups was 7 weeks post-operatively. we analyzed renal damage using a semiquantitative score for glomerulosclerosis and tubulointerstitial damage as well as for immunohistochemical analyses. the snx+gdp rats developed significantly more albuminuria (12.3 ± 4.96 mg/24 h vs. snx 5.68 ± 2.77 mg/24 h; p ≤ 0.05) and showed a significantly higher score of glomerulosclerosis index (2.18 ± 0.33 vs. 1.82 ± 0.23; p ≤0.01) and tubulointerstitial damage index (2.17 ± 0.29 vs. 1.78 ± 0.22; p ≤ 0.01). in the snx+gdp group the expression of carboxymethyllysin and methylglyoxal was significantly higher in the tubulointerstitium and the glomeruli compared to the snx rats. caspase 3 staining and tunel assay were more pronounced in the tubulointerstitium and the glomeruli of the snx+gdp group. in snx+gdp animals, the expression of the slit diaphragm protein nephrin, was significantly lower compared to snx and sham operated animals. in subtotally nephrectomized rats, administration of gdp increased albuminuria, indices of glomerular and tubulointerstitial damage significantly, specifically with a perturbation of podocytes. it is likely that gdp-free pd solutions maintain and stabilize residual renal function in pd. non-parenchymal liver cells (wu et al., hepatology, in press ). therefore, the aim of this study was to investigate whether hbv has the ability to suppress tlr-induced antiviral responses in parenchymal and non-parenchymal liver cells. we have isolated murine kc by counterflow elutriation as well as murine lsec by anti-lsec microbeads. in addition, primary murine hepatocytes were isolated by a two-step perfusion method. npcs and hepatocytes were cultivated in the presence or absence of hbsag, hbeag, hbv virions or supernatants from hbv-producing hbv-met cells, and were stimulated by tlr 1-9 ligands. supernatants were collected and tested for antiviral cytokines by viral protection assay or co-cultured with differentiated hbv-met cells. primary hepatocytes, undifferentiated hbv-met cells and highly differentiated hbv-met cells were stimulated by tlr 1-9 ligands. results: pretreatment of hepatocytes and npcs with hbv-met cell supernatants (contain hbsag, hbeag and virions), hbsag, hbeag and hbv virions almost completely abrogated tlr 3 and tlr 4 induced antiviral activity which correlated with suppressed isgs induction by poly i:c and lps on the transcriptional level. tlr 1-9 stimulation had no direct antiviral effect on hbv-met cells in contrast to primary hepatocytes. in addition, expression of pro-inflammatory cytokines (tnf-a, il-6) after tlr -3, -4, -7, -8 and -9 stimulation were significantly reduced in highly differentiated hbv-met cells compared with undifferentiated hbv-met cells. our data indicate that integrated hbv can downregulate the tlr-mediated activation of hepatocytes. furthermore, hbv can almost completely abrogate the antiviral activity of hepatocytes and npcs induced by tlr 3 and tlr 4 stimulation. this has major implications for the interaction between hbv and the immune system. schimäre im pankreas: ein glukagon-produzierendes neuroendokrines karzinom assoziiert mit hypoglykämien conclusions: dbe-ercp is an alternative method for diagnostic as well as therapeutic interventions in the biliary as well pancreatic system in the operated patient. however, it should be limited to selected patients, e.g. with contraindications for ptc, as it is a time consuming as well as a cost intensive procedure. undifferenziertes embryonales sarkom der leber -eine seltene primäre leberneoplasie beim erwachsenen patients with metabolic syndrome (ms) and type 2 diabetes are at increased risk for coronary artery disease. lipoprotein metabolism is characterized by elevated triglycerides (tg), low hdl cholesterol (hdl-c) and a predominance of atherogenic dense ldl (dldl). this is also denoted as atherogenic lipoprotein phenotype (alp). methods: multicenter, randomized, open-label cross-over study investigating the effect of combination therapy of fluvastatin/fenofibrate (80/200 mg) (ff) on the ldl-subfraction distribution compared to combination therapy of simvastatin/ ezetimibe (20/10 mg) (se) in patients with ms and type 2 diabetes. at baseline and after 6 weeks of combination therapies ldl-subfractions were measured by endpoint gradient ultracentrifugation. results: 75 patients were randomized, 69 completed the study. blood lipid samples of 56 patients could be analysed. 38 out of 56 patients (68%) showed a profile dominated by dldl. in these patients, tg were elevated and hdl-c was lower compared to non dldl patients. in all patients reduction of total and ldl cholesterol by se was stronger than by ff. the increase of hdl-c was stronger with se in the non dldl group, whereas in the dldl group there was no difference between treatments. in non dldl patients there was no difference with regard to tg reduction and no effect on calculated ldl-radius. however, in the dldl group ff was more efficient in reducing tg (p=0.020 between treatments) and se reduced ldl radius even further, whereas ff increased ldl radius to larger ldl particles (p<0.001 between treatments). conclusions: simvastatin/ezetimibe combination is more efficient in reducing total and ldl cholesterol. this is also true for hdl-c increase in patients without dldl. however, in ms patients with dldl fluvastatin/fenofibrate were more efficient in reducing tg and increasing ldl radius. overall, the number of patients with aes and myalgia was 15. 1% hintergrund: bnp und nt-probnp sind molekulare biomarker, welche ebenso für die diagnostik und prognose der chronischen herzinsuffizienz genutzt werden können wie für die optimierung einer medikamentösen therapie und das langzeit-management dieser erkrankung. erstmalig untersuchten wir den einfluss eines strukturierten, multi-modalen, stationären interventionsprogramms -wie es in deutschland in vielen kardiovaskulären rehabilitationskliniken angeboten wird -auf den zeitlichen verlauf der nt-probnp-werte während und sechs monate nach einer solchen intervention. methodik: diese studie wurde in sechs deutschen kardiovaskulären rehabilitationszentren durchgeführt mit folgenden vier interventionsmodalitäten: (1) optimierung der medikamentösen therapie, (2) erziehung zu einem und durchführung eines krankheits-bezogenen ausdauertrainings-programms, (3) intensive information über art und verlauf der erkrankung und erziehung zu einem adäquaten ernährungsverhalten, und (4) teilnahme an den physischen und psychischen entspannungsübungen. zur steigerung der motivation und dokumentation wurden speziell für diese studie entwickelte patienten-tagebücher ausgegeben, welche den gesamten sechsmonatigen studienverlauf abdeckten. nt-probnp-analysen erfolgten am anfang (r1), während (r2) und am ende der stationären rehabilitationsperiode (r3), ebenso -auf ambulanter basis -nach drei monaten (r4) und sechs monaten r5) nach entlassung. zum vergleich wurde eine gleichaltrige kontrollgruppe von patienten herangezogen, welche nicht an einem solchen intensiven programm teilnahmen, sondern in einer herzinsuffizienz-ambulanz medizinisch betreut wurden. hypothese: wir erwarteten einen positiven effekt unseres interventions-programms auf die nt-probnp-spiegel während des stationären aufenthaltes, und entweder eine konstanz dieser werte nach der entlassung oder -auf grund von mangelhafter compliance -sogar einen wiedereinstieg der werte bis zu sechs monaten nach entlassung. ergebnisse: komplette nt-probnp-werte lagen für 60 patienten vor, welche an einer chronischen herzinsuffizienz im stadium nyha ii -iii litten, eine klinisch relevante dekompensation erlitten hatten und eine lvef = 35% aufwiesen: der medianwert der nt-probnp-spiegel betrug 1985 pg/ml am anfang (r2), war geringfügig aber nicht signifikant reduziert auf 1644 (r2) und 1755 pg/dl (r3) während des stationären aufenthaltes, fiel jedoch signifikant und klinisch relevant auf 978 pg/ml nach drei monaten (r4; p<0,01) und auf 719 pg/ml nach sechs monaten ab (r5; p<0,01). der medianwert der kontrollgruppe änderte sich nicht während der sechsmonatigen beobachtungsperiode (1164 pg/ml und 1107 pg/ml; ns). schlussfolgerungen: im gegensatz zu unserer hypothese hat eine modernes, strukturiertes, stationäres rehabilitationsprogramm keinen kurzzeit-effekt auf die nt-probnp-spiegel innerhalb von wochen, sondern vielmehr einen signifikanten und größenordnungsmäßig klinisch relevanten langzeit-effekt innerhalb von monaten. dies könnte in der tatsache begründet liegen, dass die molekularen und strukturellen umbauprozesse des linken ventrikels im sinne eines umgekehrten "left ventricular remodelling" entsprechend zeit benötigen. da die nt-probnp-spiegel mit der prognose der erkrankung assoziiert sind, kann indirekt geschlossen werden, dass unsere stationäre rehabilitationsstrategie langfristig positive effekte auf hospitalisierungsrate und überlebensprognose hat. cancer fatigue und gestörte ruhe/aktivitätsregulation bei rezidivfreien mammakarzinom-patientinnen, ergebnisse einer prospektiven studie r=0, 358, p=0, 015) . vergleichbare beziehungen bestanden zwischen dem pap und ahi (r=0,39, p=0,002), dem ai (r=0,39, p=0,002) und dem zai (r=0,40, p=0,016). das hzv war bei patienten mit csa (1,93±0,5 l/ min/m 2 ) signifikant niedriger als bei osa (2,55±1,0 l/min/m 2 ) oder patienten ohne sas (2,22±0,4 l/min/m 2 ). das hzv korrelierte negativ mit dem zai (r= -0,344, p=0,008), dem ai (r= -0,31, p=0,02) und dem ahi (r= -0,21, p<0,05). bei pat mit einer osa waren solche beziehungen nicht nachweisbar. die vorliegenden befunde unterstützen die these, wonach das auftreten einer zsa ein parameter für die schwere einer hi sein kann und erhöhte pa-und pcw-drücke über pulmonal-vagale j-rezeptoren zur hyperventilation und damit entstehung und unterhaltung einer zsa beitragen können. der quotient aus serum-natrium/ urin-natrium-konzentrationen zu den serum-kalium/ urin-kalium-konzentrationen (sus:pup) im screening auf einen primären aldosteronismus hintergrund: das herzzeitvolumen (hzv) ist ein wichtiger parameter in der diagnostik und therapie kardialer erkrankungen. die aktuellen standardmethoden zur bestimmung des hzv sind jedoch entweder invasiv (rechtsherzkatheter, picco) oder technisch aufwändig bzw. teuer (magnetresonanztomographie, mrt). die bisherigen nicht-invasiven methoden zur messung des hzv mittels rückatmung von kohlendioxid oder thorakaler bioimpedanz sind zwar einfach durchzuführen, weisen aber methodisch bedingte ungenauigkeiten auf. ziel der vorliegenden prospektiven studie war es daher, eine neue methode zur bestimmung des hzv mittels cw-doppler ultraschall (cwd) zu evaluieren. methodik: bei 32 konsekutiven patienten wurde unmittelbar vor oder nach einer kardialen mrt das herzzeitvolumen (hzv) mittels cwd im liegen bestimmt (uscom, uscom ltd, sydney australia). als referenzwerte dienten die in der mrt bestimmten werte, die mit dem arithmetischen mittel aus zwei aufeinanderfolgenden cwd messungen verglichen wurden. der statistische vergleich der methoden erfolgte mittels bland-altman-analyse. ergebnisse: das patientenkollektiv bestand aus 20 männern (alter 17-80 jahre, median 51 jahre) und 12 frauen (alter 27-78 jahre, median 64 jahre). insgesamt konnte bei 29 von 32 konsekutiven patienten im untersuchungszeitraum das hzv und sv mittels cwd bestimmt werden, bei 3 patienten (9%) konnte kein ausreichendes ultraschallsignal empfangen werden. das hzv mittels mrt lag bei 5,2 +/-1,1 l/min (mittelwert +/-sd, minimum 3,0 l/min, maximum 7,5 l/min), das hzv mittels cwd bei 4,7 +/-1,1 l/min (minimum 2,8 l/min, maximum 8,0 l/min). die bland-altman-analyse ergab eine gute übereinstimmung der beiden methoden für das hzv von 0,5 +/-1,0 l/min (mittlere abweichung +/-sd) und für die bestimmung des schlagvolumens von 6,9 +/-16 ml. die methode zeigte eine gute reproduzierbarkeit mit einer mittleren abweichung von 0,1 +/-0,5 l/min für das hzv und 0,3 +/-7,7 ml für das sv. schlussfolgerung: der cw-doppler ultraschall erlaubt eine zuverlässige nichtinvasive bestimmung des hzv mit guter reproduzierbarkeit. der zukünftige stellenwert der methode in der diagnostik und therapiesteuerung muss durch weitere untersuchungen belegt werden. nichtinvasive bestimmung des herzzeitvolumens mittels inertgas-rückatmung und cw-doppler-ultraschall -sind die ergebnisse vergleichbar? bei 4 patienten (10%) war die messung mittels cwd nicht möglich, bei 2 patienten (5%) konnte die igr-messung nicht durchgeführt werden. das hzv mittels igr lag bei 4,9+/-1,1 l/min (mittelwert+/-sd, 2,7 bis 7,6 l/min) und bei 4,6+/-1,2 l/min (2,5 bis 8,0 l/min) mittels cwd. die bland-altman-analyse ergab eine gute übereinstimmung für das hzv von 0,4+/-1,1 l/min (mittlere abweichung+/-sd) und für das sv von 2+/-18 ml. es zeigte sich eine gute reproduzierbarkeit mit einer mittleren abweichung von 0,2+/-0,6 l/min für das hzv mittels igr bzw. 0,1+/-0,5 l/min mittels cwd. schlussfolgerung: die bestimmung des hzv und sv mittels igr und cwd ergab eine gute übereinstimmung und reproduzierbarkeit, so dass die methoden als vergleichbar einzuschätzen sind. zur identifikation des optimalen patientenkollektives für jede der beiden methoden sind weitere untersuchungen zur erarbeitung eines score-systems geplant. validierung einer einfach anzuwendenden neuartigen oszillometrischen methode zur bestimmung der pulswellen-geschwindigkeit obesity in humans is mainly due to high-fat intake and associated with an increased incidence of glomerular injury that is associated with low-grade inflammation and increased production of reactive oxygen species (ros). lauric acid (c12:0), a major constituent of coconut oil, has anti-inflammatory activities, however its effects on pro-inflammatory gene expression in the kidney during obesity are unknown. the aim of the study was to quantify and compare renal cortical gene expression of pro-and antioxidant enzymes and icam-1 in c57bl/6j mice fed with standard chow diet (sd), or diets rich in animal fat (afd) or vegetable fat (lauric acid, vfd) for 15 weeks. changes in body weight and glucose tolerance (ip ggt) were also determined. both high-fat diets caused weight gain and glucose intolerance to a similar degree (n.s., afd vs. vfd, both p<0.001 vs. sd). expression levels of proinflammatory genes (no synthase 2, nos2; catalytic subunit of the phagocytic nadph oxidase, nox2; intracellular adhesion molecule-1, icam-1) were more than 30-fold lower compared to antioxidant genes (gluthatione peroxidase, gpx and cu/zn superoxide dismutase, sod1) and the regulatory subunit of the nadph oxidase complex p22phox. only vfd-treatment reduced renal expression of p22phox. treatment with afd but not with vfd increased mrna expression of nos2, nox2, and icam-1. vfd, but not afd treatment was associated with upregulation of the antioxidant genes sod1 and gpx. these data suggest that fat diets rich in lauric acid, in contrast to diets containing animal fats, have distinct and possibly beneficial effects on expression of enzymes regulating cellular redox state in the kidney. contractility in the carotid artery obesity is a risk factor for diseases such as diabetes mellitus and atherosclerosis and has been associated with increased carotid artery intima thickness in obese patients. leptin has been implicated in formation of reactive oxygen species (ros), and functional leptin deficiency or impairment of leptin receptor activity result in obesity. endothelin-1 (et-1), ros and reduced no bioactivity are involved in vascular changes in obesity and diabetes. the aim of this study was to investigate the role of ros in et-1 mediated vasoreactivity in the carotid artery of obese leptin-deficient ob/ob and lean c57bl/6j wild-type mice. rings were preincubated for 30 minutes with the no synthase inhibitor, l-nitro-argininemethylester (l-name, 300 μmol/l) to block etb receptor-mediated no release. rings were exposed to et-1 (0.01-300 nmol/l) in the presence or absence of the ros antioxidant epck-1 (a combination of vitamin c and vitamin e, 0.1 mg/ml). endothelium-dependent relaxation to acetylcholine (ach) was in-vestigated in the presence of non-specific cyclooxygenase (cox) inhibitiors. maximal et-1-mediated contraction in control mice was similar to obese mice. antioxidant treatment with epc-k1 reduced et-1-induced contractions in control (from 44.7 ± 6.7% to 22.7 ± 3.8%, p<0.05), but not in obese mice (55.8 ± 11.9% vs. 49.2 ± 8.9%, n.s.) endothelium-dependent relaxation to ach remained unchanged in both groups. in conclusion, these results indicate that ros contribute to et-1-induced contractions in the carotid artery of healthy mice, an effect that disappears during obesity. leptin may modulate contraction to et-1 in the carotid artery by mechanisms that are independent of ros. high dietary intake is a major cause of obesity, a risk factor for the development of hypertension, diabetes, and atherosclerosis. the aim of this study was to investigate whether high dietary fat intake-induced vascular changes are reversible upon dietary fat restriction. body weight, glucose tolerance and vascular reactivity were measured in c57bl/6j mice, which were fed either with standard chow or with high-fat diet (hfd, 58% kcal from fat) for 6 months or with hfd for 6 months followed by fat restriction for 4 months. vascular reactivity was analyzed in the carotid artery in response to the α-adrenergic receptor agonist phenylephrine (pe; 3x10 -8 m), and to acetylcholine (ach; 10 -10 -10 -5 m) in the presence or absence of ng-nitro-l-arginine methyl ester (l-name; 3x10 -4 m), a nitric oxide (no) synthase inhibitor. high-fat diet increased body weight and impaired glucose tolerance (p<0.05 vs. control). vascular contractions in response to pe (3x10 -8 m) and ach (10 -7 m) were increased both in the absence or presence of no (p<0.05 vs. control), while the endothelium-dependent relaxation was unaffected. in contrast, dietary fat restriction normalized the body weight similar to control animals and also the relative insulin resistance (p<0.05 vs. hfd). in parallel, vascular contraction to ach was attenuated in the absence of l-name (p<0.05 vs. hfd). unexpectedly, dietary fat restriction did not reverse pe-and ach-mediated contraction in the absence of no induced by hfd. in conclusion, intake of high dietary fat has a "memory" effect on vascular reactivity, which cannot completely be reversed even upon dietary fat restriction. this may contribute to an increased risk of vascular injury seen in patients even after normalization of body weight and insulin resistance. die phagozytotische aufnahme von hcv-infizierten apoptose-körperchen durch hsc führt zur induktion der fibrogenese a minority of neuroendocrine tumors (net) present functional syndromes by uncontrolled secretion of peptide hormones or messengers. peptide receptor radiotherapy (prrt) and transcatheter arterial chemoembolisation (tace) have demonstrated efficacy as monotherapy in the treatment of functional nets. prrt has a slower and longer acting mechanism of action whereas tace can be immediately effective. however, there are no reports that both therapies have been applied in combination. we treated three consecutive patients with severe functional syndromes caused by hepatic metastasis of nets by sequential prrt and tace. the first patient (female 57 years) suffered from a pancreatic net metastasized into the liver. severe hypercalcemia with levels of 3.9 mmol/l developed leading to fatique and somnolence despite medical treatment.the patient received one course of 40 mci y-90 dota-tate prrt and 11 days later, tace was performed at the right liver lobe due to persistent hypercalcemia. after 5 days, calcium levels were normalized and the patients was able to walk with support. after demission, the patients died of progressive disease 6 weeks later. the second patient (male, 58 years) presented with somnolence and hypoglycaemia down to 1 mmol/l by disseminated insulinoma of the pancreas. the patient received two courses of prrt and three coursed of tace involving both liver lobes. he tolerated the combined treatment well and is off intravenous glucose since the first tace. the third patient (male, 62 years) presented with severe hypoglycaemia due to hyperinsulinemia caused by disseminated net of unknown origin with prominent liver metastases in both liver lobes. initially, the patient was treated by tace followed by 2 courses of prrt with 177-lu. the treatment was well tolerated, the patient is off intravenous glucose. a second tace was intended but was not be performed due to remission of the liver metastases. initial experience with combined treatment of prrt and tace demonstrated that hepatic failure did not evolve in our patients and that the treatment was well tolerated. however, prognosis remains poor due to disseminated and progressive disease. die zahl zirkulierender endothelialer progenitorzellen steigt mit zunehmendem schweregrad der peripheren arteriellen verschlusskrankheit these data indicate that the key mirna-processing enzymes dicer and drosha and consequently the overall expression of mirnas are downregulated during neointima formation in vivo. moreover, the augmented proliferative-and attenuated apoptotic response observed following knock down of drosha and dicer in vsmc implicate an important role of these enzymes and of mirnas for vsmc function during the development of vascular proliferative disease. (76) hochaktives endokrines organ. das fettgewebe produziert nämlich zahlreiche adipokinine, welche möglicherweise einen autokrinen und parakrinen einfluss auf den fettstoffwechsel, sowie eine endokrine wirkung auf andere organe haben. die entdeckung solcher faktoren eröffnet die frage, ob die adipokinine die gesuchte verbindung zwischen adipositas und den damit assoziierten erkrankungen sein könnten. im diesen zusammenhang haben wir untersucht, ob das fettgewebe die herzfunktion direkt mittels sekretion von kardioaktiven faktoren beeinflusst. wir isolierten deshalb adipozyten aus humanem fettgewebe und untersuchten anschließend den effekt ihrer sekretionsprodukte auf kardiomyozyten in zellkultur sowie mittels langendorff system auf isoliert-perfundierte rattenherzen. wie wir feststellen konnten, hatten die sekretionsprodukte der adipozyten die kontraktion der kardiomyozyten durch eine verminderung des intrazellullären kalziumstroms stark gehemmt. durch erhitzen oder trypsin-behandlung wurden die kardiodepressiven faktoren inaktiv. mittels filtration nach molekulargewicht konnten die kardiodepressiven faktoren zwischen 10 und 30kd isolierten werden. auf ähnliche weise führte die perfusion von isoliertem herz mit adipozytenfaktoren zu einer starken reduktion der kraftentwicklung sowie zu einer reduktion des koronarflusses durch eine kontraktion der koronararterien. zusammenfassend, die ermittelten daten lassen auf einen bislang unbekannten, negativen einfluss des fettgewebes auf die herzkontraktion schließen. dies geschieht einerseits direkt durch die verminderung des intrazellullären kalziumstroms in die kardiomyozyten, andererseits indirekt durch die reduktion des koronarflusses. diese daten liefern somit eine neue erklärung für den zusammenhang zwischen adipositas und herzinsuffizienz. a comparison of serum fractalkine in patients with coronary heart disease, insulin dependent diabetes, and healthy controls background: atherosclerosis is a multifactorial process that involves inflammation of the vessel wall arising from interactions of leukocytes with vascular endothelial and other local cells. these interactions are regulated by cytokines, including chemokines, as well as adhesion molecules. the chemokine fractalkine (fkn) and its receptor, cx3cr1, have emerged as interesting intermediaries in atherosclerosis. fkn is a unique chemokine because it exists in soluble and membrane-anchored forms. membrane-bound fkn contributes to adhesion of cx3cr1-expressing leukocytes, thus providing a novel pathway for leukocyte activation. soluble fkn has leukocyte chemotactic activity. therefore, serum fractalkine levels may indicate the inflammatory situation in chd patients. we investigated the serum concentrations of fkn in 50 non-diabetic patients with coronary heart disease (chd), and 50 insulin dependent diabetics (iddm) in patients attending for rehabilitation in the curschmann-clinic, timmendorfer strand. serum fkn was also determined in 50 healthy controls. fkn was analyzed by elisa. results: soluble fkn was slightly, but not significantly increased in patients with chd in comparison to healthy controls (mean ± standard deviation: 722±884 pg/ml in chd-patients versus 693±748 pg/ml in controls, p=0.833). surprisingly, soluble fkn was decreased in insulin dependent diabetics (mean±sd 430±257 pg/ml thus, iddm versus controls p=0.058, and iddm versus chd p=0.034). conclusion: previous studies described that soluble fkn is increased in chd patients compared with healthy controls and that membrane-bound fkn was increased in human carotid arteries and animal models. the decreased soluble fkn in insulin dependent diabetics compared with chd-patients and controls in this study may partly be explained by the quiescent disease state predominant in our rehabilitation patients. hintergrund: neben der jeweiligen bilddokumentation und dem deskriptiven befund des interventionalisten exsistieren keine objektiven daten zur veränderung der hämodynamik während peripheren arteriellen gefässinterventionen und dem unmittelbaren hämodynamischen erfolg nach der intervention. versuche der etablierung eines monitorings durch dopplerultraschall scheiterten in der vergangenheit offensichtlich u.a. an der aufwändigen fixierung der dopplersonden. methode: wir verwenden zur kodomo die doppler x-plore sonde (7,5 mhz, firma medi-stim, deisenhofen), welche durch einen saugnapf mit ultraschallgeldepot über einer zuvor dopplersonographisch detektierten fussarterie fixiert wird. es können so während der intervention kontinuierliche dopplersignale dokumentiert werden. ergebnisse: phänomene die mittels kodomo bei infrainguinalen eingriffen periinterventionell erfasst werden können sind: verbesserung des dopplerflusses postinterventionell, artefakte durch kontrastmittel, periphere embolien, veränderungen des dopplersignals während der angioplastie. beispielhaft wird ein fall mit filiformer superficialisstenose links demonstriert (abb. 1). schlussfolgerung: kodomo gibt dem interventionalisten über die bildgebung hinausgehende hämodynamische zusatzinformationen und ist ein verfahren zur unmittelbaren dokumentation des hämodynamischen erfolges der interventionellen therapie. es spart in konsequenter anwendung möglicherweise kontrast-mittel, durchleuchtungs-und untersuchungszeit. durch den artefiziellen gefäßverschluss zum zeitpunkt der angioplastie kann der kollateralfluss der zielläsion zukünftig gemessen werden. einfluss von shunt-verkalkungen auf das überleben von hämodialysepatienten introduction: two-dimensional strain (2ds) is a novel method to measure strain from standard two-dimensional echocardiographic images. strain imaging has been proposed as a sensitive tool for the assessment of the left ventricular myocardial function. the aim of our study was to characterize global and regional function abnormalities using this technique in patients (pts.) with constrictive pericarditis (cp) and restrictive cardiomyopathy (rcm). methods: we studied 52 consecutive patients (pts.) with heart failure of either proven pericardial (cp) or myocardial origin (rcm; biopsy proven cardiac amy-loidosis). global longitudinal strain (gls) and regional peak systolic strain (pss) was assessed by 2ds in the apical four-chamber-view using a dedicated software package (vivid 7, ge healthcare). all pts. underwent a complete echocardiographic and hemodynamic assessment. results: out of the 52 pts. (mean age: 60±12 years) 29 had cp, and 23 rcm. the thickness of the interventricular septum (ivsd) was significant increased in pts. with rcm (17±5 vs. 9±1 mm). mean gls was -11.2±3.9% in the cp-group and -7.7±2.9% in the rcmgroup (p<0.01). pts. with rcm showed a significant decreased longitudinal pss in the septal segments (basal: -5.7±4.6% vs. -17.5±6.3%, p<0.01, mid: -8.8±3.9% vs. -18.5±4.7%, p<0.01; apical: -11.5±5.7% vs. -16.2±6.7%; p<0.05) and a decreased pss in the lateral segments (basal: -8.8±5.2% vs. -12.4±6.6%, n.s.; mid: -6.9±3.4% vs. -7.7±6.3%, n.s.; apical: -6.4±3.5% vs. -10.0± 6.9%, n.s.). conclusion: two-dimensional strain is a simple and rapid method to measure gls and pss. this technique might be used as new helpful tool for the differentiation between pts. with rcm and cp, and for the detection of early regional myocardial dysfunction before the onset of congestive heart failure (chf) in patients with cardiac amyloidosis. nichtinvasive koronare plaquedifferenzierung: mehrschicht-computertomographie validiert mit intravaskulärem ultraschall über die klassischen risiken hinaus sind auch lokale faktoren an der initiation und progression von atherosklerotischen ablagerungen beteiligt. diese faktoren beinhalten multiple variablen wie wechselwirkungen zwischen gewebe und flüssigkeiten, wandspannung und flussgeschwindigkeit . die direkte messung dieser faktoren ist in-vivo nur im tierversuch möglich. entsprechende parameter können jedoch über den einsatz moderner bildgebungstechniken (cardio-cta) unter verwendung von flusssimulationen (computational fluid dynamics=cfd) berechnet werden. das ziel dieser studie war daher 1) die durchführbarkeit der in-vivo cfd-berechnung an humanen koronararterien zu demonstrieren und 2) die ergebnisse mit radiofrequenzmessungen im intravaskulären ultraschall (ivus) zu korrelieren. methoden und ergebnisse: zehn patienten mit suspektem koronarem befund wurden prospektiv in die studie einbezogen. diese erhielten innerhalb von vier wochen eine ct-gestützte koronarangiographie (dual source 64 slice ct) und eine invasive koronarangiographie. bei diesen patienten wurde die intravaskuläre ultraschalluntersuchung in allen drei hauptstammgefäßen durchgeführt, die erhebung der radiofrequenzdaten geschah simultan. mit hilfe der axialen cta-schnitte wurde ein gittermodell der jeweiligen gefäße erstellt. dieses konnte genutzt werde um die gewebeflüssigkeitinteraktionen, flussgeschwindigkeit und wandspannungsverhältnisse zu simulieren. die berechnung der cfd-parameter konnte in 24/30 herzkranzarterien erfolgreich durchgeführt werden. siebzehn koronararterien wurden zusätzlich durch ivus beurteilt. die wandspannung der gefäße korrelierte umgekehrt zu vorhandenen durch ivus ermittelte plaques(p<0,05). ein zusammenhang zwischen den erhobenen cfd-daten und den radiofrequenzdaten der gewebe konnte bisher nicht nachgewiesen werden. zusammenfassung: die ergebnisse dieser studie demonstriert die möglichkeit gewebeflüssigkeitsinteraktionen in menschlichen koronararterien, mit hilfe von modernen computertomographischer bildgebung, nichtinvasiv abzuschätzen. die bedeutung dieser zusätzlichen informationen muss prospektiv weiter untersucht werden. welchen einfluss hat die myokardiale fibrose und funktion bei patienten mit hochgradiger aortenklappenstenose auf den klinischen langzeitverlauf nach aortenklappenersatzoperation? ziel dieser studie ist es, die entwicklung des linksventrikulären remodellings sowie der regionalen myokardialen funktion bei patienten mit hochgradiger aortenklappenstenose sowohl vor als auch im verlauf nach aortenklappenersatzoperation zu untersuchen. methodik: bei 65 patienten mit hochgradiger aortenklappenstenose wurde sowohl präoperativ als auch 9 monate postoperativ eine konventionelle echokardiographie und eine strain-rate-imaging studie mit bestimmung der longitudinalen systolischen spitzen-strain-rate durchgeführt. zur beurteilung der myokardialen fibrose wurde eine magnetresonanz tomographie (mrt) mittels gadolinium late enhancement technik durchgeführt und eine biopsie aus dem linksventrikulären septum intraoperativ entnommen. ergebnisse: die patienten wurden entsprechend der nyha-klasse 9 monate nach der operation in drei gruppen aufgeteilt. gruppe1 (gutes outcome=nyha-klasse i; n=22), gruppe 2 (mittleres outcome, nyha-klasse ii; n=24), gruppe 3 (=schlechtes outcome, nyha -klasse iii bzw. iv; n=19). präoperativ zeigten sich bei allen drei gruppen eine vergleichbare ejektions-fraktion (gruppe 1=55±13%; gruppe 2=51±13%; gruppe 3=51±13%). wohingegen bei der longitudinalen strain rate signifikant höhere werte in gruppe 1 als in gruppe 3 gemessen werden konnten (gruppe 1=1,32±0,2 s -1 ; gruppe 2=0,98±0,2 s -1 ; gruppe 3=0,58±0,2 s -1 ). interessanterweise stieg die longitudinale strain rate erst mit der abnahme der wandstärken nach 9 monaten an. außerdem konnte ein signifikant höherer grad an interstitieller fibrose mittels biopsie-score (gruppe 1=0,3±0,5; gruppe 2=0,5±0,8; gruppe 3=1,6±0,5) sowie auch ein häufigeres late enhancement in der mrt bei patienten mit schlechtem klinischem outcome nachgewiesen werden (segmente mit fibrose: gruppe 1=0,6±0,4; gruppe 2=0,9±0,3; gruppe 3=1,9±0,3). zusammenfassung: diese daten lassen vermuten, dass bei patienten mit hochgradiger aortenklappenstenose sowohl eine myokardiale fibrose als auch eine reduzierte regionale myokardiale funktion präoperativ entscheidene hinweise auf den klinischen langzeit-verlauf nach aortenklappenersatzoperation geben können. indikationen und limitationen der ultraschall-elastographie bei patienten mit erkrankungen des pankreas vor dem hintergrund immer wirksamerer, aber auch sehr kostenintensiver behandlungsoptionen einerseits und dem bewusstsein der früh einsetzenden strukturellen schädigung im arthritischen prozess ("window of opportunity") andererseits, ist ein monitoring des therapieansprechens bei patienten mit rheumatoider arthritis von besonderer bedeutung. eine exakte darstellung des entzündlichen substrates gelingt besonders mit der sonographie, wodurch therapieversager frühzeitig identifiziert werden können. die arthrosonographie erlaubt eine sehr gute differenzierung zwischen exsudativen und proliferativen synovialisveränderungen sowie die beurteilung der perfusion durch die doppler-verfahren. voraussetzung für eine sonographische beurteilung des verlaufes des arthritischen prozesses ist eine quantifizierung der entzündungsaktivität. in anlehnung an den von scheel und kollegen publizierten synovitisscore, wurden patienten mit aktiver rheumatoider arthritis, die durch eine biologikatherapie (tnf-inhibitoren, rituximab, abatacept) in klinische remission gebracht werden konnten (das28: <2,6), standardisiert sonographisch nachuntersucht. bei der mehrheit der ra-patienten konnte trotz klinischer remission durch die sonographischen nachuntersuchungen 3 und 6 monate nach initiierung der biologikatherapie eine persistierende entzündungaktivität nachgewiesen werden. während eine deutliche therapiebedingte abnahme der hyperperfusion zu beobachten war, konnte nur eine geringgradige abnahme der synovialen proliferationen und ergussbildung festgestellt werden. diese ergebnisse zeigen, dass bei der mehrzahl der ra-patienten trotz klinischer remission eine sonographisch nachweisbare snovitis persistiert, was möglicherweise die ursache für den radiologischen krankheitsprogress bei einem teil dieser patienten darstellt. zielsetzung/aims: granulocyte-colony stimulating factor (g-csf) was shown to improve cardiac function after myocardial infarction (mi). recently, we histologically demonstrated enhanced arteriogenesis and reduced infarct size by g-csf treatment after mi in mice. in this study, we non-invasively, repetitively, and quantitatively investigated g-csf effects on perfusion by a pinhole single-photon emission computed tomography (spect) system after mi in mice. methoden/methods: mi was induced by lad ligation in wt mice (c57bl/6j). g-csf (100 μg/kg; n=5) or saline (n=4) was daily injected for 5 days. extent of left ventricular (lv) perfusion defects was determined with a [99mtc]-sestamibi triple headed pinhole spect system at 6 days (baseline) and 30 days after lad occlusion. polar maps were normalized by mean of a standardized reference region of interest (roi) in the septal region (100%). best threshold value for identifying infarcted areas was determined by comparing perfusion defects with the histological infarct sizes in these animals. defect size was indicated as% of lv myocardium. primary endpoint was change of defect size from baseline to 30 days after mi. ergebnisse/results: best threshold for identifying infarcted areas compared to histology was less than 65% of the septal reference roi. mean infarct size was similar in saline (20,55% ± 0,90) and g-csf group (19,16% ± 1,10) at baseline. at day 30 after mi, a slight difference (p=0,05) was found between saline (20,24 ± 0,84) and g-csf (16,54% ± 1,24) groups. however, change of defect size was significantly different (p=0,02) between g-csf (-2,63% ± 0,76) and control animals (-0,31 ± 0,14) . schlussfolgerung/conclusions: this is the first study, demonstrating the suitability of triple headed pinhole spect system for repetitive infarct size measurement in mice, offering a new non-invasive imaging technique for cardiovascular therapy monitoring. in this context, we showed that g-csf administration significantly reduces lv perfusion defects indicating positive effects on ventricular remodelling after mi in mice. endoscopy in patients with acute leukemia after intensive chemotherapy conclusions: endoscopy can be performed relatively safely in patients who received myelosuppressive chemotherapy. the procedure may induce fever and bacteremia. the percentage of patients in whom endoscopic hemostasis was applicable and effective was low. we recommend conservative treatment first. endoscopy should be reserved for patients who are unstable or refractory. impact of pregnancy on prevalence of goitre and nodular thyroid disease in women living in a region of borderline iodine deficiency purpose: an interplay of genetic, epigenetic and environmental factors contributes to thyroid disease. in a cross-sectional study we aimed to determine the actual influence of parity on goitre and nodular thyroid disease (ntd) in women living in a region with borderline iodine deficiency. methods: thyroid ultrasonography (7.5mhz; merck thyromobil) was performed by the same investigator in 731 women living in thuringia and saxony. furthermore, age and bmi were documented and all women were asked about the number of previous pregnancies, family history of thyroid disease and past or present smoking. results: goitre prevalence was 17.6%. solitary thyroid nodules were detected in 21.2%, multiple nodules in 24.6% of the study population. age was positively correlated with goitre prevalence and ntd (due to multiple but not solitary nodules). no association was found between parity and goitre prevalence. in contrast, a significant increase in both, solitary (22.2%) and multinodular thyroid disease (23.7%) was observed in women with at least one pregnancy compared to nullipara (11.9% and 16.9%, respectively). bmi in women with goitre (27.3kg/ m2) was significantly higher than in women without (24.8kg/m2). in addition, a significant correlation was detected between bmi and presence of multinodular disease (26.5kg/m2). 24,5% of women with goitre reported a positive family history for thyroid disease, as opposed to 15% of women with normal size thyroid gland. neither goitre nor ntd were associated with a present or past history of smoking. conclusion: ntd and/or goitre are present in up to 50% of woman in an area with borderline iodine deficiency. besides age, bmi and family history, parity is positively correlated with presence of ntd, whereas smoking was not associated with goitre/ntd. defizite in der medizinischen versorgung von patienten mit nebennierenkarzinom in deutschland 3 1 kardiologie, universität heidelberg, heidelberg; 2 koordinierungszentrum für klinische studien leipzig, universität leipzig, leipzig; 3 kardiologie, universität würzburg, würzburg; 4 institut für herzkreislaufforschung, dortmund; 5 kardiologie, universität marburg, marburg; 6 kardiologie, berlin; 7 institut für frauenspezifische gesundheitsforschung, deutsches herzzentrum berlin, berlin; 8 kardiologie, universität göttingen, göttingen; 9 kardiologie, essen einleitung: ältere patienten und frauen sind in großen klinischen studien zur chronischen herzinsuffizienz regelhaft unterrepräsentiert. die vorliegende arbeit analysiert daher die geschlechts-und altersabhängige leitlinien-ädhärenz im rahmen der erhebungen des kompetenznetzwerkes herzinsuffizienz. methodik: wir evaluierten die datensätze aller patienten mit systolischer herzinsuffizienz (lvef echokardiographisch ≤45%) die zwischen 01/2004 und 06/2007 in das kompetenznetzwerk herzinsuffizienz eingeschlossen wurden: n=2333; mittleres alter 62,9±13,5 jahre; 24,8% frauen. die nyha-angepasste, leitliniengerechte verschreibung der pharmakotherapie (=adhärenz) wurde über die einnahmehäufigkeit der herzinsuffizienzmedikation unter berücksichtigung von kontraindikationen erfasst. der guideline-adherence-indicator (gai) wurde für betablocker, ace-hemmer/at1-blocker und aldosteron-rezeptorblocker berechnet (=gai-3), sowie für die zusätzliche indikation für diuretikum und glykosid (=gai-5). ergebnisse: der gai-3 betrug in den nyha klassen i/ii/iii/iv 94/92/88/85% und der gai-5 94/91/72/72% (p für trend jeweils <0.001). die therapie-adhärenz (gai-3 und gai-5) war bei männern höher als bei frauen (p=0.048 und p<0.001). in der multivariablen ordinalen regression waren zwar alter (or pro dekade 0, 91, p=0, 017; und or 0, 79, p<0, 001) und nyha stadium iii-iv (or 0,59 und 0,13, p jeweils <0,001), nicht jedoch das geschlecht (p=0,24 und p=0,53) prädiktoren des gai-3 und gai-5. zusammenfassung: aktuelle daten des kompetenznetz herzinsuffizienz zeigen bei der therapie der chronischen herzinsuffizienz im vergleich zu früheren studien in deutschland hinsichtlich der verordneten substanzklassen eine klinisch hoch relevante zunahme der leitlinien-adhärenz. in zukünftigen studien wird zu klären sein, ob auch leitliniengerechte dosierungen in der klinischen praxis sinnvoll umsetzbar sind. effekt von nt-probnp purpose: the endothelial specific angiopoietin (ang)-tie ligand-receptor system has a key role in regulating vascular integrity and quiescence. the antagonistic ligands ang-1 and ang-2 control the expression of endothelial adhesion molecules and intercellular tight junctions. the role of ang-2 in anca-associated vasculitis (aav) has not been investigated yet. methods: we measured serum ang-2 in 20 healthy controls and 40 patients with aav (15 patients with active aav at initial presentation and during follow-up, 10 patients in stable long-term remission, 10 patients prior to systemic relapse, and 5 patients with active "limited" wg (ent). the disease activity was monitored by the birmingham vasculitis activity score (bvas) and the enumeration of circulating endothelial cells (cecs). for statistical analysis we used one-way anova with dunn´s correction, friedmann and wilcoxon tests, spearman´s rho test and linear regression. results: ang-2 was elevated in active aav (mean 6.1 ± 3.5 ng/ml sem) compared to controls (1.2 ± 1.0 p<0.05). most notably, ang-2 was also elevated in archival serum samples of patients in long-term remission just before systemic vasculitic relapse (2.9 ± 2.2 ng/ml p<0.05). in contrast, ang-2 was normal in patients with stable remission of aav (1.7 ± 0.7 ng/ml and "limited" granulomatous ent disease (1.3 ± 0.5 ng/ml). ang-2 was elevated at initial presentation (6.1 ± 3.5 ng/ml sem) and declined rapidly after immunosuppessive therapy at 1 month (2.4 ± 1.4 ng/ml), 2-3 months (2.7 ± 2.3 ng/ml) and 6 months (1.4 ± 1.0 ng/ml). linear regression analysis demonstrated a strong association of ang-2 with bvas (rs2 = 0.49 p<0.0001) and cecs and (rs 2 = 0.48 p<0.001). these results indicate that ang-2 might be a useful early marker to discriminate systemic vasculitic activity in aav from limited granulomatous ent disease and remission. moreover, ang-2 might also be a mediator of endothelial inflammation and detachment. further investigation of the ang/tie-system in vasculitis is crucial since pharmacologic inhibitors of ang-2 will become available shortly. warfarin-induzierte kalzifikation in mäuseneine neues modell zur untersuchung der interaktion von verkalkungsinhibitoren aim of the study: although hypercholesterolemia, a predominant risk factor of coronary heart disease, is related to cholesterol metabolism, the association between cholesterol metabolism and coronary heart disease is not well known. therefore, this study investigates effects of cholesterol metabolism on coronary heart disease and family history of cardiovascular diseases. methods: in addition to conventional coronary risk factors (age, sex, bmi, arterial hypertension, diabetes mellitus, smoking, family history of cardiovascular diseases, plasma cholesterol) campesterol and sitosterol (indicators of cholesterol absorption) and lathosterol (indicator of cholesterol synthesis) were determined in 40 consecutive men and women with severe aortic stenosis. on coronary angiograms prior to aortic valve replacement extend of coronary heart disease was determined (0, 1, 2, 3-vessel disease). furthermore, a semiquantitative score of the extend of coronary atherosclerotic lesions was determined. results: in patients with a positive history of cardiovascular diseases the ratio of campesterol to lathosterol was significantly increased (p<0.005). there was a significant increase in campesterol to lathosterol ratio in plasma with increasing extend of coronary heart disease (0, 1, 2, 3-vessel disease; p<0.05). furthermore, there was a positive correlation of coronary vessel score with the ratio of campesterol to lathosterol in plasma (r=0.52; p<0.001) and in aortic valve cusps (r=0.33; p<0.03), indicating that enhanced absorption and reduced synthesis is related to the extend of coronary heart disease. logistic regression analysis revealed that of all coronary risk factors tested the ratio of campesterol to lathosterol was the sole, significant predictor of coronary heart disease in this subset of patients (p<0.01). the results of this study suggest that in patients with severe aortic stenosis elevated ratios of campesterol to lathosterol are directly related to concomitant coronary heart disease and may serve as a predictor for coronary heart disease in humans. bei postmenopausalen frauen und bei männern mit rheumatoider arthritis und glukokortikoid-therapie besteht zu über 80 prozent die indikation für eine bisphosphonat-therapie nach dvo-leitlinie hintergrund: ein geringes ansprechen auf clopidogrel nach koronarer stentimplantation ist assoziiert mit einem erhöhten risiko für das auftreten von stentthrombosen. die cytochrom p450 (cyp) isoenzyme cyp2c19 and cyp3a4/5 sind bei der bioaktivierung von clopidogrel beteiligt. das ziel der vorliegenden studie ist es, den zusammenhang zwischen dem ansprechen auf clopidogrel und genetischer varianten der cyp isoenzyme bei patienten mit symptomatischer koronarer herzerkrankung zu untersuchen. methoden und ergebnisse: genotypisierungen für cyp2c19 (*2, *3, *17), cyp3a4*1b und cyp3a5*3 polymorphismen wurden bei konsekutiv eingeschlossenen patienten (n=237), die einer koronare stentimplantation aufgrund einer symptomatischen koronaren herzerkrankung erhielten, durchgeführt. die adenosin diphosphat (adp)-induzierte thrombozytenaggregation wurde frühestens 6 stunden nach erstgabe von 600 mg clopidogrel gemesen. träger der cyp2c19*2 variante zeigten eine signifikant höhere residuelle thrombozytenaggregation (rta) (chi-quadrat 29; p<0.0001). die übrigen untersuchten polymorphismen zeigten keinen signifikanten einfluß auf die rta. auf der grundlage des predict-score zur vorhersage einer erhöhten rta wurden der cyp2c19*2 genotyp und zuvor identifizierte nicht-genetische einflussvariablen (alter, akutes koronarsyndrom, typ 2 diabetes, reduzierte linksventrikuläre funktion, und niereninsuffizienz) untersucht. die logistische regressionsanalyse ergab eine signifikante korrelation der nicht-genetischen risikofaktoren (chi-quadrat 5,3; p=0,021) und des cyp2c19*2 polymorphismus (chi-quadrat 21,3; p<0,0001) mit einer erhöhten rta, und einen kumulative assoziation zwischen rta und der kombination aus genetischen und nicht-genetischen faktoren (chi-quadrat 25,85; p <0,0001). diskussion: träger von zumindest einem cyp2c19*2 allel haben ein ungefähr 4-faches risiko, eine erhöhte rta zu entwickeln. genotypisierung der funktionsverlust-variante cyp2c19*2 könnte zu einer verbesserten prädiktion des ansprechens auf die antithrombozytäre therapie mit clopidogrel beitragen. the shape of the glucose curve during an oral glucose tolerance test correlates with changes in glucose tolerance m. reimann 1 , j. li 1 , s. bornstein 1 , j. schulze 2 , p. schwarz 1 1 medical clinic iii, endocrinology, medical faculty carl gustav carus, technical university dresden, dresden; 2 sächsische landesärztekammer, dresden introduction and objectives: the shape of the glucose curve during an oral glucose tolerance test (ogtt) can be categorized in monophasic and biphasic. the latter has been associated with normal glucose tolerance. the aim of the study was to explore the association between the shape of the glucose curve and changes of glucose tolerance. research design and methods: ogtt data from 615 subjects with different stages of glucose tolerance were analyzed at baseline and three years follow up. the shape of the glucose curve at baseline was classified as monophasic, biphasic and unclassified. the shape index was calculated as the difference between glucose at 120 min and at 90 min and treated as continuous variable in correlation analyses. in the biphasic group, there was a significantly higher proportion of subjects with normal glucose tolerance and a lower proportion of subjects with impaired glucose tolerance and type 2 diabetes. subjects with a biphasic shape had a significant lower bmi and a better profile of carbohydrate metabolism. the shape index correlated significantly with changes of plasma glucose at baseline and at 120 min and insulinauc after adjustment for glucose tolerance state. the prevalence ratio for disease regression was significantly higher in subjects with biphasic glucose curve shape. the shape index gives additional information regarding the stage of glucose tolerance beyond the who classification. a biphasic shape is associated with improvements in plasma glucose and may predict regression of disease irrespective of glucose tolerance. die residuelle aggregation unter dualer thrombozytenhemmung beeinflusst die inzidenz schwerer kardiovaskulärer ereignisse unabhängig vom einsatz medikamentenbeschichteter stents kritisch kranke patienten weisen häufig eine charakteristische konstellation des thyreotropen regelkreises auf, die als non-thyroidal-illness-syndrom (ntis) bezeichnet wird und mit einer erhöhten morbidität und mortalität verbunden ist. trotz langjähriger forschung zu details dieses komplexes sind wichtige fragen noch immer offen. so ist bislang noch unbekannt, inwiefern vorbestehende schilddrüsenerkrankungen zur entstehung und ausprägung eines ntis beitragen. im rahmen der prospektiven aqua-fontis-studie untersuchten wir daher 164 patienten, die auf einer internistischen, einer chirurgischen und einer herzchirurgischen intensivstation des universitätsklinikums bergmannsheil versorgt wurden, bezüglich des auftretens von autoantikörpern gegen thyreoglobulin (tgak), schilddrüsen-peroxidase (tpo-ak) oder tsh-rezeptoren (trak). die antikörpertiter, die wir 24 stunden nach aufnahme auf die intensivstation bestimmten, korrelierten wir mit parametern der schilddrüsenhomöostase, der dauer des stationären aufenthaltes und dem überleben der patienten. über 98% der untersuchten patienten wiesen negative oder intermediäre tgakoder tpo-ak-titer auf, weniger als ein prozent zeigten positive antikörpertiter. ausnahmslos alle untersuchten patienten waren trak-negativ. die höhe sämtlicher antikörpertiter korrelierte weder mit dem auftreten einer thyreotropen insuffizienz noch mit der dauer des stationären aufenthaltes oder der intensivbehandlung und auch nicht mit dem überleben der betroffenen patienten. auch in der subgruppe der überlebenden patienten korrelierten die antikörpertiter nicht mit der dauer der behandlung. es kann daher festgestellt werden, dass bei kritisch kranken personen schilddrüsenautoantikörper mit annähernd der gleichen prävalenz wie bei einer normalpopulation auftreten. darüber hinaus haben autoimmunthyreopathien keinen stellenwert in der prognose von patienten mit ntis. typ-2-diabetes mittels ekg identification of markers for prediction of the clinical course remains a major challenge in the management of diabetic nephropathy. we established a proteomics approach for identification of diabetic nephropathy related biomarkers in urine. we used seldi-tof mass spectrometry and sax2 protein arrays to compare protein profiles from urine of four defined patient groups. samples from patients with type 2 diabetes (dm) (n = 45) without nephropathy and without microalbuminuria (dm-wnp), dm patients with macro-or microalbuminuria (dm-np) (n = 38), patients with proteinuria due to non-diabetic renal disease (n = 34), and healthy controls (n = 45) were analysed. anionic exchange, reversephase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. a protein with m/z 6188 (p <0.0000004) was strongly released in the urine of healthy controls, patients with proteinuria due to non-diabetic disease, and dm-wnp in contrast to dm-np-patients. a m/z 14766 protein (p <0.00008) was selectively excreted in the urine of dm-np patients, whereas the protein with m/z 11774 (p <0.000004) was significantly excreted by patients with proteinuria and dm-np. the m/z 11774 and m/z 14766 mass peaks were identified as beta-2-microglobulin and uba52, an ubiquitin ribosomal fusion protein respectively. the protein with m/z 6188 was identified as a processed form of ubiquitin. moreover the ubiquitin degradation assay confirmed the potential role of a urinary protease whose absence was specific for diabetic nephropathy. the release of high amounts of uba52 in urine of dm-np patients could serve as a diagnostic marker. the identification of the protease and longitudinal studies in larger patient groups will determine the usefulness of the short form of ubiquitin as a marker for predicting the clinical course and the potential role of the protease in the pathophysiology of diabetic renal involvement. rolle von freien fettsäuren und fettsäuretransportproteinexpression in der apoptosevermittelten pathogenese der fettlebererkrankung extravascular lung water index (elwi) has been demonstrated to predict mortality and to correlate to pao2/fio2-ratio and compliance in patients with sepsis and ards. however, with an increasing number of obese patients, there is the question which body weight should be used for indexation of extravascular lung charité -universitätsmedizin berlin, campus virchow-klinikum, berlin; 2 koordinierungszentrum für klinische studien würzburg therefore it was the aim, to investigate the correlation of elwi to pao2/ fio2-ratio and oxygenation index (mean airway pressure* 100/pao2) using different weight parameters for indexation. methods: in 25 patients of a medical icu with a body mass index >25kg/m2, 260 measurements of extravascular lung water were performed using the picco system elwi correlated to the functional parameters with high significance (p<0.001) independently of the the index used: correlation to pao2/fio2-ratio conclusions: 1.) in obese patients, extravascular lung water and its indices adjusted to abw, pbw, ibw and adpw significantly (p<0.001) correlate to pao2/ fio2-ratio and oxygenation index. 2.) the highest correlation to pao2/fio2-ratio was found using adbw unsere fallsammlung zeigt den facettenreichtum seltener pilzinfektionen. weitere zentren sind eingeladen, ihre fälle unter www.fungiscope.net zu registrieren ps47, ps60, ps99 ps3, ps15, ps16 ps31, ps42, ps46 ps254, ps255, ps258, ps259 ps237 trinkmann, frederik . . ps95, ps174 ps58 van den elsen antithrombozytäre effekte von ace-hemmern und at1-blockern: modifizierte ex-vivo-plättchenaggregation bei 418 kardiovaskulären patienten a. viktor 1 , i. tuleta 2 , m. steinmetz 2 , g. bauriedel 3 , g. nickenig 2 , d. skowasch 2 1 abteilung für kardiologie und pulmologie, zentrum für innere medizin, stuttgart; 2 innere medizin (kardiologie/pneumologie), universitätsklinikum bonn, medizinische klinik ii, bonn; 3 innere medizin (kardiologie), klinikum meiningen, medizinische klinik i, meiningen hintergrund: ace-hemmer und at1-rezeptorblocker (arbs) sind eckpfeiler in der therapie kardiovaskulärer patienten. in mehreren randomisierten und plazebo-kontrollierten studien konnte eine signifikante verminderung von kardiovaskulärer mortalität, myokardinfarkt-und schlaganfallrate nachgewiesen werden. vor diesem hintergrund untersucht diese studie mögliche antithrombozytäre effekte von ace-hemmern und arbs; vergleichskollektive sind patienten mit ass/clopidogrel und unbehandelte patienten. methoden: proben von insgesamt 418 kardiovaskulären patienten wurden mittels vollblutaggregometrie analysiert. dabei war die agonisten-induzierte plättchenaggregation (adp, kollagen) durch die zunahme der impedanz (ohm) quantifiziert. die daten wurden mit vorliegen bzw. fehlen der medikation korreliert. ergebnisse: als zentraler befund war die plättchenaggregation bei studienteilnehmern mit ace-hemmern, arbs, ass und clopidogrel vermindert. die kollagen-induzierte plättchenaggregation wurde unter ace-hemmern (23,1%, p<0,001) und arbs (33,4%, p<0,001) signifikant reduziert; unter therapie ass (35,6%, p<0,001) bzw. ass/clopidogrel (26,9%, p=0,234) nahm sie ebenfalls ab. nach adp-induktion war die plättchenaggregation unter therapie mit ace-hemmern (36,6%, p=0,003) und arbs (38,7%, p=0,011) signifikant reduziert; unter ass (38,7%, p=0,007) und ass/clopidogrel (82,8%, p=0,011) zeigte sich ebenfalls eine reduktion. eine zusätzliche verlaufsbeobachtung nach neueinstellung mit dem wirkstoff valsartan zeigte nach 28 tagen eine signifikant reduzierte kollagen-induzierte plättchenaggregation (36%, p=0,006). eine in vitro-messreihe mit der rohsubstanz valsartan konnte für äquivalente therapeutische dosen keine signifikante beeinflussung zeigen. folgerung: die therapie mit arbs und ace-hemmern führt zu einer signifikanten verminderung der plättchenaggregation ex vivo. das antithrombotische potential der arbs und ace-hemmer könnte für die reduktion der konsekutiven thrombosen und der progression atherosklerotischer organleiden mitverantwortlich sein und so die reduktion kardiovaskulärer endpunkte zumindest miterklären. kombination einer dendritischen zellvakzine mit gemcitabin im murinen pankreaskarzinom:charakterisierung der immunantwort und wirksamkeit m. dauer with or without the tlr-9 ligand cpg before (prophylactic) or after (therapeutic) tumor induction. cd8+ t cell responses were analyzed in peripheral blood. antigen uptake, activation and cytokine production of dendritic cells (dc) in lymph nodes was analyzed by flow cytometry. data: vaccine draining lymph nodes increased in size and cellularity. the iscom vaccine was effectively taken up by dc, resulting in upregulation of activation markers, production of il-12 and potent t cell stimulation. key: cord-000794-l565gha4 authors: yan, huan; zhong, guocai; xu, guangwei; he, wenhui; jing, zhiyi; gao, zhenchao; huang, yi; qi, yonghe; peng, bo; wang, haimin; fu, liran; song, mei; chen, pan; gao, wenqing; ren, bijie; sun, yinyan; cai, tao; feng, xiaofeng; sui, jianhua; li, wenhui title: sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus date: 2012-11-13 journal: elife doi: 10.7554/elife.00049 sha: doc_id: 794 cord_uid: l565gha4 human hepatitis b virus (hbv) infection and hbv-related diseases remain a major public health problem. individuals coinfected with its satellite hepatitis d virus (hdv) have more severe disease. cellular entry of both viruses is mediated by hbv envelope proteins. the pre-s1 domain of the large envelope protein is a key determinant for receptor(s) binding. however, the identity of the receptor(s) is unknown. here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-s1 specifically interacts with sodium taurocholate cotransporting polypeptide (ntcp), a multiple transmembrane transporter predominantly expressed in the liver. silencing ntcp inhibited hbv and hdv infection, while exogenous ntcp expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. moreover, replacing amino acids 157–165 of nonfunctional monkey ntcp with the human counterpart conferred its ability in supporting both viral infections. our results demonstrate that ntcp is a functional receptor for hbv and hdv. doi: http://dx.doi.org/10.7554/elife.00049.001 approximately 2 billion people have been infected with human hepatitis b virus (hbv) worldwide. over 350 million people currently are chronically infected and are at high risk for progression to cirrhosis, liver failure, or cancer. more than 50% of liver cancers worldwide are attributed to hbv infection. hbv-related liver diseases remain a major public health problem, causing approximately 1 million deaths per year. individuals coinfected with hbv and hdv are at greater risk for rapid progression and severe disease (lavanchy, 2004; hughes et al., 2011) . despite its enormous medical and social relevance, progress in hbv research has been impeded by the lack of understanding of hbv entry by which the virus specifically infects human liver cells. hbv is an enveloped virus containing a small genome of 3.2 kb of partially double-stranded dna encoding four overlapping reading frames. the hbv envelope consists of the small (s), middle (m), and large (l) envelope proteins, which are multiple transmembrane spanners sharing the same c-terminal domain corresponding to the s protein but differing at their n-terminal domains ( figure 1a ) (heermann et al., 1984; seeger et al., 2007) . hdv is a small satellite rna virus of hbv carrying all three hbv envelope proteins and can only propagate when coexisting with hbv. the mechanism of viral entry of hdv is believed to be similar to that of hbv, and hdv has been used as a surrogate for studying hbv infection at the entry level (barrera et al., 2004; sureau, 2006; hughes et al., 2011) . the l protein and integrity of s protein are critical for hbv photoreactive ligand peptides for identification of interacting protein(s) of pre-s1 domain of l envelope protein to identify the pre-s1 interacting molecule(s), we employed a photo-cross-linking approach using a synthetic peptide derived from the native pre-s1 peptide with particular residues replaced by elife digest liver diseases related to the human hepatitis b virus (hbv) kill about 1 million people every year, and more than 350 million people around the world are infected with the virus. some 15 million of these people are also infected with the hepatitis d virus (hdv), which is a satellite virus of hbv, and this places them at an even higher risk of liver diseases, including cancer. the viruses are known to enter liver cells by binding to receptors on their surface before being engulfed. both hbv and hdv have outer coats that consist of three kinds of envelope proteins, and a region called the pre-s1 domain in one of them is known to have a central role in the interaction between the viruses and the receptors and, therefore, in infecting the cells. however, the identity of the hbv receptor has remained a mystery. now yan et al. have identified this receptor to be sodium taurocholate cotransporting polypeptide. this protein, known as ntcp for short, is normally involved in the circulation of bile acids in the body. in addition to humans, only two species are known to be susceptible to infection by human hbv and hdv-chimpanzees and a small mammal known as the treeshrew. yan et al. started by isolating primary liver cells from treeshrews, and then used a combination of advanced purification and mass spectrometry analysis to show that the ntcp on the surface of the cells interacts with the pre-s1 domain in hbv. the authors then performed a series of gene knockdown experiments on liver cells of both human and treeshrew origin: when the gene that codes for ntcp was silenced, hbv infection was greatly reduced. moreover, they were able to transfect hepg2 cells-which are widely used in research into liver disease, but are not susceptible to hbv and hdv infection-with ntcp from humans and treeshrews to make them susceptible. similarly, although monkeys are not susceptible to hbv, replacing just five amino acids in monkey ntcp with their human counterparts was enough to make the monkey ntcp a functional receptor for the viruses. in the past, basic research into hbv and the development of antiviral therapeutics have both been hindered by the lack of suitable in vitro infection systems and animal models. now, the work of yan et al. means that it will be possible to use ntcp-complemented hepg2 cells for challenges as diverse as fundamental studies of basic viral entry/replication mechanisms and large-scale drug screening. it is also possible that hbv and hdv infection might interfere with some of the important physiological functions carried out by ntcp, so the latest work could also be of interest to medical scientists working on other diseases related to these infections. research article figure 1 . developing photoreactive peptide ligands and an antibody for identifying pre-s1 binding partner(s) by zero distance cross-linking. (a) schematic diagram of hbv envelope proteins and n-terminal peptides of pre-s1 domain. pre-s1 (2-47): 2-47 th residues of the pre-s1 domain of the l figure 1 . continued on next page nonnatural amino acids (l-photo-leucine, l-2-amino-4,4-azi-pentanoic acid) ( figure 1a) . l-photoleucine contains a photoactivatable diazirine ring. irradiation of ultraviolet (uv) light at 365 nm induces a loss of nitrogen of the diazirine ring and yields a reactive carbene group with short half-life for covalent cross-linking at nearly zero distance (suchanek et al., 2005) . primary hepatocytes isolated from treeshrews (tupaia belangeri), the only species susceptible to human hbv infection other than humans and chimpanzees (su et al., 1987; walter et al., 1996; glebe et al., 2003) , were used as target cells. to maximize the efficiency of photo-cross-linking, two residues (leu 11 and phe 14 ) in a region (aa 9-15) known to be critical for viral infection (schulze et al., 2010) were chosen for substitution with l-photoleucine. leu 11 is 100% conserved among hbv genotypes, and the 14th residue is a phenylalanine in most genotypes but a leucine in some hbv strains of genotypes f and g. changing phe 14 to leucine (f14l) did not significantly affect the binding of hdv virion to primary tupaia hepatocytes (pths) ( figure 1b) . the activity of the synthesized peptide ligand myr-47/wt b (or wt b hereafter) containing photo-leucines at positions 11 and 14 was also confirmed ( figure 1c,d) . wt b inhibited hdv binding to pths with efficiency comparable to myr-47/wt that is comprised of all natural amino acids ( figure 1a,c) . a peptide myr-47/n9k b (or n9k b hereafter) similar to wt b but with an additional mutation at the ninth residue (n9k) did not block hdv binding to pths ( figure 1c ). wt b but not n9k b inhibited viral infection of hbv and hdv on pths ( figure 1d ). both wt b and n9k b peptides were myristoylated at the n-terminus and conjugated with a biotin tag on a c-terminal lysine residue ( figure 1a ). n9k b differs from wt b by only one amino acid but completely lost these blocking activities. thus, n9k b was used as a negative control for wt b . in addition, a monoclonal antibody (mab) 2d3, which specifically recognizes an epitope adjacent to the critical receptor-binding region of the peptides and shared by both wt b and n9k b , was developed ( figure 1e ). identification of ntcp as a specific binding protein of pre-s1 the wt b or control n9k b peptide at 200 nm was then applied to pths in culture and near zero distance cross-linking was induced by uv irradiation. the cross-linked peptide and associated partners were precipitated by streptavidin t1 beads and separated by sds-page. western blotting using 2d3 as a probe revealed several bands including a major smeared band with apparent molecular weight of ∼65 kda in the wt b but not n9k b cross-linked sample. the 65-kda band shifted to ∼43 kda upon treatment with the deglycosylation enzyme pngase f (figure 2a, left) , indicating that it is highly n-glycosylated. the wt b cross-linked protein apparently contained no intermolecular disulfide bonds as it migrated similarly under both nonreducing and reducing conditions (figure 2a, right) . the non-photoreactive myr-47/wt peptide but not its n9k mutant peptide effectively competed with wt b for cross-linking to the 65-kda band ( figure 2b) . the cross-linked protein from pths decreased in abundance rapidly over time during culture ( figure 2c) . we also examined primary human hepatocytes (phhs) in the crosslinking experiments. bands with slightly smaller molecular weights than those seen in the pth cells were also observed in phhs ( figure 2d) . we then proceeded to identify the target protein(s) using affinity purification followed by mass spectrometry (ms) analysis. the purification procedure included three tandem steps after photo-cross-linking: protein of hbv (s472 strain, genotype c). residue numbering is based on genotype d. asterisk indicates highly conserved residues among genotypes. epitope of mab 2d3 was shaded in gray. (b) effect of alterations of the critical n-terminal residues within pre-s1 region of l protein on hdv binding to pths. both wild-type (wt) and mutant hdv virions carry hbv envelope proteins. mutant hdv carries point mutation as indicated in the pre-s1 region of l protein. pths were incubated with hdv at 16°c for 4 hr and followed by extensive wash; bound virions were quantified by qrt-pcr for virus genome rna copy, and the data are presented as percentage of virus binding, the binding of wt virus was set as 100%. (c) myr-47/wt b bait peptide dosedependently inhibited hdv virion binding. the binding assay was performed similarly as panel b except that pths were pre-incubated with indicated peptides. (d) inhibition of viral infection by the photoreactive peptides. left: pths were pre-incubated with peptides at indicated concentrations at 37°c for 1 hr and then inoculated with hdv virus. viral infection was examined by measuring viral rna in infected cells with qrt-pcr 6 days post-infection (dpi). data are presented as percentage hdv infection. right: peptides at indicated concentrations were added to pths before hbv inoculation. the cell culture medium was replenished every 2 days. secreted viral antigen hbeag was measured by elisa on 6 dpi, and the data are presented as percentage of that in the absence of peptides. (e) antibody 2d3 recognizes residues 19-33 of pre-s1. peptide nc36 (aa 4-36 of pre-s1, nlsvpnplgffpdhqldpafgansnnpdwdfnp) conjugated with keyhole limpet hemocyanin (klh) was the immunogen peptide for generating mouse mab 2d3. binding activity of 2d3 with full-length pre-s1 protein was measured by elisa in the presence of competition peptides at indicated concentrations. ld15 peptide compassing residues 19-33 of pre-s1 inhibited 2d3 binding in a dose-dependent manner, indicating that 2d3 recognizes an epitope within this region. hbv: hepatitis b virus; mab: monoclonal antibody; hdv: hepatitis d virus; pth: primary tupaia hepatocytes; hbeag: hbv e antigen. doi: 10.7554/elife.00049.003 ) or myr-47/n9k b (n9k b ), followed by streptavidin dynal t1 beads precipitation and western blot analysis using mab 2d3. the protein cross-linked by wt b is sensitive to pngase f treatment and shifted from ∼65 to ∼43 kda. right: wt b cross-linked samples were treated with 100 mm dtt and/or pngase f as indicated and detected similarly as in the left panel. (b) non-photoreactive myr-47/wt peptide (wt) but not its n9k mutant competed with 200 nm of wt b peptide for cross-linking with pths in a dose-dependent manner. (c) the abundance of the target protein(s) in pth cells decreased over time. pths on different days of in vitro culturing were photo-cross-linked with 200 nm wt b . the cross-linked samples were analyzed by western blot. the two bands at ∼65 and ∼43 kda were due to incomplete deglycosylation by pngase f. (d) wt b cross-linking with primary human hepatocytes (phh). frozen phh cells were thawed and plated 1 day before cross-linking. with same procedure as in panel a, 200 nm wt b but not n9k b cross-linked with a glycoprotein of molecular weight at ∼60 kda, which shifted to ∼39 kda upon pngase f treatment. (e) purification of target protein(s) for ms analysis. pths photo-cross-linked with 200 nm of wt b or n9k b peptide were lysed, then the peptides and their cross-linked proteins were purified in tandem with streptavidin dynal t1 beads, mab 2d3 conjugated beads, and streptavidin dynal t1 beads in 1× ripa buffer. extensive wash was applied for each purification step. the samples were treated with or without pngase f as indicated prior to the last step of streptavidin beads precipitation. the final purified samples were subjected to sds-page followed by silver staining (left). bracketed areas indicate the bands cut for ms analysis. western blot analysis (right) of the same cross-linked samples were performed similarly as in panel a. the top 10 nonredundant proteins identified in the 3 samples by ms analysis are listed in figure 2 -source data 1. the common protein hit identified by ms analysis of the ∼65and ∼43-kda bands cut from the wt b cross-linked sample was tupaia ntcp (tsntcp), and the representative ms/ms spectra and parameters of the peptide hits are shown in figure 2 -figure supplement 5. the control band cut from n9k b cross-linked sample did not generate any hits on any of these peptides. (f) predicted tsntcp protein sequence. a 30-amino acid insertion unique to tsntcp is underlined. two peptides identified by lc-ms/ms were highlighted in green. all lysine and arginine are highlighted in red to figure 2 . continued on next page capturing all biotin-labeled proteins with streptavidin t1 beads, sorting out the target protein(s) with 2d3 antibody affinity beads, and then purifying with streptavidin t1 beads again to remove residual molecules that were not covalently cross-linked with the bait peptide. the purified samples were subsequently subjected to sds-page followed by silver staining. similar to the western blotting results with the 2d3 antibody, a ∼65-kda protein band was visible by silver staining. the band was also shifted to ∼43 kda upon pngase f treatment ( figure 2e ). both the original 65-kda and the shifted 43-kda bands were subsequently excised from the gel and subjected to ltq-orbitrap velos (thermo fisher scientific, ma. usa) ms analysis after trypsin digestion. the tandem mass spectra were searched against a tupaia hepatocyte protein database, which we had established by deep sequencing of the transcriptome (figure 2-figure supplements 1-4) . two different tryptic peptide fragments, which were identified from both the ∼65-kda and ∼43-kda bands ( figure 2-figure supplement 5 ), matched to a protein homolog of human ntcp. tupaia ntcp (tsntcp) shares 83.9% protein sequence identity with its human counterpart and has an insertion of 30 aa near its c-terminus ( figure 2f ). the peptide (teetipgtlgnsth) containing 4 aa of this insertion (underlined) was one of the two peptides identified by the ms analysis at a high confidence level ( figure 2-figure supplement 5) . these data suggest that ntcp is the protein specifically interacting with the wt b bait peptide. confirmation of ntcp as a specific binding protein of pre-s1 we next cloned human and tupaia ntcps and validated the binding of the exogenously expressed ntcps with the wt b peptide and an n-terminal myristoylated pre-s1 peptide with native residues. both human ntcp (hntcp) and tsntcp could be efficiently cross-linked by wt b but not n9k b when expressed in 293t cells as shown by western blotting with the anti-wt b antibody 2d3 as well as an anti-c9 antibody recognizing the c-terminal c9 tag of the recombinant hntcp and tsntcp proteins ( figure 3a ). wt b but not the control n9k b peptide bound to 293t cells expressing a green fluorescent protein (gfp)-tagged tsntcp (tsntcp-egfp) and co-localized with tsntcp-egfp on the cell surface. this binding was readily competed off by the free myr-47/wt peptide ( figure 3b ). moreover, a native pre-s1 peptide specifically recognized the human hepatocellular carcinoma huh-7 cell line transfected with hntcp ( figure 3c ). consistently, huh-7 cells transfected with either tsntcp or hntcps had markedly increased hdv binding to the cells. the myr-47/wt peptide readily competed with binding of the wild-type hdv, whereas a noninfectious mutant hdv virus bearing a single n9k mutation in the pre-s1 domain of its l envelope protein failed to bind either hntcp-or tsntcpexpressing huh-7 cells ( figure 3d) . collectively, these data demonstrated a specific interaction between ntcp and the pre-s1 domain of the l protein, which directly mediates the binding of hdv virions to target cells. to test the requirement of endogenous expression of ntcp for hbv and hdv infection, we first examined the effect of ntcp gene silencing on viral infection of pths. pths were transfected with tsntcp-specific or a control small interfering rna (sirna) prior to viral inoculation. when tsntcp figure 3 . binding of ntcp with n-terminal peptide of pre-s1 and hdv virions. (a) 293t cells transfected with an expression vector or plasmid containing cdna of hntcp or tsntcp fused with a c9 tag at its c-terminus were cross-linked with 200 nm myr-47/wt b or myr-n9k b similarly as in figure 2a at 24 hr post-transfection. cross-linked protein samples were precipitated by streptavidin dynal beads followed by treatment with pngase f as indicated, and then analyzed by western blotting using mab 2d3 or anti-c9 tag antibody. (b) 293t cells transfected with tsntcp-egfp or a control hsdc2-egfp (encoding human heparan sulfate proteoglycan core protein fused with egfp at c-terminus) expression plasmid were incubated with wt b or n9k b in the presence or absence of 200 nm non-photoreactive myr-47/wt as indicated. bound peptides were probed with pe-streptavidin and the colocalization of peptide and ntcp on cell surface was shown in the merged images. (c) facs analysis of pre-s1 peptide binding with hntcp transiently transfected huh-7 cells. 24 hr post-transfection with hntcp or a control plasmid, the cells were stained with 200 nm fitc-pre-s1 (fitc-labeled lipopeptide corresponding to the n-terminal 59-amino acid of pre-s1). the binding was analyzed by flow cytometry. (d) huh-7 cells, after 24 hr of transfection of indicated plasmids, were incubated with wild-type hdv or hdv with a n9k mutation on its l protein. bound virions were quantified by qrt-pcr. the result is presented as fold changes of binding over the background virus binding to pcdna6-transfected cells. mab: monoclonal antibody; tsntcp: tupaia ntcp; ntcp: sodium taurocholate cotransporting polypeptide. doi: 10.7554/elife.00049.011 mrna level was reduced to ∼30% in tsntcp sirna-transfected cells ( figure 4a , upper-left), total hdv rna copies were markedly reduced in these cells comparing to those transfected with control sirna. we further quantified the hdv genome and antigenome rna copies using strand-specific reverse transcription followed by quantitative real-time polymerase chain reaction (qpcr). the hdv antigenome is a circular replication intermediate that is complementary to the genome. it is not present in the inoculum and only appears in infected cells (chen et al., 1986) . as shown in figure 4a (upper-middle panel), both hdv genomic and antigenomic rna copies were greatly reduced in cells transfected with tsntcp-specific sirna but not the control sirna, indicating that tsntcp is required for de novo hdv infection. by contrast, lenti-vsv-g virus infection, for which viral entry is mediated by glycoprotein protein g of vsv, was not affected in the tsntcp-and sirna-transfected cells ( figure 4a , upper-right). these data demonstrate that hdv viral entry requires ntcp. as hdv is enveloped by hbv envelope proteins and can only infect target cells in a single round in the absence of hbv, these data support that tsntcp functions at entry level for viral infection mediated by hbv envelope proteins. we then tested hbv infection on tsntcp knockdown pths. infection with hbv can be assessed by measuring secreted viral antigens hbv s antigen (hbsag) and hbv e antigen (hbeag). hbv inocula may contain residual hbsag that can release and interfere with the detection of newly synthesized hbsag during the first few days of infection. to differentiate de novo hbsag synthesis from the contaminating inoculum, we assayed hbsag secretion over time from days 6 to 12 after infection with the culture medium changed every 2-3 days. in addition, the kinetics of production of hbeag with minimal or no residuals in the inoculum was also examined in the same time course experiment. as shown in figure 4a (lower-left), both hbsag and hbeag levels were markedly reduced by transfection of tsntcp-specific but not a control sirna at all three time points tested, demonstrating that tsntcp expression is required for bona fide hbv infection. to confirm that tsntcp functions at the viral entry level for hbv as it does for hdv, we tested aav8-hbv virus infection on tsntcp knockdown pths. aav8-hbv is a recombinant adenovirus-associated virus containing a 1.05× overlength hbv genome, for which viral entry is mediated by aav8 capsid instead of hbv envelope proteins. aav8-hbv infection of pths can nevertheless transduce the hbv genome into cells and lead to subsequent hbv viral antigen expression. ntcp knockdown did not affect aav8-hbv infection in pths, as shown by the kinetics of hbeag ( figure 4a , lower-right). this result shows that ntcp has no effect on post-entry steps of hbv infection. we next examined the effect of silencing human ntcp on hbv and hdv infections in human hepatocytes. human hepatoma cell line heparg is the only cell line known to date to be susceptible to hbv and hdv infections upon differentiation into a mixture of hepatocyte-like and biliary-like cells (gripon et al., 2002) . heparg differentiation requires a lengthy cell culture procedure, including maintaining undifferentiated cells for 2 weeks before induction, followed by induction with corticoids and dmso for another 2-4 weeks (gripon et al., 2002) . the ntcp mrna level was low in heparg cells before induction when examined on days 5 and 10 after initial plating, but increased dramatically when the cells differentiated after induction (figure 4b, . to examine if the acquired hntcp expression on differentiated heparg cells is required for hdv and hbv infections, the cells were transfected with sirnas targeting hntcp. about 70% hdv infection was reduced by hntcp knockdown as indicated by decreased levels of hdv viral rnas ( figure 4b , upper-right). similarly, hbv infection was also inhibited as indicated by significantly reduced hbeag at multiple time points ( figure 4b , lower-left), as well as viral rnas including the 3.5 kb rna for hbv pre-c and pregenome rna (pgrna) and hbv total rna ( figure 4b , lower-right) quantified at the end of the experiment. we further validated the critical role of hntcp on hbv infection in phhs, the natural host of the virus. consistently, knockdown of hntcp significantly reduced hbv infection, which was correlated with the ntcp mrna knockdown efficiency. both viral antigens and viral rnas were decreased in cells transfected with hntcp-specific sirnas but not with the control sirna ( figure 4c ). taken together, these data demonstrate ntcp as a common key cellular receptor component necessary for hbv and hdv infections of hepatocytes. we then investigated the ability of ntcp to render nonsusceptible cells susceptible to viral infection. ntcp mrna expression is low in human hepatocarcinoma cell lines that are not susceptible to hbv or hdv infection. the levels of ntcp mrna in huh-7 and hepg2 cells were about 10,000 times lower than that in primary human and tupaia hepatocytes ( figure 5a ). we first examined if ntcp expression renders huh-7 susceptible to hdv infection. human ntcp-transfected huh-7 cells supported hdv infection with an efficiency comparable to that of pths; nearly 10% of cells were infected as shown by staining of the hdv delta antigen that mainly locates in cell nuclei, whereas huh-7 cells transfected with a vector plasmid allowed no hdv infection ( figure 5b ). moreover, the infection could be blocked by known hbv entry inhibitors, such as pre-s1 lipopeptide and hepatitis b immune globulin (hbig), demonstrating a genuine infection of hdv mediated by hbv envelope proteins on these cells ( figure 5c ). hdv rnas, including antigenomic rna that is only produced during hdv replication, rapidly increased over time in the infected cells ( figure 5d ). moreover, the infection efficiency correlated with both the inoculation dose of hdv ( figure 5e ) and the expression level of hntcp ( figure 5f ). hdv also infected hepg2 cells transiently transfected with hntcp ( figure 5 -figure supplement 1) as well as a cell line established by g418 selection of hepg2 cells after hntcp transfection, which expresses hntcp stably and could be readily stained by the fitc-pre-s1 peptide ( figure more efficient hbv infection was achieved on stable hepg2-hntcp cells with about 5-10% of the cells being infected as revealed by intracellular staining of hbsag, whereas there was no hbv infection in the parental hepg2 cells ( figure 6a ). hbv infection in the hepg2-hntcp stable cells was further evidenced by the continuously increased production of hbeag during the testing period. hbv entry inhibitors, in particular the myr-59 peptide and 17b9, efficiently inhibited the infection ( figure 6b ). the infection efficiency as evidenced by hbv total and 3.5 kb rna levels correlated with the inoculation dose. moreover, the formation of hbv covalently closed circular dna (cccdna), which is a replicative intermediate and transcriptional template for production of viral rnas, was confirmed by southern blot analysis ( figure 6d ). to further demonstrate that the replicative intermediates of hbv were synthesized de novo in hepg2-hntcp cells after infection, we performed additional time course experiments. hbv viral replicative intermediates, including cccdna, the 3.5 kb hbv rna, as well as the total hbv rna in the hbv-infected hepg2-hntcp cells were quantified at different time points. the cccdna became detectable at 24 hr post-infection. it markedly increased at day 3 post-infection and maintained a relatively stable level for the rest of the time points examined, whereas the formation of hbv cccdna was completely abolished if entry inhibitor myr-59 was included with the initial virus inoculation ( figure 6e) . consistently, hbv rna levels of the 3.5 kb transcript and the total hbv viruses aav8-hbv and lenti-vsv-g. for hdv and hbv, pths were infected at 500 and 100 genome equivalent copies per cell, respectively. the level of hdv viral rnas in infected cells was quantified by qrt-pcr on 6 dpi. strand-specific primers were used to differentiate the hdv genomic and antigenomic rnas (see 'materials and methods'). for vsv-g control virus infection, recombinant lentivirus pseudotyped by vsv-g carrying a luciferase reporter was inoculated to pths 3 days after sirna transfection. the luciferase activity was assessed on 6 dpi. for hbv infection, the kinetics of secreted viral antigens hbsag and hbeag were measured by elisa. the medium was changed every 3 days. for aav8-hbv infection, pths were infected with a recombinant aav8 carrying 1.05× overlength hbv genome. secreted hbeag was assessed on indicated days post-infection. the effect of tsntcp silencing in all viral infections was independently evaluated with a total of four sirnas against tsntcp (see 'materials and methods'). the data shown are the result of a representative sirna out of the four tested. (b) differentiated heparg cells express high level of ntcp mrna and knockdown ntcp in these cells inhibited hdv and hbv infections. hdv and hbv infection of sirna-transfected heparg cells was conducted similarly as in panel a. hdv rna levels in the infected cells were measured on 9 dpi. for hbv infection, secreted hbeag was collected every 2 days as indicated and analyzed by elisa. the copy numbers of hbv total rna and 3.5 kb rna in the infected cells were measured at the end of the experiment, 10 dpi. (c) knockdown hntcp in phhs hampered hbv infection. frozen phhs were thawed and plated 1 day before transfecting with sirnas against hntcp or a control sirna. similar to panels a and b, 3 days after transfection, phhs were inoculated with 100 genome equivalent copies of hbv per cell, and the levels of secreted hbeag were determined at indicated dpi. hbv rnas were quantified at the end of the experiment, 9 dpi. the knockdown efficiency of sirna targeting tsntcp or hntcp shown in panels a-c was determined by real time rt-pcr on day 4 after transfection. ntcp: sodium taurocholate cotransporting polypeptide; hbv: hepatitis b virus; hdv: hepatitis d virus; pth: primary tupaia hepatocytes; tsntcp: tupaia ntcp; sirna: small interfering rna; dpi: days post-infection; hntcp: human ntcp. doi: 10.7554/elife.00049.012 the huh-7 was used to normalize the relative expression levels in other cells. (b) 1 × 10 5 huh-7 cells were transfected with 100 ng hntcp/pcdna6 or a vector control in 24-well plate and maintained in pmm, 24 hr after transfection, transfected cells were infected with hdv at 500 genome equivalent copies per cell. on 8 dpi, hdv delta antigen, which typically locates in nuclei, was stained with 4g5 antibody in green, nuclei were stained with dapi in blue. (c) huh-7 cells transfected with hntcp were infected with hdv similarly as in panel b in the presence or absence of hbv entry inhibitors: hbig (hepatitis b immune globulin), myr-59, and anti-hbsag mab, 17b9. 4g5 was used as an antibody control. hdv rna copies of infected cells were quantified by real-time rt-pcr on 6 dpi. (d) huh-7 cells transfected with hntcp were infected with hdv similarly as in panel b. the hdv viral rnas in infected cells at indicated time points were quantified by real-time rt-pcr. (e) hdv infection with increasing multiplicities of genome equivalents (mge). with 100 ng hntcp/pcdna6, 1 × 10 5 figure 5 . continued on next page transcripts in the infected hepg2-hntcp cells gradually increased during first several days of infection and reached a steady level after day 5 ( figure 6f ). together these data show that ntcp contributes substantially to hbv infection. we next compared the efficiency of hbv infection in hepg2-hntcp cells with that in phhs. as shown by intracellular staining of hbv core antigen (hbcag) on day 8 post-infection, about 10% hepg2-ntcp cells were infected at multiplicities of genome equivalents (mge) of 100, which is comparable to the efficiency of hbv infection of phhs ( figure 6-figure supplement 2) . in contrast to phhs, hepg2-ntcp cells propagate in cultures, thus the actual infection efficiency of hepg2-ntcp cells may be more likely than not underestimated by the observed end-point hbcag staining. we also compared the levels of secreted hbeag and intracellular viral rnas in these two types of cells infected with three inoculation doses. the level of secreted hbeag from hepg2-ntcp appeared to be higher than that in phhs from two donors, whereas the levels of viral rnas per nanogram of total cell rna in both infected cell types are comparable ( figure 6-figure supplement 3) . this may be partially explained by their different abilities in propagation and supporting viral replication and protein expression that would require more detailed studies. efficient hbv infection of phhs or heparg cells in vitro normally requires high dose of virus inoculums, and only limited progeny viruses are produced after infection (gripon et al., 1988; gripon et al., 2002; boehm et al., 2005) . to assess viral particles released from hbv-infected hepg2-ntcp cells, we first quantified viral dna in the medium collected at different time points after the infection. as indicated by drastic decline of viral dna level on day 4 post-infection, the majority of residual viruses from inocula were removed by changing the medium and washing during the first few days of infection. the levels of viral dna in the media resulted from the ongoing infection during days 4-13 post-infection were low (equivalent to ∼1% of input viral dna copies) despite significant amount of hbeag secretion during this period. similarly, only low levels of viral dna were detected in the medium from hbv-infected phhs ( figure 6-figure supplement 4, right) . it is reasonable to speculate that some host factors that are lacking in cell cultures might be needed for efficient viral particles formation or releasing; or some cellular factors in cultures may hinder these processes during infection. the culture medium collected from infected hepg2-ntcp cells was subsequently tested for infection of phhs. in line with the low hbv viral dna level in the medium inoculum, very low number of intracellular hbv total rna copies were detected in phhs on day 13 post-infection ( figure 6-figure supplement 5) , indicating that only very limited hbv infection might have occurred, which may be attributed to the low multiplicity of infection. residues 157 to 165 of hntcp are critical for pre-s1 binding and viral infections we finally investigated the molecular determinants of ntcp for hbv and hdv infections. crab-eating monkey (macaca fascicularis) ntcp (mkntcp) shares high protein sequence identity with hntcp (96.3%) (figure 7-figure supplement 1) . however, mkntcp neither supports hdv infection nor pre-s1 peptide (myr-59) binding ( figure 7a) , consistent with the known narrow species specificity of the we then made a series of human ntcp variants to cover all the different amino acids between hntcp and mkntcp. in each variant, two or a few residues were mutated to their mkntcp counterparts (figure 7-figure supplement 1) . whereas most mutations did not significantly interfere with myr-59 binding or hdv infection, alteration of five residues of hntcp between aa 157-165 and its monkey counterpart (from kgivislvl to griilslvp, distinct residues are underlined) completely abolished myr-59 binding and the ability to support hdv infection. remarkably, replacing the motif of aa 167-156 in mkntcp with the corresponding human residues converted mkntcp to an efficient receptor for hdv infection ( figure 7a) . all the ntcp variants tested were examined for ntcp expression, and comparable levels of cell surface expression were confirmed ( figure 7b ). similar to hdv, hbv infection was also abolished on hepg2 cells expressing hntcp carrying monkey-like mutations, griilslvp, while mkntcp-bearing human residues kgivislvl within the motif of aa 157-165 largely restored hbv infection ( figure 7c) . these data show that residues between 157 and 165 of ntcp are crucial for binding to the receptor-binding region of the pre-s1 domain of the l protein of hbv, and critically contribute to ntcp-mediated hbv and hdv infections. in this study, by employing a unique approach of tandem affinity purification combined with ms analysis against a tupaia hepatocyte proteome database established by deep sequencing, we revealed that the liver bile acid transporter, ntcp, specifically interacts with a key region in the pre-s1 domain of the hbv envelope l protein. by performing a series of virological analyses, we showed that silencing ntcp expression markedly inhibited viral infection of hbv and hdv in tupaia as well as human hepatocytes. exogenous expression of ntcp rendered nonsusceptible human hepatoma cells susceptible to the viral infections. the authentic viral infections in cells complemented with ntcp were shown by the kinetic analyses of several markers of viral infections, in particular the quantification of newly synthesized viral replicative intermediates. moreover, the ntcp-rendered infections were blocked by known entry inhibitors. ntcp residues 157 to 165 were identified to be critical for pre-s1 binding and viral infections. these data clearly demonstrate that ntcp is a functional receptor for both hbv and hdv. identification of cellular receptor(s) of hbv and hdv has been challenging. in our study, we utilized a short peptide ligand, wt b , which was originated from the known receptor-binding domain of the l protein (barrera et al., 2005; glebe et al., 2005; gripon et al., 2005; engelke et al., 2006; schulze et al., 2010) , but with specially designed properties suitable for photo-cross-linking and tandem purification. form) at the mutated positions of ntcp are shown for hntcp, crab-eating monkey ntcp (mkntcp), and tsntcp. huh-7 cells were transfected with plasmids encoding tsntcp, hntcp, mkntcp, or ntcp mutants as indicated. the mutant ntcps include hntcp-bearing mutations of mkntcp residues and mkntcp-bearing mutations of human residues at indicated positions. the transfected cells were maintained in pmm for 24 hr and then either stained with 200 nm fitc-pre-s1 or infected with 500 mge hdv. hdv delta antigen in infected cells was detected with mab 4g5 on 7 dpi. replacing aa 157-165 of mkntcp with human counterpart rendered mkntcp an efficient receptor for pre-s1 binding and hdv infection. (b) all ntcp variants expressed comparable levels of ntcp. huh-7 cells transfected as in panel a were biotinylated 24 hr after the transfection, then lysed and analyzed for cell surface ntcp expression (top), total ntcp expression (middle), and gapdh (bottom), respectively. for cell surface expression, cell lysates were pulled down with streptavidin t1 dynabeads and subsequently examined by western blot with mab 1d4 recognizing a c9 tag at the c-terminus of each ntcp variant. for total ntcp expression, cell lysates were directly subjected to sds-page, followed by western blot analysis with 1d4. (c) effects of ntcp mutations on hbv infection. hepg2 cells were transfected with plasmids encoding hntcp, mkntcp, or hntcp variants bearing the indicated monkey residues, or mkntcp variants with the indicated human residues. transfected cells were maintained in pmm for 24 hr, and subsequently infected with hbv at 100 mge. hbeag and hbv 3.5 kb rna were assayed on 6 dpi. similar to panel b, comparable ntcp surface expression levels in the transfected hepg2 cells were confirmed for all the ntcp variants tested (figure 7-figure supplement 2 figure 7c were analyzed for total or cell surface ntcp expression at 24 hr post-transfection as described in figure 7b . doi: 10.7554/elife.00049.025 two photo-leucines were incorporated into the critical receptor-binding region of wt b without interfering with its receptor-binding activity, which allowed highly specific zero distance cross-linking of its direct binding partner(s) but not other neighboring molecules. a biotin moiety of wt b facilitated purification of the complex of wt b and its binding partner(s) by streptavidin beads. an mab, 2d3, was developed to recognize wt b on an epitope outside the receptor-binding site, serving as a highly specific tool for detection as well as additional affinity purification of the complex. thus, the binding partner(s) was first cross-linked by wt b , and then purified by using streptavidin and 2d3 beads in tandem. the covalent interaction between the wt b ligand and its partner(s) enabled a purification process under high-stringency conditions, and efficient isolation was achieved irrespective of the nature of the binding partner(s) even if it is a membrane protein(s) with multiple transmembrane domains, like ntcp identified here. ntcp (slc10a1) is the founding member of the slc10 family of solute carrier proteins. it is a hepatic na + bile acid symporter and is responsible for cotransportation of sodium and bile acids across cellular membranes to maintain the enterohepatic circulation of bile acids stieger, 2011) . ntcp is a multiple transmembrane glycoprotein presumed to span the cellular membrane up to 10 times with small extracellular loops (mareninova et al., 2005; hu et al., 2011) . it is mainly expressed in the liver (stieger, 2011) , consistent with the liver tropism of hbv and hdv. ntcp localizes to the sinusoidal (basolateral) plasma membrane of hepatocytes (stieger et al., 1994) , a location that fits well with its receptor role for blood-borne hbv and hdv. whereas hbv first attaches to hepatocytes mainly through heparan sulfate (schulze et al., 2007; leistner et al., 2008) , our data demonstrate that the interaction between ntcp and l protein of hbv is highly specific, and ntcp is crucial for productive viral entry of hepatocytes. consistent with previous reports on primary cultures of rat hepatocytes (liang et al., 1993; rippin et al., 2001) , ntcp expression rapidly decreased over time in cultured pths after isolation. this may at least partially explain the observations that primary hepatocytes typically remain susceptible to hbv infections in vitro for only a few days after isolation from liver tissues (gripon et al., 1988; seeger et al., 2007) . ntcp is functionally conserved in mammalians, but protein sequences of ntcp vary among species, which is likely to contribute to the narrow species tropism of viral infection. strikingly, despite the high level of protein sequence homology between human and monkey ntcp, the later did not support hbv and hdv infection. replacing a small motif of aa 157-165 of mkntcp with the corresponding hntcp residues converted mkntcp to a receptor for pre-s1 binding as well as hdv and hbv infection. further studies are warranted to determine if and how ntcp contributes to the species specificity of hdv and hbv infection in other species. it also remains to be determined if other molecule(s) additional to ntcp contributes to the cellular entry of hbv and/or hdv as a coreceptor(s) or receptor component(s), and if other host factors such as the microenvironment or architecture of hepatocytes in liver, or soluble blood components like those that have been shown to involve in infections of other viruses (shayakhmetov et al., 2005; morizono et al., 2011) , contribute to hbv and/or hdv infection. expression and subcellular distribution of ntcp are precisely regulated under physiological conditions. ntcp accounts for most, if not all, hepatic na + -dependent bile acid transport (stieger, 2011) . ntcp expression is low and inversely correlated with the degree of dedifferentiation of cancer cells in human hepatocellular carcinoma (kullak-ublick et al., 1997; zollner et al., 2005) and the severity of hbv-related liver cirrhosis (lee and kim, 2007) . the newly discovered role of ntcp as an entry receptor for hbv and hdv raises interesting questions regarding its involvement in viral pathogenesis. identification of ntcp as a functional receptor for hbv and hdv advances our understanding of their entry into host cells and may lead to new prevention and treatment strategies against these viruses and related diseases. adult tree shrews (tupaia belangeri chinensis) were housed in a tupaia animal facility at the national institute of biological science, beijing. all studies were performed in accordance with institutionally approved protocols and adherent to guidelines of the national institute of biological sciences guide for the care and use of laboratory animals. pth cells were obtained from anesthetized tupaia (100-150 g) with a two-step perfusion method as previously described (walter et al., 1996) . cell suspensions after perfusion were filtered through a 70-μm cell strainer and centrifuged at 50 g for 3 min. the cell pellet containing pths was resuspended in plating medium of williams e medium supplemented with 10% fbs, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 2 mm l-glutamine, 100 u/ml penicillin, and 100 μg/ml streptomycin. the cells were then plated on collagen-coated cell culture dishes or plates. 4 hr after plating, medium were changed to primary hepatocytes maintenance medium (pmm), that is, williams e medium supplemented with 5 μg/ml transferrin, 10 ng/ml egf, 3 μg/ml insulin, 2 mm l-glutamine, 18 μg/ml hydrocortisone, 40 ng/ml dexamethasone, 5 ng/ml sodium selenite, 2% dmso, 100 u/ml penicillin, and 100 μg/ml streptomycin. cells were maintained in 5% co 2 humidified incubator at 37°c with regular medium change every 2-3 days. phhs were purchased from becton dickinson (united states) or shanghai rild inc. (shanghai, china). the cells were cultured similarly as pths using the same plating medium and maintaining pmm medium as described above. human embryonic kidney cell lines 293 and 293t, human cervix carcinoma cell line hela, and human hepatocellular carcinoma cell line hepg2 were from american type culture collection (atcc); human hepatocellular carcinoma cell lines huh-7, smmc-7721 (smmc), and bel-7404 (bel) were from the cell bank of type culture collection, chinese academy of sciences. the cells were cultured with dulbecco's modification of eagle's medium (dmem; invitrogen, united states) supplemented with 10% fetal bovine serum, 100 u/ml penicillin, and 100 μg/ml streptomycin at 37°c in 5% co 2 humidified incubator except otherwise indicated. heparg cells were purchased from biopredic international (rennes, france) and were cultured following the product manual. differentiated heparg cells were obtained following a two-step procedure as described by gripon et al. (2002) . a plasmid containing a head to tail trimer of 1.0× hdv cdna of a genotype i virus (genebank accession number: af425644.1) under the control of a cmv promoter was constructed with de novo synthesized hdv cdna for the production of hdv rnps. a puc18 plasmid containing nucleotide 2431-1990 of hbv (genotype d, genebank accession number: u95551.1), or the same plasmid bearing mutation generated by site-directed mutagenesis, was used for expressing hbv envelope proteins under the control of endogenous hbv promoter. hdv virions were produced by transfection of the plasmids in huh-7 as previously described by sureau et al. (1992) . hbv hbv genotype b virus was obtained by ultracentrifugation of plasma from an hbv chronic carrier with written consent. hbv genotype d virus was produced by transfection of huh-7 cells with a plasmid containing 1.05 copies of hbv genome under the control of a cmv promoter similarly as previously described by blanchet and sureau (2006) . the genebank accession numbers for the viruses are jx978431 and u95501.1, respectively. recombinant adeno-associated virus 8 (aav8) carrying 1.05 copies of hbv genome was produced similarly as previously described (xiao et al., 1998) by cotransfection of 293 cells with plasmids for aav8 packaging, 1.05× overlength hbv genome (genotype d) and adenovirus helper. an hiv-1 genome-based lentivirus pseudotyped by glycoprotein of vesicular stomatitis virus and carrying a firefly luciferase reporter gene was produced by cotransfection of 293t cells with plasmids for vsv-g expression, hiv genome packaging, and luciferase reporter, respectively, as described (sui et al., 2005) . virus-related experiments were conducted in a bsl-2 facility at the national institute of biological sciences, beijing. peptides with nonnatural amino acid l-2-amino-4,4-azi-pentanoic acid (l-photo-leucine) were synthesized by american peptide company inc. (united states). other peptides corresponding to the n-terminal of pre-s1 domain of hbv l protein (genotype c, strain s472, genebank eu554535.1) were synthesized by sunlight peptides (beijing, china). mouse monoclonal antibodies (mab) 2d3, 1c10, and 4g5 were generated in the laboratory; all are of igg1 isotype. 2d3 specifically recognizes the 19-33 amino acids of the pre-s1 domain of hbv l protein; 1c10 recognizes hbcag; 4g5 targets hdv delta antigen. 17b9, a mouse mab-specific to hbv s protein, was provided by dr. lin jiang, china national biotec group. hepatitis b immune globulin (hbig) was from the national institutes for food and drug control, beijing, china. 2d3 magnetic beads were prepared by covalently cross-linking 2d3 to dynabeads m-270 epoxy following manufacturer's instructions. secondary antibodies for immunofluorescence staining and western blot were purchased from invitrogen or sigma-aldrich (united states). elisa kits for hbsag and hbeag measurement were purchased from wantai pharm inc. (beijing, china). sybr premix ex taq quantitative real-time pcr kit and reverse transcriptase (rt) kit were from takara inc. (beijing, china). streptavidin-coupled magnetic beads (dynabeads myone streptavidin t1) and magnetic beads coated in glycidyl ether (epoxy) groups (dynabeads m-270 epoxy) were purchased from invitrogen. other reagents were purchased from new england biolabs (united states), life technologies (united states), or sigma-aldrich. hbv viral antigens hbsag and hbeag were examined using 50 μl supernatants with commercial elisa kits (wantai pharmacy, beijing, china) following manufacturer's instructions. in most cases, hbsag level was normalized with who hbsag reference serum (kindly provided by dr. zhenglun liang from the national institutes for food and drug control, beijing, china) and presented as international units per milliliter. quantification of hdv total rna (genome equivalent) copies and hbv genome equivalent copies hdv viral rna was isolated with trizol reagent following manufacturer's instructions. total rna was reverse transcribed into cdna with random primers (primescript rt kit; takara) and 2 μl of the cdna was used for real-time pcr assay. primers for quantifying hdv total rna or genome equivalent copies are complemented with the delta antigen coding region of hdv rna genome: forward primer hdv-1184f, 5′-tcttcctcggtcaacctctt-3′, and backward primer hdv-1307r, 5′-acaaggagaggcaggatcac-3′. hbv viral dna was isolated by standard genomic dna isolation method. the dna was quantified using specific primers: 5′-gagtgtggattcgcactcc-3′ (forward) and 5′-gaggcgagggagttcttct-3′ (backward) by real-time pcr. the viral genome equivalent copies were calculated based on a standard curve generated with known copy numbers. real-time pcrs were performed using sybr premix ex taq kit on an abi fast 7500 real-time system instrument (applied biosystems, united states). with 5 × 10 7 copies of genome equivalent hdv, 1 × 10 5 of target cells were incubated at 16°c for 4 hr in the presence of 4% peg8000, followed by extensive wash with cold pbs for four times. the cells were then lysed directly with trizol reagent and followed by reverse transcription. rna copy numbers of viral genome and internal control glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrna were determined by real-time pcr. for hdv binding inhibition assay, peptides were pre-incubated with target cells at 16°c for 1 hr before incubating with the virus; antibodies against viral envelope protein were pre-incubated with viruses before adding to target cells. viral infections of hdv and hbv were conducted in 48-well plates at multiplicities of genome equivalents of 500 and 100, respectively. normally, 5 × 10 7 copies of genome equivalent hdv or 1 × 10 7 copies of genome equivalent hbv were inoculated in the presence or absence of entry inhibitors with 1 × 10 5 cells and incubated for 16 hr except otherwise indicated. cells were then washed with medium for three times and maintained in pmm medium with medium change every 2-3 days. for hdv infection, 4% peg 8000 was present during the 16 hours viral inoculation period similarly as described by barrera et al. (2004) . viral infection at different time points was analyzed by measuring viral dna/ rnas and viral antigen expression. quantitative real time rt-pcr was used to quantify hdv total rnas, strand-specific real time rt-pcr to determine copies of hdv genome and antigenome rna (see below). for hbv viral infection on heparg cells and phh, ∼ 4% peg 8000 was present during the inoculation period as previously described by gripon et al. (gripon et al., 1993; gripon et al., 2002; schulze et al., 2007) . viral infection of pth was conducted in the absence of peg8000. culture medium was changed every 2-3 days. secreted hbsag and/or hbeag were determined with commercial elisa kits. real-time pcr, with or without a prior reverse transcription step, was used for quantification of hbv-specific 3.5 kb pre-c and pregenomic rna, total hbv sub-genomic rna, and hbv cccdna copies. the strand-specific qrt-pcr was performed as previously described by freitas et al. (2012) . briefly, the genomic and antigenomic rnas were reverse transcribed separately with strand-specific primers into cdnas: primer hdv398r (5′-cgcttcggtctcctctaact-3′) for genomic rna; primer hdv288f (5′-gcagacaaatcacctccaga-3′) for antigenomic rna. the reverse transcribed cdnas of genmomic or antigenmoic hdv rnas were used as templates for real-time pcr using the hdv398r and hdv288f primer pair. taqman probe was 5′ fam-agagctctgacgcgcgaggagtaagc-tamra 3′. real-time pcr assays were conducted with an abi fast 7500 real-time pcr instrument. total rna from hbv-infected cells was isolated with trizol reagent (invitrogen). about 400 ng total rna was reverse transcribed into cdna with primescript rt kit (takara) in a 10 μl reaction. cdna derived from 20 ng total rna was used as template for real-time pcr amplification. in a separate realtime pcr reaction, 20 ng of total rna was directly used as template to assess the possible hbv viral dna contamination in the rna preparation. primers (hbv2270f: 5′-gagtgtggattcgcactcc-3′) and (hbv2392r: 5′-gaggcgagggagttcttct-3′) were used for hbv 3.5 kb transcripts; (hbv1805f: 5′-tcaccagcaccatgcaac-3′) and (hbv1896r: 5′-aagccacccaaggcacag-3′) were for total hbv-specific transcripts. amplification of 123-bp fragment for 3.5 kb transcripts and 92-bp product for total hbv-specific transcripts were both conducted by denaturation at 95°c for 30 s, followed by 40 cycles of 95°c denaturation for 3 s, and 60°c annealing/elongation for 30 s. real-time pcr was performed using sybr premix ex taq kit on an abi fast 7500 real-time system instrument. real-time pcr using either set of the primers generated highly specific amplification product. hbv rna copy numbers were deduced from a standard curve generated from known nucleic acid quantities. then the hbv rna copy number per nanogram rna in the infected cell cultures was calculated by subtracting the background amplification noise derived from the viral dna contamination in the rna preparation from that cdna amplification. the signal-to-noise ratio for hbv total transcripts is usually ≥50, and for 3.5 kb transcripts ≥20. the real-time pcr detection limits for total hbv-specific transcripts and 3.5 kb transcripts are ∼0.5 and ∼3.5 copies per nanogram cellular total rna, respectively. hbv cccdna southern blot was conducted following a similar procedure as described by summers et al. (1990) with modifications. briefly, to selectively extract hbv cccdna, infected hepg2-ntcp cells in 6-cm dishes were lysed with 1 ml lysis buffer at 37°c for 60 min, followed by addition of 0.25 ml of 2.5 m kcl and incubation at 4°c overnight. the lysis buffer was not supplemented with proteinase k, containing 50 mm tris-hcl, ph 7.4, 10 mm edta, 150 mm nacl, 1% sds. the lysate was then clarified by centrifugation at 12,000 g for 30 min at 4°c and extracted with phenol and phenol:chloroform. dna was precipitated with equal volume of isopropanol in the presence of 20 µg glycogen (roche) and finally dissolved in te buffer. the prepared dna sample was then treated with plasmid-safe adenosine triphosphate (atp)-dependent deoxyribonuclease dnase (epicentre technologies) following manufacturer's instructions. for southern blotting, the plasmid-safe dnase-treated dna was separated on a 1.3% agarose gel and then transferred to a nylon membrane (hybond-n + ; amersham) using a standard neutral transfer procedure. a 3280-bp plasmid constructed by inserting a 588-bp hbv dna fragment (from 1805 to 2392, genotype d, southern blot probe) into a 2692 bp pmd18t vector (takara) was also run on the same agarose gel to serve as the molecular marker for cccdna in southern blot analysis. the plasmid is of similar size of hbv genome and was mainly in supercoiled form; therefore it runs at similar size as hbv cccdna in agarose gel. the nylon membrane was hybridized with a [α-32 p] dctp-labeled hbv probe (genotype d hbv dna fragment from 1805 to 2392) prepared by random primer dna labeling kit (ver.2.0; takara). hybridization was carried out in 7 ml of perfect hyb plus hybridization buffer (sigma) with 1 hr pre-hybridization, followed by overnight hybridization at 67°c. the membrane was then washed once with 2× ssc/0.1% sds, 1× ssc/0.1% sds, and 0.5× ssc/0.1% sds at 67°c for 20 min, respectively. finally, the membrane was subjected to autoradiographic exposure. hbv cccdna (double-stranded dna without nick and gap) was quantified by real-time pcr using a protocol as previously described werle-lapostolle et al., 2004) with modifications. in particular, specific primers for cccdna detection reported by glebe et al. (2003) (ccc-1582f: 5′-tgcacttcgcttcacct-3′; ccc-2316r: 5′-aggggcatttggtggtc-3′) were validated and used for quantifying copy numbers of cccdna using real-time pcr. viral dnas other than cccdna, including single-stranded and relaxed circular dnas, were degraded prior to amplification by treatment of the dna templates with plasmid-safe adenosine triphosphate (atp)dependent deoxyribonuclease dnase (epicentre technologies). in brief, hbv-infected cells were lysed for 4 hr at 65°c in lysis buffer (50 mm tris-hcl, ph 8.0, 50 mm edta, 100 mm nacl, 1% sds) supplemented with proteinase k (200 μg/ml) and followed by phenol-chloroform extraction. a total of 250 ng of the extracted dna was digested with 5-10 units plasmid-safe dnase in a 50 μl volume for 8 hr at 37°c followed by dnase inactivation at 70°c for 30 min. 2 μl of the 50 μl reaction was then added to 20 μl of a real-time pcr reaction. amplification of 735 bp cccdna product was conducted by denaturation at 95°c for 5 min, followed by 45 cycles of denaturation at 95°c for 30 s, 62°c annealing for 25 s, and 72°c elongation for 45 s. hbv cccdna copy numbers were calculated with a standard curve from plasmid with known nucleic acid quantities. the detection limit for cccdna is ∼10 copies cccdna per reaction (equivalent to 10 ng of total cell lysate dna). realtime pcr was performed with sybr premix ex taq kit on an abi fast 7500 real-time system instrument. pth cdna library construction, deep sequencing of tupaia transcriptome, and bioinformatics analysis of illumina deep sequencing-determined transcriptome primary tupaia hepatocytes were isolated as described above. pth mrna was purified from 10 μg of total rna using oligo-dt magnetic beads. the mrna was fragmented into small pieces by incubation with divalent cations at 94°c for exactly 5 min. the first strand cdna was synthesized using random primers and superscript ii reverse transcriptase (invitrogen) with fragmented mrnas. rna template was then removed by rnase h, and double-stranded cdna was prepared with dna polymerase i. cdna with blunt ends was created by t4 dna polymerase and klenow dna polymerase and an 'a' base was subsequently added to the 3′ end of the blunt phosphorylated dna fragments by klenow fragment (3′ to 5′ exo minus). the cdna was then ligated with adapters and then ran on a 2% agarose gel. the fragments with a size range from 200 ± 25 bp were purified, followed by amplification using the manufacturer's primers. the pcr products were then purified using qiaquick pcr purification kit (qiagen), quantified and diluted for cluster generation and deep sequencing. the 72-cycle pair-end sequencing was performed with sequencing kits (version 5) on an illumina genome analyzer iix (illumina, san diego, united states). illumina casava pipeline v1.8.1 was used for sequence extraction and filtering. bioinformatics analysis of illumina deep sequencing-determined transcriptome of primary tupaia hepatocytes de novo reconstruction of transcriptome from cdna library deep sequencing data total 253,919,616-pair 72 nt sequences with 36.6g base from the sequencing results of the hepatocyte cdna library described above were fed to trinity (grabherr et al., 2011) r20110519 using pair end rna-seq protocol, with which 209,063 transcripts of average length of 1421 nt (minimum 300 nt, maximum 21,043 nt, and scaffold n50 of 3674 nt) were generated. following assembly, genscan (burge and karlin, 1997) was used with default parameters to identify coding sequences and the encoded protein sequences of these transcripts. a total of 91,479 protein sequences were identified. each chosen protein sequence was first annotated with its corresponding blastp (camacho et al., 2009 ) matches from ncbi human protein sequences. those not annotated in the first step were then submitted for similar annotation process with uniprotkb human proteome and ncbi nonredundant protein sequence database. protein sequences that were not annotated by previous steps were submitted for annotation with their corresponding transcripts. protein sequences with their corresponding transcripts that can be annotated by the blastx (camacho et al., 2009) hits of ncbi human protein sequences, uniprotkb human proteome or ncbi nonredundant protein sequence database were annotated with these hits from transcripts. all identified protein sequences were included in the hepatocyte protein sequences database. the protein sequences were labeled with corresponding functional annotation results. any identified protein sequences that were not successfully annotated are labeled with 'uncharacterized protein'. total 50,951 annotated and 40,528 uncharacterized protein sequences generated from the tupaia hepatocytes transcriptome were combined into the database. a numeric id (gi) was generated for each protein sequence in the database. the corresponding cdna sequences were deposited to ncbi transcriptome shotgun assembly (tsa) database of genbank with accession number from ju120276 to ju170736 after removal of cdnas shorter than 200 bp and a vector sequence. panther database (mi et al., 2005) was used to determine the protein class distribution of annotated transcripts and protein sequences in primary tupaia hepatocytes (pths) generated in this study and transcriptome from primary human hepatocytes (phhs) reported by hart et al. (2010). photo-cross-linking with peptide ligand and tandem purification of the target molecule(s) l-photo-leucine-bearing wild-type bait peptide (wt b ) or control bait peptide (n9k b ) was dissolved in dmso in dark. l-photo-leucine contains a photoactivatable diazirine ring, irradiation of uv light at 365 nm induces a loss of nitrogen of the diazirine ring, and yields a reactive carbene group with short half-life for covalent cross-linking at nearly zero distance. for tandem purification, wt b or n9k b at indicated concentrations was applied to ∼1 × 10 7 hepatocytes plated on collagen-coated dishes. cells were cross-linked by uv irradiation and then washed to remove residual free peptides and subsequently lysed with 1 ml radioimmunoprecipitation assay (ripa, ph 7.4) buffer containing 20 mm tris, 150 mm nacl, 0.1% sds; 0.5% sodium deoxycholate, 1% np40, and 1× protease inhibitor cocktail (roche). the cell lysates were precipitated with 100 μl streptavidin t1 magnetic beads, eluted with 50 μl nonreducing sds-page loading buffer and then diluted with cold ripa buffer to a final volume of 1 ml and was precipitated with 100 μl (1 × 10 8 ) 2d3-conjugated m-270 dynabeads, then eluted with 100 μl nonreducing loading buffer. the elute, with or without pngase f treatment, was diluted to 1 ml with ripa buffer and was precipitated again with 100 μl streptavidin t1 magnetic beads, followed by extensive washing, and finally eluted by boiling 5 min with 20 μl sds-page loading buffer. the samples were then analyzed with 12% sds-page and silver staining. for photocross-linking analysis of primary cells, cell lines, or ntcp-or control plasmid-transfected huh-7 or 293t cells, wt b or n9k b bait peptide in the presence or absence of competing peptide was applied to 2 × 10 6 cells, photo-cross-linking was conducted similarly as described above. the cross-linked samples were precipitated with streptavidin t1 magnetic beads and separated by sds-page, and followed by western blotting with mab 2d3 (recognizing bait peptides) or mab 1d4 against c-terminal tag c9. silver stained gel bands were cut, followed by in-gel reduction, alkylation, and trypsin digestion as previously described (shevchenko et al., 2006) . digested peptide mixtures containing 0.1% formic acid were loaded onto a 4 cm, 75-μm inner diameter fused silica capillary column packed with 10-μm ymc c18 material (ymc, kyoto, japan). after desalting, samples were separated with a waters nano acquity ultraperformance lc (waters, united states) and eluted to a ltq-orbitrap velos mass spectrometer (thermo fisher scientific, united states). the uplc separation gradient included a 30-min gradient from 0% to 30% acetonitrile, followed by a 10-min gradient to 80% acetonitrile, then 10 min of 80% acetonitrile and back to 0% acetonitrile within 5 min. the mass spectrometer was operated in the data-dependent mode. survey ms scans were acquired in the orbitrap with the resolution set to a value of 60,000. each survey scan (300-2000 m/z) was followed by four data-dependent cid tandem mass (ms/ms) scans at 35% normalized collision energy and four data-dependent hcd tandem mass (ms/ms) scans at 40% normalized collision energy with 15,000 resolution in orbitrap. agc target values were 500,000 for the survey scan, 10,000 for the ion trap ms/ms scan, and 50,000 for the orbitrap ms/ms scan. target ions already selected for ms/ms were dynamically excluded for 30 s. tandem mass spectra were searched against the illumina deep sequencing-determined tupaia hepatocyte protein database that was concatenated with reversed sequences to estimate false positives and was supplemented with the sequence of bait peptide under the linux operating system using the prolucid (xu et al., 2006) protein database search algorithm with peptide mass tolerance of ±100 ppm, fragment ion mass tolerance of ±400 ppm, half tryptic specificity, and a static modification of 57.0215 on cys due to carboxyamidomethylation. prolucid search results were then filtered with dta select 2.0 (tabb et al., 2002) using a cutoff of 1% for peptide false identification rate (-fp 0.01). peptides with deltamass > 10 ppm (-dm 10) were rejected; the minimum number of peptides to identify a protein was set to 1 (-p 1). immunofluorescence microscopy and facs analysis of ntcp binding with pre-s1 peptides plasmid encoding human, or tupaia ntcp, with or without a tag, or a control plasmid was transfected into cells. the cells were washed and blocked with 3% bsa 24-36 hr after transfection and then stained with biotin-labeled peptides at 4°c about 1-2 hr, followed by fixation with 4% paraformaldehyde (pfa) for 10 min. cells were then stained with pe-labeled streptavidin (ebioscience). in some cases, myr-59 peptide containing the first 59 residues of pre-s1 domain with an n-terminal myristoylation modification and labeled with fitc (fitc-pre-s1) was applied to cells directly. the cell images were captured with a nikon eclipse ti fluorescence microscope or a zeiss lsm 510 meta confocal microscope. for facs analysis, the fitc-pre-s1 stained cells without fixation were detached with 0.5 mm edta/pbs, washed and resuspended in pbs, and analyzed with a facs lsrii instrument (bd). four sirnas (tsntcp-si1: 5′-cuauguaggcauugugauadtdt-3′, tsntcp-si2: 5′-guguuauccu ggugguuaudtdt-3′, tsntcp-si3: 5′-ggacaugaaucucagcauudtdt-3′, tsntcp-si4: 5′-gggcaagagcaucauguuudtdt-3′) specifically targeting tupaia slc10a1 were designed through sidesign center (www.thermo.com/sidesign). the specificities of these sirnas were examined by searching against ncbi cdna databases and the in-house tupaia hepatocyte transcriptome to ensure they are free of off-target. sirna with sequence ctrl-si: 5′-uucuccgaacgugucacgudtdt-3′ that is a scramble sequence with no known mammalian target sequence was used as a negative control. 20 nm sirna were transfected into pths 24 h after initial seeding with lipofectamine 2000 (invitrogen). tsntcp mrna level in the sirna-transfected cells was quantified by real-time rt-pcr 3 days after sirna transfection. transfected pth cells were infected on day 4 after sirna transfection with hbv, hdv, aav8-hbv (carrying 1.05 copies of hbv genome), or lenti-vsv-g-luc viruses. the secreted viral antigen hbsag or hbeag from hbv-and aav8-hbv-infected cells were examined as indicated. for lenti-vsv-g-luc virus-infected cells, luciferase activity was determined on 6 days post-infection. for gene knockdown experiment on heparg cells slc10a1 from heparg cells was cloned, and the sequence was deposited to genebank (accession number: jq814895). differentiated heparg cells in 48-well plate were transfected using rnaimax (invitrogen) with 20 nm of a sirna pool (qiagen) containing four specific sirnas targeting different regions of human slc10a1 (5′-ggaucguccucaaauccaadtdt-3′, 5′-ggagucagccggagaacaadtd t-3′, 5′-ggacaaggugcccuauaaadtdt-3′, 5′-ggugcuaugagaaauucaadtdt-3′) or a negative control sirna (ctrl-si: 5′-uucuccgaacgugucacgudtdt-3′) as indicated. gene knockdown efficiency was examined 4 days after transfection. cells were then inoculated for 16 hr with hbv in the presence of 3.6% peg8000. the secreted hbeag and the 3.5 kb hbv rna were determined on indicated days after infection. frozen phh cells were thawed and subsequently transfected as that of pth with human slc10a1specific sirna 5′-cacaagugcuguagaauuadtdt-3′ (sir405) or a sirna pool containing four slc10a1 specific sirnas from qiagen (5′-ggaucguccucaaauccaadtdt-3′, 5′-ggagucagccggagaacaadtdt-3′, 5′-ggacaaggugcccuauaaadtdt-3′, 5′-ggugcua ugagaaauucaadtdt-3′). control sirna 5′-uucuccgaacgugucacgudtdt-3′ (ctrl-si) has a scramble sequence with no known mammalian target sequence. total 20 nm sirna was transfected into ∼1.4 × 10 5 phh cells per well in 48-well plate 24 h after initial cell seeding; the cells were then inoculated with hbv 72 hr after transfection. hntcp mrna level in the research article sirna-transfected cells was quantified by qrt-pcr 3 days after sirna transfection. the secreted antigens hbsag and hbeag, and the intracellular hbv rnas were determined on indicated days after infection. hepg2 or huh-7 cells were transfected with a plasmid expressing human, treeshrew, monkey ntcp, or an ntcp variant, or a vector control. stable cell line expressing hntcp was established by transfection of hepg2 cells with a plasmid encoding hntcp (hntcp/pcdna3.1) followed by selection with 500 μg/ml g418 and maintained in dmem supplemented with 10% fbs, 500 μg/ml g418, 100 u/ml penicillin, and 100 μg/ml streptomycin. the transfected cells, or hepg2-hntcp stable cells, were cultured in pmm 24 hr before infection. with 5 × 10 7 genome equivalent copies of hdv, 1 × 10 5 cells were incubated, or otherwise indicated, for 24 hr in the presence or absence of entry inhibitors. hdv inoculation was conducted in the presence of 4% peg8000. pmm was replenished every 2 days. on indicated days postinfection, cells were treated with 100% methanol for 10 min, followed by incubating with 5 μg/ml fitc-labeled mab 4g5 for 1 hr at rt to stain hdv delta antigen. the nuclei were stained with 4′-6-diamidino-2-phenylindole (dapi) before analyzing. hdv viral rna copies in cell lysates were quantified by qpcr. with ∼1 × 10 7 genome equivalent copies of hbv, 1 × 10 5 cells were inoculated, or indicated otherwise, in the presence of ∼4% peg8000 as reported for primary human hepatocyte and heparg cell (gripon et al., 1993; gripon et al., 2002; schulze et al., 2007) . the cells were maintained subsequently in pmm and the medium was changed every 2-3 days. for immunofluorescence microscopy analysis, hbv-infected cells, with or without replating on glass coverslips for imaging, were fixed with 4% paraformaldehyde (pfa) and permeabilized with 0.5% tritionx-100, and then stained either with 10 μg/ml 17b9 against hbsag followed by fitc-labeled goat anti-mouse igg, or with 5 μg/ml 1c10 against hbcag followed by qdot 655 vivid donkey anti-mouse igg. 1 μg/ml of dapi was added to stain the nucleus before analyzing. the cell images were captured with a nikon eclipse ti fluorescence microscope or a zeiss lsm 510 meta confocal microscope. secreted viral antigens and intracellular viral replication intermediates cccdna and/or rnas were examined on indicated days after infection. for cell surface expression, the transfected cells expressing ntcps or mutants were surfacebiotinylated with sulfo-nhs-lc-biotin (pierce) following the manufacturer's instruction manual. the biotinylated cells were then lysed in 600 μl of 1× ripa buffer supplemented with 1× protease inhibitor cocktail (roche) and then treated with pngaes f. total cellular protein in the lysate was determined by using bio-rad dc protein assay. about 300 μg streptavidin t1 dynabeads (invitrogen) were then used to pull down surface-biotinylated proteins in the supernatants containing ∼160 μg of total cellular protein each sample. after extensive washing with 1× ripa buffer, bound proteins were eluted, and separated by sds-page and subsequently examined with anti-c9 mab 1d4 by western blotting. for total ntcp expression, the same transfected cells expressing wild-type or mutant ntcps were lysed with 1× ripa buffer and treated with pngase f. each sample containing same amount of total cellular protein (∼8 μg) were loaded for sds-page followed by western blotting analysis with mab 1d4 that recognizes the c9 tag fused at the c-terminus of ntcps. all experiments were repeated 2-6 times with duplicate or triplicate samples for each condition unless indicated otherwise. a representative result of multiple independent experiments is present (n = 2-6) in each figure. error bars shown in all figures represent standard deviation of the mean (n = 2-4). dotted lines show detection limit except otherwise specified. reporting standards: we followed the reporting standards for the transcriptome shotgun assembly sequence database of ncbi, which are: submitted sequences must be assembled from data experimentally determined by the submitter. screened for vector contamination and any vector/linker sequence removed. this includes the removal of nextgen sequencing primers. sequences cannot be less than 200 bp. sequences should have no more than 10% n's or greater than 14 n's in a row. if the submission is a single-step, unannotated assembly and the output is a bam file(s) these should be submitted as a tsa project to sra. conception and design, acquisition of data, analysis and interpretation of data; gz, conception and design, acquisition of data, analysis and interpretation of data acquisition of data, analysis and interpretation of data acquisition of data, analysis and interpretation of data acquisition of data, analysis and interpretation of data acquisition of data, analysis and interpretation of data acquisition of data, analysis and interpretation of data; yq, acquisition of data acquisition of data acquisition of data acquisition of data acquisition of data; pc, acquisition of data acquisition of data acquisition of data; ys, acquisition of data acquisition of data, analysis and interpretation of data analysis and interpretation of data; js, conception and design, analysis and interpretation of data, drafting or revising the article; wl, conception and design, analysis and interpretation of data, drafting or revising the article ethics human subjects: this study was approved by the institutional review board (irb) of national institute of biological sciences (irbs030901) and consent was obtained; see the materials and methods section for details. the helsinki guidelines were followed. animal experimentation: the institutional animal care and use committee (iacuc) of the national institute of biological sciences and the approved animal protocol is 09001t analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis d virus pseudotypes mapping of the hepatitis b virus pre-s1 domain involved in receptor recognition analysis of the cytosolic domains of the hepatitis b virus envelope proteins for their function in viral particle assembly and infectivity infectivity determinants of the hepatitis b virus pre-s domain are confined to the n-terminal 75 amino acid residues primary human hepatocytes as an in vitro model for hepatitis b virus infection quantification of hbv covalently closed circular dna from liver tissue by real-time pcr prediction of complete gene structures in human genomic dna blast+: architecture and applications structure and replication of the genome of the hepatitis delta virus a short n-proximal region in the large envelope protein harbors a determinant that contributes to the species specificity of human hepatitis b virus characterization of a hepatitis b and hepatitis delta virus receptor binding site hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks pre-s1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres1 lipopeptides and tupaia hepatocytes viral and cellular determinants involved in hepadnaviral entry full-length transcriptome assembly from rna-seq data without a reference genome hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide infection of a human hepatoma cell line by hepatitis b virus reproducible high level infection of cultured adult human hepatocytes by hepatitis b virus: effect of polyethylene glycol on adsorption and penetration myristylation of the hepatitis b virus large surface protein is essential for viral infectivity efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein molecular cloning, chromosomal localization, and functional characterization of a human liver na+/bile acid cotransporter a comparison of whole genome gene expression profiles of heparg cells and hepg2 cells to primary human hepatocytes and human liver tissues large surface proteins of hepatitis b virus containing the pre-s sequence crystal structure of a bacterial homologue of the bile acid sodium symporter asbt hepatitis delta virus chlorambucil-taurocholate is transported by bile acid carriers expressed in human hepatocellular carcinomas hepatitis b virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures the pre-s1 and antigenic loop infectivity determinants of the hepatitis b virus envelope proteins are functionally independent infection process of the hepatitis b virus depends on the presence of a defined sequence in the pre-s1 domain gene regulations in hbv-related liver cirrhosis closely correlate with disease severity role of glycosaminoglycans for binding and infection of hepatitis b virus parallel decrease of na(+)-taurocholate cotransport and its encoding mrna in primary cultures of rat hepatocytes topography of the membrane domain of the liver na+-dependent bile acid transporter the panther database of protein families, subfamilies, functions and pathways the soluble serum protein gas6 bridges virion envelope phosphatidylserine to the tam receptor tyrosine kinase axl to mediate viral entry cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes hepatitis b virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans fine mapping of pre-s sequence requirements for hepatitis b virus large envelope protein-mediated receptor interaction hepadnaviruses adenovirus binding to blood factors results in liver cell infection and hepatotoxicity in-gel digestion for mass spectrometric characterization of proteins and proteomes in situ localization of the hepatocytic na+/taurocholate cotransporting polypeptide in rat liver the role of the sodium-taurocholate cotransporting polypeptide (ntcp) and of the bile salt export pump (bsep) in physiology and pathophysiology of bile formation photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells evaluation of human monoclonal antibody 80r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants hepadnavirus envelope proteins regulate covalently closed circular dna amplification production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis b virus pre-s antigens the role of the hbv envelope proteins in the hdv replication cycle dtaselect and contrast: tools for assembling and comparing protein identifications from shotgun proteomics hepatitis b virus infection of tupaia hepatocytes in vitro and in vivo persistence of cccdna during the natural history of chronic hepatitis b and decline during adefovir dipivoxil therapy production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus prolucid, a fast and sensitive tandem mass spectra-based protein identification program hepatobiliary transporter expression in human hepatocellular carcinoma we thank dr. she chen and lin li at national institute of biological sciences (nibs) proteomic center for the mass spectrometry analysis, dr. cheng zhan at imaging center, and zhihua qiu and ping qu at animal facility of nibs for their excellent technical assistances. we thank dr. michael r. farzan for helpful discussions and drs. xiaodong wang and hyeryun choe for critical comments on the article. we are grateful for drs. lin jiang, zhenglun liang, and fengming lu for providing us with valuable reagents. grant reference number author the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-016704-99v4brjf authors: nicholson, felicity title: infectious diseases: the role of the forensic physician date: 2005 journal: clinical forensic medicine doi: 10.1385/1-59259-913-3:235 sha: doc_id: 16704 cord_uid: 99v4brjf infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the 21st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in 2003. despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. infections have plagued doctors for centuries, in both the diagnosis of the specific diseases and the identification and subsequent management of the causative agents. there is a constant need for information as new organisms emerge, existing ones develop resistance to current drugs or vaccines, and changes in epidemiology and prevalence occur. in the 21st century, obtaining this information has never been more important. population migration and the relatively low cost of flying means that unfamiliar infectious diseases may be brought into industrialized countries. an example of this was an outbreak of severe acute respiratory syndrome (sars), which was first recognized in 2003. despite modern technology and a huge input of money, it took months for the agent to be identified, a diagnostic test to be produced, and a strategy for disease reporting and isolation to be established. there is no doubt that other new and fascinating diseases will continue to emerge. for the forensic physician, dealing with infections presents two main problems. the first problem is managing detainees or police personnel who have contracted a disease and may be infectious or unwell. the second problem is handling assault victims, including police officers, who have potentially been exposed to an infectious disease. the latter can be distressing for those involved, compounded, in part, from an inconsistency of management guidelines, if indeed they exist. with the advent of human rights legislation, increasing pressure is being placed on doctors regarding consent and confidentiality of the detainee. therefore, it is prudent to preempt such situations before the consultation begins by obtaining either written or verbal consent from the detainee to allow certain pieces of information to be disclosed. if the detainee does not agree, then the doctor must decide whether withholding relevant details will endanger the lives or health of those working within custody or others with whom they may have had close contact (whether or not deliberate). consent and confidentiality issues are discussed in detail in chapter 2. adopting a universal approach with all detainees will decrease the risk to staff of acquiring such diseases and will help to stop unnecessary overreaction and unjustified disclosure of sensitive information. for violent or sexual assault victims, a more open-minded approach is needed (see also chapter 3) . if the assailant is known, then it may be possible to make an informed assessment of the risk of certain diseases by ascertaining his or her lifestyle. however, if the assailant is unknown, then it is wise to assume the worst. this chapter highlights the most common infections encountered by the forensic physician. it dispels "urban myths" and provides a sensible approach for achieving effective management. the risk of exposure to infections, particularly blood-borne viruses (bbvs), can be minimized by adopting measures that are considered good practice in the united kingdom, the united states, and australia (1) (2) (3) . forensic physicians or other health care professionals should wash their hands before and after contact with each detainee or victim. police officers should be encouraged to wash their hands after exposure to body fluids or excreta. all staff should wear gloves when exposure to body fluids, mucous membranes, or nonintact skin is likely. gloves should also be worn when cleaning up body fluids or handling clinical waste, including contaminated laundry. single-use gloves should only be used and must conform to the requirements of european standard 455 or equivalent (1) (2) (3) . a synthetic alternative conforming to the same standards should also be available for those who are allergic to latex. all staff should cover any fresh wounds (<24 hours old), open skin lesions, or breaks in exposed skin with a waterproof dressing. gloves cannot prevent percutaneous injury but may reduce the chance of acquiring a bloodborne viral infection by limiting the volume of blood inoculated. gloves should only be worn when taking blood, providing this does not reduce manual dexterity and therefore increase the risk of accidental percutaneous injury. ideally, a designated person should be allocated to ensure that the clinical room is kept clean and that sharps containers and clinical waste bags are removed regularly. clinical waste must be disposed of in hazard bags and should never be overfilled. after use, the clinical waste should be doublebagged and sealed with hazard tape. the bags should be placed in a designated waste disposal (preferably outside the building) and removed by a professional company. when cells are contaminated with body fluids, a professional cleaning company should be called to attend as soon as possible. until such time, the cell should be deemed "out of action." there is a legal requirement in the united kingdom under the environmental protection act (1990) and the control of substances hazardous to health regulations 1994 to dispose of sharps in an approved container. in the united states, the division of health care quality promotion on the centers for disease control and prevention (cdc) web site provides similar guidance. in custody, where sharps containers are transported off site, they must be of an approved type. in the united kingdom, such a requirement is contained within the carriage of dangerous goods (classification, packaging and labelling) and use of transportable pressure receptacles regulations 1996. these measures help to minimize the risk of accidental injury. further precautions include wearing gloves when handling sharps and never bending, breaking, or resheathing needles before disposal. sharps bins should never be overfilled, left on the floor, or placed above the eye level of the smallest member of staff. any bedding that is visibly stained with body fluids should be handled with gloves. there are only three acceptable ways of dealing with contaminated bedding: the bbvs that present the most cross-infection hazard to staff or victims are those associated with persistent viral replication and viremia. these include hbv, hcv, hepatitis d virus (hdv), and hiv. in general, risks of transmission of bbvs arise from the possible exposure to blood or other body fluids. the degree of risk varies with the virus concerned and is discussed under the relevant sections. figure 1 illustrates the immediate management after a percutaneous injury, mucocutaneous exposure, or exposure through contamination of fresh cuts or breaks in the skin. hbv is endemic throughout the world, with populations showing a varying degree of prevalence. approximately two thousand million people have been infected with hbv, with more than 350 million having chronic infection. worldwide, hbv kills about 1 million people each year. with the development of a safe and effective vaccine in 1982, the world health organization (who) recommended that hbv vaccine should be incorporated into national immunization programs by 1995 in those countries with a chronic infection rate of 8% or higher, and into all countries by 1997. although 135 countries had achieved this goal by the end of 2001, the poorest countries-often the ones with the highest prevalence-have been unable to afford it. in particular these include china, the indian subcontinent, and sub-saharan africa. people in the early stages of infection or with chronic carrier status (defined by persistence of hepatitis b surface antigen [hbsag] beyond 6 mo) can transmit infection. in the united kindgom, the overall prevalence of chronic hbv is approx 0.2-0.3% (6, 7) . a detailed breakdown is shown in table 1 . the incubation period is approx 6 weeks to 6 months. as the name suggests, the virus primarily affects the liver. typical symptoms include malaise, anorexia, nausea, mild fever, and abdominal discomfort and may last from 2 days to 3 weeks before the insidious onset of jaundice. joint pain and skin rashes may also occur as a result of immune complex formation. infections in the newborn are usually asymptomatic. * in the united kingdom, written consent from the contact must be sent with the sample, countersigned by the health care practitioner and, preferably, an independent police officer. the majority of patients with acute hbv make a full recovery and develop immunity. after acute infection, approx 1 in 300 patients develop liver failure, which may result in death. chronic infection develops in approx 90% of neonates, approx 50% of children, and between 5 and 10% of adults. neonates and children are usually asymptomatic. adults may have only mild symptoms or may also be asymptomatic. approximately 15-25% of chronically infected individuals (depending on age of acquisition) will develop cirrhosis over a number of years. this may also result in liver failure or other serious complications, including hepatocellular carcinoma, though the latter is rare. the overall mortality rate of hbv is estimated at less than 5%. a person is deemed infectious if hbsag is detected in the blood. in the acute phase of the illness, this can be as long as 6 months. by definition, if hbsag persists after this time, then the person is deemed a carrier. carriers are usually infectious for life. the degree of infectivity depends on the stage of disease and the markers present table 2 . the major routes include parenteral (e.g., needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized), mucous membrane exposure (including mouth, eyes, and genital mucous membranes), and contamination of broken skin (especially when <24 hours old). hbv is an occupational hazard for anyone who may come into contact with blood or bloodstained body fluids through the routes described. saliva alone may transmit hbv. the saliva of some people infected with hbv contains hbv-dna concentrations 1/1000-1/10,000 of that found in their serum (8) . this is especially relevant for penetrating bite wounds. infection after exposure to other body fluids (e.g., bile, urine, feces, and cerebrospinal fluid) has never been demonstrated unless the fluids are contaminated with blood. intravenous drug users who share needles or other equipment are also at risk. hbv can also be transmitted through unprotected sexual contact, whether homosexual or heterosexual. the risk is increased if blood is involved. sexual assault victims should be included in this category. evidence has shown that the virus may also be spread among members of a family through close household contact, such as through kissing and sharing toothbrushes, razors, bath towels, etc. (9) (10) (11) . this route of transmission probably applies to institutionalized patients, but there are no available data. studies of prisoners in western countries have shown a higher prevalence of antibodies to hbv and other bbvs than the general population (12) (13) (14) ; the most commonly reported risk factor is intravenous drug use. however, the real frequency of transmission of bbvs in british prisons is unknown owing to the difficulty in compiling reliable data. hbv can be transmitted vertically from mother to baby during the perinatal period. approximately 80% of babies born to mothers who have either acute or chronic hbv become infected, and most will develop chronic hbv. this has been limited by the administration of hbv vaccine to the neonate. in industrialized countries, all prenatal mothers are screened for hbv. vaccine is given to the neonate ideally within the first 12 hours of birth and at least two more doses are given at designated intervals. the who recommends this as a matter of course for all women in countries where prevalence is high. however, the practicalities of administering a vaccine that has to be stored at the correct temperature in places with limited access to medical care means that there is a significant failure of vaccine uptake and response. in industrialized countries, hbv vaccination is recommended for those who are deemed at risk of acquiring the disease. they include the following: 1. through occupational exposure. 2. homosexual/bisexual men. 3. intravenous drug users. 4. sexual partners of people with acute or chronic hbv. 5. family members of people with acute or chronic hbv. 6. newborn babies whose mothers are infected with hbv. if the mother is hbsag positive, then hepatitis b-specific immunoglobulin (hbig) should be given at the same time as the first dose of vaccine. 7. institutionalized patients and prisoners. ideally, hbv vaccine should be administered before exposure to the virus. the routine schedule consists of three doses of the vaccine given at 0, 1, and 6 months. antibody levels should be checked 8-12 weeks after the last dose. if titers are greater than10 miu/ml, then an adequate response has been achieved. in the united kingdom, this is considered to provide protection for 5-10 years. in the united states, if an initial adequate response has been achieved, then no further doses of vaccine are considered necessary. vaccine administration after exposure varies according to the timing of the incident, the degree of risk involved, and whether the individual has already been partly or fully vaccinated. an accelerated schedule when the third dose is given 2 months after the first dose with a booster 1 year later is used to prevent postnatal transmission. where risks are greatest, it may be necessary to use a rapid schedule. the doses are given at 0, 7, and 21-28 days after presentation, again with a booster dose at 6-12 months. this schedule is currently only licensed with engerix b. hbig may also be used either alone or in conjunction with vaccine. the exact dose given is age dependent but must be administered by deep intramuscular injection in a different site from the vaccine. in an adult, this is usually into the gluteus muscle. hbig is given in conjunction with the first dose of vaccine to individuals who are deemed at high risk of acquiring disease and the incident occurred within 72 hours of presentation. it is also used for neonates born to mothers who are hbeag-positive. between 5 and 10% of adults fail to respond to the routine schedule of vaccine. a further full course of vaccine should be tried before deeming the patients as "nonresponders." such individuals involved in a high-risk exposure should be given two doses of hbig administered 1 mo apart. ideally, the first dose should be given within 48 hours after exposure and no later than 2 weeks after exposure. other measures include minimizing the risk of exposure by adopting the safe working practices outlined in subheading 2. any potential exposures should be dealt with as soon as possible. in industrialized countries blood, blood products, and organs are routinely screened for hbv. intravenous drug users should be encouraged to be vaccinated and to avoid sharing needles or any other drug paraphernalia (see subheading 6.9.2.). for staff or victims in contact with disease, it is wise to have a procedure in place for immediate management and risk evaluation. an example is shown in fig. 1 . although forensic physicians are not expected to administer treatment, it is often helpful to inform persons concerned what to expect. tables 3 and 4 outline treatment protocols as used in the united kingdom. detainees with disease can usually be managed in custody. if the detainee is bleeding, then the cell should be deemed out of action after the detainee has left until it can be professionally cleaned. contaminated bedding should be dealt with as described in subheading 2.2. if the detainee has chronic hbv and is on an antiviral agent (e.g., lamivudine), then the treatment course should be continued, if possible. hcv is endemic in most parts of the world. approximately 3% (200 million) of the world's population is infected with hcv (15) . for many countries, no reliable prevalence data exist. seroprevalence studies conducted among blood donors have shown that the highest prevalence exists in egypt (17-26%). this has been ascribed to contaminated needles used in the treatment of schistosomiasis conducted between the 1950s and the 1980s (16) . intermediate prevalence (1-5%) exists in eastern europe, the mediterranean, the middle east, the indian subcontinent, and parts of africa and asia. in western europe, most of central america, australia, and limited regions in africa, including south africa, the prevalence is low (0.2-0.5%). previously, america was included in the low prevalence group, but a report published in 2003 (17) indicated that almost 4 million americans (i.e., 1.8% of the population) have antibody to hcv, representing either ongoing or previous infection. it also states that hcv accounts for approx 15% of acute viral hepatitis in america. the lowest prevalence (0.01-0.1%) has been found in the united kingdom and scandinavia. however, within any country, there are certain groups that have a higher chance of carrying hcv. these united kingdom figures are given in table 5 . after an incubation period of 6-8 weeks, the acute phase of the disease lasts approx 2-3 years. unlike hepatitis a (hav) or hbv, the patient is usually asymptomatic; therefore, the disease is often missed unless the individual has reported a specific exposure and is being monitored. other cases are found by chance, when raised liver enzymes are found on a routine blood test. a "silent phase" follows the acute phase when the virus lies dormant and the liver enzymes are usually normal. this period lasts approx 10-15 years. reactivation may then occur. subsequent viral replication damages the hepatocytes, and liver enzymes rise to moderate or high levels. eighty percent of individuals who are hcv antibody-positive are infectious, regardless of the levels of their liver enzymes. approximately 80% of people develop chronic infection, one-fifth of whom progress to cirrhosis. there is a much stronger association with hepatocellular carcinoma than with hbv. an estimated 1.25-2.5% of patients with hcv-related cirrhosis develop liver cancer (18) . less than 2% of chronic cases resolve spontaneously. approximately 75% of cases are parenteral (e.g., needle-stick, etc.) (19) . transmission through the sexual route is not common and only appears to be significant if there is repeated exposure with one or more people infected with hcv. mother-to-baby transmission is considered to be uncommon but has been reported (20) . theoretically, household spread is also possible through sharing contaminated toothbrushes or razors. because the disease is often silent, there is a need to raise awareness among the general population on how to avoid infection and to encourage high-risk groups to be tested. health care professionals should also be educated to avoid occupationally acquired infection. an example of good practice blood or blood-stained body fluids need to be involved for a risk to occur. saliva alone is not deemed to be a risk. the risk from a single needlestick incident is 1.8% (range 0-7%). contact through a contaminated cut is estimated at 1%. for penetrating bite injuries, there are no data, but it is only considered a risk if blood is involved. blood or blood-stained body fluids have to be involved in transmission through mucous membrane exposure. this may account for the lower-than-expected prevalence among the gay population. follow the immediate management flow chart, making sure all available information is obtained. inform the designated hospital and/or specialist as soon as possible. if the contact is known and is believed to be immunocompromised and he or she has consented to provide a blood sample, it is important to tell the specialist, because the antibody tests may be spuriously negative. in this instance, a different test should be used (polymerase chain reaction [pcr] , which detects viral rna). the staff member/victim will be asked to provide a baseline sample of blood with further samples at 4-6 weeks and again at 12 weeks. if tests are negative at 12 weeks but the risk was deemed high, then follow-up may continue for up to 24 weeks. if any of the follow-up samples is positive, then the original baseline sample will be tested to ascertain whether the infection was acquired through the particular exposure. it is important to emphasize the need for prompt initial attendance and continued monitoring, because treatment is now available. a combination of ribavirin (antiviral agent and interferon a-2b) (18) or the newer pegylated interferons (15) may be used. this treatment is most effective when it is started early in the course of infection. unless they are severely ill, detainees can be managed in custody. special precautions are only required if they are bleeding. custody staff should wear gloves if contact with blood is likely. contaminated bedding should be handled appropriately, and the cell cleaned professionally after use. this defective transmissible virus was discovered in 1977 and requires hbv for its own replication. it has a worldwide distribution in association with hbv, with approx 15 million people infected. the prevalence of hdv is higher in southern italy, the middle east, and parts of africa and south america, occurring in more than 20% of hbv carriers who are asymptomatic and more than 60% of those with chronic hbv-related liver disease. despite the high prevalence of hbv in china and south east asia, hdv in these countries is rare. hdv is associated with acute (coinfection) and chronic hepatitis (superinfection) and can exacerbate pre-existing liver damage caused by hbv. the routes of transmission and at-risk groups are the same as for hbv. staff/victims in contact with a putative exposure and detainees with disease should be managed as for hbv. interferon-î± (e.g., roferon) can be used to treat patients with chronic hbv and hdv (21) , although it would not be practical to continue this treatment in the custodial setting. hiv was first identified in 1983, 2 years after the first reports were made to the cdc in atlanta, ga, of an increased incidence of two unusual diseases (kaposi's sarcoma and pneumocystis carinii pneumonia) occurring among the gay population in san francisco. the scale of the virus gradually emerged over the years and by the end of 2002, there were an estimated 42 million people throughout the world living with hiv or acquired immunodeficiency syndrome (aids). more than 80% of the world's population lives in africa and india. a report by the joint united nations programme on hiv/aids and the who in 2002 stated that one in five adults in lesotho, malawi, mozambique, swaziland, zambia, and zimbabwe has hiv or aids. there is also expected to be a sharp rise in cases of hiv in china, papua new guinea, and other countries in asia and the pacific during the next few years. in the united kingdom, by the end of 2002, the cumulative data reported that there were 54,261 individuals with hiv, aids (including deaths from aids) reported, though this is likely to be an underestimate (22) . from these data, the group still considered at greatest risk of acquiring hiv in the united kingdom is homosexual/bisexual men, with 28,835 of the cumulative total falling into this category. among intravenous drug users, the overall estimated prevalence is 1%, but in london the figure is higher at 3.7% (6, 23) . in the 1980s, up to 90% of users in edinburgh and dundee were reported to be hiv positive, but the majority have now died. individuals arriving from africa or the indian subcontinent must also be deemed a risk group because 80% of the world's total cases occur in these areas. the predominant mode of transmission is through unprotected heterosexual intercourse. the incidence of mother-to-baby transmission has been estimated at 15% in europe and approx 45% in africa. the transmission rates among african women are believed to be much higher owing to a combination of more women with end-stage disease with a higher viral load and concomitant placental infection, which renders it more permeable to the virus (24, 25) . the use of antiretroviral therapy during pregnancy, together with the advice to avoid breastfeeding, has proven efficacious in reducing both vertical and horizontal transmission among hiv-positive women in the western world. for those in third-world countries, the reality is stark. access to treatment is limited, and there is no realistic substitute for breast milk, which provides a valuable source of antibodies to other life-threatening infections. patients receiving blood transfusions, organs, or blood products where screening is not routinely carried out must also be included. the incubation is estimated at 2 weeks to 6 months after exposure. this depends, to some extent, on the ability of current laboratory tests to detect hiv antibodies or viral antigen. the development of pcr for viral rna has improved sensitivity. during the acute phase of the infection, approx 50% experience a seroconversion "flu-like" illness. the individual is infectious at this time, because viral antigen (p24) is present in the blood. as antibodies start to form, the viral antigen disappears and the individual enters the latent phase. he or she is noninfectious and remains well for a variable period of time (7-15 years). development of aids marks the terminal phase of disease. viral antigen reemerges, and the individual is once again infectious. the onset of aids has been considerably delayed with the use of antiretroviral treatment. parenteral transmission included needlestick injuries, bites, unscreened blood transfusions, tattooing, acupuncture, and dental procedures where equipment is inadequately sterilized. risk of transmission is increased with deep penetrating injuries with hollow bore needles that are visibly bloodstained, especially when the device has previously been in the source patient's (contact) artery or vein. other routes include mucous membrane exposure (eyes, mouth, and genital mucous membranes) and contamination of broken skin. the higher the viral load in the contact, the greater the risk of transmission. this is more likely at the terminal stage of infection. hiv is transmitted mainly through blood or other body fluids that are visibly blood stained, with the exception of semen, vaginal fluid, and breast milk. saliva alone is most unlikely to transmit infection. therefore, people who have sustained penetrating bite injuries can be reassured that they are not at risk, providing the contact was not bleeding from the mouth at the time. the risk from a single percutaneous exposure from a hollow bore needle is low, and a single mucocutaneous exposure is even less likely to result in infection. the risk from sexual exposure varies, although it appears that there is a greater risk with receptive anal intercourse compared with receptive vaginal intercourse (26). high-risk fluids include blood, semen, vaginal fluid, and breast milk. there is little or no risk from saliva, urine, vomit, or feces unless they are visibly bloodstained. other fluids that constitute a theoretical risk include cerebrospinal, peritoneal, pleural, synovial, or pericardial fluid. management in custody of staff/victims in contact with disease includes following the immediate management flow chart (fig. 1 ) and contacting the designated hospital/specialist with details of the exposure. where possible, obtain a blood sample from the contact. regarding hbv and hcv blood samples in the united kingdom, they can only be taken with informed consent. there is no need for the forensic physician to go into details about the meaning of the test, but the contact should be encouraged to attend the genitourinary department (or similar) of the designated hospital to discuss the test results. should the contact refuse to provide a blood sample, then any information about his or her lifestyle, ethnic origin, state of health, etc., may be useful for the specialist to decide whether postexposure prophylaxis (pep) should be given to the victim. where only saliva is involved in a penetrating bite injury, there is every justification to reassure the victim that he or she is not at risk. if in doubt, then always refer. in the united kingdom, the current recommended regime for pep is combivir (300 mg of zidovudine twice daily plus 150 mg of lamivudine twice daily) and a protease inhibitor (1250 mg of nelfanivir twice daily) given for 4 weeks (27) . it is only given after a significant exposure to a high-risk fluid or any that is visibly bloodstained and the contact is known or is highly likely to be hiv positive. ideally, treatment should be started within an hour after exposure, although it will be considered for up to 2 weeks. it is usually given for 4 weeks, unless the contact is subsequently identified as hiv negative or the "victim" develops tolerance or toxicity occurs. weekly examinations of the "victim" should occur during treatment to improve adherence, monitor drug toxicity, and deal with other concerns. other useful information that may influence the decision whether to treat with the standard regimen or use alternative drugs includes interaction with other medications that the "victim" may be taking (e.g., phenytoin or antibiotics) or if the contact has been on antiretroviral therapy or if the "victim" is pregnant. during the second or third trimester, only combivir would be used, because there is limited experience with protease inhibitors. no data exist regarding the efficacy of pep beyond occupational exposure (27) . pep is not considered for exposure to low-or no-risk fluids through any route or where the source is unknown (e.g., a discarded needle). despite the appropriate use and timing of pep, there have been reports of failure (28, 29) . unless they are severely ill, detainees can be kept in custody. every effort should be made to continue any treatment they may be receiving. apply universal precautions when dealing with the detainee, and ensure that contaminated cells and/or bedding are managed appropriately. cases of this highly infectious disease occur throughout the year but are more frequent in winter and early spring. this seasonal endemicity is blurring with global warming. in the united kingdom, the highest prevalence occurs in the 4-to 10-years age group. ninety percent of the population over the age of 40 is immune (30) . a similar prevalence has been reported in other parts of western europe and the united states. in south east asia, varicella is mainly a disease of adulthood (31) . therefore, people born in these countries who have moved to the united kingdom are more likely to be susceptible to chicken pox. there is a strong correlation between a history of chicken pox and serological immunity (97-99%). most adults born and living in industrialized countries with an uncertain or negative history of chicken pox are also seropositive (70-90%). in march 1995, a live-attenuated vaccine was licensed for use in the united states and a policy for vaccinating children and susceptible health care personnel was introduced. in summer 2002, in the united kingdom, glaxosmithkline launched a live-attenuated vaccine called varilrix. in december 2003, the uk department of health, following advice from the joint committee on vaccination and immunisation recommended that the vaccine be given for nonimmune health care workers who are likely to have direct contact with individuals with chicken pox. any health care worker with no previous history of chicken pox should be screened for immunity, and if no antibodies are found, then they should receive two doses of vaccine 4-8 weeks apart. the vaccine is not currently recommended for children and should not be given during pregnancy. following an incubation period of 10-21 days (this may be shorter in the immunocompromised), there is usually a prodromal "flu-like" illness before the onset of the rash. this coryzal phase is more likely in adults. the lesions typically appear in crops, rapidly progressing from red papules through vesicles to open sores that crust over and separate by 10 days. the distribution of the rash is centripetal (i.e., more over the trunk and face than on the limbs). this is the converse of small pox. in adults, the disease is often more severe, with lesions involving the scalp and mucous membranes of the oropharynx. in children, the disease is often mild, unless they are immunocompromised, so they are unlikely to experience complications. in adults (defined as 15 yr or older), the picture is rather different (32) . secondary bacterial infection is common but rarely serious. there is an increased likelihood of permanent scarring. hemorrhagic chicken pox typically occurs on the second or third day of the rash. usually, this is limited to bleeding into the skin, but lifethreatening melena, epistaxis, or hematuria can occur. varicella pneumonia ranges from patchy lung consolidation to overt pneumonitis and occurs in 1 in 400 cases (33) . it can occur in previously healthy individuals (particularly adults), but the risk is increased in those who smoke. immunocompromised people are at the greatest risk of developing this complication. it runs a fulminating course and is the most common cause of varicella-associated death. fibrosis and permanent respiratory impairment may occur in those who survive. any suspicion of lung involvement is an indication for immediate treatment, and any detainee or staff member should be sent to hospital. involvement of the central nervous system includes several conditions, including meningitis, guillain-barre, and encephalitis. the latter is more common in the immunocompromised and can be fatal. this is taken as 3 days before the first lesions appear to the end of new vesicle formation and the last vesicle has crusted over. this typically is 5-7 days after onset but may last up to 14 days. the primary route is through direct contact with open lesions of chicken pox. however, it is also spread through aerosol or droplets from the respiratory tract. chicken pox may also be acquired through contact with open lesions of shingles (varicella zoster), but this is less likely because shingles is less infectious than chicken pox. nonimmune individuals are at risk of acquiring disease. approximately 10% of the adult population born in the united kingdom and less than 5% of adults in the united states fall into this category. therefore, it is more likely that if chicken pox is encountered in the custodial setting, it will involve people born outside the united kingdom (particularly south east asia) or individuals who are immunocompromised and have lost immunity. nonimmune pregnant women are at risk of developing complications. pneumonia can occur in up to 10% of pregnant women with chicken pox, and the severity is increased in later gestation (34) . they can also transmit infection to the unborn baby (35) . if infection is acquired in the first 20 weeks, there is a less than 3% chance of it leading to congenital varicella syndrome. infection in the last trimester can lead to neonatal varicella, unless more than 7 days elapse between onset of maternal rash and delivery when antibodies have time to cross the placenta leading to either mild or inapparent infection in the newborn. in this situation, varicella immunoglobulin (vzig) should be administered to the baby as soon as possible after birth (36). staff with chicken pox should stay off work until the end of the infective period (approx 7-14 days). those in contact with disease who are known to be nonimmune or who have no history of disease should contact the designated occupational health physician. detainees with the disease should not be kept in custody if at all possible (especially pregnant women). if this is unavoidable, then nonimmune or immunocompromised staff should avoid entering the cell or having close contact with the detainee. nonimmune, immunocompromised, or pregnant individuals exposed to chickenpox should seek expert medical advice regarding the administration of vzig. aciclovir (or similar antiviral agent) should be given as soon as possible to people who are immunocompromised with chicken pox. it should also be considered for anyone over 15 years old because they are more likely to develop complications. anyone suspected of severe complications should be sent straight to the hospital. after chicken pox, the virus lies dormant in the dorsal root or cranial nerve ganglia but may re-emerge and typically involves one dermatome (37) . the site of involvement depends on the sensory ganglion initially involved. shingles is more common in individuals over the age of 50 years, except in the immunocompromised, when attacks can occur at an earlier age. the latter are also more susceptible to secondary attacks and involvement of more than one dermatome. bilateral zoster is even rarer but is not associated with a higher mortality. in the united kingdom, there is an estimated incidence of 1.2-3.4 per 1000-person years (38). there may be a prodromal period of paraesthesia and burning or shooting pains in the involved segment. this is usually followed by the appearance of a band of vesicles. rarely, the vesicles fail to appear and only pain is experienced. this is known as zoster sine herpete. in individuals who are immuno-compromised, disease may be prolonged and dissemination may occur but is rarely fatal. shingles in pregnancy is usually mild. the fetus is only affected if viremia occurs before maternal antibody has had time to cross the placenta. the most common complication of shingles is postherpetic neuralgia, occurring in approx 10% of cases. it is defined as pain lasting more than 120 days from rash onset (39) . it is more frequent in people over 50 years and can lead to depression. it is rare in children, including those who are immunocompromised. infection of the brain includes encephalitis, involvement of motor neurones leading to ptosis, paralysis of the hand, facial palsy, or contralateral hemiparesis. involvement of the oculomotor division of the trigeminal ganglion can cause serious eye problems, including corneal scarring. shingles is far less infectious than chicken pox and is only considered to be infectious up to 3 days after lesions appear. shingles is only infectious after prolonged contact with lesions. unlike chickenpox, airborne transmission is not a risk. individuals who are immunocompromised may reactivate the dormant virus and develop shingles. people who have not had primary varicella are at risk of developing chickenpox after prolonged direct contact with shingles. despite popular belief, it is untrue that people who are immunocompetent who have had chicken pox develop shingles when in contact with either chicken pox or shingles. such occurrences are merely coincidental, unless immunity is lowered. staff with shingles should stay off work until the lesions are healed, unless they can be covered. staff who have had chickenpox are immune (including pregnant women) and are therefore not at risk. if they are nonimmune (usually accepted as those without a history of chicken pox), they should avoid prolonged contact with detainees with shingles. pregnant nonimmune women should avoid contact altogether. detainees with the disease may be kept in custody, and any exposed lesions should be covered. it is well documented that prompt treatment attenuates the severity of the disease, reduces the duration of viral shedding, hastens lesion healing, and reduces the severity and duration of pain. it also reduces the likelihood of developing postherpetic neuralgia (40) . prompt treatment with famciclovir (e.g., 500 mg three times a day for 7 days) should be initiated if the onset is 3 d ays or less. it should also be considered after this time if the detainee is over age 50 years. pregnant detainees with shingles can be reassured that there is minimal risk for both the mother and the unborn child. expert advice should be given before initiating treatment for the mother. this tiny parasitic mite (sarcoptes scabiei) has infested humans for more than 2500 years. experts estimate that in excess of 300 million cases occur worldwide each year. the female mite burrows into the skin, especially around the hands, feet, and male genitalia, in approx 2.5 min. eggs are laid and hatch into larvae that travel to the skin surface as newly developed mites. the mite causes intense itching, which is often worse at night and is aggravated by heat and moisture. the irritation spreads outside the original point of infection resulting from an allergic reaction to mite feces. this irritation may persist for approx 2 weeks after treatment but can be alleviated by antihistamines. crusted scabies is a far more severe form of the disease. large areas of the body may be involved. the crusts hide thousands of live mites and eggs, making them difficult to treat. this so-called norwegian scabies is more common in the elderly or the immunocompromised, especially those with hiv. after a primary exposure, it takes approx 2-6 weeks before the onset of itching. however, further exposures reduce the incubation time to approx 1-4 days. without treatment, the period of infectivity is assumed to be indefinite. with treatment, the person should be considered infectious until the mites and eggs are destroyed, usually 7-10 days. crusted scabies is highly infectious. because transmission is through direct skin-to-skin contact with an infected individual, gloves should be worn when dealing with individuals suspected of infestation. usually prolonged contact is needed, unless the person has crusted scabies, where transmission occurs more easily. the risk of transmission is much greater in households were repeated or prolonged contact is likely. because mites can survive in bedding or clothing for up to 24 hour, gloves should also be worn when handling these items. bedding should be treated using one of the methods in subheading 2.2. professional cleaning of the cell is only warranted in cases of crusted scabies. the preferred treatment for scabies is either permethrin cream (5%) or aqueous malathion (0.5%) (41) . either treatment has to be applied to the whole body and should be left on for at least 8 hours in the case of permethrin and 24 hours for malathion before washing off. lindane is no longer considered the treatment of choice, because there may be complications in pregnancy (42) . treatment in custody may not be practical but should be considered when the detainee is believed to have norwegian scabies. like scabies, head lice occur worldwide and are found in the hair close to the scalp. the eggs, or nits, cling to the hair and are difficult to remove, but they are not harmful. if you see nits, then you can be sure that lice are also present. the latter are best seen when the hair is wet. the lice bite the scalp and suck blood, causing intense irritation and itching. head lice can only be passed from direct hair-to-hair contact. it is only necessary to wear gloves when examining the head for whatever reason. the cell does not need to be cleaned after use, because the lice live on or near skin. bedding may be contaminated with shed skin, so should be handled with gloves and laundered or incinerated. the presence of live lice is an indication for treatment by either physical removal with a comb or the application of an insecticide. the latter may be more practical in custody. treatment using 0.5% aqueous malathion should be applied to dry hair and washed off after 12 hours. the hair should then be shampooed as normal. crabs or body lice are more commonly found in the pubic, axillary, chest, and leg hair. however, eyelashes and eyebrows may also be involved. they are associated with people who do not bath or change clothes regularly. the person usually complains of intense itching or irritation. the main route is from person to person by direct contact, but eggs can stick to fibers, so clothing and bedding should be handled with care (see subheading 6.5.3.). staff should always wear gloves if they are likely to come into contact with any hirsute body part. clothing or bedding should be handled with gloves and either laundered or incinerated. treatment of a detainee in custody is good in theory but probably impractical because the whole body has to be treated. fleas lay eggs on floors, carpets, and bedding. in the united kingdom, most flea bites come from cats or dogs. the eggs and larvae fleas can survive for months and are reactivated in response to animal or human activity. because animal fleas jump off humans after biting, most detainees with flea bites will not have fleas, unless they are human fleas. treatment is only necessary if fleas are seen. after use, the cell should be vacuumed and cleaned with a proprietary insecticide. any bedding should be removed wearing gloves, bagged, and either laundered or incinerated. bedbugs live and lay eggs on walls, floors, furniture, and bedding. if you look carefully, fecal tracks may be seen on hard surfaces. if they are present for long enough, they emit a distinct odor. bedbugs are rarely found on the person but may be brought in on clothing or other personal effects. bedbugs bite at night and can cause sleep disturbance. the detainee does not need to be treated, but the cell should deemed out of use until it can be vacuumed and professionally cleaned with an insecticide solution. any bedding or clothing should be handled with gloves and disposed of as appropriate. staphylococcus aureus is commonly carried on the skin or in the nose of healthy people. approximately 25-30% of the population is colonized with the bacteria but remain well (43) . from time to time, the bacteria cause minor skin infections that usually do not require antibiotic treatment. however, more serious problems can occur (e.g., infection of surgical wounds, drug injection sites, osteomyelitis, pneumonia, or septicemia). during the last 50 years, the bacteria have become increasingly resistant to penicillin-based antibiotics (44) , and in the last 20 years, they have become resistant to an increasing number of alternative antibiotics. these multiresistant bacteria are known as methicillinresistant s. aureus (mrsa). mrsa is prevalent worldwide. like nonresistant staphylococci, it may remain undetected as a reservoir in colonized individuals but can also produce clinical disease. it is more common in individuals who are elderly, debilitated, or immunocompromised or those with open wounds. clusters of skin infections with mrsa have been reported among injecting drug users (idus) since 1981 in america (45, 46) , and more recently, similar strains have been found in the united kingdom in idus in the community (47) . this may have particular relevance for the forensic physician when dealing with idus sores. people who are immunocompetent rarely get mrsa and should not be considered at risk. the bacteria are usually spread via the hands of staff after contact with colonized or infected detainees or devices, items (e.g., bedding, towels, and soiled dressings), or environmental surfaces that have been contaminated with mrsa-containing body fluids. with either known or suspected cases (consider all abscesses/ulcers of idus as infectious), standard precautions should be applied. staff should wear gloves when touching mucous membranes, nonintact skin, blood or other body fluids, or any items that could be contaminated. they should also be encouraged to their wash hands with an antimicrobial agent regardless of whether gloves have been worn. after use, gloves should be disposed of in a yellow hazard bag and not allowed to touch surfaces. masks and gowns should only be worn when conducting procedures that generate aerosols of blood or other body fluids. because this is an unlikely scenario in the custodial setting, masks and gowns should not be necessary. gloves should be worn when handling bedding or clothing, and all items should be disposed of appropriately. any open wounds should be covered as soon as possible. the cell should be cleaned professionally after use if there is any risk that it has been contaminated. during the last decade, there has been an increasing awareness of the bacterial flora colonizing injection sites that may potentially lead to life-threatening infection (48) . in 1997, a sudden increase in needle abscesses caused by a clonal strain of group a streptococcus was reported among hospitalized idus in berne, switzerland (49) . a recent uk study showed that the predominant isolate is s. aureus, with streptococcus species forming just under one-fifth (50% î²-hemolytic streptococci) (50) . there have also been reports of both nonsporing and sporing anerobes (e.g., bacteroides and clostridia species, including clostridia botulinum) (51, 52) . in particular, in 2000, laboratories in glasgow were reporting isolates of clostridium novyi among idus with serious unexplained illness. by june 12, 2000, a total of 42 cases (18 definite and 24 probable) had been reported. a definite case was defined as an idu with both severe local and systemic inflammatory reactions. a probable case was defined as an idu who presented to the hospital with an abscess or other significant inflammation at an injecting site and had either a severe inflammatory process at or around an injection site or a severe systemic reaction with multiorgan failure and a high white cell count (53) . in the united kingdom, the presence of c. botulinum in infected injection sites is a relatively new phenomenon. until the end of 1999, there were no cases reported to the public health leadership society. since then, the number has increased, with a total of 13 cases in the united kingdom and ireland being reported since the beginning of 2002. it is believed that these cases are associated with contaminated batches of heroin. simultaneous injection of cocaine increases the risk by encouraging anerobic conditions. anerobic flora in wounds may have serious consequences for the detainee, but the risk of transmission to staff is virtually nonexistent. staff should be reminded to wear gloves when coming into contact with detainees with infected skin sites exuding pus or serum and that any old dressings found in the cell should be disposed of into the yellow bag marked "clinical waste" in the medical room. likewise, any bedding should be bagged and laundered or incinerated after use. the cell should be deemed out of use and professionally cleaned after the detainee has gone. the health care professional managing the detainee should clean and dress open wounds as soon as possible to prevent the spread of infection. it may also be appropriate to start a course of antibiotics if there is abscess formation or signs of cellulites and/or the detainee is systemically unwell. however, infections can often be low grade because the skin, venous, and lymphatic systems have been damaged by repeated penetration of the skin. in these cases, signs include lymphedema, swollen lymph glands, and darkly pigmented skin over the area. fever may or may not be present, but septicemia is uncommon unless the individual is immunocompromised (e.g., hiv positive). co-amoxiclav is the preferred treatment of choice because it covers the majority of staphylococci, streptococci, and anerobes (the dose depends on the degree of infection). necrotizing fasciitis and septic thrombophlebitis are rare but life-threatening complications of intravenous drug use. any detainee suspected of either of these needs hospital treatment. advice about harm reduction should also be given. this includes encouraging drug users to smoke rather than inject or at least to advise them to avoid injecting into muscle or skin. although most idus are aware of the risk of sharing needles, they may not realize that sharing any drug paraphernalia could be hazardous. advice should be given to use the minimum amount of citric acid to dissolve the heroin because the acid can damage the tissue under the skin, allowing bacteria to flourish. drugs should be injected at different sites using fresh works for each injection. this is particularly important when "speedballing" because crack cocaine creates an anerobic environment. medical help should be requested if any injection site become painful and swollen or shows signs of pus collecting under the skin. because intravenous drug users are at increased risk of acquiring hbv and hav, they should be informed that vaccination against both diseases is advisable. another serious but relatively rare problem is the risk from broken needles in veins. embolization can take anywhere from hours to days or even longer if it is not removed. complications may include endocarditis, pericarditis, or pulmonary abscesses (54, 55) . idus should be advised to seek medical help as soon as possible, and should such a case present in custody, then send the detainee straight to the hospital. the forensic physician may encounter bites in the following four circumstances: a detailed forensic examination of bites is given in chapter 4. with any bite that has penetrated the skin, the goals of therapy are to minimize soft tissue deformity and to prevent or treat infection. in the united kingdom and the united states, dog bites represent approximately three-quarters of all bites presenting to accident and emergency departments (56) . a single dog bite can produce up to 220 psi of crush force in addition to the torsional forces as the dog shakes its head. this can result in massive tissue damage. human bites may cause classical bites or puncture wounds (e.g., impact of fists on teeth) resulting in crush injuries. an estimated 10-30% of dog bites and 9-50% of human bites lead to infection. compare this with an estimated 1-12% of nonbite wounds managed in accident and emergency departments. the risk of infection is increased with puncture wounds, hand injuries, full-thickness wounds, wounds requiring debridement, and those involving joints, tendons, ligaments or fractures. comorbid medical conditions, such as diabetes, asplenia, chronic edema of the area, liver dysfunction, the presence of a prosthetic valve or joint, and an immunocompromised state may also increase the risk of infection. infection may spread beyond the initial site, leading to septic arthritis, osteomyelitis, endocarditis, peritonitis, septicemia, and meningitis. inflammation of the tendons or synovial lining of joints may also occur. if enough force is used, bones may be fractured or the wounds may be permanently disfiguring. assessment regarding whether hospital treatment is necessary should be made as soon as possible. always refer if the wound is bleeding heavily or fails to stop when pressure is applied. penetrating bites involving arteries, nerves, muscles, tendons, the hands, or feet, resulting in a moderate to serious facial wound, or crush injuries, also require immediate referral. if management within custody is appropriate, ask about current tetanus vaccine status, hbv vaccination status, and known allergies to antibiotics. wounds that have breached the skin should be irrigated with 0.9% (isotonic) sodium chloride or ringer's lactate solution instead of antiseptics, because the latter may delay wound healing. a full forensic documentation of the bite should be made as detailed in chapter 4. note if there are clinical signs of infection, such as erythema, edema, cellulitis, purulent discharge, or regional lymphadenopathy. cover the wound with a sterile, nonadhesive dressing. wound closure is not generally recommended because data suggest that it may increase the risk of infection. this is particularly relevant for nonfacial wounds, deep puncture wounds, bites to the hand, clinically infected wounds, and wounds occurring more than 6-12 hours before presentation. head and neck wounds in cosmetically important areas may be closed if less than 12 hours old and not obviously infected. â�¢ dog bites-pasteurella canis, pasteurella multocida, s. aureus, other staphylococci, streptococcus species, eikenella corrodens, corynebacterium species, and anerobes, including bacteroides fragilis and clostridium tetani â�¢ human bites-streptococcus species, s. aureus, e. corrodens, and anerobes, including bacteroides (often penicillin resistant), peptostreptococci species, and c. tetani. tuberculosis (tb) and syphilis may also be transmitted. â�¢ dog bites-outside of the united kingdom, australia, and new zealand, rabies should be considered. in the united states, domestic dogs are mostly vaccinated against rabies (57) , and police dogs have to be vaccinated, so the most common source is from racoons, skunks, and bats. â�¢ human bites-hbv, hbc, hiv, and herpes simplex. antibiotics are not generally needed if the wound is more than 2 days old and there is no sign of infection or in superficial noninfected wounds evaluated early that can be left open to heal by secondary intention in compliant people with no significant comorbidity (58) . antibiotics should be considered with high-risk wounds that involve the hands, feet, face, tendons, ligaments, joints, or suspected fractures or for any penetrating bite injury in a person with diabetes, asplenia, or cirrhosis or who is immunosuppressed. coamoxiclav (amoxycillin and clavulanic acid) is the first-line treatment for mild-moderate dog or human bites resulting in infections managed in primary care. for adults, the recommended dose is 500/125 mg three times daily and for children the recommended does is 40 mg/kg three times daily (based on amoxycillin component). treatment should be continued for 10-14 days. it is also the first-line drug for prophylaxis when the same dose regimen should be prescribed for 5-7 days. if the individual is known or suspected to be allergic to penicillin, a tetracycline (e.g., doxycycline 100 mg twice daily) and metronidazole (500 mg three times daily) or an aminoglycoside (e.g., erythromycin) and metronidazole can be used. in the united kingdom, doxycycline use is restricted to those older than 12 years and in the united states to those older than 8 years old. specialist advice should be sought for pregnant women. anyone with severe infection or who is clinically unwell should be referred to the hospital. tetanus vaccine should be given if the primary course or last booster was more than 10 years ago. human tetanus immunoglobulin should be considered for tetanus-prone wounds (e.g., soil contamination, puncture wounds, or signs of devitalized tissue) or for wounds sustained more than 6 hours old. if the person has never been immunized or is unsure of his or her tetanus status, a full three-dose course, spaced at least 1 month apart, should be given. penetrating bite wounds that involve only saliva may present a risk of hbv if the perpetrator belongs to a high-risk group. for management, see subheadings 5.1.6. and 5.1.7. hcv and hiv are only a risk if blood is involved. the relevant management is dealt with in subheadings 5.2.5. and 5.4.6. respiratory tract infections are common, usually mild, and self-limiting, although they may require symptomatic treatment with paracetamol or a nonsteroidal antiinflammatory. these include the common cold (80% rhinoviruses and 20% coronaviruses), adenoviruses, influenza, parainfluenza, and, during the summer and early autumn, enteroviruses. special attention should be given to detainees with asthma or the who are immunocompromised, because infection in these people may be more serious particularly if the lower respiratory tract is involved. the following section includes respiratory pathogens of special note because they may pose a risk to both the detainee and/or staff who come into close contact. there are five serogroups of neisseria meningitidis: a, b, c, w135, and y. the prevalence of the different types varies from country to country. there is currently no available vaccine against type b, but three other vaccines (a+c, c, and acwy) are available. overall, 10% of the uk population carry n. meningitidis (25% in the 15-19 age group) (59) . in the united kingdom, most cases of meningitis are sporadic, with less than 5% occurring as clusters (outbreaks) amongst school children. between 1996 and 2000, 59% of cases were group b, 36% were group c, and w135 and a accounted for 5%. there is a seasonal variation, with a high level of cases in winter and a low level in the summer. the greatest risk group are the under 5 year olds, with a peak incidence under 1 year old. a secondary peak occurs in the 15-to 19-year-old age group. in sub-saharan africa, the disease is more prevalent in the dry season, but in many countries, there is background endemicity year-round. the most prevalent serogroup is a. routine vaccination against group c was introduced in the united kingdom november 1999 for everybody up to the age of 18 years old and to all firstyear university students. this has since been extended to include everyone under the age of 25 years old. as a result of the introduction of the vaccination program, there has been a 90% reduction of group c cases in those younger than under 18 years and an 82% reduction in those under 1 year old (60, 61) . an outbreak of serogroup w135 meningitis occurred among pilgrims on the hajj in 2000. cases were reported from many countries, including the united kingdom. in the united kingdom, there is now an official requirement to be vaccinated with the quadrivalent vaccine (acwy vax) before going on a pilgrimage (hajj or umra), but illegal immigrants who have not been vaccinated may enter the country (62). after an incubation period of 3-5 days (63,64) , disease onset may be either insidious with mild prodromal symptoms or florid. early symptoms and signs include malaise, fever, and vomiting. sever headache, neck stiffness, photophobia, drowsiness, and a rash may develop. the rash may be petechial or purpuric and characteristically does not blanche under pressure. meningitis in infants is more likely to be insidious in onset and lack the classical signs. in approx 15-20% of cases, septicemia is the predominant feature. even with prompt antibiotic treatment, the case fatality rate is 3-5% in meningitis and 15-20% in those with septicemia. (65). a person should be considered infectious until the bacteria are no longer present in nasal discharge. with treatment, this is usually approx 24 hour. the disease is spread through infected droplets or direct contact from carriers or those who are clinically ill. it requires prolonged and close contact, so it is a greater risk for people who share accommodation and utensils and kiss. it must also be remembered that unprotected mouth-to-mouth resuscitation can also transmit disease. it is not possible to tell if a detainee is a carrier. nevertheless, the risk of acquiring infection even from an infected and sick individual is low, unless the individual has carried out mouth-to-mouth resuscitation. any staff member who believes he or she has been placed at risk should report to the occupational health department (or equivalent) or the nearest emergency department at the earliest opportunity for vaccination. if the detainee has performed mouth-to-mouth resuscitation, prophylactic antibiotics should be given before receiving vaccination. rifampicin, ciprofloxacin, and ceftriaxone can be used; however, ciprofloxacin has numerous advantages (66) . only a single dose of 500 mg (adults and children older than 12 years) is needed and has fewer side effects and contraindications than rifampicin. ceftriaxone has to be given by injection and is therefore best avoided in the custodial setting. if the staff member is pregnant, advice should be sought from a consultant obstetrician, because ciprofloxacin is not recommended (67) . for anyone dealing regularly with illegal immigrants (especially from the middle east or sub-saharan africa) (e.g., immigration services, custody staff at designated stations, medical personnel, and interpreters), should consider being vaccinated with acwy vax. a single injection provides protection for 3 years. detainees suspected of disease should be sent directly to the hospital. human tb is caused by infection with mycobacterium tuberculosis, mycobacterium bovis, or mycobacterium africanum. it is a notifiable disease under legislation specific to individual countries; for example, in the united kingdom, this comes under the public health (control of disease) act of 1984. in 1993, the who declared tb to be a global emergency, with an estimated 7-8 million new cases and 3 million deaths occurring each year, the majority of which were in asia and africa. however, these statistics are likely to be an underestimate because they depend on the accuracy of reporting, and in poorer countries, the surveillance systems are often inadequate because of lack of funds. even in the united kingdom, there has been an inconsistency of reporting particularly where an individual has concomitant infection with hiv. some physicians found themselves caught in a dilemma of confidentiality until 1997, when the codes of practice were updated to encourage reporting with patient consent (68) . with the advent of rapid identification tests and treatment and the use of bacillus calmette-guã©rin (bcg) vaccination for prevention, tb declined during the first half of the 20th century in the united kingdom. however, since the early 1990s, numbers have slowly increased, with some 6800 cases reported in 2002 (69) . in 1998, 56% of reported cases were from people born outside the united kingdom and 3% were associated with hiv infection (70, 71) . london has been identified as an area with a significant problem. this has been attributed to its highly mobile population, the variety of ethnic groups, a high prevalence of hiv, and the emergence of drug-resistant strains (1.3% in 1998 ) (phls, unpublished data-mycobnet). a similar picture was initially found in the united states, when there was a reversal of a long-standing downward trend in 1985. however, between 1986 and 1992, the number of cases increased from 22,201 to 26,673 (72) . there were also serious outbreaks of multidrug-resistant tb (mdr-tb) in hospitals in new york city and miami (73) . factors pertinent to the overall upswing included the emergence of hiv, the increasing numbers of immigrants from countries with a high prevalence of tb, and perhaps more significantly, stopping categorical federal funding for control activities in 1972. the latter led to a failure of the public health infrastructure for tb control. since 1992, the trend has reversed as the cdc transferred most of its funds to tb surveillance and treatment program in states and large cities. from 1992 to 2001, the annual decline averaged by 7.3% (74) , but the following year this was reduced to 2%, indicating that there was no room for complacency. the who has been proactive and is redirecting funding to those countries most in need. in october 1998, a global partnership called stop tb was launched to coordinate every aspect of tb control, and by 2002, the partnership had more than 150 member states. a target was set to detect at least 70% of infectious cases by 2005. the acquisition of tb infection is not necessarily followed by disease because the infection may heal spontaneously. it may take weeks or months before disease becomes apparent, or infection may remain dormant for years before reactivation in later life especially if the person becomes debilitated or immunocompromised. contrary to popular belief, the majority of cases of tb in people who are immunocompetent pass unnoticed. of the reported cases, 75% involve the lung, whereas nonrespiratory (e.g., bone, heart, kidney, and brain) or dissemination (miliary tb) are more common in immigrant ethnic groups and individuals who are immunocompromised (75) . they are also more likely to develop resistant strains. in the general population, there is an estimated 10% lifetime risk of tb infection progressing to disease (76) . there has been an increase in the number of cases of tb associated with hiv owing to either new infection or reactivation. tb infection is more likely to progress to active tb in hiv-positive individuals, with a greater than50% lifetime risk (77) . tb can also lead to a worsening of hiv with an increase in viral load (78) . therefore, the need for early diagnosis is paramount, but it can be more difficult because pulmonary tb may present with nonspecific features (e.g., bilateral, unilateral, or lower lobe shadowing) (79). after an incubation period of 4-12 weeks, symptoms may develop (see table 6 ). the main route is airborne through infected droplets, but prolonged or close contact is needed. nonrespiratory disease is not considered a risk unless the mycobacterium is aerosolized under exceptional circumstances (e.g., during surgery) or there are open abscesses. a person is considered infectious as long as viable bacilli are found in induced sputum. untreated or incompletely treated people may be intermittently sputum positive for years. after 2 weeks of appropriate treatment, the individual is usually considered as noninfectious. this period is often extended for treatment of mdr-tb or for those with concomitant hiv. patient compliance also plays an important factor. the risk of infection is directly proportional to the degree of exposure. more severe disease occurs in individuals who are malnourished, immunocompromised (e.g., hiv), and substance misusers. people who are immunocompromised are at special risk of mdr-tb or mycobacterium avium intracellulare (mai). staff with disease should stay off work until the treatment course is complete and serial sputum samples no longer contain bacilli. staff in contact with disease who have been vaccinated with bcg are at low risk of acquiring disease but should minimize their time spent in the cell. those who have not received bcg or who are immunocompromised should avoid contact with the detainee wherever possible. detainees with mai do not pose a risk to a staff member, unless the latter is immunocompromised. any staff member who is pregnant, regardless of bcg status or type of tb, should avoid contact. anyone performing mouth-to-mouth resuscitation with a person with untreated or suspected pulmonary tb should be regarded as a household contact and should report to occupational health or their physician if no other route exists. they should also be educated regarding the symptoms of tb. anyone who is likely to come into repeated contact with individuals at risk of tb should receive bcg (if he or she has not already done so), regardless of age, even though there is evidence to suggest that bcg administered in adult life is less effective. this does not apply to individuals who are immunocompromised or pregnant women. in the latter case, vaccination should preferably be deferred until after delivery. detainees with disease (whether suspected or diagnosed) who have not been treated or treatment is incomplete should be kept in custody for the minimum time possible. individuals with tb who are immunocompromised are usually too ill to be detained; if they are, they should be considered at greater risk of transmitting disease to staff. any detainee with disease should be encouraged to cover his or her mouth and nose when coughing and sneezing. staff should wear gloves when in contact with the detainee and when handling clothing and bedding. any bedding should be bagged after use and laundered or incinerated. the cell should be deemed out of action until it has been ventilated and professionally decontaminated, although there is no hard evidence to support that there is a risk of transmission from this route (70). on march 14, 2003 , the who issued a global warning to health authorities about a new atypical pneumonia called sars. the earliest case was believed to have originated in the guandong province of china on november 16, 2002. the causative agent was identified as a new corona virus-sars-cov (80, 81) . by the end of june 2003, 8422 cases had been reported from 31 different countries, with a total of 916 deaths. approximately 92% of cases occurred in china (including hong kong, taiwan, and macao). the case fatality rate varied from less than 1% in people younger than 24 years, 6% in persons aged 25-44 years, 15% in those aged 44-64 years, and more than 50% in persons 65 years or older. on july 5, 2003, the who reported that the last human chain of transmission of sars had been broken and lifted the ban from all countries. however, it warned that everyone should remain vigilant, because a resurgence of sars is possible. their warning was well given because in december 2003, a new case of sars was detected in china. at the time of this writing, three more cases have been identified. knowledge about the epidemiology and ecology of sars-cov and the disease remains limited; however, the experience gained from the previous outbreak enabled the disease to be contained rapidly, which is reflected in the few cases reported since december 2003. there is still no specific treatment or preventative vaccine that has been developed. the incubation period is short, approx 3-6 days (maximum 10 days), and, despite the media frenzy surrounding the initial outbreak, sars is less infectious than influenza. the following clinical case definition of sars has been developed for public health purposes (82) . a person with a history of any combination of the following should be examined for sars: â�¢ fever (at least 38â°c); and â�¢ one of more symptoms of lower respiratory tract illness (cough, difficulty in breathing, or dyspnea); and â�¢ radiographic evidence of lung infiltrates consistent with pneumonia or respiratory distress syndrome or postmortem findings of these with no identifiable cause; and â�¢ no alternative diagnosis can fully explain the illness. laboratory tests have been developed that include detection of viral rna by pcr from nasopharyngeal secretions or stool samples, detection of antibodies by enzyme-linked immunosorbent assay or immunofluorescent antibody in the blood, and viral culture from clinical specimens. available information suggests that close contact via aerosol or infected droplets from an infected individual provide the highest risk of acquiring the disease. most cases occurred in hospital workers caring for an index case or his or her close family members. despite the re-emergence of sars, it is highly unlikely that a case will be encountered in the custodial setting in the near future. however, forensic physicians must remain alert for the sars symptoms and keep up-to-date with recent outbreaks. information can be obtained from the who on a daily basis from its web site. if sars is suspected, medical staff should wear gloves and a surgical mask when examining a suspected case; however, masks are not usually available in custody. anyone suspected of sars must be sent immediately to the hospital, and staff who have had prolonged close contact should be alerted as to the potential symptoms. the most consistent feature of diseases transmitted through the fecaloral route is diarrhea (see table 7 ). infective agents include bacteria, viruses, and protozoa. because the causes are numerous, it is beyond the remit of this chapter to cover them all. it is safest to treat all diarrhea as infectious, unless the detainee has a proven noninfectious cause (e.g., crohn's disease or ulcerative colitis). all staff should wear gloves when in contact with the detainee or when handling clothing and bedding, and contaminated articles should be laundered or incinerated. the cell should be professionally cleaned after use, paying particular attention to the toilet area. this viral hepatitis occurs worldwide, with variable prevalence. it is highest in countries where hygiene is poor and infection occurs year-round. in temperate climates, the peak incidence is in autumn and winter, but the trend is becoming less marked. all age groups are susceptible if they are nonimmune or have not been vaccinated. in developing countries, the disease occurs in early childhood, whereas the reverse is true in countries where the standard of living is higher. in the united kingdom, there has been a gradual decrease in the number of reported cases from 1990 to 2000 (83, 84) . this results from, in part, improved standards of living and the introduction of an effective vaccine. the highest incidence occurs in the 15-to 34-year-old age group. approximately 25% of people older than 40 years have natural immunity, leaving the remainder susceptible to infection (85) . small clusters occur from time to time, associated with a breakdown in hygiene. there is also an increasing incidence of hav in gay or bisexual men and their partners (86) . an unpublished study in london in 1996 showed a seroprevalence of 23% among gay men (young y et al., unpublished). the clinical picture ranges from asymptomatic infection through a spectrum to fulminant hepatitis. unlike hbv and hcv, hav does not persist or progress to chronic liver damage. infection in childhood is often mild or asymptomatic but in adults tends to be more severe. after an incubation period of 15-50 days (mean 28 days) symptomatic infection starts with the abrupt onset of jaundice anything from 2 days to 3 weeks after the anicteric phase. it lasts for approximately the same length of time and is often accompanied by a sudden onset of fever. hav infection can lead to hospital admission in all age groups but is more likely with increasing age as is the duration of stay. the overall mortality is less than 1%, but 15% of people will have a prolonged or relapsing illness within 6-9 months (cdc fact sheet). fulminant hepatitis occurs in less than 1% of people but is more likely to occur in individuals older than 65 years or in those with pre-existing liver disease. in patients who are hospitalized, case fatality ranges from 2% in 50-59 years olds to nearly 13% in those older than 70 years (84). the individual is most infectious in the 2 weeks before the onset of jaundice, when he or she is asymptomatic. this can make control of infection difficult because the disease is not recognized. the main route is fecal-oral through the ingestion of contaminated water and food. it can also be transmitted by personal contact, including homosexuals practicing anal intercourse and fellatio. there is a slight risk from blood transfusions if the donor is in the acute phase of infection. it should not be considered a risk from needlestick injuries unless clinical suspicion of hav is high. risk groups include homeless individuals, homosexuals, idus, travellers abroad who have not been vaccinated, patients with chronic liver disease and chronic infection with hbv and hcv, employees and residents in daycare centers and hostels, sewage workers, laboratory technicians, and those handling nonhuman primates. several large outbreaks have occurred among idus, some with an epidemiological link to prisons (87, 88) . transmission occurs during the viremic phase of the illness through sharing injecting equipment and via fecal-oral routes because of poor living conditions (89) . there have also been reports of hav being transmitted through drugs that have been carried in the rectum. a study in vancouver showed that 40% of idus had past infection of hav, and they also showed an increased prevalence among homosexual/bisuexual men (90). staff with disease should report to occupational health and stay off work until the end of the infective period. those in contact with disease (either through exposure at home or from an infected detainee) should receive prophylactic treatment as soon as possible (see subheading 8.3.7.). to minimize the risk of acquiring disease in custody, staff should wear gloves when dealing with the detainee and then wash their hands thoroughly. gloves should be disposed of only in the clinical waste bags. detainees with disease should be kept in custody for the minimum time possible. they should only be sent to the hospital if fulminant hepatitis is suspected. the cell should be quarantined after use and professionally cleaned. any bedding or clothing should be handled with gloves and laundered or incinerated according to local policy. detainees reporting contact with disease should be given prophylactic treatment as soon as possible (see subheading 8.3.7.). contacts of hav should receive hav vaccine (e.g., havrix monodose or avaxim) if they have not been previously immunized or had disease. human normal immunoglobulin (hnig), 500 mg, deep intramuscular in gluteal muscle should be used in the following circumstances: â�¢ has the detainee traveled to africa, south east asia, the indian subcontinent, central/south america, or the far east in the last 6-12 months? â�¢ ascertain whether he or she received any vaccinations before travel and, if so, which ones. â�¢ ask if he or she took malaria prophylaxis, what type, and whether he or she completed the course. â�¢ ask if he or she swam in any stagnant lakes during the trip. â�¢ if the answer to any of the above is yes, ask if he or she has experienced any of the following symptoms: a fever/hot or cold flushes/shivering. diarrhea â± abdominal cramps â± blood or slime in the stool. a rash. persistent headaches â± light sensitivity. nausea or vomiting. aching muscles/joints. a persistent cough (dry or productive) lasting at least 3 weeks. â�¢ take temperature. â�¢ check skin for signs of a rash and note nature and distribution. â�¢ check throat. â�¢ listen carefully to the lungs for signs of infection/consolidation. staff at higher risk of coming in to contact with hav should consider being vaccinated before exposure. two doses of vaccine given 6-12 months apart give at least 10 years of protection. there is no specific treatment for hav, except supportive measures and symptomatic treatment. although the chance of encountering a tropical disease in custo1dy is small, it is worth bearing in mind. it is not necessary for a forensic physician to be able to diagnose the specific disease but simply to recognize that the detainee/staff member is ill and whether he or she needs to be sent to the hospital (see tables 8-10) . this is best achieved by knowing the right questions to ask and carrying out the appropriate examination. tables 8-10 should be used as an aide to not missing some more unusual diseases. guidance for clinical health care workers: protection against infection with blood-borne viruses; recommendations of the expert advisory group on aids and the advisory group on hepatitis guidelines for hand hygiene in health care settings. recommendations of the healthcare infection control practices advisory committee and the hicpac/ shea/apic/idsa hand hygiene task force national model regulations for the control of workplace hazardous substances. commonwealth of australia, national occupational health and safety committee good practice guidelines for forensic medical examiners and the hospital infection control practices advisory committee. guideline for infection control in health care personnel report from the unlinked anonymous surveys steering group. department of health a strategy for infectious diseases-progress report. blood-borne and sexually transmitted viruses: hepatitis. department of health universal precautions for prevention of transmission of human immuno-deficiency virus, hepatitis b virus and other bloodborne pathogens in health-care settings risk factors for horizontal transmission of hepatitis b in a rural district in ghana familial clustering of hepatitis b infection: study of a family intrafamilial transmission of hepatitis b in the eastern anatolian region of turkey hepatitis b outbreak at glenochil prison during european network for hiv/aids and hepatitis prevention in prisons. second annual report. the network prevalence of hiv, hepatitis b and hepatitis c antibodies in prisoners in england and wales; a national survey the epidemiology of acute and chronic hepatitis c the role of the parenteral antischistosomal therapy in the spread of hepatitis c virus in egypt chronic hepatitis c: disease management. nih publication no. 03-4230. february department of health hepatitis c virus: eight years old laboratory surveillance of hepatitis c virus in england and wales: 1992-1996 last update aids/hiv quarterly surveillance tables provided by the phls aids centre (cdsc) and the scottish centre for infection and environmental health hiv and aids in the uk in 2001. communicable disease surveillance centre. an update mode of vertical transmission of hiv-1. a metanalysis of fifteen prospective cohort studies vertical transmission rate for hiv in the british isles estimated on surveillance data hiv post-exposure prophylaxis after sexual assault: the experience of a sexual assault service in london guidance from the uk chief medical officer's expert advisory group on aids. uk health department failures of zidovudine post exposure prophylaxis seroconversion to hiv-1 following a needlestick injury despite combination post-exposure prophylaxis department of health, immunisation against infectious disease. united kingdom: her majesty's stationery office chickenpox-disease predominantly affecting adults in rural west bengal prevention of varicella: recommendations of the advisory committee on immunization practices varicella-zoster virus epidemiology. a changing scene? use of acyclovir for varicella pneumonia during pregnancy outcome after maternal varicella infection in the first 20 weeks of pregnancy outcome in newborn babies given anti-varicella zoster immunoglobulin after perinatal maternal infection with varicella zoster virus varicella-zoster virus dna in human sensory ganglia epidemiology and natural history of herpes zoster and post herpetic neuralgia clinical applications for changepoint analysis of herpes zoster pain treatment of scabies with permethrin versus lindane and benzoyl benzoate treatment of ectoparasitic infections; review of the english-language literature nasal carriage of staphylococcus aureus: epidemiology and control measures centers for disease control and prevention. community-acquired methicillinresistant staphylococcus aureus infections-michigan methicillinresistant staphylococcus aureus, epidmiologic observations during a community acquired outbreak emergence of pvl-producing strains of staphylococcus aureus bacteriology of skin and soft tissue infections: comparison of infections in intravenous drug users and individuals with no history of intravenous drug use outbreak among drug users caused by a clonal strain of group a streptococcus. dispatchesemerging infectious diseases bacteriological skin and subcutaneous infections in injecting drug users-relevance for custody wound botulism associated with black tar heroin among injecting drug users isolation and identification of clostridium spp from infections associated with injection of drugs: experiences of a microbiological investigation team greater glasgow health board, scifh. unexplained illness among drug injectors in glasgow embolization of illicit needle fragments right ventricular needle embolus in an injecting drug user: the need for early removal departments of emergency medicine and pediatrics, lutheran general hospital of oak brook, advocate health system. emedicine-human bites prevention and treatment of dog bites human bites. department of plastic surgery guidelines for public health management of meningococcal diseases in the uk planning, registration and implementation of an immunisation campaign against meningococcal serogroup c disease in the uk: a success story efficacy of meningococcal serogroup c conjugate vaccine in teenagers and toddlers in england quadrivalent meningoimmunisation required for pilgrims to saudi arabia risk of laboratory-acquired meningococcal disease cluster of meningococcal disease in rugby match spectators immunisation against infectious disease. her majesty's stationery office ciprofloxacin as a chemoprophylactic agent for meningococcal diseaselow risk of anaphylactoid reactions joint formulary committee 2002-03. british national formulary notification of tuberculosis an updated code of practice for england and wales statutory notifications to the communicable disease surveillance centre. preliminary annual report on tuberculosis cases reported in england, wales, and the prevention and control of tuberculosis in the united kingdom: uk guidance on the prevention and control of transmission of 1. hiv-related tuberculosis 2. drug-resistant, including multiple drug-resistant, tuberculosis. department of health, scottish office control and prevention of tuberculosis in the united kingdom: code of practice epidemiology of tuberculosis in the united states nosocomial transmission of multi-drug resistant tuberculosis among hiv-infected persons-florida the continued threat of tuberculosis tuberculosis-a clinical handbook the white plague: down and out, or up and coming? a prospective study of the risk of tuberculosis among intravenous drug users with human immunodeficiency virus infection influence of tuberculosis on human immunodeficiency virus (hiv-1): enhanced cytokine expression and elevated b 2 -microglobulin in hiv-1 associated tuberculosis the chest roenterogram in pulmonary tuberculosis patients seropositive for human immunodeficiency virus type 1 coronavirus as a possible cause of severe acute respiratory syndrome epidemiological determinants of spread of causal agents of severe acute respiratory syndrome in hong kong alert, verification and public health management of sars in post-outbreak period age-specific antibody prevalence to hepatitis a in england: implications for disease control phls advisory committee on vaccination and immunisation. guidelines for the control of hepatitis a infection control of a community hepatitis a outbreak using hepatitis a vaccine seroprevalence of and risk factors for hepatitis a infection among young homosexual and bisexual men outbreaks of hepatitis a among illicit drug users identifying target groups for a potential vaccination program during a hepatitis a community outbreak multiple modes of hepatitis a transmission among metamphetamine users past infection with hepatitis a among vancouver street youth, injection drug users and men who have sex with men implications for vaccination programmes key: cord-296979-8r851j4t authors: zhong, ying; liu, dong-ling; ahmed, mohamed morsi. m.; li, peng-hao; zhou, xiao-ling; xie, qing-dong; xu, xiao-qing; han, ting-ting; hou, zhi-wei; zhong, chen-yao; huang, ji-hua; zeng, fei; huang, tian-hua title: host genes regulate transcription of sperm-introduced hepatitis b virus genes in embryo date: 2017-10-31 journal: reproductive toxicology doi: 10.1016/j.reprotox.2017.08.009 sha: doc_id: 296979 cord_uid: 8r851j4t abstract hepatitis b virus (hbv) can invade the male germline, and sperm-introduced hbv genes could be transcribed in embryo. this study was to explore whether viral gene transcription is regulated by host genes. embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the hbv genome. total rna extracted from test and control embryos were subjected to smart-pcr, ssh, microarray hybridization, sequencing and blast analysis. twenty-nine sequences showing significant identity to five human gene families were identified, with csh2, eif4g2, pcbd2, psg4 and ttn selected to represent target genes. using qrt-pcr, when csh2 and pcbd2 (or eif4g2, psg4 and ttn) were silenced by rnai, transcriptional levels of hbv s and x genes decreased (or increased). this is the first report that host genes participate in regulation of sperm-introduced hbv gene transcription in embryo, which is critical to prevent negative impact of hbv infection on early embryonic development. hepatitis b is a potentially life-threatening liver infection caused by the hepatitis b virus (hbv). transmission of hbv through blood transfusion [1] , body fluids [2] , intrauterine infection [3] , cell, tissue and organ transplantation [4] , and others including hemodialysis units or intravenous drug injection or occupational exposure [5, 6] have been documented. in recent years some studies reported a true vertical transmission of hbv via germline and found that hbv has a negative effect on sperm motility in vivo and that couples in which the male is infected with hbv have a high risk of a low fertilization rate after in vitro fertilization (ivf) [7] . huang et al. provided direct evidence that hbv dna is able to integrate into the chromosomes of patient sperm, and that such hbv-carrying sperm can fertilize oocytes [8] . after fertilization, the sperm-introduced hbv c, s and x genes retain their functions of replication and expression in embryonic cells [9, 10] . subsequently, some studies showed that the hbv s protein can cause a series of apoptotic events resulting in reduced sperm motility, loss of sperm membrane integrity, sperm dysfunction, decreased fertility, and sperm death [11] [12] [13] . hu et al. collected 578 embryos from couples with at least one hbsag seropositive partner, and found that hbv dna was present in 14.4% (83/578) of embryos [14] . thus the effects of hbv infection on human reproduction and early embryonic development and their mechanisms have attracted the attention of researchers. sperm samples from healthy donors were divided into two groups: the test group was transfected with plasmid containing the full-length hbv genome (t), and the control group was non-transfected (c). zona-free hamster ova were fertilized by transfected or non-transfected sperm to obtain two-cell embryos. total rna was extracted from t-and c-sperm-derived embryos, respectively and then subjected to smart amplification, followed by ssh in both forward (t as tester) and reverse (c as tester) directions to enrich for up-regulated genes (t-c amplicon) or down-regulated genes (c-t amplicon). after t-a cloning and bacterial amplification, the forward and reverse libraries were cloned by pcr to obtain single insert-containing clones that were used for microarray assay. clones with a fold change value greater than 2 or less than 0.5 when compared with the average cy5/cy3 intensity ratio, the differential expression of which was statistically significant, were selected for sequencing. for the acquired sequences, blast was used to search for sequences homologous to human genes in the genbank nucleotide database. of the sequences, five were selected as the target genes and their expression in two-cell embryos was confirmed by rt-pcr. b: sperm in the test group was co-transfected with plasmid containing the full-length hbv genome and the target gene-specific shrna/sirna. sperm in the control group was transfected with plasmid containing the full-length hbv genome alone. the transcription levels of hbv s and x genes in the two-cell embryos were assessed by real time qrt-pcr to identify whether the target genes participated in regulation of hbv gene expression. same procedures listed above were hepatitis b infection and disease pathogenesis are known to be influenced by a number of factors, including host genetic factors [15] . many cellular proteins that possibly regulate hbv gene transcription in hepatocytes have been identified, including liver-enriched proteins, such as hnf1, hnf3, c/ebp, and klf15, ubiquitous factors, such as sp1, rfx1, nf-y, and ap1, and members of the nuclear receptor superfamily, such as hnf4, rxra, ppara and coup-tfs [16] [17] [18] [19] . hepatotropism is a prominent feature of hbv infection, and virus infection appears to be restricted to hepatocytes [20] . because sperm-derived embryos differ from hepatic cells, this study was undertaken to explore whether host genes participate in regulation of hbv gene transcription in embryonic cells, which is critical to reveal the regulation mechanism of viral gene transcription in these cells and to prevent the negative impact of hbv infection on early embryonic development. although using embryo produced by fertilization of human oocyte with human sperm carrying hbv genes would be an ideal model, such a model presents major moral, ethical and legal problems. the interspecific ivf between human sperm and zona-free golden hamster ova is highly associated with human in vitro fertilization [21] and has no the aforementioned problems, which has been included in "who laboratory manual for the examination and processing of human semen" [22] and widely used in the research of reproductive biology and medicine [23] [24] [25] , thus the interspecific ivf was employed to obtain two-cell embryos in the current study. as hbv dna has been detected in patient sperm [7, 8] , donor sperm transfected with recombinant plasmid containing the fulllength hbv genome was used instead of patient sperm to explore whether host genes participate in regulation of sperm-introduced hbv gene transcription in embryos. three measures were taken to ensure reliable and accurate results (fig. 1) . first, switching mechanism at 5 end of rna template (smart) amplification was employed because, unlike cells from other tissues, limited number of two-cell embryos made recovery of significant amounts of rna very difficult. smart amplification allows the synthesis of highquality cdna for suppression subtractive hybridization (ssh) from sub-microgram levels of rna [26] . combined with t-a cloning and bacterial amplification, we obtained sufficient subtraction products for array probe preparation, microarray assay and cdna sequencing. next, we combine ssh with microarray to obtain the cdnas highly enriched for differentially expressed genes of both high and low abundance and greatly reduce tedious work for screening of subtraction libraries, as well as the likelihood of false-positive clones enriched via ssh [27] . finally, we silenced the target genes and a control gene by rna interference (rnai) to detect effects of the silencing of these genes on transcriptional level of hbv genes to determine whether host genes participate in regulation of hbv gene transcription. semen samples were obtained from healthy donors. written, informed consent was obtained from all study subjects who allowed their sperm samples to be used for research. all protocols used in the current study involving human subjects were approved by the institutional ethical review boards of chengdu jingjiang hospital for maternal and child health care (approval number: cjhmchc-0010) and by the ethics committee of shantou univeralso used to isolate and identify a control gene with a fold change value greater than 0.5 and less than 2 when compared with the average cy5/cy3 intensity ratio, the differential expression of which was not statistically significant. sity medical college (approval no. sumc-00-0031) according to the world medical association declaration of helsinki: ethical principles for medical research involving human subjects [28] . the animal protocol was designed to minimize pain or discomfort. the animals were acclimatized to laboratory conditions (23 • c, 12 h/12 h light/dark, 50% humidity, ad libitum access to food and water) for two weeks prior to experimentation. all animals were euthanized by barbiturate overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for oocyte collection, and then their bodies were subjected to harmless treatment. all procedures involving animals were reviewed and approved by the medical animal care & welfare committee in shantou university medical college (iacuc protocol number: sumc2015-152) according to the recommended guide for the care and use of laboratory animals published by the national research council (us) [29] . semen samples were incubated in a humidified incubator (37 • c, 5% co 2 in air) for 30 min to allow liquefaction. motile sperm were selected using the swim-up method in biggers-whittem-whittingham (bww) medium supplemented with 0.3% bovine serum albumin (bsa) [30] . after washing, these sperm samples were used for subsequent experiments. recombinant plasmid pbr322-hbv containing the full-length hbv genome was kindly provided by professor yi-ping hu (the open laboratory for molecular genetics, the second military medical university, china). construction and transfection of plasmid pires2-egfp-hbv ( fig. 2 ) was performed as described previously [31] . briefly, a full-length cdna of hbv was isolated from pbr322-hbv by digestion of the restriction enzyme ecori, and purified using purification extraction kit (takara biotechnology (dalian) co., ltd, china). plasmid pires2-egfp vector (promega, madison, wi, usa) was linearized and mixed with the hbv cdna at 1:3-1:10 molar ratio in a ligation reaction and incubated at 16 • c overnight. the ligation mixture was used directly to transform the ecoli competent cells dh5␣ according to the manufacturer's instruction (takara). the right orientation and authenticity of the hbv coding sequence were confirmed by restriction digestion. each sperm sample from a healthy donor was divided into two groups: a test group and a control group. a 100-l aliquot of a mixture comprised of 1 l of plasmid pires2-egfp-hbv (1.5 g/l) containing the full-length hbv genome, 6 l of fugene hd, and 93 l of hepes-buffered saline was incubated at room temperature for 15 min, and then added to the sperm in the test group, which was incubated for 1.5 h. the sperm in the control group were not transfected. to measure the transfection efficiency, a nick translation kit (roche, basel, switzerland) and fluorescein-12-dutp (thermo fisher scientific, waltham, ma, usa) were used. the plasmid was labeled with fluorescein-12-dutp by nick translation according to the manufacturer's instructions. transfected sperm were detected by green fluorescence and counted under a fluorescence microscope. in our preliminary experiment, the transfection efficiency was 39.51 ± 2.72%, and the proportion of embryos derived from hbv transfected sperm was 27.66%. oocyte preparation and insemination were performed as described in reference [22] . briefly, zona-free hamster ova were fertilized by sperm from the test and control groups and then incubated in a humidified incubator (37 • c, 5% co 2 in air) for 24 h to obtain two-cell embryos. two-cell embryos exhibiting green fluorescence, derived from the hbv-transfected sperm (the test group), and those without green fluorescence, derived from the non-transfected sperm (the control group), were collected under a fluorescence microscope (dmil led, leica, germany). after washing, total rna from the two-cell embryos was extracted for cdna library construction by smart amplification. smart amplification and ssh were carried out using smarter tm pcr cdna synthesis kit and clontech pcr-select tm cdna subtraction kit (clontech laboratories, inc, ca, usa) according to the manufacturer's instructions. control mouse liver total rna (the positive group), deionized h 2 o (the negative group) and the primers (3 smart cds primer ii a and 5 pcr primer ii a) were provided by the kit. briefly, 3.5 l (50 ng) total rna extracted from two-cell embryos of the test and control groups were successively subjected to first-strand cdna synthesis, amplified by long-distance pcr, purified by column chromatography, digested with rsai, diluted to a final concentration of 300 ng/l in 1x tne buffer, and subjected to adaptor ligation. ssh materials consisted of cdna from the test two-cell embryos as the tester and cdna from the control two-cell embryos as the driver for forward subtraction, and vice versa for reverse subtraction. after the first and second hybridizations, hybridized samples 1 and 2 were mixed and then incubated at 68 • c overnight, followed by a primary pcr and a secondary pcr. after an agarose/ethidium bromide gel analysis, the products were used for dna ligation. the same procedures listed above were also performed for reverse subtraction and control subtraction. t/a cloning and amplification of subtraction products were performed using pgem ® -t easy vector system (promega) according to the manufacturer's instructions. briefly, ligation reactions containing pgem ® -t easy vector, 2x rapid ligation buffer, t4 dna ligase and the secondary pcr products of ssh were incubated overnight at 4 • c. two microliters of ligation reaction was added into 50 l of jm109 high efficiency competent cells, which was placed on ice for 20 min followed by heat-shocking at 42 • c for 45 s, putting on ice for 2 min, and then adding into 950 l of luria-bertani (lb) medium and incubation at 37 • c for 1.5 h with shaking. one hundred microliters of the transformation culture was plated onto lb/ampicillin/iptg/x-gal plates followed by incubation overnight at 37 • c. the 174 white (positive) colonies obtained were separately resuspended in 1.5 ml lb medium with ampicillin (100 g/ml) and grown at 37 • c for 6 h. pcr amplifications were carried out using 0.3 l bacterial suspension and premix taq tm (takara), and 5 l from each pcr reaction was subjected to 1% agarose gel electrophoresis. positive bands were detected for 152 of 174 colonies. microarray hybridization and data analysis were performed by takara biotechnology (dalian) co., ltd. briefly, the pcr products for differentially expressed genes from 152 clones, obtained from the subtraction libraries, were purified using 2-propanol precipitation and then spotted in triplicate onto takara glass slides (takara) using an affymetrix arrayer 417 (takara). the pcr products from the forward-and reverse-subtracted libraries (2 g each) were labeled with cy3 and cy5 fluorescent dyes and separately used to probe the glass slides containing the pcr-amplified cdna. the slides were hybridized overnight at 65 • c with labeled purified probes. quality control of the microarray chips was performed according to takara's method and standard. array slides were scanned using an affymetrix 428 array scanner (affymetrix, buckinghamshire, uk). the measured intensities are expressed as a ratio of cy5/cy3 intensities, which were background-corrected and normalized to the average cy5/cy3 ratio. the ratios were log 2 -transformed, and the following clones were selected for sequencing: 1. clones with a fold change value greater than 2 or less than 0.5 when compared with the average cy5/cy3-intensity ratio, the differential expression of which was statistically significant, were used to select target genes; 2. a clone with a fold change value greater than 0.5 and less than 2, the differential expression of which was not statistically significant, was chosen as the control [32] . dna sequencing and analysis were performed by beijing genomics institute shenzhen co., ltd. (shenzhen, china) using a 3730 xl dna analyzer (applied biosystems, ca, usa). sequencing reactions were carried out with bigdye v3.1 mix and pop-7 tm polymer (applied biosystems). for the acquired sequences, basic local alignment search tool (blast) was used to search for sequences homologous to human genes in the genbank nucleotide database. of the sequences, five with statistically significant differential expression were selected as the target genes, and a non-statistically significant sequence without statistical significance of differential expression was selected as a control. the transcriptions of both target and control genes in the two-cell embryos were confirmed using rt-pcr. the extraction and reverse transcription of total rna was performed using an ambion cells-to-cdnatm ii kit (life technologies, ca, usa) according to the manufacturer's instructions. pcr amplification was performed in a 25 l reaction mixture containing cdna (5 l), premixed taq polymerase (12.5 l) (takara), forward and reverse primer (10 m, 0.5 l each) and ddh 2 o (6.5 l), and the ˇactin was co-amplified as an internal control [33] . the gene-specific primers were designed using primer-blast (table 1 ). cycling conditions were as follows: 2 min at 94 • c; 35 cycles of 30 s at 94 • c and 30 s at 57 • c; and one cycle of 1 min at 72 • c. the amplified pcr fragments were subjected to 1.5% agarose gel electrophoresis and stained with ethidium bromide. eighteen healthy donors were randomly divided into six groups, and their sperm samples were used to fertilize zona-free hamster oocytes in vitro and assess the effects of the silencing of five target genes (csh2, eif4g2, pcbd2, psg4 and ttn) and a control gene (esrrg) on transcription of hbv s and x genes by real-time quantitative (q)rt-pcr. the sperm samples from three donors were individually used for assaying each gene, and each assay was repeated three times. gene-specific short hairpin (sh)rna or short interfering (si)rna used for silencing the target and control genes, and non-interfering scrambled oligonucleotides used as the negative controls, were designed and synthesized by shanghai genepharma co., ltd. or qiagen china (shanghai) co., ltd, respectively (table s1 ). each sperm sample from a healthy donor was divided into two groups: a test group, which was co-transfected with plasmids containing the full-length hbv genome and the gene-specific shrna/sirna or a non-interfering scrambled oligonucleotide; and a control group, which was transfected with the plasmid containing the full-length hbv genome alone. real-time qrt-pcr was performed using an abi 7300 real-time pcr system (applied biosystems, ca, usa) to compare the transcriptional level of hbv genes in the two-cell embryos of the test and control groups, with gapdh as an internal control [33] . total rna was extracted from the two-cell embryos and cdna synthesis was performed as described previously [34] . a total volume of 20 l of reaction mixture contained cdna (2 l), 2 x quantifast sybr green pcr master mix (10 l) (qiagen, hilden, germany), forward and reverse primers (10 m, 0.2 l each) for s and x genes and gapdh (table 1) , and rnase-free water (7.6 l). cycling conditions were as follows: 5 min at 95 • c; 40 cycles of 15 s at 95 • c and 30 s at 60 • c; and one cycle of 15 s at 95 • c, 2 min at 55 • c, and 15 s at 95 • c. the data was analyzed quantitatively using the 2 − ct method. in the 2 − ct analysis, individual data were converted to a linear form using the 2 −ct calculation [35] and then subjected to a paired-sample t-test using spss 16.0 software to determine a significant differences in average transcription levels of s and x genes between the test and control groups. a p-value of less than 0.05 was considered statistically significant. smart amplification showed that dispersed positive bands were detected in the test, control and positive groups, and not in the negative group, indicating successful amplification. after ssh, t/a cloning and bacterial amplification, 174 white (positive) colonies from subtracted libraries (123 from the forward and 51 from the reverse ssh libraries) were amplified by pcr, resulting in 152 positive insert-containing clones (103 from the forward-and 49 from the reverse-subtracted libraries). the insert sizes ranged from 500 to 1500 bp, and the majority of insert sizes was approximately 1000 bp (fig. 3) , in agreement with the statistical prediction of rsai digestion. table 1 the primers were used to amplify the target genes, a control gene, an internal control genes and hbv s and x genes. forward reverse in the current study, the two-fold average ratio of cy5/cy3 intensities was 5.740, and the 0.5-fold average ratio of cy5/cy3 intensities was 1.4349. both are the thresholds for determining whether gene differential expression is statistically significant. of 152 positive insert-containing clones, 29 showed fold change values greater than 2 or less than 0.5, and 123 exhibited fold change values greater than 0.5 and less than 2. these 29 clones and one of the 123 clones were selected for sequencing (table s2 ). blast analysis revealed 29 acquired sequences showing significant identity to five human gene families (average identity = 94.59%, ranging from 82% to 100%, e-value: 0.0) (table s2 ). of these, five representative genes (one from each family, identity: ≥96%, e-value: 0.0) were selected as target genes that were chorionic somatomammotropin hormone 2 (csh2), eukaryotic translation initiation factor-4␥, 2 (eif4g2), pterin-4␣-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1␣ (tcf1)2 (pcbd2), pregnancy-specific ␤-1-glycoprotein 4 (psg4) and titin (ttn) ( table 2 ). the selection criteria were as follows: first, three sequences showed significant identity to three human gene families, and each gene family had one sequence only, thus they (eif4g2, psg4 and ttn) were chosen as representative genes in these gene families. next, seven sequences showed significant identity to two members (nadh and pcbd2) of a human gene fam-ily. because nadh is located within mitochondria and not within cytoplasm, it is difficult to determine entry efficiency of nadhspecific shrna into mitochondria and to evaluate its silencing effects, thus pcbd2 was chosen as representative gene in this gene family. finally, 19 sequences showed significant identity to one human gene family, in which a sequence from the clone f123 with the highest ratio of cy5/cy3-intensities showed the highest identity (99%) to the members (csh1 and csh2) of this gene family, thus the csh2 was chosen as representative gene. in addition, a sequence (f110) from 123 clones showed significant identity to human estrogen receptor-related receptor (esrrg) family and was chosen as a control gene ( table 2, table s2 ). their transcriptions in the two-cell embryos were confirmed by rt-pcr (fig. 4) . the transcriptional levels of hbv s and x genes in two-cell embryos after the silencing of target genes and a control gene by rna interference (rnai) are shown in table 3 . when csh2 and pcbd2 were silenced, s and x genes expression in two-cell embryos was down-regulated. csh2 knockdown reduced the transcription of s and x genes by 11.1-and 8.33-fold in two-cell embryos, respectively, as compared to those in the control group. knockdown of pcbd2 reduced s and x gene transcription by 4.35-and 3.70-fold, respectively. when eif4g2, psg4 and ttn were silenced, s and x genes expression in two-cell embryos was up-regulated between 2.52-to 14.94-fold. there was no significant difference in the levels table 2 the clones were selected as the target genes and a control gene. both are the thresholds for determining whether gene differential expression is statistically significant. §2 identities: the extent to which two nucleotide sequences have the same residues at the same positions in an alignment, often expressed as a percentage [36] . §3 e value: the expectation value or expect value represents the number of different alignments with scores equivalent to or better than s that is expected to occur in a database search by chance. the lower the e value, the more significant the score and the alignment [36] . * the target genes. ¤the control gene. effects of the silencing of the target and control genes on transcriptional levels of hbv s and x genes in two-cell embryos. eighteen healthy donors were randomly divided into six groups, and their sperm sample were used to fertilize zona-free hamster oocytes in vitro and assess the effects of the silencing of five target genes (csh2, eif4g2, pcbd2, psg4 and ttn) and a control gene (esrrg) on transcription of hbv s and x genes in two-cell embryos using real time qrt-pcr and 2 − ct method. the sperm samples from three donors were individually used for assaying each gene, and each assay was repeated three times. each sperm sample from a donor was divided into two groups: a test group (t), which was co-transfected with plasmids containing the full-length hbv genome and the gene-specific shrna or sirna; and a control group (c), which was transfected with plasmid containing the full-length hbv genome alone. the data were presented as a fold change value (fcv) in transcription of s and x genes in the test group normalized to internal control gene (gapdh) and relative to those in the control group. individual data were converted to a linear form using 2 −ct calculation [35] and then subjected to a paired-sample t-test using 16.0 software to determine a significant difference in average transcriptional levels of s and x genes between the test and control groups. a p-value of less than 0.05 was considered statistically significant. * p < 0.05; ¤p > 0.05. of s and x gene transcription in two-cell embryos silenced for esrrg compared to non-silenced two-cell embryos (table 3 ). in the current study, two non-interfering scrambled oligonucleotides (niso) were used as the negative controls. there was no significant difference in the levels of s and x gene transcription in the two-cell embryos between the test (niso treated) and control (niso non-treated) groups (table 4 ). rnai is a biological process in which rna molecules inhibit gene expression or translation, by neutralizing targeted mrna molecules. oligonucleotides are short dna or rna molecules, of which sirna and shrna are central to rnai. it has been demon-strated that hamster and human sperm have a strong tendency to interact with exogenous dna and are able to transfer dna to oocytes [37] . zhang et al. injected plasmid rnai-ready-psiren-retroq-zsgreen, which was constructed for interrupting the zfx gene, into testis in the test group mice, and then the male mice were mated individually with females, resulting in 78.75 ± 7.50% of the male offspring, significantly higher than the offspring derived from the control groups (p < 0.01), suggesting that rnai could be as a tool to control the sex ratio of mouse offspring by interrupting zfx/zfy genes in sperm cells [38] . in the current study, the two-cell embryos derived from sperm co-transfected with plasmids containing the full-length hbv genome and gene-specific shrna/sirna for interrupting the target genes were as the test group, and those derived from sperm transfected with plasmid containing the full-length table 4 effects of the non-interfering scrambled oligonucleotides on transcriptional levels of hbv s and x genes in two-cell embryos. each sperm sample from a donor was divided into two groups: a test group (t), which was co-transfected with plasmids containing the full-length hbv genome and a non-interfering scrambled oligonucleotide (niso); and a control group (c), which was transfected with plasmid containing the full-length hbv genome alone. the data were presented as a fold change value (fcv) in transcription of s and x genes in the test group normalized to internal control gene (gapdh) and relative to those in the control group. individual data were converted to a linear form using 2 −ct calculation [35] and then subjected to a paired-sample t-test using 16.0 software to determine a significant difference in average transcriptional levels of s and x genes between the test and control groups. ap-value of less than 0.05 was considered statistically significant. ¤p > 0.05. hbv genome alone were as the control group. the transcription levels of hbv genes between the test and control groups are significantly different (p < 0.05), which suggested that in the test group the target genes have been silenced by rnai and participated in transcriptional regulation of hbv genes, causing the change of hbv gene transcription levels. we found that csh2, pcbd2, eif4g2, psg4 and ttn to be involved in transcriptional regulation of hbv s and x genes in two-cell embryos. the protein encoded by csh2 is a member of the somatotropin/prolactin family of hormones and plays an important role in growth control [39] . pcbd2 is a protein coding gene and associated with tetrahydrobiopterin biosynthesis [39] . silencing of csh2 and pcbd2 by rnai decreased the levels of s and x gene transcription in two-cell embryos, indicating that both genes up-regulated transcription of s and x genes. the protein encoded by eif4g2 is a subunit of eif4f, a cap binding protein complex that mediates translation initiation by specific recognition [39] . psg4 is a member of the carcinoembryonic antigen (cea) gene family and may play a role in regulation of the innate immune system [39] . ttn encodes a large abundant protein that is a key component in the assembly and functioning of vertebrate striated muscles, and in non-muscle cells, seems to play a role in chromosome condensation and chromosome segregation during mitosis [39] . when eif4g2, psg4 and ttn were silenced by rnai, the transcriptional levels of s and x genes in two-cell embryos increased, suggesting that these genes down-regulate s and x gene transcription. these results suggest that certain host genes participate in regulating hbv gene transcription in two-cell embryos. however, the interplay between the virus and host cells is complex, and it remains largely unknown how these genes interact with each other or act independently to allow differential transcription of hbv genes. some clues may help in understanding their interaction. first, certain previously identified host hbv-regulatory genes share high similarity in function and signaling pathways with genes (identified herein) that regulate hbv gene transcription. for example, sp1 is a ubiquitous factor that binds to gc-rich motifs in many promoters, can regulate transcription of hbv genes [16, 17] , and is involved in many cellular processes, including cell differentiation, growth, and apoptosis. the protein encoded by csh2 also plays an important role in growth control and provides avenues for developmental regulation and tissue specificity. both genes function in the prolactin signaling pathway, suggesting possible regulation of hbv gene transcription by a similar mechanism. next, some host genes might indirectly participate in regulating hbv gene transcription through general activation of transcription regulators or factors. for example, hnf1 is a liver-enriched transcription factor that can regulate transcription of hbv genes [16, 17] . pcbd2, identified herein, regulates dimerization of hnf1␣ and enhance its transcriptional activity. finally, our identified genes might directly contribute to regulation of hbv gene expression. for example, eif4g2, besides down-regulating transcription of s and x genes, might repress viral rna translation by forming translationally inactive complexes [39] . there are still significant questions. human embryonic genome activation (ega) occurs at the 4-8 cell stage [40, 41] , why host genes can regulate hbv gene expression in two-cell embryos? some studies offered the useful clues, which may help shed light on this question. first, it has been demonstrated that ega in mouse occurs at the two-cell stage, which is controlled by maternally deposited rnas and proteins. yu et al. identified lately that oocyte-expressed yes-associated protein is a key activator of ega in mouse [42] . in the current study, the two-cell embryos were produced by ivf of zona-free golden hamster oocyte with human sperm. ega in golden hamster also occurs at the two-cell stage [43] , whether certain activator (s) deposited in golden hamster oocyte activate human genome? next, in development of human preimplantation embryo, there are four unique embryonic stagespecific patterns (essps) of gene expression [44] . essp1 (maternally inherited oocyte mrnas) were expressed at high levels at the zygote stage and declined during development to the blastocyst stage. essp2 includes embryonic-activated genes, first transcribed at approximately the 8-cell stage. essp3 comprises genes not expressed until the blastocyst stage. essp4 includes persistent transcripts that maintained stable expression from the zygote to blastocyst stages [41] . it is unknown whether the target genes identified in the current study are included in essp4? undoubtedly, clarification of these questions would explore many mysteries in early embryonic development. in addition to the above question, hbv s gene encodes hbsag, which packages the viral components. hbv x gene encodes hbx protein, whose activity is absolutely required for in vivo replication and spread of the virus [44] . can the target genes affect virion assembly, or affect replication and spread of the virus through regulating viral transcription to block its transmission? moreover, outcome of hbv infection is markedly heterogeneous, varying from acute asymptomatic self-limiting infection to fulminant hepatic failure or to decompensated cirrhosis and hepatocellular carcinoma. it has been recognized that host genetic background influences the outcome of viral hepatitis infection [45] . in the current study, we found that some host genes upregulate or downregulate transcription of s and x genes. can we assume that these host genes together function to maintain homeostasis? disturbance of such homeostasis could decrease or increase host susceptibility to hbv infection or allow infection to develop in different directions, leading to different clinical outcomes. these questions will be evaluated in future clinical studies. human embryo development is more fragile than that of many other species [41] . human fecundity rates are relatively low, largely due to pre-and post-implantation embryo loss [41] . in vitro, 50-70% of ivf embryos fail to reach the blastocyst stage [41, 46] . certain factors that may contribute to abnormal development before ega include inherited genetic mutations, aneuploidy, environmental insult to germ cells, events during fertilization and sperm-related factors [41] . in our previous study, it was detected that hbv infection induces various chromosomal abnormalities in patient sperm, including aneuploidy, acentric fragment and deletion, ring chromo-some, triradial, dicentric chromosome and pulverization [8] . sperm with these chromosomal abnormalities can achieve normal fertilization and introduce these aberrations into the embryo, which may increase the risk of abortion, stillbirth, or birth defects [8] . in a recent clinical literature, hbv mrna was found in abandoned ivf embryos of hbv-infected fathers, which confirmed that hbv could not only enter early cleavage embryos via sperm but also replicate in embryos, resulting in early abortion [47] . the aformentioned findings suggested that hbv may interfere with early embryonic development and thus affect pregnancy outcome. therefore it is very important to investigate hbv gene transcription and its regulation mechanism in embryos. this study, for the first time, provides experimental evidence that transcription of hbv genes occurs in early embryonic cells and is regulated by host genes. it is worth mentioning that, besides hbv, there are more emerging infectious diseases virus, such as hepatitis c virus (hcv), human immunodeficiency virus (hiv), severe acute respiratory syndrome virus (sars), ebola virus and zika virus (zikv), which pose a serious threat to human health [48] [49] [50] [51] [52] . lately, some studies begin to investigate the true vertical transmission of hcv and hiv via germline [34, [53] [54] [55] [56] , but the research on such transmission of sars, ebola and zika viruses is still a blank. to clarify whether the aforementioned viral genes are transmitted via germline and their expressional regulation in embryo would make a great contribution to exploring the interplay mechanism between the viruses and host cells and to maintaining human reproductive health. the authors declare no conflict of interest. the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. refining the risk estimate for transfusion-transmission of occult hepatitis b virus epidemiological and molecular features of hepatitis b and hepatitis delta virus transmission in a remote rural community in central africa #38: hepatitis b in pregnancy screening, treatment, and prevention of vertical transmission hepatitis b transmission by cell and tissue allografts: how safe is safe enough? hepatitis b reverse seroconversion and transmission in a hemodialysis center: a public health investigation and case report incidence of percutaneous injury in taiwan healthcare workers adverse effects of hepatitis b virus on sperm motility and fertilization ability during ivf effects of hepatitis b virus infection on human sperm chromosomes expression of hepatitis b virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome detection and expression of hepatitis b virus x gene in one and two-cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying hbv dna hepatitis b virus s protein enhances sperm apoptosis and reduces sperm fertilizing capacity in vitro effects of hepatitis b virus s protein exposure on sperm membrane integrity and functions effects of hepatitis b virus s protein on human sperm function the presence and expression of the hepatitis b virus in human oocytes and embryos host genetic factors in hepatitis b infection, liver cancer and vaccination response: a review with a focus on africa core promoter: a critical region where the hepatitis b virus makes decisions regulatory elements of hepatitis b virus transcription kruppel-like factor 15 activates hepatitis b virus gene expression and replication genotype-dependent activation or repression of hbv enhancer ii by transcription factor coup-tf1 transcriptional regulation of hepatitis b virus by nuclear hormone receptors is a critical determinant of viral tropism correlation between the zona-free hamster egg sperm penetration assay and human in vitro fertilization world health organization, zona-free hamster oocyte penetration test, in: who cpg methylation participates in regulation of hepatitis b virus gene expression in host sperm and sperm-derived embryos the sperm penetration assay for the assessment of fertilization capacity sperm penetration assay as an indicator of bull fertility cdna amplification by smart-pcr and suppression subtractive hybridization (ssh)-pcr suppression subtractive hybridization world medical association declaration of helsinki: ethical principles for medical research involving human subjects guide for the care and use of laboratory animals world health organization, direct swim-up, in: who investigation of recombinant mouse sperm protein izumo as a potential immunocontraceptive antigen distributional fold change test − a statistical approach for detecting differential expression in microarray experiments both beta-actin and gapdh are useful reference genes for normalization of quantitative rt-pcr in human ffpe tissue samples of prostate cancer in vitro study on vertical transmission of the hiv-1 gag gene by human sperm analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta c(t)) method sperm-mediated gene transfer into oocytes of the golden hamster: assessment of sperm function rnai as a tool to control the sex ratio of mouse offspring by interrupting zfx/zfy genes in the testis functional genomics of 5-to 8-cell stage human embryos by blastomere single-cell cdna analysis non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage oocyte-expressed yes-associated protein is a key activator of the early zygotic genome in mouse golden hamster embryonic genome activation occurs at the two-cell stage: correlation with major developmental changes x region mutations of hepatitis b virus related to clinical severity understanding the host genetics of chronic hepatitis b and c, semin does severe teratozoospermia affect blastocyst formation, live birth rate, and other clinical outcome parameters in icsi cycles? relationship between the mechanism of hepatitis b virus father-infant transmission and pregnancy outcome the impact of emerging infectious diseases on chinese blood safety sars-like wiv1-cov poised for human emergence hepatitis delta and hiv infection effect of ebola virus disease on maternal and child health services in guinea: a retrospective observational cohort study model-informed risk assessment for zika virus outbreaks in the asia-pacific regions factors affecting sperm fertilizing capacity in men infected with hiv the integrated hiv-1 provirus in patient sperm chromosome and its transfer into the early embryo by fertilization research on the vertical transmission of hepatitis c gene from father-to-child via human sperm effects of hepatitis c virus infection on human sperm chromosomes this work was financially supported by the national natural science foundation of china (grant number 30972526) and the applied basic research programs of sichuan province of china (grant number 2014jy0110). the authors thank professor stanley lin for his assistance in revising the final draft of manuscript and editing for english grammar and syntax. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reprotox.2017. 08.009. key: cord-340503-zwdewiu1 authors: mokhtarzadeh, ahad; eivazzadeh-keihan, reza; pashazadeh, paria; hejazi, maryam; gharaatifar, nasrin; hasanzadeh, mohammad; baradaran, behzad; de la guardia, miguel title: nanomaterial-based biosensors for detection of pathogenic virus date: 2017-10-13 journal: trends analyt chem doi: 10.1016/j.trac.2017.10.005 sha: doc_id: 340503 cord_uid: zwdewiu1 viruses are real menace to human safety that cause devastating viral disease. the high prevalence of these diseases is due to improper detecting tools. therefore, there is a remarkable demand to identify viruses in a fast, selective and accurate way. several biosensors have been designed and commercialized for detection of pathogenic viruses. however, they present many challenges. nanotechnology overcomes these challenges and performs direct detection of molecular targets in real time. in this overview, studies concerning nanotechnology-based biosensors for pathogenic virus detection have been summarized, paying special attention to biosensors based on graphene oxide, silica, carbon nanotubes, gold, silver, zinc oxide and magnetic nanoparticles, which could pave the way to detect viral diseases and provide healthy life for infected patients. viruses are the smallest infectious agents that can cause many diseases such as influenza, chicken pox, flu, acquired immune deficiency syndrome (aids), severe acute respiratory syndrome (sars), ebola and etc. viruses are intracellular parasites unlike other microorganisms. these agents have only a few number of responsible genes for the synthesis of new viruses and most of them used different enzymes which created by the host cell [1] . so, fast and well-timed diagnosis of these diseases is very important due to the limited treatment options. virus detection usually requires specific methodologies and equipment as: cell culturing, antigen or antibody detection (elisa, immunofluorescence, immunoperoxidase) hemagglutination assay, nucleic acid detection (polymerase chain reaction) and gene sequencing [2, 3] . most of these methods are time consuming, labor intensive and often expensive. table 1 offers a comparison of advantages and limitations of some of the most commonly used methods for virus detection. therefore, we need rapid, simple, sensitive, and accurate methods for detection of pathogenic agents [4] . so in order to find alternative methodologies without the mentioned limitations, recently there has been growing attention for using biosensors in biomedical applications based on the advantages of these methods such as high speed results, excellent sensitivity and suitable selectivity [5] . moreover, since viral and bacterial diseases are real menaces to human life, there is a growing demand to detect them meticulously by advanced methods [6] . biosensors are highly accurate, sensitive and specific measurement systems that can determine very low analyte concentrations in biological samples. a biosensor is an analytical device which made up biological elements; such as microorganisms, organelles, cell receptor, enzymes, antibodies, nucleic acids, etc. that produces signals (electrical, optical or thermal) based on the interaction with tested element and a transducer that converts these signals into a measurable electrical parameter [7] . due to the use of specific biological elements, these measurement systems have unique properties for the identification and measurement of target analytes. the importance of biosensor components is to assure a high level of specificity based on particular binding sites. different medical and clinical applications are expected for biosensors such as: i) fast diagnosis and treatment of illness like cancer or diabetes, ii) detection of pathogens, iii) measurement of drugs and their metabolites, iv) discovery of new drugs and v) evaluation of drug activity, vi) assessment and measurement of analytes in biological samples and early detection of diseases using rapid tests [7e9] . considering special properties (physical, chemical, mechanical, magnetic, etc.), various kinds of nanomaterials, such as gold nanoparticles, carbon nanotubes (cnts), magnetic nanoparticles and quantum dots (qds), are being merged to biosensors. recent developments in nanoscience and nanotechnology and the possibility of making electrodes on a very small scale, nanoscale sensors make possible and resulted in a new set of diagnostic biosensors called nano-biosensors. continuous decrease in material dimension, from large scale to small one in the range between 1 and 100 nm, does not change biosensor properties but significantly improves their applicability. the surface interaction of the sensors with analyte becomes highly efficient due to the extremely large surface to volume ratios in nano-size devices [15] . therefore, nanoscale materials show unique features, functionality, and effects. nowadays, nanotechnology is focused on the elimination of disadvantages of existing methods for virus detection to minimize costs and time consuming. furthermore, the use of nanomaterials in the construction of biosensors resulted in increasing of efficiency and sensitivity of these systems [16] . by designing the interaction between biological element and nanomaterial-based transducer, we can have biosensors by widely usage for the detection of biomolecules and disease diagnostics. these devices can detect patients physiological status at the moment and safety analyze the food and environmental samples for pesticides and water pollution quickly [17] . several nanomaterials; such as nanorods, nanotubes, nanowires, thin films, and nanoparticles have been explored for biomedical applications due to their functional electrical and mechanical features for biomedical applications [18] . in this review, we will focus on the use of different nanomaterials such as quantum dots, carbon nanotubes, graphene oxide, silica and metal nanoparticles for the fabrication of pathogenic virus biosensors (scheme 1). qds are nanoscale semiconductor crystals (with a diameter of 2e10 nm) with unique optical and electrical properties. qds have high versatility because of their small size. qds includes: 1) a core that made up of group iievi atoms, e.g. cadmium selenide (cdse), or group iiiev atoms of the periodic table, like indium phosphide, 2) a shell made of another semiconductor that covers the core, in most cases zinc sulfide (zns), to improve optical properties and increase stability and reduce cytotoxicity and 3) an organic coating applied to change nanoparticle into hydrophilic compound to become a place to connect of biomolecules; such as, oligonucleotides, proteins, peptides and also small molecules [19] . by using ultraviolet radiation to qds, different wavelengths of visible light are emitted from them. the emitted wavelength depends on the size of the qds, the distance between energy bands in small qds being higher than in larger ones. so, by illuminating small qds with ultraviolet radiation, electrons move to high energy bar and loss additional energy by return to a steady state. finally, visible light emitted from them has high energy and tends to blue. also by using ultraviolet radiation to large qds, visible light is emitted from them, in the reddish range, due to the loss of excess energy and return to a steady state, because of the small energy gap and less energy losing. so, by enlarging the size of the qds, the light spectrum tends toward red from blue [20] . growing interest for chemical and biological detection by quantum dot (qd)-based sensors, leads to development of several ways to prepare qds such as colloidal synthesis, plasma synthesis, viral assembly, electrochemical assembly, etc. since the qds have unique properties when compared to organic fluorescent dyes, such as wide absorption range, symmetric size photoluminescence, in addition to broad excitation range, multiplexed staining, long florescent lifetime and high resistance to photo bleaching, therefore, bioconjugation of these nano-particles to sensor structure has an increasing interest. qds light emission is a useful property for a lot of medical labeling, imaging or sensing applications such as to distinguish between normal and tumor cells [21] gene therapy studies [22] , proteomics [23] , etc. from the perspective of virology, qds are useful tools for providing rapid and sensitive virus detection to facilitate early treatment and monitoring of viral disease. additionally, the combination of qds and platforms with immobilized antibodies results in high sensitivity and selectivity [6] . [10] electron microscopy viral particle hours broad spectrum; rapid method necessity for presence of around 10 6 virus particles/ml for detection; similarity of morphologies [11] hemagglutination assay viral protein hours easy; inexpensive poor sensitivity; necessity for fresh reagents [12] elisa viral protein hours only one incubation step; no hook effect at high analyte concentrations limited concentration range in which the analyte can be quantified without sample dilution; and that the antigen or antibody produce the same response and not distinguishable in a one step [13] pcr viral nucleic acid hours extremely high sensitivity; easy to set up extremely liable to contamination; not easy to quantitate results; high degree of operator skill required [14] as an example for hiv, a type of virus that gradually attacks the immune system and makes it harder to fight off infections and diseases in infected body, a qds-based rapid capture and imaging system was developed by kim et al. this method is a type of dual-stain imaging technique, based on streptavidin conjugated qdot525 and qdot655, for hiv1 gp120 envelope glycoprotein and its highmannose glycan capturing by an anti-gp120 antibody, immobilized on a microfluidic chip (see fig. 1 ). the capabilities of this system are not only selective for capturing and detecting hiv virus in whole blood but also makes possible to obtain countable imaging. considering the analytical features, it provides high detection speed, due to the lack of blood sample pre-processing (<10 min), even with 10 ml of blood sample, cost effectively, and portability, thus providing a new and effective tool for valid identification of hiv [24] . wang et al. developed a fret-based qds-dna system for rapid, easy and sensitive detection of hbv-dna and single-base mutation of this virus that presents increased benefits when virus try to become resistance to drug treatment. bioconjugation of dna to cdse/zns qds accrued by carbodiimide cross-linker chemistry in this method, and after addition of cy5-modified signal dnas and complementary dna targets into the qds-dna conjugates. a sandwiched hybrids with cy5 fluorophore, as the acceptor, and qds, as the donor of the fluorescence resonance energy transfer system, were performed. the oligonucleotide ligation assay was used for detection of single base mutation in this system which ligase recognize a mismatch base. therefore, the ligation will not make and, subsequently, there is no cy5 emission. the validity of this system was examined by synthetic 30-mer oligonucleotide targets of hbv dna with a sensitivity of 4.0 nm by a multilabel counter [25] . krejcova and coworkers [29] proposed an influenza virus detection sensor as a pandemic threat by using a 3d microfluidic chip, applied for influenza hemagglutinin. this method was based on two different steps, (i) specific isolation, after labeling ha with cds-qds, and conjugate them on a surface of glycan-modified mps and (ii) voltammetry based detection step. considering unique properties of qds, these nanoparticles are a key part of this system that makes it a rapid sensitive and specific way for detection of isolated influenza virus. table 2 summarizes some of biosensing methods for detection of viruses based on the use of various types of qds. in short, according to their unique optical properties, which covered a wide range of light absorbance, qds are one of the best choices for the design of sensing instruments, especially optical ones. another noticeable aspect about these nanoparticles is their biocompatibility, which make them suitable for sensing samples in biological environments. also, in some cases, the detection process could be very rapid. in conclusion, between carbon based nanostructures, graphene oxide is the best choice which introduced to detecting systems due to especial variety of properties like having natural source, biocompatible and cost effective. one of the eye catching advantages of this carbon-based nanostructure is the ability of surface treatment to be a good hostage for immobilizing of ligands, nanoparticles and single-strand dna in aptasensors. graphene is a well-known carbon based material that is made by mechanical exfoliation of graphite [30] . go, which includes carboxylic, phenol hydroxyl and epoxide groups, can be produced from graphite oxide. beside unique electronic, mechanical and thermal properties of go, some noticeable applications in nanosensors and nanomedicine, make these materials in the center of attention [31] . size controllability of go nano sheets and changes in oxidation level are other interesting properties of these carbon based materials [32] . recently a research has been conducted by hu et al. that shows the antibacterial activity of graphene oxide towards escherichia coli [33] . these compounds have been used in sensors, especially for the detection of biomolecules. qds fluorescence quenching has been employed, together with go, in an optical biosensing platform [34] . this property of go leads to use of them in identifying some especial viruses. nowadays there is a high demand of novel methods of diagnosing and disinfecting of viruses, due to the low specificity and sensitivity of traditional diagnostic methods [35] . one of the novel tools for virus diagnostics is based on the use of bio and immune sensors which employed nanotechnology in their structure [36] . the world health organization (who) reported that h5n1 influenza virus has caused infection of 650 people and death of 386 since 2003 [37] and it is a good example on the need of sensitive and selective methods for detection of viruses. as an example, xie and colleagues proposed an immunosensor for detection of viruses based on go [38] . according to this report, this immunosensor improved virus sensing based on immobilization of h5-polychonal antibody (pab) on go. the nanocomposite leaded to amplify the signals. in this electrochemical immunosensor, for the loading of go-pab-bsa nanocomposite on gold electrode, thiourea, gold nanoparticles, h5 antigens and h5-monoclonal antibodies (mab) were used as linkers (fig. 2) . the limit of detection (lod) and linear range were respectively 2 à15 ha unit/50 ml and 2 à15 to 2 à8 ha unit/ 50 ml. another procedure was used to enhance the sensitivity of hiv virus detection. wang et al. synthesized an electrochemical biosensor based on a nanocomposite which contained go and carbon nanotubes to employ it in electrochemical biosensor [39] . one of the positive point in this modified electrode was the encapsulation of horseradish peroxidase enzyme by silica-carbon nanotubes, which grafted to go (fig. 2) , which enhanced the sensitivity, the recoverability and stability of the system to be used to made measurements in human plasma. the lod was 0.15 pg ml à1 , that showed a progress in comparing with traditional and common electrochemical procedures, and the linear range was 0.5 pg ml à1 to 8.5 ng ml à1 . one of the unique properties of go is the ability of being host of stranded dna to make aptasensors. a research conducted by bao et al. [40] about immobilizing dna strand on go provided the way to make a label free aptasensor for detection of viruses this novel procedure for the designing of sensors had some significant points; (i) there was no need to decoration of go towards immobilization, (ii) comparing with other apatasensors, it was unnecessary to label [29] dna. (iii) the device was also used for the biosensing of nanostructures. two parts of dna were used in this study, probe part and immobilization part. in the presence of human immunedeficiency virus (hiv), the probe part was linked to double helix dna. therefore, negative charge was formed on go which was detected by electrochemical impedance spectroscopy (fig. 3) . in this assay, the linear range and lod were 10 à12 to 10 à6 m and 1.1 â 10 à13 , respectively. recently functionalized go has been used in biosensors for enhancement of sensitivity. seo and coworkers [41] used go, decorated by aunps, to make the go sheets suitable for being a photoluminescence quencher. the target in this biosensor was rotavirus and the amine groups were the linkage of go and antibody. due to the unique structure of modified electrode in this biosensor, selectivity and sensitivity were impressive. lod of this biosensor was 105 pfu ml à1 . to sum up, go based biosensors decorated with nanoparticles, like aunps, are more precise to detect viruses than conventional methods. between carbon based nanostructures, graphene oxide seems to be the best choice to be employed in detection systems due to their especial variety of properties as their natural source, biocompatibility and cost effective relationship. one of the eye catching advantages of this carbon-based nanostructures is the ability of surface treatment to provide a good hostage for immobilizing of ligands, nanoparticles and single-strand dna in aptasensors (see table 3 ). according to specific properties and activities; such as thermal, electrical, chemical and mechanical behavior, cnts provided a great area of scientific research. the biomedical utility of these carbon based nanomaterials is of special interest in the field of biosensors. cnts are playing an important role in preparing biosensors which can detect target molecules in trace amounts. this powerful aspect of cnts is sourced from transduction of physical/chemical interactions and high surface area-to-volume ratio [47] . in the case of virus detection, pu et al. [48] used the ability of surface decorating of cnts for making them ready to immobilize on gold sheets to synthesize a modified electrode. horseradish peroxidase enzyme was set on carbon nanotubes which contain capture probes, and then joined to the gold layers by au nanoparticles. this biosensor was used for the detection of hiv virus effects in human body by investigation of nuclear paraspeckle assembly transcript 1 (neat1). because of unique structure designing in this biosensor, the target capturing was very high due to the great surface area of cnts. the lod was considerably low (0.8863 fm ml à1 ). also the linear calibration ranged from 1 fm ml à1 to 100 nm ml à1 . in another study which has been done recently by yang et al. [49] dna enzyme of hemin/g-quadruplex was produced which trigger oxidation of the 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb). this biosensor was based on detection of hepatitis b virus (hbv) dna portion. the result of setting aunps in the structure was responsible for linking hairpin capture probes to the carbon based nanocomposite (goecarboxyl multi-walled carbon nanotube), and provided the enhancement in amplification effects with a linear range from 10 pm to 10 nm. due to selective identifying between dna of target molecules from those which were incompatible in one or two-base, the specificity and selectivity of this sensor was strongly high. also the lod achieved for the target dna was about 0.5 pm. to prepare a modified cnt-based electrode containing aunps, electrochemical impedance was used by wang et al. [50] to deposit aunps on single walled carbon nanotube (swcnts) previously prepared by in situ procedure. the target molecules, which were the dna of hepatitis b and papilloma virus, were captured by probe dna (single stranded (ssdna)) and immobilized on swcnts/au. the experimental lod for the hepatitis b and papilloma virus were respectively 0.1 pmol and 1 amol. early diagnosis of diseases related to gene and high sensitivity and specificity are some of the advantages of combination of aunps and swcnts. the lod of swcnts/au/ssdna biosensor for detection of hepatitis b and papilloma virus were 0.1 pm and 1 am, respectively. so, it can be seen that carbon nanoparticles provide a simple, rapid and precise sensing method to recognize viruses. cnt-based systems have introduced a new generation of biosensors which resulted in high sensitivity and selectivity according to their high surface area, the ability of easy functionalization and the fact that they provide a good hostage for immobilizing nanoparticles. silica nanoparticles have wide surface area, stability in critical thermal and chemical conditions, good compatibility with biomolecules, like proteins, being green about environmental challenges [55] . there are many biomolecules which could be linked to silica nanoparticles, including antigen-antibodies, peptides and dna and that, subsequently, make these nanomaterials as unique elements to be incorporated to bio-and immunosensors. in addition to their biocompatibility, some considerable properties about optoelectronic aspects such as visible luminescence activities [56] , make them important in bioanalytical research. the new generation of biosensors, called microcantilevers, have shown outstanding applications in viruses detection. the research group of kim et al. used microcantilevers for the detection of hepatitis b virus (hbv) [57] . in this research, a high sensitivity was obtained being improved the limit of detection from picomolar (pm) hbv target dna in the absence of sinps till femtomolar (fm) level in the presence of nanoparticles. due to harmful effect of viruses in our body, it is necessary to detect some species of them before being late. for example eps-teinebarr virus-derived latent membrane protein 1 (lmp-1) make disturbances in morphology and growing of cells which causes irreparable effects in human body [58] . a research was done by liu et al. about detection of this virus using a multi nanolayer structure to enhance the sensitivity of the detection [59] . using silica nanospheres together with qds of cadmium telluride (cdte/qds) and the possibility of bringing these nanoparticles on gold layer it was prepared a biosensor to amplify the signals in order to obtain a limit of detection of 1 pg ml à1 and a linear range of 0.001e10 ng ml à1 . hybridization of organic and inorganic compounds is another solution for improving biosensors applications. enrichi et al. [60] combined sinps with commercial qds and organic dyes in the structure of a biosensor. result was significant about sensitivity in order to achieve a sensor to detect traces of targets. a dna microarray was added to this structure for improving selectivity and increasing optical signal. they compared fluorescence activity of qds, silica nps and free dyes separately, the best lod was related to the dye doped silica nanoparticles. the detection limit for free dyes, qds and dye doped sinps were 150, 250 and 20 pm, respectively. table 4 summarizes applications of sinps on the construction of different biosensors for viruses detection. the noticeable advantages of silica nanoparticles concern their capability for designing unique nanolayer structures including sinps compatible with organic compounds in biological conditions. it favors their use in different types of bio and immunosensors. 6. metal and metal oxide nanoparticles the size of silver nanoparticles is in the range from 1 to 100 nm at one dimension which make these nps suitable for bio applications. by decreasing the size of agnps, the ratio of surface area to volume surprisingly increases considerably which results to noticeable changes in biological, physical and chemical activities [61] . recently, agnps have been tested in novel and high-tech diagnostic instruments such as bio and immunosensors. the compatibility of mixing ag nanoparticles with other nano metal patterns, like gold nanoarray, permitted to design a new generation of biosensors using surface-enhanced raman scattering (sers). wu et al. [62] prepared an optical biosensor for detection of hepatitis b viruse. the outcome of this coupling was to produce a wide plasmonic hot spots, which increased accessibility of raman labels to produce great electromagnetic field. the lod and linear range of this biosensor for the detection of hepatitis b virus dna were 50 am and 0.5e100 fm, respectively. the fluorescence characteristics of agnps provided optical biosensors with high sensitivity. a biosensor has been prepared by wang et al. based on detection of target dna sequence of (hiv), (hbv) and (htlv-i) gene by fluorescence activity of silver nanoclusters (agcns) [63] . before conjugation of hairpin probe with mentioned viruses dna, as target molecules, the fluorescence activity of agcns are high and bright, but after binding between probe strand and dna of target molecules, the structure of hairpin probe was disordered thus, decreasing the fluorescence intensity of nanoparticles. the advantages of this optical-based biosensor were the high sensitivity and low lods for the detection of hiv, hbv and htlv-i, which were 4.4 nm, 6.8 nm and 8.5 nm, respectively. in another study two-dimensional (2d) coreeshell structure of (au@ag) nps arrays was used as substrate for sers based biosensor [64] . in this report, the target molecule was influenza a virus, being obtained a lod of 6 tcid50/ml and a linear range between 5 and 56 tcid50/ml using a unique sers substrate (well-tuned au@ag 2d array) (fig. 4) . table 5 summarizes the main figures of merit of several optical and electrochemical agnps-based biosensors proposed in the literature for detection of viruses using different bio-receptors. aunps have been extensively used in the field of virus detection owing to their unique optical/electrical properties [68] . for instance, darbha et al. [69] detected hiv-1 viral dna sequence with a sensitivity of about 100 pmol l à1 by taking advantage of secondorder nonlinear optical properties of gold nanorods (aunrs). this method can be applied to in-vitro selected aptamers for detecting a wide range of analytes such as small organic molecules and divalent cations. lu et al. [70] proposed a fret system, containing aunrs and fluorescein (fam), for the detection of hepatitis b virus dna sequences. in this method aunrs were synthesized and the surface of the aunrs wrapped with a thin layer of cetyltrimethylammonium bromide (ctab), leading to the positive charge of aunrs. their designed, structure led to a fluorescence resonance energy transfer (fret) process from fam to aunrs. the fluorescence intensity of fam was consequently quenched. the decline of the fluorescence intensity of fam (df) was linear with the concentration of the complementary dna from 0.045 to 6.0 nmol l à1 and the lod was as low as 15 pmol l à1 (signal/noise ratio of 3). a highly sensitive and selective hepatitis b dna biosensor using aunps has been developed by mashhadizadeh and talemi [71] . in this work, mercapto-benzaldehyde was used for the enhanced detection of a short dna sequence of hbv virus. the fabrication process offered a very simple and convenient methodology for the preparation of a self-assembled monolayer and covalent immobilization of nh2-hbv-ss-dna. a detection limit of 7.6(±0.1) â 10 à12 mol l à1 was estimated for target hbv-dna. the successful discrimination between the complementary hbv dna, three-base mismatched, and non-complementary ss-dna displayed good selectivity for the biosensor. a sandwich immunoassay with aunps was developed by escosura-muñiz et al [72] , that allowed to distinguish 3 miu ml à1 , a_hbs ag igg antibodies in human serum samples. the platform of assay was made of magnetic beads. the a_hbs ag igg antibodies were captured from human sera and the further signaling with aunps tags was made. 3 miu ml à1 of a-hbsag igg antibodies in human serum can be detected by this biosensor. in comparison with meia method this method provided a deviation of 6.5%. in another report, an immune assay based on label-free electrochemical method was proposed by ma et al. for detecting the core antigen of the hepatitis c virus [73] . in this work, using synergetic effect of aunps, zirconia nps and chitosan, an electrochemical immunosensor was built. this immunosensor exhibited board liner range between 2 and 512 ng ml à1 , a detection limit of 0.17 ng ml à1 , high consistence and cost-effective way to detect hcv core antigen, which pave the way to early diagnosis of hcv infection in clinical. although, immunoassays have a very good selectivity, they require long time of analysis to get results going from 2 h to 4e26 days [74] . in order to overcome immunoassays limitations and enhance sensitivity, sophisticated assays based on dna detection were exploited for virus detection. therefore, improved sensitive and specific hbv genomic dna assay, utilizing rolling circle amplification (rca)-based quartz crystal microbalance (qcm) assay [54] has been proposed for direct dna detection of hbv in clinical samples. during the assay the covalent bonding between the capture probes and the gold electrode surface maintains the connection between probes and amplified rca products, being possible to determine less than 10 4 copies/ml concentration of hbv genomic dna [75] . mnp includes a wide variety of materials and properties, including magnetic fluids, catalysis activity and magnetic resonance imaging [76] that made them useful in the field of biosensing. because of being controllable by external magnet, these mnps, are widely used in hybrid catalysts, drug delivery systems and reusable biosensor platforms [77] . kamikawa et al. took advantage of mnps to detect surface glycoprotein hemagglutinin (ha) from the influenza a virus (fluav) h5n1. electrically active polyaniline coated magnetic nanoparticles (eapm) are the basis of this biosensors [78] . compared to other nanoparticles employed in the field, this system provided increased assay kinetics due to the close proximity to targets, easy magnetic manipulation of the nanoparticles, and minimized matrix interference from complex samples as food and clinical specimens [79] . this assay is able to distinguish recombinant h5 ha at 1.4 mm in 10% mouse serum, in fig. 4 . design of a modified electrode with two-dimensional (2d) coreeshell structure of (au@ag) nps array as substrate. this figure was obtained with permission from ref. [64] . optical aptamer 2 ng ml à1 in buffer and 3.5 ng ml à1 in human serum 2e100 ng ml à1 in buffer and 3.5e100 in human serum [67] which specificity for h5 is highly than for h1. novel eam nanoparticles provide delicate, specific, inexpensive, and easy-to-use biosensor with applications in disease monitoring and biosecurity [78] . since viruses are considered a greet menace to human life it is necessary to develop sophisticated diagnostic technique to effectively control pandemic strains. krejcova et al. utilized paramagnetic particles to detect viral a/h5n1/vietnam/1203/2004 protein-labeled qds. the use of these particles being able to detect a wide panel of influenza virus strains. the lod of viral protein was estimated as 0.1 mg ml à1 [80] . krejcova et al. used mnps, with covalently boundoligooligo (dt25) for isolation of complementary h5n1 chains, in order to do point mutation detection of h5n1 in neuraminidase gene. oligonucleotide chains lengths were 12 (þ5 adenine) or 28 (þ5 adenine) bp that were labeled with qds (136). the merit of this assay is its capability to multi-target diagnosis of nais-resistant influenza sub-types with qds. these point mutations are resistant to nais, which is of vital importance for accurately treating severe human influenza cases, and to avoid the occurrence of severe influenza diseases, especially based on hpai h5n1 [81] . recently, amino functional carbon coated mnps have been used to distinguish hybridization of hbv nucleic acids. altay et al., in 2016 made possible to transfer immobilized molecules from one solution into the appropriate environment with magnetic separation. the omitted signal would be useful in highly selective labelfree hbv dna hybridization. in this assay pre-treatment was not required before separation which reduced the cost and time required [82] . the limit of detection of 1.15 mg ml à1 (20 pmol in 110 ml solution) was obtained with a linear target dna concentration range of 5e25 mg ml à1 . zinc oxide (zno) is the main zinc species used widely in nanostructures. zno has been employed in different types of transducers, instruments of surface acoustic wave, gas sensors and also photonic devices. having piezoelectric properties, zno plays an important role in some special sensors called mechanochemicals [83] . additionally, being zno an innocuous material, biocompatible and environmentally friendly, it can be used without special requirements in the clinical field. as it is summarized in fig. 5 , cao and coworkers used the piezotronic effect of zno nps is nanowire template to design a sensitive biosensor for detection of human immunodeficiency virus (hiv). the zno nanowire provided an in situ selective dna detection of hiv virus. composing zno nanoparticles with a natural and carbon based polymer, like graphene, could provide a new way to design a novel modified electrode or platform to achieve a sensitive biosensor for measuring target molecules at trace levels. tan et al. synthesized pure graphene and composing it with zinc oxide nanoparticles produced by hydrothermal process [85] . because of its unique structure design (graphene/zinc oxide nanocomposite) and using hydrogen peroxide, with noticeable electrocatalytic activity, the sensitivity of the biosensor made was very high in comparing with conventional methods (p < 0.05) for influenza h5 gene detection: the lod being 7.4357 mm (fig. 6) . from different species of al nps, nanoporous morphology is the most famous and attractive one for the scientists involved in biosensing. some considerable chemical, optical and physical properties like chemical and thermal stability, being compatible in bioconditions like human body and having high surface area make this nanostructure suitable to be used it in analytical methods [86] . one of the advantages of having porous structure is the increasing in surface to volume ratio which resulted in an increased number of target molecules inside nanopores. therefore, alumina nanoporous structures could be a rational option to be employed as platforms in modified electrodes for biosensing. from pathogenic viruses, ebola is a mortal species which is spread quickly by fatality results. recently, yang and coworkers [87] designed a high sensitive biosensor for detection of ebola oligonucleotide. in this luminescence-based biosensor, nanoparticles which are up conversion and contains bagdf 5 :yb/er, were used. nanoparticles were connected with oligonucleotide probe and the oligonucleotide of ebola virus as a target linked to aunps. a homogeneous system was prepared with an increased specificity and sensitivity. the novelty of this research was adding the nanoporous alumina in the structure of prepared biosensor which was resulted in considerable lod enhancing from pm to fm level and forming a heterogeneous system. the reason was the efficient light interaction by means of nanoporous alumina walls (fig. 7) . in this report, before adding alumina nanoporous to the biosensor structure, the lod was about 7 pm. but after employing this nanoparticle in the proposed biosensor, the lod was improved to fm. in another study, thin film of anodic aluminum oxide (aao) was used as a substrate of electrode by wu et al. [88] . in this work, aunps were immobilized by an electrochemical method to provide a suitable hostage for deposition of hbv genome probe. polymerase chain reaction (pcr) was the process of detection in this biosensor by using two linear ranges, 10 2 to 10 3 and 10 3 to 10 5.1 copies ml à1 and the lod of the method was 111 copies ml à1 . because of the nanoporous unique structures, with a wide surface area, it leads to make a sensitive biosensor by providing opportunities to capture a great amount of target molecules. toh and coworkers used nanoporous alumina and designed a biosensor for detection of dengue virus [89] . they immobilized considerable amount of antibodies on nanoporous alumina to make impedance changing in mentioned nanostructure by binding antibodies to dengue virus (fig. 8) . by existence of channel capacitance in this system, the biosensor provided a special specificity by responding to just dengue virus in the presence of other virus like west nile and chikungunya viruses. a concentration of 1e900 pfu ml à1 dengue virus was distinguished by this system, thus, producing a sensitive anodic aluminum biosensing application based on simple, cost-effective device. cunps have attracted much consideration because of its massive potential for replacing other expensive nanoparticles. novel nanotechnology reveals the new opportunities for exploring the viral effect of cunps. small size and high surface to volume ratio of copper nanoparticles, are able to interact closely with virus and detect their easily. a major drawback of this particles is their inherent tendency to oxidize in ambient conditions. however, the properties of copper nanoparticles are well-established and base on their use, several biosensors for virus detection have been developed [90] . also, from the chemical point of view, the process of producing cunps from its various salts is cost effective and simple such as hydrothermal procedures. chen et al. fabricated an ultrasensitive electrochemical biosensor for distinguishing influenza a virus utilizing copper nanoparticles. this biosensor is able to detect single stranded dna (ss-dna) of influenza a virus with a detection limit down to fm levels. they utilized cuhcf nanoparticles on the electrode surface through introducing gox-a into the system via biotineavidin interaction to amplify the dna hybridization. a detection limit of~10 3 copies (1 fm in a sample volume of 2 ml) was successfully achieved by this system. meanwhile, the previous reported limits of detection were around 10 5 e10 7 copies ml à1 [91] . mao et al. took advantage of copper nanoclusters to develope a colorimetric biosensing method. in this biosensor, the dna of hepatitis b virus can be distinguished by naked eyes. this method has great potential in comparison to conventional methods; such as the detection of three base-pair mismatches target dna, high sensitivity and selectivity, precise diagnosis of genetic disease and a favorable cost effective. moreover, the detection limit of this assay is 12 â 10 9 molecules. to sum up, this assay is a highly attractive candidate in dna analysis which do not requires the use of sophisticate and expensive solvents [92] . viruses are real menace to human safety since they infect host cells and cause various acute and chronic diseases. aids, smallpox, polio, influenza, diarrhea, and hepatitis are examples of the devastating effects of viral diseases that can affect the safety of people. therefore, an initial warning system for proper detection and recognition of virus is required for eradicating viruses. extensive investigation has been conducted for this purpose over decades. meanwhile, the potential of nanotechnology in onset recognition of viruses is in the eye caching. these particles enhance mechanical, electrochemical, optical and magnetic properties of biosensors, and pave the way for an increased precision and selectivity of their detection. herein we have critically discussed, the use of nanomaterials; such as nanoparticles and nanotubes in fabrication of different biosensors to detect pathogenic viruses. several immobilization procedures, immunosensing schemes and their applications in various virus detection have been also mentioned. although, biosensor technologies is highly promising, they present many challenges in order to move from the bench to their use in the point of care. nanotechnologies offer new tools to overcome these challenges and perform direct detection of molecular targets in real time. emerging graphene oxide, silica, carbon, gold, magnetic nanoparticles and nanotubes provide delicate and accurate platform in this field. apart from mentioned advantages, to guarantee the profits of biosensors, further efforts should be considered concerning the appropriate selection of nanomaterials. the immobilization method of the concerned nanomaterials and biological elements is critical in order to minimize the risk of mistakes and errors in virus detection. another important issue is lifetime of the assay, which is sometimes considerable. indeed, great effort is required to provide portable and reusable devices capable for discriminating of viruses with high selectivity and sensitivity levels. to sum up, advances in nanotechnology will be in a near future a great breakthrough in medicine to halt viral diseases and provide a healthy life to potentially infected patients. detection of influenza virus by a biosensor based on the method combining electrochemiluminescence on binary sams modified au electrode with an immunoliposome encapsulating ru (ii) complex hepatitis a and b immunizations of individuals infected with human immunodeficiency virus detection of the frequency of the novel tt virus by pcr and its role in the induction of hepatic injuries in blood donors in west azarbaijan advanced biosensors for detection of pathogens related to livestock and poultry a review of molecular recognition technologies for detection of biological threat agents vibrio cholerae detection: traditional assays, novel diagnostic techniques and biosensors nano-materials for use in sensing of salmonella infections: recent advances poly-dopamine-beta-cyclodextrin: a novel nanobiopolymer towards sensing of some amino acids at physiological ph poly arginineegraphene quantum dots as a biocompatible and non-toxic nanocomposite layer-by-layer electrochemical preparation, characterization and non-invasive malondialdehyde sensory application in exhaled breath condensate traditional and modern cell culture in virus diagnosis a systematic approach to novel virus discovery in emerging infectious disease outbreaks porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods a review of traditional and contemporary assays for direct and indirect detection of equid herpesvirus 1 in clinical samples current trends in nanobiosensor technology membrane-based electrochemical nanobiosensor for the detection of virus nakedeye nanobiosensor for therapeutic drug monitoring of methotrexate biosensing with quantum dots: a microfluidic approach signatures of dynamical tunneling in semiclassical quantum dots tumortargeted multimodal optical imaging with versatile cadmium-free quantum dots noninvasive theranostic imaging of hsv-tk/gcv suicide gene therapy in liver cancer by folate-targeted quantum dot-based liposomes effect of surface capping of quantum dots (cdte) on proteomics quantum dot-based hiv capture and imaging in a microfluidic channel qds-dna nanosensor for the detection of hepatitis b virus dna and the single-base mutants labeling the nucleocapsid of enveloped baculovirus with quantum dots for single-virus tracking detection of epsteinebarr virus infection in cancer by using highly specific nanoprobe based on dbsa capped cdte quantum dots electrochemical detection of hepatitis b and papilloma virus dnas using swcnt array coated with gold nanoparticles beads-based electrochemical assay for the detection of influenza hemagglutinin labeled with cdte quantum dots two dimension (2-d) graphene-based nanomaterials as signal amplification elements in electrochemical microfluidic immune-devices: recent advances graphene in mice: ultrahigh in vivo tumor uptake and efficient photothermal therapy graphene quantum dot as an electrically conductive material toward low potential detection: a new platform for interface science anti-bacterial activity of graphene oxide as a new weapon nanomaterial to combat multidrug-resistance bacteria graphene oxide as an optical biosensing platform evaluation of a rapid test for detection of h5n1 avian influenza virus recent advances in nanomaterial-mediated bio and immune sensors for detection of aflatoxin in food products avian influenza a (h5n1) infection in humans ultrasensitive electrochemical immunoassay for avian influenza subtype h5 using nanocomposite an enhanced sensitive electrochemical immunosensor based on efficient encapsulation of enzyme in silica matrix for the detection of human immunodeficiency virus p24 simple and label-free electrochemical assay for signal-on dna hybridization directly at undecorated graphene oxide a graphene oxide based immunobiosensor for pathogen detection electrochemical monitoring of nucleic acid hybridization by single-use graphene oxide-based sensor double stranded aptamer-anchored reduced graphene oxide as target-specific nano detector a graphene oxide platform for the assay of biomolecules based on chemiluminescence resonance energy transfer micropatterned reduced graphene oxide based field-effect transistor for real-time virus detection upconversion fluorescence resonance energy transfer biosensor for sensitive detection of human immunodeficiency virus antibodies in human serum carbon nanotube based biosensors a novel label free long non-coding rna electrochemical biosensor based on green l-cysteine electrodeposition and auerh hollow nanospheres as tags a label-free hemin/gquadruplex dnazyme biosensor developed on electrochemically modified electrodes for detection of a hbv dna segment electrochemical immunosensor with graphene quantum dots and apoferritin-encapsulated cu nanoparticles double-assisted signal amplification for detection of avian leukosis virus subgroup j label-free electrochemical detection of short sequences related to the hepatitis b virus using 4, 4 0 -diaminoazobenzene based on multiwalled carbon nanotube-modified gce, oligonucleotides electrochemical detection of avian influenza virus h5n1 gene sequence using a dna aptamer immobilized onto a hybrid nanomaterial-modified electrode dna sensor development based on multi-wall carbon nanotubes for label-free influenza virus (type a) detection hybridization biosensor based on the covalent immobilization of probe dna on chitosanemutiwalled carbon nanotubes nanocomposite by using glutaraldehyde as an arm linker magnetic control of an electrochemical microfluidic device with an arrayed immunosensor for simultaneous multiple immunoassays the structural and luminescence properties of porous silicon detection of hepatitis b virus (hbv) dna at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor multiplex measurement of seven tumor markers using an electrochemical protein chip sensitive detection of epsteinebarr virusderived latent membrane protein 1 based on cdte quantum dots-capped silica nanoparticle labels signal enhancement in dna microarray using dye doped silica nanoparticles: application to human papilloma virus (hpv) detection state of the science literature review: everything nanosilver and more, us environmental protection agency plasmonic nanorice antenna on triangle nanoarray for surface-enhanced raman scattering detection of hepatitis b virus dna a label-free fluorescent molecular beacon based on dna-ag nanoclusters for the construction of versatile biosensors extrinsic surface-enhanced raman scattering detection of influenza a virus enhanced by two-dimensional gold@ silver coreeshell nanoparticle arrays surface-enhanced raman scattering (sers) detection of multiple viral antigens using magnetic capture of sers-active nanoparticles sensitive dna biosensor improved by luteolin copper (ii) as indicator based on silver nanoparticles and carbon nanotubes modified electrode a fluorescent aptasensor for h5n1 influenza virus detection based-on the coreeshell nanoparticles metal-enhanced fluorescence (mef) plasmonics-based detection of virus using sialic acid functionalized gold nanoparticles gold-nanorod-based sensing of sequence specific hiv-1 virus dna by using hyper-rayleigh scattering spectroscopy a gold nanorods-based fluorescent biosensor for the detection of hepatitis b virus dna based on fluorescence resonance energy transfer a highly sensitive and selective hepatitis b dna biosensor using gold nanoparticle electrodeposition on an au electrode and mercaptobenzaldehyde gold nanoparticle-based electrochemical magnetoimmunosensor for rapid detection of anti-hepatitis b virus antibodies in human serum label-free sandwich type of immunosensor for hepatitis c virus core antigen based on the use of gold nanoparticles on a nanostructured metal oxide surface performance evaluation of three automated human immunodeficiency virus antigeneantibody combination immunoassays sensitive and specific hbv genomic dna detection using rca-based qcm biosensor method of preparing iron oxide nanoparticles coated with hydrophilic material, and magnetic resonance imaging contrast agent using the same ligands for label-free detection of whole bacteria on biosensors: a review nanoparticle-based biosensor for the detection of emerging pandemic influenza strains electrically active polyaniline coated magnetic (eapm) nanoparticle as novel transducer in biosensor for detection of bacillus anthracis spores in food samples paramagnetic particles coupled with an automated flow injection analysis as a tool for influenza viral protein detection development of a magnetic electrochemical bar code array for point mutation detection in the h5n1 neuraminidase gene development of amino functionalized carbon coated magnetic nanoparticles and their application to electrochemical detection of hybridization of nucleic acids a comprehensive review of zno materials and devices piezotronic effect enhanced label-free detection of dna using a schottky-contacted zno nanowire biosensor facile hydrothermal growth graphene/zno nanocomposite for development of enhanced biosensor nanoporous anodic alumina platforms: engineered surface chemistry and structure for optical sensing applications ultrasensitive detection of ebola virus oligonucleotide based on upconversion nanoprobe/nanoporous membrane system polymerase chain reaction-free detection of hepatitis b virus dna using a nanostructured impedance biosensor electrochemical impedance spectroscopy characterization of nanoporous alumina dengue virus biosensor copper nanoparticles for printed electronics: routes towards achieving oxidation stability an ultrasensitive dna biosensor based on enzyme-catalyzed deposition of cupric hexacyanoferrate nanoparticles colorimetric detection of hepatitis b virus (hbv) dna based on dna-templated copper nanoclusters key: cord-353467-wbtzvm4i authors: lambert, carsten; thomé, nicole; kluck, christoph j.; prange, reinhild title: functional incorporation of green fluorescent protein into hepatitis b virus envelope particles date: 2004-12-05 journal: virology doi: 10.1016/j.virol.2004.09.031 sha: doc_id: 353467 cord_uid: wbtzvm4i the envelope of hepatitis b virus (hbv), containing the l, m, and s proteins, is essential for virus entry and maturation. for direct visualization of hbv, we determined whether envelope assembly could accommodate the green fluorescent protein (gfp). while the c-terminal addition of gfp to s trans-dominant negatively inhibited empty envelope particle secretion, the n-terminal gfp fusion to s (gfp.s) was co-integrated into the envelope, giving rise to fluorescent particles. microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of gfp.s, required for particle export were rescued by interprotein interactions with wild-type s. thereby, a dual location of gfp, inside and outside the envelope, was observed. gfp.s was also efficiently packaged into the viral envelope, and these gfp-tagged virions retained the capacity for attachment to hbv receptor-positive cells in vitro. together, gfp-tagged virions should be suitable to monitor hbv uptake and egress in live hepatocytes. hepatitis b virus (hbv), a human hepadnavirus, is an enveloped dna virus that infects hepatocytes and causes acute and chronic liver disease. despite considerable understanding of the details of hepadnaviral genome replication, fundamental insights into the early and late steps of an hbv infection are still lacking. infection initiates by virus attachment to the hepatocyte and is determined by the hbv envelope. among the three related large l, middle m, and small s envelope proteins, l has been shown to play the key role in receptor recognition and cell attachment (le seyec et al., 1999; neurath et al., 1986) . the route of subsequent hbv uptake is a yet poorly identified process and may proceed by direct fusion of the viral envelope with the plasma membrane, receptor-mediated endocytosis, or alternate mechanisms (for review, see cooper et al., 2003) . in favor of a fusion mechanism at the cell surface is the previous demonstration of a ph-independent internalization of hbv in primary human hepatocytes (hagelstein et al., 1997) . by contrast, infectivity studies performed with the related duck hbv in duck liver cells have demonstrated that viral entry depends on endocytosis (breiner et al., 1998) . in the late stages of an hbv infection, progeny virions are formed by budding of the pre-assembled cytosolic nucleocapsids, enclosing the partially double-stranded dna genome 3.2 kb in length and the viral polymerase through intracellular membranes accommodating the viral envelope proteins (for review, see nassal, 1996) . during this process, an excess of envelope proteins is not incorporated into virion envelopes but self-assembles into secreted subviral lipoprotein particles referred to as hepatitis b surface antigen (hbsag) particles or empty envelopes. these particles have been shown to mature by budding into intralumenal cisternae of post-endoplasmic reticulum (er)-pre-medial-golgi compartments and to exit the cell by the constitutive secretory pathway (huovila et al., 1992) . in contrast, the intracellular budding site of viral particles has not been defined to date. the three l, m, and s envelope proteins contribute differently to subviral and viral particle formation. assembly and secretion of subviral particles are solely driven by the s protein and are initiated by co-translational integration of s molecules into the er membrane (simon et al., 1988) . the current models for the transmembrane structure of s assume a lumenal disposition (i.e., external in the mature particles) of both the n-and c-termini and four membranespanning segments (berting et al., 1995; stirk et al., 1992) . following dimerization, about 100 transmembrane s monomers then self-assemble with host-derived lipids into spherical empty envelopes, 20 nm in diameter, which are secreted from the infected liver and transfected cell lines with high efficiency. this process is also sustained by the m protein that shares the sequence of s but differs in its nterminal pres2 extension. by contrast, the l protein along with its n-terminal pres1 plus pres2 domains blocks subviral particle production but is the key player in virion formation (bruss and ganem, 1991; chisari et al., 1986) . the pivotal role of the l protein in the viral life cycle is related to its dual transmembrane topology (bruss et al., 1994; ostapchuk et al., 1994; , as its internal (cytosolic) pres domain is needed for envelopment of cytosolic nucleocapsids , while the same external (lumenal) pres domain mediates receptor binding during host cell attachment (le seyec et al., 1999) . in the present study, we addressed whether the hbv envelope could be tagged with the green fluorescent protein (gfp) without impairing its functionality. we reasoned that such gfp-tagged particles should be a promising tool to study the early and late stages of the hbv life cycle in live cells. for tagging of the hbv envelope with gfp, the s protein was chosen as the target because this protein is an essential and abundant constituent of the virion envelope. two chimeras were constructed by fusing either a fluorescenceenhanced gfp in frame to the n-terminus of s (gfp.s) or the yellow-shifted yfp variant to the c-terminus of s (s.yfp) (fig. 1a) . the fusion constructs were transiently expressed in cos-7 cells and analyzed by gfp-specific western blotting. the unfused s protein with a c-terminal ha-tag (sha) was included as reference. as shown in fig. 1b , lysates of cells contained sha in its characteristic doublet of a non-glycosylated (p24) and single-glycosylated (gp27) form as a consequence of partial modification at asn-146. n-linked glycosylation was confirmed by treatment with peptide/n-glycosidase f (pngase f), which converted the gp27 form of s to its faster migrating p24 form. the gfp.s and s.yfp fusion proteins were stably expressed as 51-kda polypeptides in accordance with the molecular masses calculated for their non-glycosylated forms (p51) (fig. 1b) . as evidenced by enzymatic deglycosylation, both fusion proteins appeared in glycosylated forms (gp54) in addition, indicating that they were fig. 1 . the n-terminal gfp.s fusion is co-secreted with wild-type s. (a) schematic representation of the hbv s protein and the gfp.s and s.yfp fusion constructs. numbers below the domains refer to the corresponding amino acid positions of s (hatched bar) and gfp/yfp (white bars). for gfp.s, the two domains are linked by the amino acid sequence sglrsraqasn and terminated by the extra sequence wdppdldn, while s and yfp are interspersed by the sequence dppvat. (b) synthesis, glycosylation, and secretion of sha and the fusion constructs. lysates (c) from transfected cos-7 cells were divided into two portions and either left mock treated or digested with pngase f, as indicated. cellular supernatants (m) were analyzed without enzymatic de-glycosylation. proteins were processed by sds-page and ha-specific (lanes 1-3) or gfp-specific (lanes 4-9) immunoblotting. nonglycosylated (p) and glycosylated (gp) forms are indicated on the left with the numbers referring to the molecular masses of the polypeptides. (c) secretion of gfp.s is rescued by coexpressed wild-type s molecules. supernatants of cells synthesizing gfp.s in the absence (à) or presence (+) of s were assayed by gfp-specific western blotting. competent for co-translational integration of the s region into the er lumen. however, unlike wild-type sha, each chimera failed to be secreted from transfected cells into the supernatants (fig. 1b) . although these results implicated some misfolding of gfp.s and s.yfp, we nonetheless assessed whether their block to secretion could be abrogated by co-expression of wild-type s. cos-7 cells were co-transfected with each fusion construct, and the wild-type vector at a ratio of 1:1 and cellular supernatants were assayed by gfp-specific immunoblotting. the s.yfp fusion turned out to act in a trans-dominant-negative manner and even inhibited export of co-expressed wt s chains (data not shown). by contrast, gfp.s was secreted in the presence of s (fig. 1c) , and thus was chosen for further analysis. for quantitative evaluation of the secretion efficacy of gfp.s, lysates and supernatants of gpf.s + s co-transfected cells were analyzed with a gfp-specific elisa. this determination revealed that 68 f 5.2% of the intracellular gfp.s was released into the supernatant when co-expressed with wt s. in order to get insights how s rescued the secretion of gfp.s, we comparatively examined the intracellular distribution of gfp.s in the presence and absence of sha. when expressed alone, gfp.s surprisingly yielded an intense micropunctate fluorescence (fig. 2a) . these bdotsq partially overlapped with er structures, as shown by co-staining of the cells with antibodies to the er-resident protein disulfide isomerase (pdi) (figs. 2b and c) . although the nature of these structures remained to be determined, they might present aggregates of malfolded gfp.s destined for degradation. importantly, however, on co-expression of gfp.s with sha, the chimera now exhibited a widely dispersed membranous staining pattern together with a significant colocalization with the er marker pdi (figs. 2d-f ). under the same conditions, a nearly 100% co-localization of gfp.s with sha was observed when cells were stained with haspecific antibodies . from these data we concluded that the correct distribution of gfp.s, required for export, was warranted by interaction with co-expressed s chains. to determine whether the co-secreted gfp.s and sha proteins resembled authentic subviral hbv envelope particles, the culture supernatant of transfected cells was fractionated by isopycnic cscl gradient centrifugation and fractions were analyzed by an s-specific elisa. as shown in fig. 3a , the co-secreted proteins banded at a buoyant density of about 1.22 g/ml, typical for wild-type s lipoprotein particles (heermann and gerlich, 1991) . peak fractions were then subjected to specific western blot analysis that confirmed the presence of both the gfp.s fusion and the sha protein (fig. 3a ). in addition, when fig. 2 . gfp.s co-localizes with wild-type s at the er. shown is the intracellular distribution of gfp.s expressed either alone (squares a-c) or together with sha (squares d-i) in cos-7 cells. cells were fixed, permeabilized, and examined by fluorescence microscopy. (a, d, and g) gfp fluorescence (green); (b and e) immunostaining with a mouse antibody to pdi followed by alexafluor 494-conjugated goat anti-mouse igg (red); (h) immunostaining with a mouse anti-ha antibody followed by alexafluor 494-conjugated goat anti-mouse igg (red) to visualize sha. squares c, f, and i are the corresponding merged images so that overlapping red and green signals appear yellow. gfp.s + sha and wild-type sha particles were analyzed by sucrose gradient velocity centrifugation, their sedimentation profiles were nearly coincident with a buoyant density of about 1.130 g/ml in sucrose (fig. 3b) . together, these data demonstrated that gfp.s was successfully incorporated into subviral particles that closely resembled s spheres with respect to density and size. because the fluorescence of gfp depends on its native three-dimensional structure that might be altered within the chimeric context, we next examined whether purified gfp.s + sha particles were fluorescent. as imaged by fluorescence microscopy, a strong vesicular gfp staining was observed, indicating a native display of gfp within the particle (fig. 3c ). the intensity of fluorescence and hence the apparent size of the particles appeared slightly variable and brighter than expected, perhaps because of some particle aggregation or variation in the amount of gfp.s incorporation. the production of fluorescent subviral particles indicated that the individual constituents of the gfp.s chimeric protein were properly folded, at least when co-expressed with wild-type s chains. nonetheless, for further studies outlined below, it was important to know its precise topology, that is, the orientation of the gfp domain relative to the membrane. according to current models for the transmembrane structure of s, its n-terminus and hence the gfp fusion site are located to the lumenal side of intracellular membranes that is topologically equivalent to the virion surface ( fig. 4a ) (berting et al., 1995; stirk et al., 1992) . to analyze the topological features of gfp.s, expressed in the absence or presence of s molecules, we used proteinase k protection experiments of microsomes prepared from transfected cos-7 cells. both gfp.s and s proteins were synthesized in ha-tagged form to enable their simultaneous detection by immunoblotting. consistent with previous works , proteinase k did not cleave the p24/gp27 forms of sha unless the protecting microsomal membrane was disrupted by detergent ( fig. 4b , lanes 4-6). by contrast, gfp.sha synthesized in the absence of sha chains was accessible to cleavage with the protease in intact microsomes (fig. 4b , lanes 1-3). thereby, two ha-reactive fragments were generated that closely resembled the full-length p24 and gp27 forms of sha. therefore, cleavage of gfp.sha must have occurred at least at a very distal site within the nterminal fused gfp domain. in support of a fully cytoplasmic location of the gfp domain, we were unable to detect gfp-reactive proteolysis products by western blotting (data not shown). when expressed together with sha, the topology of gfp.sha surprisingly changed in such that a fraction of polypeptides was now protected from cleavage with proteinase k by the microsomal membrane (fig. 4b, lanes 4-6) . these results indicated that the presence of extra sha chains caused a partial topological change of the n-terminal gfp domain from cytosolic to lumenal, likely as a consequence of gfp.sha/sha interprotein subunit interaction. given the strict correlation between partial n-tail translocation and secretion competence and vice versa, we finally investigated the topological properties of gfp.sha complemented in trans with a secretion-defective s mutant protein. to this aim, we took advantage of sdtm1ha that lacks the first transmembrane domain and is blocked in the assembly and secretion of subviral particles (prange et al., 1992) . consistent with our previous results (prange et al., 1992) , the deletion mutant predominantly appeared in a glycosylated 24-kda form (gp24; fig. 4b, lane 7) . importantly, sdtm1ha failed to impart a partial lumenal orientation of gfp.sha (fig. 4b , lanes 7-9). together, these data indicated that the topological change of gfp.sha in the presence of extra s molecules was likely established during the formation and extracellular release of the gfp.sha/sha mosaic particles. if this notion was correct, we would expect the gfp tag located outside and inside of the secreted particle. for a formal proof of a surface display of the gfp domain, we tried to immunoprecipitate the particles under non-denaturing conditions using two monoclonal a-gfp antibodies directed against different epitopes. however, neither antibody brought down the particles in the absence of detergent (data not shown; and see below). although these results might argue against an exterior location of gfp, it seemed equally possible that the epitopes were not accessible in the bulky beta barrel of gfp on the intact particle. incorporation of gfp.s into envelopes allows viral particle formation next we examined whether gfp.s would also be incorporated into the viral envelope. for virus production, human hepatoma huh-7 cells were transiently transfected with a cloned over-length copy of the hbv genome. on cotransfection with the gfp.s expression plasmid at a 1:1 ratio, virus secretion into the cellular supernatant was reduced about 3-fold (data not shown), whereas no inhibitory effect of gfp.s on the virus secretion profile was observed if a 4-fold excess of the hbv encoding vector was used for co-transfection. because transfected huh-7 cells released viral and subviral particles into the culture medium, virions were separated from empty envelope particles by isopycnic cscl gradient centrifugation. a resolution of these particle types was verified by assaying of fractions for the presence of hbv dna by the endogenous polymerase reaction. as shown in fig. 5 (a and b) , dna-containing virions banded at a density near 1.24 g/ml around fractions 18-22, with the strongest signal in fraction 20, while empty envelopes were found at their typical density of 1.22 g/ml. importantly, when probed with a gfp-specific elisa, the virus-containing fractions were clearly reactive, thus giving first hints to an incorporation of the gfp-tagged s protein during virus assembly. as a final proof, supernatants of hbv + gfp.s co-transfected huh-7 cells were immunoprecipitated with a gfp-specific antibody prior to detection of the viral genome by dot-blot analysis. for reasons mentioned above, such a precipitation, however, required the presence of detergent (0.5% np-40) that decreased the limits of detection, likely because some nucleocapsids might leak from the disordered envelope network. nonetheless, despite these restraints, we could specifically detect the viral dna in a-gfp-precipitated supernatants (fig. 5c) . to investigate whether these fluorescent hbv virions supported attachment to hepatocytes, cell binding assays were performed. although most permanent cell lines are not permissive to hbv infection, the human hepatoblastoma hepg2 cell line is able to bind the virion (lu et al., 1996) . for binding, we applied the same assay conditions as validated previously for in vitro infection of primary human hepatocytes (gripon et al., 1993) . accordingly, recombinant hbv + s.gfp particles secreted from co-transfected huh-7 cells (see above) were precipitated with polyethylene glycol (peg) and reacted with hepg2 cells grown on glass bottom fig. 4 . gfp.s forms a mixed topology when co-expressed with wild-type s. (a) model of the transmembrane topology of s at the er membrane and the virion envelope. the predicted four membrane spanning segments of s project its n-and c-terminus into the er lumen that is topologically equivalent to the virus outside. the n-glycosylation site at asn-146 is indicated by w. (b.) proteinase k protection assay of gfp.s. cos-7 cells were transfected with ha-tagged gfp.s either alone (lanes 1-3) or together with ha-tagged s (lanes 4-6) or the secretion-defective sdtm1 mutant (lanes 7-9). two days after transfection, microsomes were prepared and either left untreated or digested with proteinase k (prot. k) in the absence (à) or presence (+) of np-40, as denoted above each lane. samples were analyzed by ha-specific immunoblotting. the gfp.s-and s-specific p and gp forms are indicated on the left; the corresponding forms of sdtm1ha are denoted on the right. dishes. after incubating cell cultures overnight, followed by several washes, a strong gfp fluorescence almost evenly distributed on the plasma membrane of the hepatocytes was observed (figs. 6a and b) . to test the specificity of adsorption, we analyzed the binding properties of gfp.s + s subviral particles that lacked the l envelope protein and thus the receptor binding site. as expected, these particles failed to attach to hepg2 cells thereby yielding only some diffuse background staining (figs. 6c and d) . however, because infected liver cells and hbv-producing cell lines are known to also produce subviral particles containing the l protein (nassal, 1996) , we could not totally exclude the possibility that the gfp fluorescence, shown in figs. 6a and b, might be also generated by binding of this particle type. in recent years, the adaptation of the aequorea victoria gfp for visualization of protein expression and protein localization in living organisms has provided a powerful new tool. here we applied this approach to hepatitis b virus and obtained fluorescent subviral and viral particles by incorporation of the viral s envelope protein, tagged with gfp, in trans, thereby preserving all the functions necessary for the viral life cycle. because hbv empty envelope particles, built from the s protein, provide a safe antigen delivery system, their use as a carrier for the presentation of various antigens is a long established practice. to date, chimeric particles have been produced in mammalian cells with inserts in the range of 10-80 amino acids (delpeyroux et al., 1986; michel et al., 1988; . for example, the n-terminal ectodomain of the s protein has been shown to tolerate 84 foreign residues while its c-terminus accommodated 42 residues without effects on particle assembly and secretion (michel et al., 1988; . in agreement and extension, the current study demonstrated that the nterminus of s is more permissive for insertions than the c-terminus and is even amenable for addition of the complete gfp protein. hence, proteins of at least up to 238 amino acids can be displayed on hbv envelope containing virion particles were further subjected to an envelope-specific immunoprecipitation and radioactive labeling of the viral genome by the endogenous viral polymerase. the migration of the hbv dna genome as visualized by agarose electrophoresis and phosphorimaging is indicated. (c) pooled extracellular viral particle fractions from hbv + gfp.s cotransfected cells were immunoprecipitated with either an envelope-specific (a-hbsag) or a gfp-specific (a-gfp) antibody prior to extraction of the viral genome and dna dot blot analysis. for control of nonspecific immunoprecipitation, native hbv particles (control) were reacted with the a-gfp mab (third panel from the left). in addition, the control material was probed with a-hbsag, as shown in the right panel. fig. 6 . gfp-tagged hbv binds to hepg2 cells. in squares a and b are shown the adsorption of gfp-tagged hbv particles (hbv + gfp.s) to live hepg2 cells, grown on glass bottom dishes, as detected by gfp autofluorescence (magnification, â1600). under the same assay conditions, gfp-tagged subviral particles (s + gfp.s) did not bind to cells (squares c and d). particles, raising intriguing perspectives for their future use as a carrier system. particle formation of the gfp.s chimeric protein, however, depended on the presence of the wild-type s protein, indicating that the fusion construct itself failed to fold into a functional conformation required for assembly. nonetheless, as judged by the autofluorescence of gfp.s in cells, at least the gfp-tag must have folded properly. thereby, gfp.s surprisingly appeared in sequestered speckles that overlapped with the er compartment. the formation of similar structures, termed concentric membranous er bodies, has been recently observed upon expression of a misfolded mutant of the cystic fibrosis transmembrane conductance regulator that accumulates in these bodies prior to erassociated degradation (okiyoneda et al., 2004) . accordingly, the s moiety of gfp.s might not attain its native transmembrane structure thereby tending to aggregate into discrete speckles. on co-expression with wild-type s, the intracellular distribution of gfp.s completely changed in such that it now yielded a typical membranous er staining pattern and a high degree of co-localization with s. this result indicates that the proper folding and localization of gfp.s were warranted by interprotein interactions with wildtype s chains. as a consequence thereof, highly fluorescent mosaic particles were formed and secreted, which closely resembled authentic spherical s lipoprotein particles according to density and size. furthermore, the gfp.s fusion protein could be also natively incorporated into the virion envelope. gfp, by virtue of its properties, has been successfully used to tag various viruses like, for example, herpes simplex virus type i, vesicular stomatitis virus, vaccinia virus, hiv, and mouse hepatitis coronavirus (bosch et al., 2004; dalton and rose, 2001; desai and person, 1998; stauber et al., 1999; ward and moss, 2001) . however, unlike these viruses, hbv is a very small particle with a diameter of 42 nm and a dense packed structure. even so, it can apparently accommodate multiple copies of gfp, indicating that there is substantial space available between the viral envelope and the nucleocapsid as well as sufficient flexibility allowing the incorporation of proteins in trans. although we were unable to unequivocally define the location of the gfp-tag within the secreted hbv envelope, our topological analysis of gfp.s suggested an interior (i.e., cytosolic side of microsomes) and exterior (i.e., lumenal side of microsomes) display of gfp. the formation of such a mixed topology is rare among membrane proteins but is a prominent feature of the large l envelope protein of hbv (bruss et al., 1994; ostapchuk et al., 1994; . in that case, it is established post-translationally at the er membrane and is uncoupled from envelope assembly (lambert and prange, 2001) . by contrast, the two differently orientated isoforms of gfp.s were only generated upon coexpression with secretion-competent wild-type s molecules, thus hinting to a link between envelope subunit interactions and topogenesis of gfp.s. while the underlying mechanism remains to be determined, it is tempting to speculate that the dual location of the gfp domain might be beneficial for the functionality of gfp-tagged hbv. by splitting the tag to both sides of the virion envelope, the local density of gfp might be lowered thereby limiting the risk of sterical hindrance of the envelope/nucleocapsid interactions at the interior and the envelope/receptor binding at the exterior. by using hepg2 cells, an established hepatocyte-derived cell line that is permissive for hbv attachment but nonpermissive to a productive infection (lu et al., 1996) , a specific binding of gfp-tagged viral particles was observed. hence, these particles must imitate the natural virus and viral attachment appeared to proceed through the authentic pathway. however, whether these particles even retained their infectivity remains to be addressed. in vitro hbv infection studies had been restricted to primary human hepatocytes that are not readily available. more recently, however, successful experimental infections of tree shrews were carried out that might present a new animal model (köck et al., 2001) . in addition, a human hepatoma cell line has been established that supports hbv infection (gripon et al., 2002) . therefore, the gfp-tagged hbv envelope might be useful for the investigation of the early events of virus penetration such as monitoring entry and uncoating in living infected cells and tracing the fate of the envelope structure. with gfp as a marker, it should also be possible to approach sites of viral assembly and to follow the intracellular trafficking and egress of (sub)viral particles during the late stages of an hbv infection. the mammalian expression vectors carrying the hbv s gene with or without a c-terminally tagged influenza virus hemagglutinin (ha) epitope under the control of the human metallothionein iia promoter had been described (pni2.-sha and pni2.s, respectively) (hartmann-stühler and . for construction of the gfp.s chimera, plasmid pegfp-c1 (bd biosciences clontech) was cut within the multiple cloning site by using ecori and smai. to introduce an ecori restriction site downstream of the start codon of the hbv s gene, site-directed mutagenesis with a recombinant m13mp19 bacteriophage carrying a 2.3kb bglii-bglii fragment (nucleotide [nts] 2839 to 1986, as referred of the hbv genome, subtype ayw) with the antisense oligonucleotide 5v-gaatcctgaattcatgttctc-3v (the ecori site is italic) was performed. the newly created ecori site, cutting between codon positions 4 and 5 (nts 170) of the s gene, together with the cognate filled-in acci site (nts 827) was then used to generate an s encoding fragment for in-frame insertion into pegfp-c1, giving rise to plasmid pegfp.s. in parallel, the ha-tagged version of the s gene was similarly cloned, giving rise to gfp.sha. a reciprocal chimera, containing the yellow-shifted yfp colinearly fused to the c-terminus of s (s.yfp), was created using plasmid peyfp-n1 (bd biosciences clontech), which was opened with xhoi and bamhi. the ha-tagged s gene was derived from pni2.sha by cleavage with xhoi (nts 127) and psii (cutting between the ha-tag and the translational stop codon) and inserted into peyfp-n1 after filling-in of its bamhi site. plasmid pni2.sdtm1ha carries an in-frame deletion of tm1 that was achieved by removal of a styi (nts 180)-xbai (nts 249) fragment from pni2.s. to produce viral envelope proteins, transient transfection of cos-7 cells by electroporation was used. unless otherwise indicated, 5 â 10 6 cells were transfected with 12 ag of plasmid dna, while in the case of co-transfections, 12 ag of each dna was used. three days posttransfection, cellular supernatants were harvested and clarified by low-speed centrifugation. cells were washed twice in tris-buffered saline (tbs, 50 mm tris-cl [ph 7.5], 150 mm nacl) and lysed with 1 ml of tbs-0.5% nonidet p-40 (np-40), supplemented with a protease inhibitor cocktail (roche), for 30 min on ice. after centrifugation for 5 min at 13 000 â g and 4 8c, proteins of lysates and cell supernatants were precipitated with 10% trichloroacetic acid (tca), washed twice with 5% tca, and once with acetone. tca precipitates were resolved by sds-page and western blotted to nitrocellulose membranes. enzymatic n-deglycosylation of proteins with pngase f (new england biolabs) was done according to the instructions of the supplier. immunoblots were incubated with a mouse monoclonal antibody (mab) against the ha epitope (babco), diluted 1:2000 in blotting buffer (pbs with 5% skim milk) or a mouse mab specific for gfp (jl-8; bd biosciences clontech) applied in 1:4000 dilution. peroxidase-labeled secondary antibodies (dianova) were diluted as instructed by the manufacturer, and the blots were developed with enhanced chemiluminescence detection reagents (amersham biosciences). in parallel, cell lysates and supernatants were analyzed by elisas. hbsag was measured using the auszyme ii diagnostic kit (abbott laboratories). for simultaneous detection of gfp and hbsag, a sandwich elisa with gfp-specific antibodies in the solid phase and s-specific antibodies in the detection phase was employed. briefly, the a-gfp-mab jl-8 was coated to microtiter plates (1:500 dilution in tbs) for 3 h at room temperature. nonspecific binding sites were blocked with tbs-1% bovine serum albumin for 18 h at 4 8c, followed by three washes with tbs-0.1% tween 20. samples were reacted for 2 h at 37 8c in the presence of 0.5% np-40, washed out, and incubated with peroxidase-labeled hbsag-specific mabs (auszyme ii) for 1 h at 37 8c. transfected cos-7 cells on glass cover-slips were fixed and permeabilized with ice-cold methanol containing 2 mm egta for 15 min at à20 8c. for staining of internal antigens, cells were incubated with the ha-specific mab (1:200 dilution in pbs) or a mouse mab specific for protein disulfide isomerase (spa-891, stressgen biotechnologies) (1:2000 dilution in pbs) prior to incubation with an alexafluor 594-conjugated goat anti-mouse immunoglobulin g (2 ag/ml in pbs; molecular probes). spontaneous gfp fluorescence and immunostaining were visualized with a fluorescence microscope (axiovert 200m, zeiss), and images obtained with a zeiss axiocam digital camera were processed using the zeiss axiovision software. to assess for fluorescent particles, cellular supernatants were concentrated 50-fold by ultracentrifugation (see below). a 10-al aliquot was spotted on glass bottom dishes (bd biosciences), briefly air dried, and imaged with a â100 objective. three days after transfection of cos-7 cells, microsomes were prepared essentially as described . briefly, cells were disrupted by dounce homogenization, and microsomes were recovered by ultracentrifugation prior to proteolysis with proteinase k (100 ag/ml) in the presence or absence of 0.5% np-40. after incubation on ice for 60 min, proteinase k was inactivated by the addition of 10 al/ml phenylmethylsulfonyl fluoride. each sample was then adjusted to 0.5% np-40 and solubilized for 20 min on ice. cleared samples were precipitated with 10% tca and subjected to western blot analysis. cesium chloride and sucrose gradient purification of particles subviral particles of cellular supernatants were pelleted through a 1-ml cushion of 20% sucrose in tne (10 mm tris-cl [ph 7.5], 150 mm nacl, 10 mm edta) using a sw 40 rotor (beckman) at 37 000 rpm for 4 h at 4 8c. the resuspended pellets were separated by isopycnic gradient centrifugation through 10-50% (wt/vol) cesium chloride in tne. after centrifugation at 35 000 rpm and 10 8c (sw 40 rotor), fractions were collected from the top and screened for chimeric particles by elisa, immunoblotting, and fluorescence analysis. for velocity sedimentation, pelleted particles were layered on top of a 12-ml linear sucrose gradient (10 to 50% [wt/wt] sucrose in tne buffer) with a 68% sucrose cushion, centrifuged at 35 000 rpm and 10 8c for 16 h (sw 40 rotor), and processed as above. for replication of hbv in the huh-7 liver cell line, plasmid phbv was used that carries a 1.1 mer of the hbv dna genome (radziwill et al., 1990) . this plasmid was cotransfected with pegfp.s by the calcium phosphate precipitation technique. four days after transfection, extracellular virions were separated from subviral empty envelopes and non-enveloped core particles by isopycnic cscl gradient centrifugation. to this aim, 3.8 g of cscl was dissolved in 10 ml cleared supernatant and 28 ml of this solution was spun for 20 h at 45 000 rpm at 10 8c in a beckman 50vti rotor. fractions were collected from the bottom, supplemented with the protease inhibitor cocktail, and were screened for hbsag and gfp by immunoassays. following dialysis of fractions against 10 mm tris-cl [ph 7.5], 150 mm nacl at 4 8c, virions were isolated by an envelope-specific immunoprecipitation, as described previously (löffler-mary et al., 2000) . for detection of gfp.s within the virion envelope, particles were immunoprecipitated with the a-gfp mab jl-8 that had been coated to a 10% (wt/vol) suspension of protein g-sepharose (10-al antibody was bound to 100 al sepharose solution in an overnight reaction at 4 8c). immunoprecipitation was performed in the presence of 0.5% np-40 and precipitates were then washed two times with ten buffer (50 mm tris-hcl [ph 7.5], 1 mm edta, 75 mm nh 4 cl). detection of the encapsidated viral progeny dna by radioactive labeling of the partially double-stranded genome with 10 aci [a 32 p]datp (amersham biosciences) by the endogenous polymerase, isolation, and separation of the dna by agarose gel electrophoresis have been described (löffler-mary et al., 2000) . the radioactive signal was detected by phosphorimager scanning. for detection of hbv dna on dot-blots, an ecorilinearized unit-length hbv genome was labeled with digoxygenin-dutp by random priming as instructed by the manufacturer (roche). after extraction of the dna genomes from the immunoprecipitated samples as described, they were denatured by boiling in 0.4 m naoh-10 mm edta and filtered with a dot-blot manifold (schleicher and schqll) onto nylon membranes. the membrane was baked at 120 8c for 30 min and hybridized with the labeled probe according to the roche dig dna labeling and detection kit. for binding analysis, human hepatoma hepg2 cells were cultivated in glass bottom dishes in dulbecco's modified eagle's medium with 10% fetal calf serum (fcs). hbv and subviral particles secreted from huh-7 (co)-transfected cells were precipitated from 10-ml culture supernatant in the presence of 6% polyethylene glycol 8000 (peg; sigma). the pellet was resuspended in 200-al phosphate-buffered saline (pbs) containing 25% fcs. hepg2 cells were incubated with 50 al of this concentrate, diluted in 1 ml culture medium with 4% peg, for 20 h at 37 8c. cells were washed three times with pbs and analyzed for gfp fluorescence by microscopy. computer-aided studies on the spatial structure of the small hepatitis b surface protein coronavirus spike glycoprotein, extended at the carboxy terminus with green fluorescent protein, is assembly competent carboxypeptidase d (gp180), a golgi-resident protein, functions in the attachment and entry of avian hepatitis b viruses the role of envelope proteins in hepatitis b virus assembly post-translational alterations in transmembrane topology of the hepatitis b virus large envelope protein functions of the large hepatitis b virus surface protein in viral particle morphogenesis expression of hepatitis b virus large envelope polypeptide inhibits hepatitis b surface antigen secretion in transgenic mice the earliest steps in hepatitis b virus infection vesicular stomatitis virus glycoprotein containing the entire green fluorescent protein on its cytoplasmic domain is incorporated efficiently into virus particles a poliovirus neutralization epitope expressed on hybrid hepatitis b surface antigen particles incorporation of the green fluorescent protein into the herpes simplex virus type 1 capsid reproducible high level infection of cultured adult human hepatocytes by hepatitis b virus: effect of polyethylene glycol on adsorption and penetration infection of a human hepatoma cell line by hepatitis b virus phindependent uptake of hepatitis b virus in primary human hepatocytes hepatitis b virus large envelope protein interacts with g2-adaptin, a clathrin adaptor-related protein surface proteins of hepatitis b viruses hepatitis b surface antigen assembles in a post-er, pre-golgi compartment efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis b virus dual topology of the hepatitis b virus large envelope protein: determinants influencing post-translational pre-s translocation infection process of the hepatitis b virus depends on the presence of a defined sequence in the pre-s1 domain hepatitis b virus assembly is sensitive to changes in the cytosolic s loop of the envelope proteins protease-induced infectivity of hepatitis b virus for human hepatoblastoma cell line induction of anti-human immunodeficiency virus (hiv) neutralizing antibodies in rabbits immunized with recombinant hiv-hepatitis b surface antigen particles hepatitis b virus morphogenesis. cmti identification and chemical synthesis of a host cell receptor binding site on hepatitis b virus df508 cftr pool in the endoplasmic reticulum is increased by calnexin overexpression a dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis b viral morphogenesis novel transmembrane topology of the hepatitis b virus envelope proteins deletions in the hepatitis b virus small envelope protein: effect on assembly and secretion of surface antigen particles properties of modified hepatitis b virus surface antigen particles carrying pres epitopes mutational analysis of the hepatitis b virus p gene product: domain structure and rnase h activity secreted hepatitis b surface antigen polypeptides are derived from a transmembrane precursor direct visualization of hiv-1 entry: mechanisms and role of cell surface receptors a topological model for hepatitis b surface antigen visualization of intracellular movement of vaccinia virus virions containing a green fluorescent protein-b5r membrane protein chimera characterization of early hepatitis b virus surface protein oligomers we acknowledge the perfect technical assistance of tatjana dfring and thank rolf e. streeck, martin sapp, and hans-christoph selinka for helpful discussion. this work was supported by grants to r. p. from the deutsche forschungsgemeinschaft (sfb 490-a1, pr305/1-1). key: cord-266105-8avkjc84 authors: li, qiang; feng, wei; quan, ying-hui title: trend and forecasting of the covid-19 outbreak in china date: 2020-02-27 journal: j infect doi: 10.1016/j.jinf.2020.02.014 sha: doc_id: 266105 cord_uid: 8avkjc84 by using the public data from jan. 20 to feb. 11, 2020, we perform data-driven analysis and forecasting on the covid-19 epidemic in mainland china, especially hubei province. our results show that the turning points of the daily infections are predicted to be feb. 6 and feb. 1, 2020, for hubei and china other than hubei, respectively. the epidemic in china is predicted to end up after mar. 10, 2020, and the number of the total infections are predicted to be 51600. the data trends reveal that quick and active strategies taken by china to reduce human exposure have already had a good impact on the control of the epidemic. a novel coronavirus from wuhan in central china, named 2019-ncov, has recently caused an epidemic of pneumonia in humans and posed a huge threat to global public health. 1 , 2 to the date 06/02/2020, 2019-ncov has led to more than 31,0 0 0 confirmed cases and 637 deaths in china according to national health commission of the people's republic of china ( http://en.nhc.gov. cn/index.html ). cases have also been documented in a growing number of other international locations, including the united states ( https://www.cdc.gov/coronavirus/2019-ncov/index.html ). as a consequence, it is urgent to develop effective measures to control this novel coronavirus on the basis of its pathogenesis. host receptor recognition is a determinant for virus infection. during the time of this letter preparation, three works have just been published to explore the receptor usage of 2019-ncov. a work by zheng-li shi et al. has shown that angiotensin-converting enzyme 2 (ace2), the receptor for severe acute respiratory syndrome coronavirus (sars-cov), 3 from human, rhinolophus sinicus bat, civet, swine but not mouse mediate 2019-ncov infection in vitro , while the detailed mechanisms are not yet determined. 4 the other two works have reported or predicted human ace2 usage of 2019-ncov in a similar way to sars-cov mainly based on the coronavirus spike (s) glycoproteins. 5 , 6 considering the fact that the s proteins mutate and gain capability to recognize host receptors among species, 7 , 8 there is still a lack of analyses on receptor usage of 2019-ncov from the receptor perspective, which does not evolve as quickly as viruses. here, we firstly performed amino acid sequence alignment of ace2 from different species, including human, five non-human primates (gibbon, green monkey, macaque, orangutan and chimpanzee), two companion animals (cat and dog), six domestic animals (bovine, sheep, goat, swine, horse and chicken), three wild animals (ferret, civet and chinese horseshoe bat) and two rodents (mouse and rat). the alignment by clustal w 2.1 shows that they share a high sequence similarity except chicken (data not shown). the result suggests that 2019-ncov of probable bat origin may not interact with chicken ace2 and subsequently infect them, which were not considered in the following analyses. in ace2, the regions at position 30-41, 82-84 and 353-357 are demonstrated to be involved in the interaction with sars-cov s protein, where the residues at positions 31, 35, 38, 82 and 353 are critical. 9 therefore, we took a close comparison in these regions and residues. as shown in fig. 1 , human and non-human primates share the identity sequences in the regions and residues, implying that ace2 from non-human primates may recognize 2019-ncov and medi-ate its infection. as a result, non-human primates may be susceptible to 2019-ncov and serve as animal models for antiviral research or intermediate hosts for cross-species transmission. in fig. 1 , the residues of most companion, domestic and wild animals are conserved, especially for the critical ones stated above, while certain ones are variable. for example, lys31, glu35, asp/glu38 and lys353 are conserved, which probably form salt bridges. interestingly, the changes at positions 31, 38 and 82 are observed. these changes suggest steric hindrance and electrostatic interference for host-virus interaction. taking civet ace2 as an example, the change of lys31 to thr31 is likely to form a hydrogen bond instead of a salt bridge. in addition, the polar side chain of thr82 may influence the hydrophobic interaction of the original met82. all these changes may result in a lower binding affinity. however, an additional region covering residues 90-93 has been shown to be involved in civet ace2 binding to sars-cov and enhance their interaction. 10 consequently, we can't preclude the existence of other regions to compensate for the residue changes. with most residues in human ace2, the ones from these compaion, domestic and wild animals may be favorable for 2019-ncov recognition, which is in consistent with the recent work by zheng-li shi et al. in case cross-species transmission, close contact with sick or asymptomatic companion, domestic and wild animals should be cautious, such as for workers in livestock farming and travellers in the wild. in contrast, certain significant changes occur in the mouse and/or rat ace2 compared to the human one ( fig. 1 ) . the asn31 and ser82 in mouse ace2 may not form favorable interactions with 2019-ncov due to their electrostatic or hydrophilic characteristics. importantly, the change into his353 in both mouse and rat ace2 does not form a strong salt bridge as lys353 does. since the structural information for mouse and rat ace2 is unavailable, we carried out homology modeling using human ace2 (pdb code 2ajf) as template on online ( https://swissmodel.expasy.org ) for further analyses. in fig. 2 a, the change into ser82 in mouse ace2 may interfere with the hydrophobic interaction of the original met82. additionally, the changes into asn31 and his353 definitely affect the salt bridge formation and electrostatic potential. the change into his353 in rat ace2 is similar in the effect on receptor-virus interaction ( fig. 2 b) . these analyses partially explain why mouse ace2 does not mediate 2019-ncov infection reported by zheng-li shi et al. and assume that rodents are not likely to be the susceptible host. in conclusion, we conducted sequence and structural analyses of angiotensin-converting enzyme 2 (ace2) from different species, which sheds some light on cross-species receptor usage of 2019-ncov. all these analyses raise an alert on a potential interspecies transmission of 2019-ncov and propose further surveillance in other animal populations. structural studies on human and other species ace2 in complex with 2019-ncov spike protein will con the authors declare no conflict of interest. very recently, a letter in journal of infection reported the outbreak of the novel cornonavirus from dec. 2019 in china, especially in hubei province. 1 this novel cornonavirus may originate from the bat, 2 is just named as the covid-19 by the world health organization (who). the covid-19 outbroke from wuhan, the capital of hubei province, has spread to other provinces of china and even other countries. 3 strong human-to-human transmission is established. 4 until feb. 11, 2020, there have been 44653 cases of covid-19 infections confirmed in mainland china, including 1113 deaths. to prevent and control the spread of the epidemic, many strategies are needed. 5 predicting the trend of the epidemic are quite important to the allocation of medical resources, the arrangement of production activities, and even the domestic economic development all over china. therefore, it is very urgent to use the latest data to establish an efficient and highly suitable epidemic analysis and prediction model according to the actual situation, and then to give reliable predictions, which could provide an important refer-ence for the government to formulate emergency macroeconomic decisions and medical resources allocation. recently, the susceptible-exposed-infectious-recovered (seir) or other similar models 6,7 are used to forecast the potential domestic and international spread of this covid-19 epidemic with parameters estimated from other sources.the real situation could be much more complicated and changing all the time. especially, with the implementation of the chinese government's multiple epidemic control policies, the control of nationwide epidemic has become obvious. however, the medical supplies in hubei will still affect the implementation of national policies. in this letter, we present the current situation of the epidemic, predict the ongoing trend with data driven analysis, and estimate the outbreak size of the covid-19 in both hubei and other areas in mainland china. the data of the epidemic are listed in table 1 and also graphically shown in fig. 1 , in which "china" is used to denote the mainland china, and "other" mainland china other than hubei province. the data includes the daily confirmed(suspected) infections, totally confirmed(suspected) infections, daily deaths, and total deaths from jan. 20, to feb. 11, 2020, reported by the national health commission of the republic of china (nhc), 8 , and health commission of hubei province (hch). 9 jan. 20, 2020, containing all the cases reported from 0 to 24, is the zeroth day in this letter, and then others are implied. the total number of suspected cases reaches the peak value on the 19th day (feb. 8), and then drops rapidly. notice that, until feb. 11, 2020, almost all the cases of deaths (1068/1113, 96%,) locates in hubei province, which reveals the epidemic in hubei is much more serious than that in the other areas of china. on the hand, it states the strict quarantine and limitation on population mobility have effectively prevented outbreaks in other provinces of china. scribe the data of daily infections and deaths in hubei, where x = (t + 0 . 5 − t t ) with t denoting the day, and t t representing the turning point; a and k are the parameters and determined by the data together with t t . the cumulative data of infections or deaths are obtained by the integration over h ( t ). for the epidemic in the other areas of china, the data of infections shows an asymmetric character, and then will be described as where x = t − t t ; the parameters b, k 1 , and k 2 together with t t , are then determined by fitting to the data. fig. 2 shows the fit and trend predictions to the total infections and deaths in hubei and china other than hubei. the extracted turning point of the infections in hubei is the 17th day, namely, feb. 6, 2020. the epidemic in hubei is predicted to end after mar. 10, 2020. we estimated that the epidemic is to end up with a total of 39, 0 0 0 infections in hubei, not including the clinically diagnosed cases since feb. 12, which may enlarge the prediction by 1.4 times. with considered data, namely, data from jan. 20 to feb. 11, the average errors are bout 166 and 190 for the fits to describe the daily and cumulative infections in hubei, respectively, corresponding to 8.6% and 1.6% for the average relative errors, respectively. fig. 2 (b) and (e) shows the estimations of the total and daily deaths in hubei. the predicted turning point is feb. 12, 2020. the total deaths is estimated to be 2250. notice the distribution of the daily deaths is delayed about 5 ∼6 days compared with the that of the daily infections. the average errors are bout 4 and 22 for the model to describe the daily and cumulative death numbers, respectively, corresponding to the relative errors 8.6% and 6.2%, respectively. the numbers of the daily and total infections in china other than hubei are showed in fig. 2 (c) and 2 (f), respectively. the extracted turning point is feb. 1, 2020 and the epidemic is expected to end on the 45th day, namely, on mar. 5, 2020. the estimated number of cumulative infections is about 12,600 in china other table 1 the data of epidemic caused by the covid-19 pneumonia in the mainland china and hubei, including (a) daily infections, (b) daily deaths, (c) total infections, (d) total deaths, (e) daily and (f) total suspected cases. china hubei a b c d e f a b c d 2020/1/20 77 2 291 6 27 54 72 2 270 6 2020/1/21 149 3 440 9 26 37 105 3 375 9 2020/1/22 131 8 571 17 257 393 69 8 4 4 4 17 2020/1/23 259 8 830 25 680 1072 105 7 549 24 2020/1/24 4 4 4 16 1287 41 1118 1965 180 15 729 39 2020/1/25 688 15 1975 56 1309 2684 323 13 1052 52 2020/1/26 769 24 2744 80 3806 5794 371 24 1423 76 2020/1/27 1771 26 4515 106 2077 6973 1291 24 2714 100 2020/1/28 1459 26 5974 132 3248 9239 840 25 3554 125 2020/1/29 1737 38 7711 170 4148 12,167 1032 37 4586 162 fig. 1 (f)), we did not parameterize this data, and hence did not give a trend prediction. the covid-19 epidemic in china is predicted to end after mar. 20, 2020, and cause 52,0 0 0-68,0 0 0 infections and about 240 0 deaths. however, the data trends show that the quick and active strategies to reduce human exposure taken in china, such as limi-tation on population mobility and interpersonal contact rates, strict quarantine on migrants, have already had good impacts on control of the epidemic. now the outbreak and deaths of the covid-19 epidemic are mainly in hubei province. after this letter has been written, the hubei reported 14,840 confirmed infections (including 13,332 clinically diagnosed cases) on feb. 12, 2020, which is almost 9 times greater than the data of the previous day. the huge fluctuation is due to the changing of diagnostic criteria in hubei. and this clinical criteria taken in hubei is expected to play an active and important role in controlling the outbreak and death rate. the authors declare no conflict of interest. dear editor , recently, several studies in this journal have highlighted the threat of avian influenza virus (aiv) to humans, poultry, and other animals. [1] [2] [3] [4] [5] [6] in equines, there was only one reported influenza outbreak caused by aiv, which occurred in 1989-1990 in china. however, aiv still poses a potential threat to equines. in contrast to aiv, equine influenza virus (eiv) is a commonly known causative pathogen of acute respiratory disease in equines. to date, two subtypes of eivs have been determined in the equine population worldwide: h7n7 and h3n8. 7 h7n7 eiv was initially identified in prague in 1956, and the last outbreak caused by h7n7 eiv occurred in 1979. 7 h3n8 eiv was first isolated in miami in 1963 and is currently responsible for ei outbreaks worldwide. during continuous transmission and evolution in equines, h3n8 eiv strains diverged genetically into three distinct lineages: predivergent, european, and american. 7 historically, there have been four main ei outbreaks in mainland china, occurring in 1974 china, occurring in , 1989 china, occurring in -1990 china, occurring in , 1994 , and 20 07-20 08. in the first, third, and fourth outbreaks, the isolated eiv strains were of the h7n7 subtype, h3n8 subtype european lineage, and h3n8 subtype american lineage, respectively. in the second ei outbreak, the causative pathogen (a/equine/jilin/1/1989) was determined to be h3n8 subtype influenza a virus (iav) by antigenicity characterization. however, after genetic sequencing, all eight segments of a/equine/jilin/1/1989 were genetically closer to those of avian influenza virus than those of eiv, indicating an interspecies transmission event. the 1989-1990 ei outbreaks in china contain two major outbreaks. the first ei outbreak occurred in jilin and heilongjiang provinces between march and june 1989, with a morbidity of 81% and a mortality of up to 20% in some herds. the second ei outbreak occurred in heilongjiang province in april 1990, with a morbidity of 41% and no mortality. the high mortality and unique antigenicity of a/equine/jilin/1/1989 attracted the attention of eiv researchers. however, after the 1989-1990 ei outbreaks in china, the virus disappeared from the equine population for unknown reasons. although no evidence in the epidemiological investigation worldwide supports the continuous circulation of a/equine/jilin/1/1989-like eiv in equines, we should not underestimate the potential of interspecies aiv transmission to equines and the possibility of future ei outbreaks caused by aiv. the haemagglutinin (ha) of iavs recognizes and binds the cell surface sialic acid (sa) receptor of the host respiratory tract, and then the virus enters into cells and replicates. the distribution of the sa receptor influences the host range of iavs. human influenza viruses preferentially bind the saa2,6 gal receptor, while aivs preferentially bind the saa2,3 gal receptor. it has been reported that the saa2,3 gal receptor is abundant in the epithelial cells of the horse trachea, and animal experiments indicate that iavs with an ha recognizing the saa2,3 gal receptor could replicate in horses. this finding provides a prerequisite for cross-species transmission of aiv to equines. in fact, there were several pieces of direct molecular evidence supporting the interspecies transmission of aiv to equines, in addition to the 1989-1990 ei outbreaks in china. in 2010, abdel-moneim et al. reported one h5n1 eiv strain (a/equine/egypt/ av1/2009) isolated from donkeys with cough, fever and serous nasal discharge in egypt. 8 sequencing results indicated that the virus had a close genetic relatedness to h5n1 aiv. in addition, h5 seroconversion was observed in 25.71% (27/105) of the examined donkeys. in 2011, he collected 284 equine lung tissue samples in china and isolated one aiv-derived h9n2 eiv strain (a/equine/guangxi/3/2011) ( https://kns.cnki.net/ kcms/detail/detail.aspx?dbcode=cmfd&dbname=cmfd201301& filename=1012496506.nh&v=mtmxmzc3ck5wrji2sexleedove1x wkviuelsogvymux1efltn0romvqzcvryv00xrnjdvvjmt2vazvpt rknua1u= ). among the tested equine serum samples in china, 0.54% (7/1110) of samples were positive for anti-h9n2 antibody. in the 2015-2016 ei outbreaks in pakistan, khana et al. reported that the isolated eiv strain (a/equine/pakistan/16) was reassorted from aiv. 9 the common characteristic for the reported cross-species transmission events of aiv to equines in china, pakistan, and egypt is that they occur in farming equines. 8 , 9 in the mixed farming system, equines and domestic poultry often live in close proximity. compared with racehorses, farming equines have more opportunities to contact domestic poultry and experience long-term environmental exposure to poultry. aiv infections in poultry increase exposure risks to equines. recently, frequent reports of cross-species transmission events of aiv to farming dogs in china and korea also indicate the potential threat of aiv to farming animals with close contact with poultry. 10 another problem is that the farming equines in china, pakistan, and egypt had no vaccination history. even in some racehorse populations, vaccinations for eiv are not routinely performed as recommended by the world organization for animal health (oie) expert surveillance panel on ei vaccine composition. although h3n8 eiv is antigenically distinct from a/equine/jilin/1/1989, animal experiments indicated that high doses of ei vaccines still provided complete protection against challenge with a/equine/jilin/1/1989. accordingly, routine vaccination with h3n8 ei vaccines in equines might prevent aiv infection to some degree, at least for h3n8 subtype aiv. several strategies may help reduce the threat of aiv to equines, including reducing exposure of equines to poultry, birds, and other hosts of iav, especially animals with clinical signs of influenza virus infection; monitoring aiv prevalence in domestic poultry around equines and routinely vaccinating domestic poultry with aiv vaccines; vaccinating susceptible equines with ei vaccines, especially farming equines in close contact with domestic poultry; and monitoring the prevalence of multiple aiv subtypes in equines, not merely that of those restricted to h3n8 subtype. none. we read with interest recent articles in this journal regarding the utility of next-generation sequencing for the diagnosis bacterial meningitis. 1 , 2 bacterial meningitis causes substantial morbidity and mortality worldwide. 3 rapid identification of the microorganisms is essential for early initiation of appropriate antimicrobial therapy, thereby improving clinical outcome. yet routine diagnostic methods fail to identify the bacteria in the majority of patients. over the last decade, advanced sequencing technologies have greatly improved our capacity to detect the causative agents of infectious diseases in clinical samples. 4 , 5 of these, the single molecule real-time sequencing developed by oxford nanopore technologies (ont) is a promising tool for diagnostic setting because of its short turnaround time. in late april 2019, a 59-year old seller of fish-noodles was referred to our hospital with a 1-day history of headache, fever and vomiting. he had a history of heavy alcohol use and hepatitis c infection, and had cirrhosis and diabetes mellitus. on admission, he was unconsciousness (a glasgow coma scale of 8), with a body temperature of 40 °c, a blood pressure of 140/80 mmhg and neck stiffness. initial gram-stain and microscopy of csf showed grampositive cocci, 8449 white cells/ul with 91% neutrophils, elevated protein and low glucose level, and high lactate concentration ( fig. 1 a) . routine bacterial culture, plus streptococcus pneumoniae and s. suis pcrs were all negative. he was diagnosed with bacterial meningitis, and given a combination of ceftriaxone (2 g/12 h) and dexamethasone (0.4 mg/kg/12 h). his clinical condition steadily improved. his second and third csf samples became negative by gram stain. the other csf parameters also improved, except the glucose, which remained low ( fig. 1 a) . on day 20 of hospitalization, the patient suddenly became unconsciousness with fever. brain magnetic resonance imaging showed bifrontal abscesses ( fig. 1 b) . after consulting a local neurosurgeon, aspiration of the brain abscesses was not advised and the patient was treated empirically with meropenem (2 g/8 h) and vancomycin (1 g/8 h). due to continued diagnostic uncertainty, we performed 16s rrna sequencing of the admission csf, stored as part of an going clinical study (supplementary materials), using an established sangersequencing based 16s rrna method. 6 subsequently, analysis of the obtained sequences revealed evidence of s. agalactiae (supplementary figure 1 ). given this new diagnostic result of the admission csf and because the patient had recovered clinically, the patient was given 24 million units of penicillin g for every 4 h. after day 43 of hospitalization, all csf parameters had normalised ( fig. 1 a) . likewise, on ct scan the brain abscess was now significantly improved ( fig. 1 c) . the patient was discharged with full clinical recovery. additionally, minion sequencing of complete 16s rrna gene was retrospectively carried out on the extracted nucleic acid of the admission csf yielded a total of 14,848 reads after 100 min of sequencing run. of these, 11,556 reads (79%) were successfully aligned to s. agalactiae ( fig. 1 d) . the remaining reads were assigned to other streptococcus species (mostly s. dysgalacticiae ( n = 2.145, 14%)), likely attributed to a combination of the high level of sequence similarities of the 16s rrna region between them and the sequencing errors introduced by the minion systems. analysis of sequencing data generated during the 25, 50 and 75 min of sequencing run time also yielded the same results (supplementary figure 2 ). details about the minion procedure are presented in supplementary materials. to further assess of the utility of csf minion sequencing of 16s rrna gene for the detection of bacterial meningitis pathogens, six csf samples from patients with confirmed bacterial meningitis enrolled in the abovementioned clinical study were tested ( table 1 ) . analysis of the minion reads obtained after two hours of the sequencing run showed that the majority of reads were correctly assigned to the corresponding bacterial species ( s. pneumoniae and s. suis) or genus ( neisseria ) found in the csf samples by diagnostic work up of the clinical study ( fig. 1 e and table 1 ). additional analysis of the obtained reads generated at two earlier time points (20 min and 60 min) of the sequencing run generated the same results ( table 1 ) . collectively, we report the first application of minion sequencing of 16s rrna gene to detect bacterial meningitis causing pathogens in csf samples from a low and middle-income country. the assay was able to detect the bacterial causes in all of the seven tested csf samples. meanwhile, gram stain and culture, the two most commonly used methods in clinical microbiology laboratories worldwide, were negative in 3/7 samples. ( fig. 1 and table 1 ). in addition to csf samples described in the present study and a recent pilot study from korea, 7 successful detections of haemophilus influenzae in sputum and campylobacter fetus in culture materials by minion sequencing of 16s rrna have recently been reported. 8 together, the data suggest that minion sequencing of 16s rrna is a sensitive method for rapid and accurate detection of pan-bacterial pathogens, including unexpected microorganisms, in clinical samples. additionally, the bacterial species information generated by the analysis of 16s rrna sequences can be useful for disease surveillance and vaccine evaluation. thus, the application of the method would be relevant for both patient management and epidemiological research. indeed, to the best of our knowledge the present study represents the first report of s. agalactiae associated meningitis in vietnam. because the incidence of invasive diseases (including meningitis) caused by s. agalactiae has been reported with increased frequency in recent years, 9 s. agalactiae should be considered as an important differential owing to the unavailability of the reagents at the time of patient admission, we were not able to perform real-time diagnosis using minion sequencing on the collected csf samples. however, same day diagnosis is theoretically achievable, because the current workflow takes 5 -6 h to operate. prospective study is urgently needed to assess its translational potential in the diagnosis of bacterial meningitis. since september 2017, a prospective observational study aiming at exploring the utility potential of next-generation sequencing in patients presenting with central nervous system (cns) infections has been conducted in the brain infection ward of the hospital for tropical diseases (htd) in ho chi minh city, vietnam. htd is a tertiary referral hospital for patients with infectious diseases from southern provinces of vietnam, serving a population of > 40 million. any patient ( ≥16 years) with an indication for lumbar puncture was eligible for enrolment. patient was excluded if no written informed consent was obtained. as per the study protocol, csf, plasma and urine samples were collected at presentation alongside demographic, meta-clinical data and results of routine diagnosis. after collection, all clinical specimens were stored at −80 °c until analysis. the clinical study received approvals from the institutional review board of the htd and the oxford tropical research ethics committee of the university of oxford. written informed consent was obtained from each study participant or relative (if the patient was unconsciousness). sequencing of complete 16s rrna gene was retrospectively performed using minion nanopore sequencer (ont), following the manufacturer's instructions. in brief, amplification of the complete 16s rrna gene and library preparation were carried out on extracted nucleic acid using 16s barcoding kit (sqk-rab204, ont) and primers (27f 5 -agagtttgatcctggctcag-3 and 1492r 5 -ggttaccttgttacgactt-3 ), followed by the sequencing of the amplified product using r9.4 flow cells (ont). minion reads were first basecalled using albacore v2.1.7 (ont), followed by demultiplexing using porechop ( https://github.com/rrwick/porechop ). determination of bacterial genus/species composition in the obtained reads was then carried out using epi2me interface (metrichor, oxford, uk), a platform for cloud-based analysis of minion data. overall, the whole procedure of minion sequencing of 16s rrna gene takes 5-6 h to complete (supplementary figure 3) . we, the author of the submitted manuscript declare that we do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding). dear editor, several aspects of influenza have been highlighted recently, including its global, comparative seasonality, 1 and issues around rapid point-of-care testing. 2 in addition, the uk has a national surveillance programme, the uk severe influenza surveillance system (usiss) to monitor and investigate severe cases of influenza across the country, 3 including severe cases of influenza admitted to intensive care (icu) and high dependency units (hdu). 4 specifically, the aim of this latter arm was to "monitor and estimate the impact of seasonal influenza on the population" and to "describe the epidemiology of severe disease." this surveillance began in the 2011-2012 influenza season and has continued to the present, with mandatory participation by all nhs trusts. leicester is one of 5 nhs commissioned centres in the uk providing extra-corporeal membrane oxygenation (ecmo) support for severe acute respiratory failure in adults. this process involves draining deoxygenated venous blood from the superior and inferior vena cavae, pumping this blood through a membrane lung, where oxygenation and carbon dioxide elimination take place. oxygenated blood is delivered back into the right atrium, therefore replacing the function of the native lung. in this way, ecmo can be used to support patients with respiratory failure of any cause. acceptance for admission for ecmo support follows referral to the ecmo service via structured questionnaire and discussion with the ecmo consultant on-call. patients are commenced on ecmo where benefits are deemed to outweigh the risks in patients with potentially reversible respiratory failure, who are already on maximal conventional therapy at their referring centre, and who are not achieving lung protective ventilation. 5 here, as part of our national usiss role, we describe severe influenza cases that required ecmo support in whom the predominant indications were severe hypoxia with a pao 2 :fio 2 ratio of < 100 despite maximal conventional therapy, and/or hypercapnoeic respiratory failure with a ph < 7.2 despite ventilation pressures > 30 cm h 2 o. during the 2018-2019 influenza season 33 cases of severe influenza were admitted to glenfield hospital for ecmo from our referring centres. most were male (23/33, 69.7%, 28-62 years, bmi: 17-47; female 10/33, 27-51 years, bmi: 21-38), and of white british ethnicity (30/33, 91.0%; with 1 each of chinese, asian, african ethnicity). comorbidities included, obesity, hypertension, asthma, copd, diabetes, anxiety, depression, epilepsy, a history of smoking and alcohol use (or abuse). all cases except for one influenza a(h3n2) infection (possibly two as subtyping was not performed for another sample) were due to influenza a(h1n1)pdm09. only one case had a history of influenza vaccination. various 'on referral' ecmo-related parameters were extracted, as well as contemporaneous laboratory results. these were statistically compared between patients who died ( n = 8) and those who survived using t -test or mann-whitney test for continuous variables and fisherexact test for categorical variables. correlation between duration on ecmo and laboratory parameters was assessed using spearman's rank correlation coefficient. ( n = 24) ( tables 1 and 2 ) . surprisingly, it was found that on direct comparison, most of the referral parameters for patients starting ecmo were not statistically different between those influenza-infected patients that eventually survived ( n = 24) versus those who died ( n = 8) -a case fatality rate of 25%. one patient was dropped from this analysis due to some missing data. only the respiratory rate (rr, p = 0.041) and the lactate ( p = 0.008) showed statistically significant differences between the two groups, with higher values being found in the patients who ecmo -extra-corporeal membrane oxygenation; sofa -sequential organ failure assessment; peep -positive end expiratory pressure; h 2 o -water; bpm -breaths per minute; pao 2 /paco 2 -partial pressure of arterial oxygen/carbon dioxide; altalanine aminotransferase. * rank correlation coefficient measures the strength and direction of a relationship between two variables. the coefficient ranges from −1 to 1 with 1 indicating a strong positive relationship between two variables and −1 indicating a strong negative relationship. a correlation coefficient close to 0 means the relationship between the two variables is very weak. died ( table 1 ) . however, clinically, these differences are of doubtful significance, as the rr is at the discretion of the parent clinical team prior to referral to ecmo, and the lactate levels are normal in the survivors and only marginally elevated in those who died which will again be dependent on use of cvvh. the higher pao 2 :fio 2 (p:f) ratio was statistically significantly correlated ( p = 0.020) with a shorter duration of ecmo, which suggests those with less severe disease recover quicker ( table 2 ) . many studies have reported on intensive care patient outcomes of the 2009 influenza a(h1n1)pdm09 pandemic, with or without the use of ecmo. in one study from australia, where ecmo was used, of 68 patients with confirmed influenza a (53 a(h1n1)pdm09, 8 un-subtyped) infection, 14 (21%) had died mainly due to intracranial and other forms of haemorrhage ( n = 10), or intractable respiratory failure ( n = 4). 6 another study from the usa, where influenza a(h1n1)pdm09-infected patients had non-ecmo icu admission, of 154 cases, 48 (37%) developed acute respiratory distress syndrome (ards), of whom 37 (24%) died. 7 a more recent study from spain that reviewed influenza a(h1n1)pdm09 cases admitted to icu (non-ecmo) from 2009-2015, found that of a total of 2421 cases, the mortality ranged from 18.8% (for community-acquired influenza) to 32.9% (for hospitalacquired influenza). 8 these ecmo-icu case fatality rates of severe influenza a(h1n1)pdm09 infection are similar to ours of 25% reported here, though compared to the specific ecmo patient cohort, 6 the causes of death in our patients were more variable, including intracranial and other haemorrhage ( n = 3), sepsis and multi-organ failure ( n = 2), respiratory failure ( n = 1), post-cardiopulmonary resuscitation hypoxic brain injury ( n = 1), and ischaemic bowel associated with atrial fibrillation ( n = 1). thus, even after 10 years of experience with this 'new' pandemic influenza a(h1n1)pdm09 virus, across almost the entire adult age-range ( ∼25-70 years in these previous studies), 6 -8 it appears that for severe cases, globally, the survival of such patients appears not to have improved. this may be somewhat surprising as the world's populations have become more immunologically experienced with this virus, which is now considered as a seasonal influenza virus that should be conferring some degree of persisting, cross-reactive individual and herd immunity over consecutive seasons. 9 this may be due to some predisposing genetic or environmental factors in individual patients, which should reinforce the general message that seasonal influenza immunisation is still recommended to minimise the number of people needing icu or ecmo support for severe influenza infection. more detailed monitoring on how these physiological parameters change over time (perhaps including more complex cytokine studies), in these severely ill, influenza a(h1n1)pdm09-infected patients admitted to icu-ecmo units, may eventually yield data to improve their management and clinical outcomes. none. we read with interest the report by stalenhoef and colleagues in this journal who show that biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection (ref). 1 here we report on a prospective observational analysis of the biomarker: cd5 antigen like protein (cd5l), in serum samples collected from 64 patients diagnosed with pneumonia 2 and 44 healthy adults between 2018 and 2019. the demographic and clinical characteristics of the 64 adults (males 70.3%; mean age 66 ± 20 years) with pneumonia were summarized in table 1 . 57 (89.1%) bacterial pneumonia patients (including 34 patients with confirmed bacterial pneumonia 2 and 23 patients with suspected bacterial pneumonia 3 ) and 7(10.9%) viral pneumonia patients were studied. there were significant differences in age, sex, wbc, neu%, crp, pct, cd5l, apache ii scores and length of icu stay between bacterial pneumonia and viral pneumonia. the pathogens responsible for pneumonia were described in supplementary table 1 . globally, gram-negative bacteria infection is more common than gram-positive bacteria infection in pneumonia. among them, acinetobacter baumannii (16, 59.3%) was considered the major pathogen in gram-negative bacteria and staphylococcus aureus (3, 42.9%) was considered the major pathogen in gram-positive bacteria. in viral pneumonia, influenza a (h1n1) was detected as the unique pathogen. in the study of the diagnostic performance of serum cd5l to identify etiology of pneumonia, as we found in supplementary fig. 1 , no matter in total bacterial pneumonia, suspected bacterial pneumonia or confirmed bacterial pneumonia, the serum of cd5l levels on day of pneumonia diagnosis were significantly higher than those in viral pneumonia and healthy control subjects. for evaluating the diagnostic performance of cd5l to differentiate bacterial from viral infection in pneumonia, roc analysis was conducted for the above bacterial pneumonia (supplementary fig. 2 ) and compared with routine laboratory markers ( table 2 ). by horizontal comparison, we found that the best auc was for cd5l (auc = 0.89), better than neu% (auc = 0.79), wbc (auc = 0.79), crp (auc = 0.78) and even pct (auc = 0.85). interestingly, by longitudinal comparison, the auc was observed highest in confirmed bacterial pneumonia (auc = 0.92), intermediate in all bacterial pneumonia (auc = 0.89), and lowest in suspected bacterial pneumonia (auc = 0.84), which may better elucidate the diagnostic performance of cd5l for etiology diagnosis in pneumonia patients. although there are 23 patients with suspected bacterial pneumonia for which no definitive pathogen was found, our result here may still provide a new treatment for bacterial infection identification in pneumonia patients for the current limitations in direct pathogen testing make it difficult to identify the pathogen at the time of diagnosis 4 . besides, as a potential biomarker to distinguish pathogens 5-8 , cd5l levels were also significantly correlated with wbc, neu%, crp and pct ( supplementary fig. 3 ) in patients with pneumonia. in the study of the diagnostic performance of serum cd5l to predict mortality in adults with pneumonia, mortality was defined as death occurring within 30 days after the onset of pneumonia. as we observed in supplementary fig. 4a , serum cd5l levels on day of pneumonia diagnosis were significantly higher in non-survivors ( n = 18) than survivors ( n = 46) ( p < 0.001). to ensure that serum cd5l levels were not influenced by the class of the infection in the specimens, spearman's rank correlation analysis was performed between serum cd5l levels and poor prognosis related scores 9 , 10 (sofa and apache ii scores) in patients with total bacterial pneumonia, suspected bacterial pneumonia, confirmed bacterial pneumonia and viral pneumonia, respectively. our correlation analysis between serum cd5l levels and sofa or apache ii scores shows that no matter for bacterial pneumonia or viral pneumonia, the cd5l levels showed positive correlation with sofa and apache ii scores ( supplementary figs. 5 and 6) . furthermore, the auc of cd5l for identifying 30-day mortality in adult pneumonia patients was 0.79 ( supplementary fig. 4b) , a value means good diagnostic performance, which is consistent with gao's study 9 before. therefore, we found that determining serum cd5l concentrations on day of pneumonia diagnosis was of great value in identifying bacterial infection from viral infection and predicting 30day mortality in adult patients with pneumonia, which suggests that cd5l may work for the etiologic diagnosis and could represent a novel biomarker for identification of a group of patients with pneumonia presenting with higher risk of death. these findings encourage further effort s aimed at exploring the clinical value of circulating cd5l to help early clinical decision-making in human pneumonia. none. high morbidity and mortality (up to 100%). asf is a devastating threat to pig agriculture and is responsible for serious production and economic losses. the asfv genome is 170-193 kilo base pairs in length 2 and has been divided into 24 different genotypes based on their b646l gene sequence, a gene which encodes the capsid protein p72. 3 , 4 our previous study showed that the p72 gene located in a very low genetic diversity region of the genome. 5 ye and colleagues, however, identified two novel genotypes xxv and xxvi, with genotype xxvi being especially divergent from all other genotypes. 1 to examine this unexpected result, in this study, we reanalyzed their data to evaluate the reliability of these two genotypes. we collected 716 available p72 gene sequences from ncbi ( http://www.ncbi.nlm.nih.gov/ ), which we then aligned with mafft software, a fast multiple sequence alignment program. 6 the alignments show that genotype xxv (accession number fr668409) has a single amino acid change at position 506 compared to genotype i, while for genotype xxvi (accession numbers fr668404 and fr668418), the region coding for amino acid residues 509 to 528 differs greatly compared to all other genotypes ( fig. 1 a) . to examine this in greater detail, we calculated genetic distance across the p72 coding sequence in 60 bp sliding windows, with a step size of 10 bp, between these two sequences and the 24 other genotypes ( fig. 1 b) . this sliding window analysis shows these two sequences for genotype xxvi have a region that is very divergent, with genetic distance up to 0.5067 from the other genotypes, while the genetic distances of the remaining regions are less than 0.1 ( fig. 1 b) . genetic distances for asfvs were calculated for 182 coding gene sequences, from 42 available complete genomes of asfvs, to assess the overall divergence. pairwise genetic distances of each gene for all asfv strains were calculated. the mean pairwise genetic distance for asfv genes was 0.041 ( fig. 2 ) , which is much lower than this divergent region of these two sequences for genotype xxvi (0.5067). therefore, it seems highly unlikely that a gene has such a highly divergent region. since recombination frequently occurs in asfvs, 5 we conducted a recombination analysis with the rdp4 program, which used the 3seq, bootscan, chimaera, genecov, lard, maxchi, rdp and siscan detection methods, 7 to determine whether recombination might have occurred in the xxvi genotype. we found reliable evidence for recombination events in both p72 genotype xxvi sequences ( fig. 1 c) . this suggests that the very highly divergent region of this p72 genotype is not homologous with other p72 genotypes. we used blastn, from ncbi ( https://blast.ncbi.nlm.nih.gov/blast.cgi ), to identify a source for this highly divergent region, however, no similarity sequence was found. we then mapped the multiple amino acid changes found in genotype xxvi to the 3d structure of p72 ( fig. 3 ) . the changed sites (marked in red in fig. 3 ) are located in the dec loop, a region which plays an important role in the formation of the trimer spike. 8 the amino acid mutations result in changes of electrostatic potential, hydrophobicity, and steric hindrance. it seems unlikely to have so many changes in such a functionally important region. in our previous studies we noticed that sequences directly submitted by individual laboratories to genbank often contain errors such as misidentification of species, sampling error, contamination, or are pseudogenes, which can lead to sequence analysis problems and erroneous conclusions. 9 , 10 sequences that deviate from the overall intraspecific pairwise divergence are potentially erroneous. 9 , 10 since genotype xxvi deviates from other genotypes not only in genetic distance, but also in protein structure, and mutations occur in the flanking regions of the sequences, we deduced that these reported mutations might be due to low quality sequencing. we identified the original manuscript reporting the three sequences of genotypes xxv and xxvi, 11 where the authors stated that the "alignment and translation of sequences obtained from sardinian isolates revealed that the c-terminal end of p72 gene was completely conserved between the sequences compared". this statement indicates that these sequences did not considerably diverge from previously reported asfv p72 sequences, and thus, suggests that an error in these sequences had been introduced upon submitting them to genbank. further examination of the original samples and sequences is needed. we contacted the corresponding author of this manuscript to check these three sequences, and were told that these three p72 sequences were all genotype i, and had some errors when submitted to genbank. in conclusion, the two novel genotypes of asfvs (xxv and xxvi) identified by ye and colleagues 1 are misled by problematic sequences. as reminded by our previous studies, many problematic sequences are present in genbank, which can lead to problems in downstream analyses. 9 , 10 thus, when published data is used for new analyses, the first set in the process of data analyses should be to filter these sequences for potential errors to reduce the possibility of reaching incorrect conclusions. if conclusions are based on obviously strange sequences, then we should trace back the data to their original source, and manuscript, and contact the corresponding authors before making conclusions based on these sequences. the authors declare no conflict of interest. fig. 1 . the analyses of phylogenetic tree, recombinant, and nucleotide divergence between 6xi and the other known 6 subtypes (6a-6xh) based on full-length hbv genome sequences. (a) the known hcv subtype 6 reference sequences (6a-6xh) from the previous report were used. phylogenetic analysis was performed by the maximum-likelihood method, based on the gtr + g + i substitution model, with 10 0 0 bootstrap replicates using the software mega v6. the sequences of hcv 6xi (ynkh261 and ynkh284) are marked in red dot. (b) bootscan plots were constructed using simplot 3.5.1 software based on 100 replicates with a 300-bp sliding window moving in steps of 50 bases. (c) pairwise comparisons of nucleotides similarities between 3 hcv 6xi strains and 30 reference genotype 6 sequences. 399,0 0 0 deaths per year. 2 currently, daa treatment regimens that target ns3/ns4a protease, ns5a phosphor-protein and the ns5b polymerase have shown high safe and high rates of sustained virologic response in hcv chronically infected patients ( > 90%). 3 however, under selective pressure from these drugs, drug resistance-associated substitutions (ras) can emerge during this therapy and result in treatment failure in 2 −10% of patients. 4 therefore, hcv infection is still a major global health concern. to date, eight confirmed genotypes have been characterized based on > 30% sequence divergence in the complete hcv genome, and genotypes are further classified into > 80 subtypes with a sequence divergence of > 15% to other subtypes of the same geno-type. 5 in the current study, we characterized a new hcv subtypes among chronic hepatitis c patients in yunnan, china, initially designated as 6xi, further analyzed its evolutionary history and investigated its baseline ras by next generation sequencing (ngs) method. plasma samples were collected between january 2018 and october 2018 from 160 chronic hepatitis c patients from kunming city in yunnan, china (fig. s1a) . the samples met the following inclusion criteria: (1) hepatitis c antibody-positive for 6 months with normal serum alanine aminotransferase (alt) levels; (2) subject was residing in yunnan province and was over 18 years old; (3) complete demographic information and clinical data were available; (4) consented to the use of patient information in studies on hcv epidemics; and (5) were treatment-naïve during sampling. there was no epidemiologic link among these individuals. the study was approved by the first people's hospital of yunnan province ethics committee. written informed consent was obtained from all participants prior to the study. out of a total of 160 chronic hepatitis c patients, 152 partial ns5b gene fragments were successfully amplified and sequenced with a success rate of 95.0% (152/160). multiple subtypes were identified in 152 subjects, including subtype 3b (36.2%, 55/152), 3a (23.7%, 36/152), 1b (19.1%, 29/152), 2a (8.5%, 13/152), 6n (7.9%, 12/152), 6a (2.0%, 3/152), and 1a (1.3%, 2/152) (fig. s1b) . interestingly, the remaining two strains (1.3%, 2/152) involving ynkh261 and ynkh284 together with the isolate km35 reported formed a novel separate cluster in the genotype 6 with an 82% bootstrap value, indicating a potential new hcv subtype 6. to confirm that the two strains belong to a novel hcv subtype 6, their complete genome sequences were successfully amplified and sequenced with 12 overlapping fragments. further, phylogenetic analysis was performed along with hcv reference sequences of representative subtypes 6a-6xh. the result showed that the two strains and isolate km35 formed a distinct monophyletic cluster supported by a high bootstrap value of 100%. the three strains were isolated from three hiv-1 infected patients without obvious epidemiological linkage in yunnan and showed no evidence of recombination using bootscan analysis ( fig. 1 (b) ). moreover, the intergroup nucleotide divergence (mean ± sd) % over the fulllength genome sequences of the isolates (ynkh261, ynkh284, and km35) were compared to that of representative subtypes (6a-6xh) ( fig. 1 (c) ). the results revealed that the three strains were different from known hcv subtypes of 6a-6xh by 16.5-28.5%. therefore, the three strains are initially designated 6xi. to better understand the time of emergence of hcv 6xi, we performed bayesian molecular clock analyses using full-length genome sequences to estimate the time to the most recent common ancestor (tmrca). as shown in fig. 2 (a) , the estimated tmr-cas for the genotype 6xi was 1971.1 [95% highest probability density (hpd): 1951. 1, 1991.4 ]. in addition, to further investigate baseline ras of subtype 6xi, naturally occurring resistance-associated substitutions (ras) were analyzed for the ns3, ns5a and ns5b sequences using next generation sequencing (ngs) method. strikingly, hcv 6xi strains contain the substitution 28 v with a 100% frequency of mutations in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor, 6 suggesting that the subtype 6xi maybe basically resistant to ns5a inhibitors ( fig. 2 (b) ). among the hcv eight genotypes, genotype 6 exhibits a high degree of genetic complexity and diversity, and 31 subtypes have been confirmed by the international committee on taxonomy of viruses. 7 in china, hcv genotype 6 is common, and subtype 6a is the most prevalent subtype, primarily distributed in guangdong, 6n is the second most prevalent subtype, mainly found in yunnan, followed by subtypes 6xa, 6 g, 6v, 6 w, 6e, 6b, 6j, 6q,and 6r among genotype 6 isolates. 8 -10 to our knowledge, 6xi is the eighth detection of novel hcv subtypes 6 in china combined with previously identified 6a, 6e, 6n, 6v, 6xa, 6xe and 6xh, resulting in genotype 6 to expand to 32 subtypes in the world. our findings again demonstrated that hcv genotype 6 was more complex and diverse. in summary, we characterized a new hcv subtype 6xi based on the characteristics of a monophyletic cluster, > 15% genetic distances, no significant evidence of recombination, and no epidemiologic link among individuals. in addition, bayesian analyses showed that 6xi may originate around the year 1971, and the strains of hcv 6xi naturally contain the substitution 28 v in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor. the present finding again highlights the genetic characteristics and hcv strains in yunnan, and the urgent need for continuous molecular screening and epidemic surveillance in yunnan to implement effective measures to reduce hcv transmission. the authors declare no competing financial interests. we recently read the article by corma-gómez a. et al. on which the authors described a higher probability of relapses with sofosbuvir/ledipasvir 8 weeks compared with 12 weeks of hcv (hepatitis c virus) among hiv (human immunodeficiency virus)/hcv coinfected patients. 1 in this regard, coinfections of hiv/ hcv also with hepatitis b virus (hbv) is associated with high mortality and comorbidity too. the persistence of hbv dna within the core cell in the absence of hbsag and even after clearance of the infection has been described previously in immunosuppressed patients, where hbv screening and prophylaxis is recommended. 2 interestingly, hbv reactivation has recently emerged during or after hcv treatment with direct-acting antivirals (daa). [3] [4] [5] the us food and drug administration issued a warning about this risk in 2016, 6 until that moment 24 cases were reported, two of which died. although specific mechanisms of this event are not well known, it has been suggested that hcv core proteins could inhibit hbv replication and hbsag production as well as production of envelope proteins, being hbcab the only marker of the presence of hbv. 2 in this context, treatment with daa would produce a drastic and rapidly blocking of hcv replication providing the opportunity to hbv to emerge and produce an immune reconstitution syndrome. the interference hbv-hcv has been outlined in 24% of patients with chronic hbv infection (hbsag-positive serology) and 1.4% of patients with resolved hbv infections (hbsag-negative and hbcabpositive) in a recently published systematic review and metaanalysis of patients with hcv-hbv. 7 in most cases, these reports have focused on coinfection hcv-hbv, but there is still a concern about patients triple-infected with hcv, hbv and hiv. although most cases reported occur in hbsag-positive, the main concern affects to patients with a basal serology showing hbcab-positive, hbsag-negative and hbsab-negative. hbsagpositive was associated with higher rates of hbv reactivations compared with hbsag-negative and hbcab-positive patients. 8 there is no clear information about this issue in hbsag-negative, hbcab-positive, and, when occurs, it is considered an uncommon event. 9 reactivation of hbv is characterized by reappearance or increase in hbv dna levels, and could also be accompanied by symptoms of hepatitis. the cases reported previously did show difference in the time of presentation of clinical symptoms, they used to start either during or after daa treatment. it seems that hbv reactivation usually occurs early after daa initiation treatment (4-8 weeks) while otherwise can occur after treatment completion. in that way, it seems necessary that hbv infection should be monitored early after daa initiation. the most recent european association for the study of the liver (easl) guideline about hbv infection, recommends that patients hbsag-negative and hbcab-positive undergoing daa treatment should be monitored and tested for hbv reactivation only in case of alt elevation. 10 they also recommend performing hbv dna levels only in case of alt increase. at the same time, the american association for the study of liver diseases (aasld) and the infectious disease society of america (idsa) guideline has been recently updated recommendations to monitor hbv dna levels only in patients hbsag-positive, but they emphasize that there is not enough data to monitoring dna among patients hbcabpositive or hbcab-positive and hbsab-positive. they remark that a reactivation should be considered if an unexplained increase in liver enzyme is present. in hiv patients, they provide the same recommendations. 11 in a recent review of the literature, the authors also recommend to perform an hbv dna only in patients with altered alt. 9 a meta-analysis recommended not to perform an hbv dna test in this case due to low rate of incidence and the associated cost which needs to be considered especially in an endemic hbv areas. 7 few data exist in hiv patients. the fact that many triple-infected patients are receiving antiretroviral therapy including (art) nucleoside/nucleotide analogous, as tenofovir disoproxil fumarate (tdf) or tenofovir alafenamide (taf), both active against hbv, suggest that hbv reactivation rate could be underestimated. in a recently published review, chang et al., recommend to perform an hbv dna test at baseline in triple-infected patients. if positive, an hbv-active antiretroviral therapy (art) should be started and hbv dna needs to be monitored every 4 weeks during treatment and until week 12 after completion of treatment. however, if baseline hbv dna is negative they match with aasld/idsa and easl recommendations. 12 one of our patients has a basal serology with hbcab-iggpositive, both hbsag-and hbsab-negatives, and undetectable hbv dna levels, prior to treatment with daas. regarding hiv infection, he has an analogues-free regimen. one month after having finished treatment with daas, the patient consulted because of abdominal pain, nausea and jaundice. he had a total bilirubin level of 11 mg/dl, ast 1025 iu/l, alt 642 iu/l and inr 1.30. a hbv viral load of 6,193,455 iu/ml was detected; hbsag, hbcab-igm and hbeag were positive. there were no reasons to believe that he was re-infected. treatment with entecavir was initiated; however, the clinical evolution was unfavorable and died as a result of an acute liver failure. the hbv viral load was requested from the stored samples drawn during the treatment of hcv showing that hbv viral load was undetectable at the beginning of treatment and in week 2, but progressively increased to 98 iu/ml in week 8 and up to 82,700 iu/ml in week 4 after treatment completion. during the follow-up of daa treatment alt and ast remained normal. this case changed our daily practice, since then all hiv patients and more specifically those not treated with either tdf or taf and presenting a previous hbcab-positive, are followed using dna levels and not only monitoring hepatic enzymes, as recommended by guidelines. we consider that this case may reflect the necessity of change the current guidelines. we recommend to perform a periodic monitoring of hbv reactivation using hbv dna during and after daa therapy in hbsag-negative and hbcab-positive patients, independently of hbsab presence, hepatic enzymes and clinical symptoms, particularly in hiv patients who are not receiving active treatment of hbv. the study was not funded. authors declare that there is no conflict of interest. recently, a notable pattern of synchrony of influenza a and b virus, and respiratory syncytial virus incidence peaks globally was reported in this journal. 1 a previous study characterized seasonal pattern of influenza a and b in china and identified three epidemiological regions featured by distinct seasonality. 2 on the basis of laboratory surveillance data from 30 chinese provinces spanning about 12 years from october 2004 through january 2017, our study further characterized seasonal patterns of circulating influenza a subtypes and influenza b lineages in the three defined epidemiological regions. our study revealed that pre-2009 a(h1n1) and a(h3n2) displayed wintertime and summertime epidemics in midlatitude and southernmost chinese provinces with subtropical climate, wavelet analysis demonstrated the two subtypes displayed twice-annual cycle in some years in mid-latitude chinese provinces ( fig. 1 (a)-(h) ). however, the two subtypes peaks in the winter with annual or longer cycle in northern chinese provinces. influenza a(h1n1)pdm09 b/victoria and b/yamagata virus all displayed epidemics in the winter or winter-spring with annual or longer cycle in all three epidemiological regions. we developed univariate and multivariate regression models to evaluate the association between climatic factors and the presence or absence of epidemics of each influenza subtype and lineage (positive proportion ≥5%) in the southernmost provinces where heating system is not generally used in the winter so temperature and relative humidity in external conditions in winter are close to those in indoor environment where people spend most of the time. we fitted the mixed-effects logistic regression model to control for the repeated measurements in each province of the region cluster. we kept one of temperature and absolute humidity (representative of vapor pressure) with smaller akaike information criterion in the model to reduce of multi-collinearity due to a high degree of correlation between the two factors. our analysis indicated temperature, humidity and rainfall were environmental predictors of influenza subtype/lineage-specific epidemics in the southernmost provinces when a 1-week lag of influenza epidemics behind climate was considered, which was similar to the findings from some ecological studies ( table 1 ). 3 our study indicated the u-shaped relationship between absolute humidity (ah) and pre-2009 a(h1n1) or a(h3n2) epidemics in the southernmost provinces, and suggest that high levels of ah in the summer, and low levels of ah in the winter increased the possibility of epidemics of the two subtypes. 3 in our analysis, lower temperature was an environmental driver of a (h1n1)pdm09 and b/yamagata epidemics in the southernmost provinces while there were bimodal associations between temperature, rainfall and b/victoria epidemics with highest probability of b/victoria epidemics at 11.7 °c of daily average temperature and 11.3 mm of daily average rainfall. although seasonal changes in human behaviors, such as school attendance or crowding indoors, and seasonal variations in immunity, such as melatonin and vitamin d levels have been proposed to account for the seasonal nature of influenza, 4 our findings suggest that the heterogeneity in influenza subtype/lineage-specific seasonality patterns could be driven by seasonal variations in virus survival, transmission and adaptive immunity by influenza subtype and lineage because of the same behavior modes and background of non-adaptive immunity in the same regions and seasons. we propose a hypothesis: under humid and hot condition the dominant transmission mode(s) for a(h1n1)pdm09, b/victoria and b/yamagata might have reduced efficiency, however, there could be effective transmission mode(s) for a(h3n2) and pre-2009 a(h1n1) virus. some experimental studies were performed to establishing a causal link between humidity, temperature and influenza virus survival/transmission. 5 transmission of influenza a (h3n2), a(h1n1)pdm09, b/victoria and b/yamagata virus by respiratory droplets or aerosols in the guinea pig model proceeds most readily under cold, dry conditions. 5 low humidity and temperature increased the stability of influenza virus in aerosols and on surfaces. furthermore, aerosol transmission of a (h3n2) virus in the guinea pig model was almost completely blocked at 30 °c, but contact transmission of a (h3n2) virus seemed to be efficient at different level of humidity and 30 °c. 5 it remains unclear if high temperature and humidity levels have effect on aerosol and contact transmission of pre-2009 a(h1n1), a(h1n1)pdm09, b/victoria and b/yamagata virus among hosts and their stability on surfaces. of note, an experimental study found the survival durations of a(h3n2) strains on swiss banknotes were significantly longer than pre-2009 a(h1n1) and b/victoria virus. 6 further studies are needed to understand how efficient are these transmission modes for different influenza subtypes or lineages, which is/are dominant mode(s) of transmission among hosts, and which of potential mechanisms is at play under humid and hot condition. one of our findings was the mutual inverse association between a(h3n2) and a(h1n1)pdm09 epidemics, which provided the evidence on interference between the two influenza a subtypes perhaps possibly due to multiple immune mechanisms. 7 however, our study showed b/yamagata epidemics were positively correlated with a(h3n2) epidemics, suggesting b/yamagata epidemics across over 12 study years that were weak could get well along with simultaneous weak h3n2 epidemics. understanding of influenza seasonality is important to define optimal timing of influenza vaccination campaigns. our study indicated that a(h3n2) virus brought about twice-annual epidemics in some years in mid-latitude chinese provinces and more frequent summertime epidemics in southernmost chinese provinces, which questions if a single annual influenza vaccination campaign starting in october can offer optimal protection against summertime epidemics of a (h3n2) virus in mid-latitude and southernmost chinese provinces. in recent years, it has been reported that mismatches of a(h3n2) virus between the influenza vaccine strains and circulating strains were identified frequently, 8 and vaccine effectiveness of a(h3n2) virus declined within 4-6 months postvaccination. 9 in conclusion, we identified the heterogeneity of seasonality pattern of pre-2009 a(h1n1) or a(h3n2) virus in three epidemiological regions of china, and different environment predictors for influenza subtypes and lineages in the southernmost provinces. further work should focus on understanding difference in virus survival, transmission by influenza subtype and lineage under humid and hot conditions. bjc has received research funding from medimmune inc. and sanofi pasteur, and consults for crucell nv. the authors report no other potential competing interests. as this study included data from the national influenza surveillance system, ethics approval was not required. not applicable. the datasets at national level analyzed during the current study are available in the world health organization flunet. the datasets with more specific information analyzed during the current study are available in chinese national influenza surveillance informatio system, but they are not open-access datasets. these influenza surveillance data can be available from chinese national influenza center on reasonable request. this study was supported by the national mega-projects for infectious diseases (grant number 2018zx102010 02-0 08-001 ), national natural science foundation of china (grant number 81302476 ) and emergency prevention and control project of ministry of science and technology (grant number 10600100000015001206 ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. reported in zhejiang in 2017. 8 in this study, we identified ten cases of imported chikv infection in travelers returning to yunnan from southeastern asia in 2019. out of the ten patients with imported chikv infection examined in this study, nine patients had traveled back from myanmar and one patient had travelled back from thailand ( fig. 1 (a) ). all the patients displayed different degrees of symptoms, such as fever, cough, muscle pain, and rash. the details of all the symptoms are shown in table 1 . chikv infection was diagnosed using specific real-time reverse transcription-pcr. serum specimens were collected from the ten patients that tested positive for chikv by real-time pcr analysis, in yunnan between may 7, 2019 and august 1, 2019. the current study was approved by the medical ethics committee of kunming university of science and technology. written informed consent was obtained from all the participants. a new coronavirus associated with human respiratory disease in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin emergence of sars-like coronavirus poses new challenge in china bat origin of a new human coronavirus: there and back again transmission of 2019-ncov infection from an asymptomatic contact in germany early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia what to do next to control the 2019-ncov epidemic? nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) national health commission of people's republic of china prevention and control of novel coronavirus pneumonia a hospital cluster combined with a family cluster of avian influenza h7n9 infection in anhui province first human infection by a novel avian influenza a(h7n4) virus contact reductions from live poultry market closures limit the epidemic of human infections with h7n9 influenza comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds death of a very young child infected with influenza a (h5n6) equine influenza-a global perspective isolation and characterization of highly pathogenic avian influenza virus subtype h5n1 from donkeys molecular epidemiology of a novel re-assorted epidemic strain of equine influenza virus in pakistan in 2015-16 avian-origin h3n2 canine influenza virus circulating in farmed dogs in guangdong detection of pediatric bacterial meningitis pathogens from cerebrospinal fluid by next-generation sequencing technology next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center community-acquired bacterial meningitis rapid bacterial identification by direct pcr amplification of 16s rrna genes using the minion tm nanopore sequencer campylobacter fetus meningitis confirmed by a 16s rrna gene analysis using the minion nanopore sequencer bacterial diversity assessment in antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation rapid diagnosis of bacterial meningitis by nanopore 16s amplicon sequencing: a pilot study diagnosis of haemophilus influenzae pneumonia by nanopore 16s amplicon sequencing of sputum epidemiology of invasive group b streptococcal infections among nonpregnant adults in the united states comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses impact of a poorly performing point-ofcare test during the 2017-2018 influenza season uk severe flu surveillance system icu-hdu influenza surveillance nhse) service specification 170110s. extra corporeal membrane oxygenation (ecmo) for respiratory failure in adults australia and new zealand extracorporeal membrane oxygenation (anz ecmo) influenza investigators extracorporeal membrane oxygenation for 2009 influenza a(h1n1) acute respiratory distress syndrome intensive care unit patients with 2009 pandemic influenza a (h1n1pdm09) virus infection -united states characteristics of patients with hospital-acquired influenza a (h1n1)pdm09 virus admitted to the intensive care unit cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection guidelines for the management of adults with community-acquired pneumonia. diagnosis, assessment of severity, antimicrobial therapy, and prevention cytokine markers as predictors of type of respiratory infection in patients during the influenza season procalcitonin as a marker of etiology in adults hospitalized with community-acquired pneumonia cd5l contributes to the pathogenesis of methicillin-resistant staphylococcus aureus-induced pneumonia orchestrating role of apoptosis inhibitor of macrophage in the resolution of acute lung injury aim inhibits apoptosis of t cells and nkt cells in corynebacterium-induced granuloma formation in mice the macrophage soluble receptor aim/api6/cd5l displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression assessment of apoptosis inhibitor of macrophage/cd5l as a biomarker to predict mortality in the critically ill with sepsis plasma long pentraxin 3 (ptx3) concentration is a novel marker of disease activity in patients with community-acquired pneumonia the updated analysis of african swine fever virus genomes: two novel genotypes are identified african swine fever virus replication and genomics phylogeographic patterns of the african swine fever virus genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype the recombination hot spots and genetic diversity of the genomes of african swine fever viruses parallelization of the mafft multiple sequence alignment program rdp4: detection and analysis of recombination patterns in virus genomes structure of the african swine fever virus major capsid protein p72 detection of potential problematic cytb gene sequences of fishes in genbank assessing dna barcoding as a tool for species identification and data quality control genetic characterisation of african swine fever viruses from recent and historical outbreaks in sardinia consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group hepatitis c virus infection in children and adolescents challenges and perspectives of direct antivirals for the treatment of hepatitis c virus infection whole-genome characterization and resistance-associated substitutions in a new hcv genotype 1 subtype identification of 19 novel hepatitis c virus subtypes-further expanding hcv classification hepatitis c virus drug resistance associated substitutions and their clinical relevance: update identification of a new hcv subtype 6xg among injection drug users in kachin hepatitis c virus genotypes and subtypes circulating in mainland china higher relapse rate among hiv/hcv-confected patients receiving sofosbuvir/ledipasvir for 8 vs 12 weeks significance of anti-hbc alone serological status in clinical practice evaluation of hepatitis b reactivation among 62,920 veterans treated with oral hepatitis c antivirals acute fulminant hepatitis b during hepatitis c virus therapy with direct-acting antivirals in a patient co-infected with hiv spontaneous remission of hepatitis b virus reactivation during direct-acting antiviral agent-based therapy for chronic hepatitis c adverse events reporting system (faers): the public's stake in adverse event reporting hepatitis b virus reactivation during direct-acting antiviral therapy for hepatitis c: a systematic review and meta-analysis reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals controversies in hepatitis c therapy: reactivation of hepatitis b virus clinical practice guidelines on the management of hepatitis b virus infection monitoring patients who are starting hcv treatment, are on treatment, or have completed therapy. hcv guidance: recommendations for testing, managing, and treating hepatitis c. electronic access significance and management of isolated hepatitis b core antibody (anti-hbc) in hiv and hcv: strategies in the daa era comparative global epidemiology of inßuenza, respiratory syncytial and parainßuenza viruses characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data environmental predictors of seasonal influenza epidemics across temperate and tropical climates global influenza seasonality: reconciling patterns across temperate and tropical regions roles of humidity and temperature in shaping influenza seasonality survival of influenza virus on banknotes a systematic approach to virus-virus interactions comparing influenza vaccine efficacy against mismatched and matched strains a systematic review and meta-analysis i-move multicentre case-control study 2010/11 to 2014/15: is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination? metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers chikungunya virus infections in military deployments in tropical settings-a narrative minireview chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen east/central/south african genotype in a chikungunya outbreak caribbean and la réunion chikungunya virus isolates differ in their capacity to induce proinflammatory th1 and nk cell responses and acute joint pathology new insights into chikungunya virus emergence and spread from southeast asia mosquito-associated viruses in china chikungunya fever outbreak enhanced molecular surveillance of chikungunya virus chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes this work was supported by henan emergency project for prevention and control of 2019 novel coronavirus, the earmarked fund for modern agro-industry technology research system of china (cars-35) and the special fund for henan agriculture research system (s2012-06). the funders had no role in study de-sign, data collection and interpretation, or the decision to submit the work for publication. we thank jing li and hao-nan wang for the helpful discusses and suggestions. this work is supported by the open research fund of key laboratory of digital earth science ( 2019lde005 ), and by the fundamental research funds for the central universities under grant no. 310201911qd054 . this work was supported by the national natural science foundation of china ( 31702271 ) and the guangdong provincial natural science foundation ( 2017a030310367 ). we thank le kim thanh, le nguyen truc nhu, and lam anh nguyet for their logistic support. we are indebted to patients for their participations in this study.this study was funded by the wellcome trust of great britain ( 10 6 680/b/14/z and 204904/z/16/z ). supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.011 . we would like to thank the patients and healthy volunteers in the first affiliated hospital of chongqing medical university for their cooperation and support. this work has been financially supported by national natural science foundation of china (no. 81902134 and no. 81722001 ). we thank chinese national influenza surveillance network for contribution in influenza epidemiological and laboratory surveillance. we thank the members of the yunnan international travel healthcare center and kunming changshui airport customs for the data and sample collection. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.003 . supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.11.019 . recent correspondence in this journal has highlighted the current threat posed by recently-emerging imported chikungunya virus (chikv) in febrile returning travellers. 1 chikungunya fever is infection caused by the chikv and is characterized by fever, arthralgia, myalgia, headache, and rash. chikv belongs to the genus alphavirus within the togaviridae family and is transmitted to humans by the bite of infected mosquitoes-ae. aegypti and ae. albopictus . 2 since the first report of chikv infection in humans in tanzania, intermittent outbreaks have been documented in africa, south america, southern and southeastern asia, and the indian ocean islands; thus, its outbreak has become a global public health problem. 3 to date, three evolutionary distinct chikv genotypes, namely west african (wa), east/central/south african (ecsa), and asian have been identified, based on phylogenetic analyses. 4 a novel lineage of chikv-the indian ocean lineage (iol)-has also been reported, which descended from the ecsa genotype during an outbreak on the island of la reunion between 2005 and 2006. 5 recently, it has been reported that iol of chikv has spread to malaysia, singapore, thailand, and indonesia. 6 in china, the first case of imported chikv infection was found in yunnan in 1987. subsequently, several sporadic cases of nonindigenous chikv infection have been described. in 2010, the first outbreak of chikv fever with 129 cases was documented in guangdong. 7 recently, another outbreak of chikungunya fever was table 1 epidemiological information on ten febrile returning travelers infected with chikv in this study. in this study, nine complete genome sequences isolated from serum samples were successfully amplified and sequenced with 12 overlapping fragments, and then the sequence obtained was deposited in genbank under accession no. mn402883-mn402892. compared with the nucleotide sequences of the available from the ncbi database, the nine strains shared the highest 99.61-99.74% nucleotide identity with the east/central/south african lineage strain thail 2019 reported previously in thailand, 2019. further, bayesian maximum-clade-credibility tree for full-length nucleotide sequences were constructed using the beast package v.1.7.3. phylogenetic analyses revealed that the ten chikv strains clustered into the homogeneous indian ocean clade of the ecsa genotype ( fig. 1 (b) ).notably, the ten chikv strains isolated from serum samples possessed the mutation k211e in the e1 gene and v264a in the e2 gene; these mutations are associated with significant increase in viral infectivity in ae. aegypti . 9 the strains also possessed g60d and i211t substitutions in the e2 gene; these mutations contribute to increased chikv fitness in ae. albopictus. 9 however, the a226v mutation in the e1 gene that is related to significant increase in viral infectivity in ae. albopictus was not observed in any of the strains. 10 in summary, we characterized ten cases of human infection caused by imported ecsa genotype chikv in yunnan, china and successfully isolated nine infectious chikvs from the chikvpositive serum samples. the mutations associated with significant increase in viral infectivity for ae. aegypti or ae. albopictus were also observed in these strains. geographically, the yunnan province is in southeastern china and shares its border with southeast asian countries (laos, vietnam, and myanmar) that are most affected by chikv. with the increase of tourism and trade with southeast asian countries, cases of imported chikv infection are constantly increasing and may have the potential for re-emergence and autochthonous transmission to yunnan. the present study highlights the urgent need for continuous molecular screening and epidemic surveillance for chikv and its vectors to prevent future outbreaks of chikv infection among the human population of yunnan. this work was supported by the national natural science foundation of china (nsfc) ( u1702282 ), the reserve talents project for young and middle-aged academic and technical leaders of yunnan province (2019hb012), and youth talent program of yunnan "ten-thousand talents program" (ynwr-qnbj-2018-054). the authors declare no competing financial interests. key: cord-331289-02411gfv authors: di minno, giovanni; perno, carlo federico; tiede, andreas; navarro, david; canaro, mariana; güertler, lutz; ironside, james w. title: current concepts in the prevention of pathogen transmission via blood/plasma-derived products for bleeding disorders() date: 2015-07-20 journal: blood rev doi: 10.1016/j.blre.2015.07.004 sha: doc_id: 331289 cord_uid: 02411gfv the pathogen safety of blood/plasma-derived products has historically been a subject of significant concern to the medical community. measures such as donor selection and blood screening have contributed to increase the safety of these products, but pathogen transmission does still occur. reasons for this include lack of sensitivity/specificity of current screening methods, lack of reliable screening tests for some pathogens (e.g. prions) and the fact that many potentially harmful infectious agents are not routinely screened for. methods for the purification/inactivation of blood/plasma-derived products have been developed in order to further reduce the residual risk, but low concentrations of pathogens do not necessarily imply a low level of risk for the patient and so the overall challenge of minimising risk remains. this review aims to discuss the variable level of pathogenic risk and describes the current screening methods used to prevent/detect the presence of pathogens in blood/plasma-derived products. acute bleeding episodes can arise either because of inherited bleeding disorders (e.g. haemophilia, von willebrand disease), acquired deficiency of haemostatic components (e.g. due to infection, malignancy or autoimmune disease), trauma, surgery or as a result of infection with an organism that causes haemorrhagic disease (e.g. ebola or marburg virus) [1] . various treatment options exist for preventing or treating acute bleeding episodes, including fresh-frozen plasma/cryoprecipitate, platelets and plasma-derived/recombinant clotting factor concentrates [2, 3] . the use of blood-derived and recombinant haemostatic products has increased markedly over recent years, as exemplified by the global use of factor viii products ( fig. 1) [4] . this increased use has been driven by improved availability of clotting factors, increased life expectancy of people with bleeding disorders [5, 6] , increased use of prophylaxis for severe bleeding disorders [7, 8] and decreased risk of transmission of infectious agents. historically, the risk of transmission of infectious agents via blood/ plasma-derived products has been of great concern to the medical community. this risk has reduced dramatically since the implementation of stricter donation screening/donor selection procedures and improved purification procedures, but cannot be fully eradicated. furthermore, the implementation of pathogen inactivation technology for blood/plasma-derived products has further reduced the risk of transmission of both known and emerging pathogens, although results can be variable according to the methods used [9, 10] . however, it is important to note that patient risk is highly dependent on the circumstances under which blood products are collected, handled and used. in general, clinicians assess the level of risk associated with the use of blood/ plasma-derived products by evaluating factors such as patient characteristics (e.g. age, immune status, geographical location, lifestyle) and the nature of the pathogen (e.g. physical characteristics, level of virulence, chronicity of infection, prevalence). the presence of a particular pathogen within blood/plasma-derived products may pose a significant threat to specific patient groups (e.g. the elderly or immunocompromised), while being of low risk to the general population (e.g. hev). while the clinical assessment of risk is based on a variety of factors, the virological assessment of risk is based solely on the presence or absence of pathogens. the presence of pathogens implies the possibility of infection, so only pathogen-free products can be described as entirely risk-free. adopting the virological approach (i.e. discarding all products a large number of pathogenic agents (including viruses, protozoan parasites and prions) can be transmitted via blood/plasma-derived products and are capable of causing disease in humans ( table 1) . the presence of viruses in plasma-derived products became a concern in the 1980s, when 60-70% of patients with severe haemophilia became infected with human immunodeficiency virus (hiv-1) [6] . this concern continued with the discovery that 80% of patients treated with plasmaderived products prior to 1992 had become infected with hepatitis c virus (hcv) [5] . current donor selection and screening practices have improved our ability to detect or reduce the presence of pathogens in blood/plasma-derived products; for example, the residual risk of transfusion-transmitted infection (tti) with hiv/hbv/hcv has fallen to near or less than 1 per million transfused units [14, 15] . despite this success, however, a residual risk still remains. the pathogenic agents shown in table 1 (and the supplementary appendix) do not form an exhaustive list. many microorganisms that are normally non-pathogenic have the potential to cause disease when responding to changes in the biological environment, or when transfused to an immunosuppressed patient. in addition, there is still a risk that new and emerging pathogens may enter the blood supply ( table 2) . the standard assays commonly used for blood screening are nucleic acid amplification technology (nat) and immunoassays for detection of antibody and/or antigen. immunoassays are frequently used for screening purposes as multiple samples can be processed together and they may yield semi-quantitative results. nat assays allow earlier pathogen detection than with immunoassays, but they are also more costly and complex. assay selection is generally determined by the level of accuracy/speed required, but factors such as the resources available (e.g. staff, infrastructure), assay complexity and cost considerations (e.g. consumables) must also be considered. most assays for blood donation screening are mandatory (particularly in europe and north america) and the world health organization (who) recommends that all whole blood (and blood which has been processed by apheresis) should undergo pathogen screening before it is used for clinical or manufacturing purposes. screening for hiv-1, hiv-2, hbv, hcv and treponema pallidum subspecies pallidum (t. pallidum; the causative agent of syphilis) is strongly advised. the who and world federation of hemophilia (wfh) suggest that countries should carry out individual routine screening for further pathogens based on epidemiological information for their region e.g. htlv-1 and trypanosoma cruzi [16] . the wfh also acknowledges the positive impact of hiv, hbv and hcv screening on global blood safety and recommends that these screening tests be implemented whenever possible [2] . details of the serological tests carried out on individual donor plasma and nat testing of plasma mini-pools are shown in tables 3 and 4 [17] . it is recommended that the minimum evaluated sensitivity/specificity level of any assay used for blood donation screening should be 99.5% or higher [16] . however, not all assays fulfil these criteria as sensitivity/ [196] . specificity levels vary according to assay type and type of microorganism being tested for (see table 1 for details). therefore, the general advice is to screen donated blood to as high a standard as possible [16] . in the early stages of infection with hiv, hbv or hcv, viraemia occurs in the host's bloodstream at variable levels. viral antigens may appear at the same time as dna/rna, but more often become detectable several weeks later. specific antibodies are measurable 2 to 6 weeks after infection; the time between initial infection and the appearance of detectable parameters of infection (e.g. viral nucleic acid/antigens/ antibodies) is known as the 'window period' [18] [19] [20] . 4.1.1.1. hiv. when screening blood for the presence of hiv-1/hiv-2, the use of a combined antigen/antibody assay is advised (combined hiv p24 ag and anti-hiv-1 + anti-hiv-2 antibodies) as it allows earlier detection of infection. the performance of nat testing is mandatory in many countries and further reduces the window period (from around 20 to 11 days) [20] [21] [22] . 4.1.1.2. hbv. the majority of diagnostic laboratories focus on the detection of hbsag, which is the first detectable serological marker of infection. however, there is a risk that the hbsag concentration may decline to undetectable levels during the course of infection, yielding a false negative result [23] . screening for antibodies to hbc is the most conservative approach for identifying potentially exposed donors, as this identifies all individuals who have ever immunologically experienced any type of hbv infection (either current, chronic or resolved) and who may experience viral reactivation during their lifetime (particularly under conditions of immunosuppression). however, assays to measure hbc antibodies are relatively non-specific and do not always correlate with the presence of hbv virus in plasma [18] . also, we cannot exclude the possibility that donors who test positive for anti-hbc do not have pulsed recurrences of virus replication, resulting in the presence of low levels of hbv-dna in plasma. for these reasons, national transfusion services do not always routinely screen donations for anti-hbc. nat assays can be carried out, but their use is restricted by potentially low levels of viral dna [16, 18] . the combined use of hbsag screening tests and nat assays has reduced the window period for detection of hbv infection from approximately 60 to 35.5 days [20, 24] . mutant hbv strains (escape variants) should also be considered, as they may occasionally escape serological detection (although most can be detected by nat assays) [24, 25] . these hbv-variants are rare, but therefore more likely to enter and contaminate the blood supply as they are more difficult to detect [25] . in summary, the screening of blood supplies for the presence of hbv is effective, but an optimal screening system has not yet been defined. 4.1.1.3. hcv. hcv (both recent and chronic infection) can be detected by screening blood for the presence of both hcv antigen and hcv antibody (anti-hcv). seroconversion occurs at approximately 6-8 weeks postinfection; however, steady improvements in screening technology (including the adoption of nat assays) have reduced the window period to approximately 1-3 weeks [20, 26] . as with hiv, nat assays are more useful for detecting early infection, although the issue of low viral rna concentration persists [27, 28] . htlv-1 and htlv-2 are endemic in some regions, but very rare in others, and therefore screenings are conducted on a geographical or at-risk basis. the presence of virus is mostly inferred by the detection of virus-specific antibodies, using sensitive immunoassays [16] . in this instance, screening involves detection of non-specific, nontreponemal or specific treponemal antibodies. nat assays are generally not used [29] . specific antibody tests identify all individuals who have ever been exposed to this bacterium (and may continue to yield positive results for more than ten years following initial infection), while the non-specific tests (e.g. vdrl or cardiolipin tests) are primarily of use in identifying donors who may have an active infection. since t. pallidum is heat-sensitive and cannot readily withstand extended storage at low temperatures, storage at 4°c for more than three days is sufficient to render the pathogen non-infectious. however, blood components (e.g. platelets) that are stored at temperatures of around 20°c do present a risk of t. pallidum persistence. therefore screening for antibodies of this organism is recommended [16] . blood can be screened for further pathogens as appropriate, according to geographical location, seasonal activity of the vector and also patient risk factors. a current viral pathogen of interest is the mosquitoborne flavivirus west nile virus (wnv), which was confirmed to have been transmitted via transfusion in 2002 [30] . an immediate screening policy was put in place in the usa in order to reduce the risk of further transmission. this policy included deferral of any individual displaying symptoms of infection, quarantine of plasma collected during periods of high mosquito activity (when wnv is most prevalent) and the rapid development/use of wnv-specific nat and serological assays. these measures were highly effective and caused a significant reduction in the number of confirmed cases of wnv transfusion-related transmission. however, wnv outbreaks still occur within the americas, indicating a potential need for seasonal blood screening for wnv [31] . wnv outbreaks have also occurred in europe (including italy and greece), prompting the implementation of seasonal blood screening procedures in the affected regions of those countries [32] . this is another mosquito-borne pathogen that could potentially pose a risk to transfusion safety, although to date reports of transfusionrelated transmission of this virus are rare [33] . a mutated form of the chikungunya virus has been responsible for several epidemics in the past decade, spreading to the reunion islands in the indian ocean (2005), italy (2007) and the caribbean area (2012/2013) [34] [35] [36] . the virus may be detected in blood donors by nat, which will help to reduce the level of transmission risk [35] . there is also concern about the possibility of parvovirus b19 in the blood supply. b19 is prevalent worldwide, with seroprevalence in blood donors varying from between 0.2-1.3% in the usa, europe and africa and 25-40% in asia [37] . the risk of parvovirus transmission is higher when units of blood are pooled (e.g. to create batches of clotting factor concentrates, albumin etc.) and so individuals with bleeding disorders are at a higher risk of infection. b19 dna was detected in 26% of clotting factor concentrates in a recent german study [38] , and another study found that populations receiving blood-derived products were 1.7 times more likely to display antibodies to b19 than populations who had not received blood products [39] . b19 lacks a lipid envelope, which renders it highly resistant to some methods of pathogen inactivation [40] . screening of blood donations for b19 dna is not currently routine, but many manufacturers only process plasma that has been screened for the absence of b19 dna in order to reduce the risk of transmission [41] . given the prevalence of b19 in different populations, it is difficult to define the residual tti risk of this virus. however, it is clear that a transmission risk does exist. trypanosoma brucei gambiense/rhodiense africa, 10% at risk [160] direct microscopic visualisation/antibody/nat nat: b100 trypanosomes/ml [161] nat: 1-10 trypanosomes/ml [162] antibody: catt -87-98% [161] nat: 88% [163] antibody: catt -95% [161] nat: 99.2% [163] (continued on next page) the potential presence of hepatitis e virus (hev) in blood or bloodderived products is relevant. currently there seems to be a discrepancy between the number of hev-rna positive blood donations in europe (ranging from 1 in 1240 in germany to 1 in 1761 donations in the netherlands) [42] , and the low number of confirmed cases of hepatitis e in blood transfusion recipients (one confirmed case in the uk [2006] , one in france [2007] and two in germany [2014]) [43] [44] [45] . this indicates that the subject of hev infectivity and pathogenicity needs to be investigated further [46] [47] [48] [49] . there is also a debate over the necessity of introducing screening blood for the presence of hev; the virus is not currently screened for in the uk and other european countries, although one study of english donors found hev to be widespread (1 in 2848 donations) within the donor population [50] . since infection with this virus can be harmful to immunocompromised individuals, the potential need for introducing hev screening should be considered [42] . the residual risk for tti of hbv, hcv, hiv and hev in selected countries is given in table 5 . despite recent advances in methods for the detection of prions, no single method has been developed as a screening test for blood, although several methods in animal models show great promise [51] [52] [53] . in humans, a protocol for the evaluation of a blood-based test for its suitability in the diagnosis of variant creutzfeldt-jakob disease (vcjd) has been established, but no test yet appears to satisfy the requirements of sensitivity and specificity [54] . the only report so far of a blood-based diagnostic test for vcjd claimed an assay sensitivity of 71.4% and a specificity of 100% in symptomatic patients and its potential applicability as a screening test to detect asymptomatic vcjd infection has recently been investigated [55] . there is currently no strategy for confirming a positive screening result, although the protein misfolding cyclical amplification technique has recently been shown to yield positive results in buffy coat/white blood cell samples from a small number of patients with vcjd [56] . extensive use of blood donor selection and testing does not always guarantee a safe product. if the test is insufficiently sensitive, then false negatives may occur. alternatively, tests which are not sufficiently specific (e.g. anti-hbc assay for hbv) may cause false positive results, leading to an unnecessary decrease in the number of clotting products available [16] . torque tenovirus (ttv) is an example where testing specificity has been an issue, as this virus exists in various divergent forms (23 distinct genotypes have been identified thus far) [57] . since ttv is often detected in healthy individuals and is not associated with any particular disease, routine screening for this virus is not considered to be necessary; even a test with excellent sensitivity/specificity would not contribute to increase the level of safety of blood/plasma-derived products with regard to ttv. insufficient assay sensitivity remains a rare but potential problem when blood donations are screened during the window period of initial hbv, hcv or hiv infection. an increase in testing sensitivity threshold is needed to prevent hbv transmission via blood/plasma-derived products and by blood transfusion, as extremely low concentrations of hbv (e.g. 1.6 copies/ml) is capable of viral transmission (see above section on anti-hbc positive patients) [58] . in the case of hiv and hcv, even nat testing may not always be sufficient to ensure sufficiently high levels of safety as virus transmission has previously occurred after transfusion of blood with undetectable levels of viraemia [59] . recent reports have highlighted concerns about the inability of nat assays to detect different variants of hiv. there have been at least four cases in which the presence of hiv-1 rna was undetected by nat assay screening, potentially putting transfusion recipients at risk [21] . two of these false-negative results occurred due to genetic mutation in the viral rna regions targeted by nat assay primers (although in in cases where no overall figures are available, specificity/sensitivity has been described as low, medium or high (as appropriate). catt, card agglutination test for trypanosomiasis; id-nat, individual donation nat; mp-nat, mini-pool nat; nat, nucleic acid testing; pcr, polymerase chain reaction; pfu, plaqueforming unit. the majority of cases serology yielded a positive result) [60, 61] . falsenegative results can be avoided by designing nat assays that target a minimum of two amplification regions; such testing will be mandatory in germany from 2015 [21, 62] . it is hoped that as the sensitivity and specificity of nat tests continue to improve, cases of undetected infection may become less of an issue in the future. a recent report suggests that it is not necessary to carry out serological screening for multiple hbv markers, and that nat based screening is preferred [58] . however, there is a risk that the practice of relying upon a single method of screening may lead to a higher incidence of false-negative results. in one case of transfusion-associated parvovirus b19 transmission, donated blood was screened for the presence of b19 antigen and deemed to be safe since no antigen was detected. since the recipient subsequently developed b19 infection, the donation was re-analysed and found to contain b19 dna [63] . reports such as this support the argument for multiple parameter testing. the localisation of pathogens within blood may also influence ease of detection. for example, wnv is present at 10-fold higher levels in whole blood than in plasma in viraemic seropositive donors. the situation is reversed in viraemic seronegative donors, who display higher wnv levels (4-fold) in plasma than in whole blood [64] . also, cellassociated viruses such as cmv and htlv-1 are less frequently transmitted with the use of leukoreduced products [65, 66] , indicating that these viruses are less likely to be found in blood/plasma-derived products. the minimum infective dose (mid: the lowest number of pathogenic particles required to successfully infect a host) of a particular pathogen is more likely to be reached in whole blood or blood-derived components, making the use of plasma-derived or recombinant clotting factors the safest option. to date, studies attempting to measure human mid values have generally determined the viral concentration needed to infect a particular percentage of the exposed population (e.g. 50%). this value (the human infective dose for 50% of the population) is referred to as hid 50 and is often described as the human mid [67] . the hid 50 value varies greatly between pathogens (even if they are physically similar) [68] and also varies depending upon the immune status of the recipient, as immunocompromised individuals, neonates and the elderly are at greater risk of infection than healthy individuals [67] . when this finding and the prevalence of immunocompromised patients receiving blood products are both taken into account, it implies a need for screening tests to have the highest sensitivity possible. the impact of transmission on morbidity and mortality is dependent on patient characteristics. for example, although the vast majority of cases of cmv infection are not clinically important, influencing factors such as genetic predisposition, malnutrition and pre-existing infection can lead to the development of severe disease [69] . national screening programmes do not currently screen for cmv as standard, but nat assays exist and may be carried out if required, e.g. if blood is specifically intended for vulnerable recipients, such as pregnant women or transplant patients [70] . it is the opinion of the authors that both serological assays and nat tests should be used in order to reach the highest level of safety possible, particularly when in the case of immunosuppressed patients. as well as blood donation testing, a range of other measures are used to increase the pathogen safety of blood-and plasma-derived products. these include donor selection and screening, recipient vaccination and the use of blood product purification/inactivation methods. the choice of inactivation method also impacts upon the level of risk. questionnaires are often used to attempt to assess donors' health status and their potential exposure to various risks. donors can be accepted or rejected on the basis of these answered questionnaires, or alternatively their blood may be put through additional screening tests as appropriate [16, 31] . patients with bleeding disorders should be vaccinated against hav and hbv. european studies have reported that universal hbv vaccination of blood donors could be cost-effective as this measure would reduce the risk of hbv transmission in general and might even remove the necessity for general hbv nat testing; however, this would not reduce the risk posed by hbv escape variants (as described earlier) [71, 72] . blood/plasma-derived products typically undergo various procedures designed to reduce the pathogen level, although these procedures are not effective against all pathogens. plasma donations undergo quarantine (approximately 4-6 months) prior to fractionation and when the donor is again screened negative ffp can then be subjected to chromatographic fractionation, solvent-detergent treatment, nanofiltration and/or heat inactivation [73, 74] . prolongation of product storage time can be effective in reducing the infectivity of temperature-sensitive pathogens (such as t. pallidum). production of recombinant products also follows strict protocols to remove and inactivate any viruses that might be present, even though the risk of viral presence is minimal. although current purification/inactivation techniques (such as solvent-detergent treatment, nanofiltration and heat activation) do reduce the risk of pathogen transmission, they are not always sufficient to render blood/plasma-derived products safe [75] . small nonenveloped viruses (e.g. hav, b19 and picornavirus) are often highly resistant to inactivation procedures and may still be infectious in some plasma-derived concentrates [75, 76] . the presence of prions is also a concern. attempts to remove prions from plasma-derived products have involved several techniques, including ion-exchange chromatography and nanofiltration [77, 78] . several problems exist with these approaches, in particular the use of exogenous "spikes" derived from prion-infected brain homogenates to measure prion clearance, which may result in an overestimation of the amount of prion removal, and the methods used for the estimation of the reduction in prion load, which ideally should involve bioassay to measure infectivity [79] . further developments in this field are required to address these issues. a recently proposed approach for the inactivation of infectious agents in blood is whole-blood treatment with ultraviolet (uv) light in combination with a photosensitiser such as riboflavin or amotosalen [80, 81] . the main disadvantage of this approach is that uv treatment has been linked to the formation of neoantigens, which may be generated via modification of the surface antigens of platelets. the presence of neoantigens may provoke a recipient immune response during transfusion of uv-treated platelets, causing them to be rapidly cleared from the circulation [81] . while this approach to pathogen inactivation is currently used for platelets and is effective, it needs further refinement for the inactivation of whole blood [81, 82] . the wfh strongly recommends the use of plasma-derived or recombinant products in preference to cryoprecipitate or fresh frozen plasma, as the infectious load of any infectious human pathogen is lower in plasma-derived products than in cryoprecipitate, and even lower in recombinant products [2] .in some european countries, recombinant products have almost completely replaced plasmaderived products [12] . the use of recombinant products, which have been manufactured and formulated with minimal addition of human/animal-derived materials, greatly reduces the risk of recipient exposure to plasma-derived infectious agents and after they have undergone virus removal/inactivation processes, recombinant products can be considered to be as safe as currently possible [83] . despite these benefits, the use of recombinant products may be limited by higher costs and perceived problems of inhibitor formation [83] . for indications where no recombinant factor concentrates are available, the use of inactivated plasma-derived concentrates is safer than fresh frozen plasma and will reduce the risk of other adverse effects such as hypervolemia, transfusion-related lung injury (trali) and hypersensitivity [84] . a minimal risk approach would ensure that patients receive effective treatment with the lowest possible risk, but this is difficult to achieve in practical terms. regulatory needs in different european countries are usually based on recommendations from the medical community, so in order to achieve minimal risk, it would be ideal for these regulations to be standardised and mandatory in all countries. directives issued by the european union commission describe the regulatory requirements for the safety of whole blood and plasma, stating that "all precautionary measures during their collection, processing, distribution and use need to be taken making appropriate use of scientific progress in the detection and inactivation and elimination of transfusion transmissible pathogenic agents" [85] . however, the relative safety of different screening tests, products and processing methods is not discussed and so individual countries may adopt different approaches towards minimising risk. although recombinant products are associated with the highest level of pathogen safety, higher costs for development and production may make them too expensive for some healthcare systems [86] . inhibitor formation also remains an important element of concern with both plasma-derived and recombinant products, particularly with regard to fviii in haemophilia a [87] . the risk of inhibitor formation was shown to be greater with recombinant versus plasma-derived factor viii concentrates in some cohort studies [88] , but similar in others [89] . in light of these considerations (availability, adverse reactions and cost), it appears that the issue of pathogen safety of blood/plasmaderived products is highly important but may not be the limiting factor with respect to overall patient safety. the benefits of treatment with a hypothetically 'unsafe' plasma-derived product may outweigh the risk of a negative outcome (e.g. bleeding, inhibitor formation), although we suggest that it may be clinically simpler to deal with inhibitor formation than to combat an infection from an unknown or untreatable pathogen. there are still significant knowledge gaps and areas of unmet need with respect to the pathogen safety of blood/plasma-derived and recombinant products. the incidence of hbv, hcv and hiv tti has fallen to near or below 1 per million transfused units in the industrialised world, indicating that current donor selection and blood screening strategies have had a positive impact on blood safety [14] . however, it is clear that screening both donors and donated blood cannot exclude all known pathogens or eliminate all risks from emerging pathogens [63, 90] . historical precedent indicates that the blood supply is always vulnerable to contamination by hitherto non-prevalent/unknown pathogens, and that this risk cannot be accurately gauged [91] . as we identify new infectious agents of concern and develop new tests for their detection, it will also be necessary to clearly define the infectious dose range for each agent and use appropriately sensitive tests for their identification. for example, in suspected cases of vcjd infection, considerable challenges remain in the development of screening and confirmatory tests that have sufficient sensitivity and specificity to be of use in both a clinical setting and within blood banks [92] . surveillance of people with haemophilia is required to monitor pathogen safety issues related to blood and plasma products. the european haemophilia safety surveillance system (euhass), which began in 2008, is a pharmacovigilance programme which spans 25 european countries and is designed to detect, monitor and investigate adverse drug reactions. reports of adverse events (such as acute/allergic events, ttis and inhibitors) are submitted to euhass by participating centres and cumulative patient and clotting factor data are recorded annually [93] . a formal coordinated risk-assessment and management action plan, in addition to a task force, should be developed to respond quickly to any emerging infections. such a plan should include long-term storage of samples from produced batches (for retesting in the case of outbreaks with known or recently emerged infectious agents) and guidance on responsibility for developing/performing tests for emerging pathogens (industry vs. regulatory agencies). guidance on approaching patients who have potentially been infected and surveillance strategies for patients at high risk would also be beneficial. the majority of evidence indicates that the concept of clinical safety of blood/plasma derivatives does not necessarily correspond to the concept of pathogen safety; blood can only be classed as microbially safe in reference to the infectious agents that are known and have been screened for. establishing whether the presence of undetected microbes in the blood is clinically relevant will require further long-term, detailed studies. it should also be noted that even though the risk of transmission of key detectable viruses (such as hiv, hbv and hcv) via transfusion has fallen significantly, transmission does still occur. in general, balancing safety, efficacy and practicalities is a difficult goal to achievepatient safety is typically the key driver, but striving for near-complete safety at the expense of the patient's health or quality of life may not be the best course of action for patients or clinicians. the lack of a cohesive international strategy for blood donation and screening is a pressing concern that needs to be addressed. furthermore, a formal coordinated continuous risk-assessment and management action plan needs to be developed to deal with the constant potential risk of emerging infections. establishing an international registry (or harmonising data collection in national registries) and dedicated task force may help to identify newly emerging pathogens more rapidly than in the past and to further improve pathogen safety of blood/plasma-derived products and the blood supply in general. • the use of blood/plasma-derived products for the treatment of bleeding disorders carries a risk of pathogen transmission. • blood donations are screened for key pathogens such as hbv, hcv, hiv and the causative agent of syphilis, but other screening tests should be conducted as required according to geographical location and patient risk factors. • screening tests for pathogens may lack sensitivity/specificity and so false negatives may occur, resulting in a residual pathogen risk to patients. • in terms of pathogen safety, recombinant products (products which have had minimal exposure to blood/plasma-derived proteins) are considered to pose the lowest level of risk to patients. research agenda • regional and international rates of transfusion-transmitted infection for key pathogens and emerging pathogens • safety and efficacy of blood/plasma-derived products and recombinant products for treatment of bleeding disorders. pathogenesis of the viral hemorrhagic fevers guidelines for the management of hemophilia plasma components: properties, differences, and uses a study of reported factor viii use around the world mortality and causes of death in patients with hemophilia, 1992-2001: a prospective cohort study past, present and future of hemophilia: a narrative review the management of hemophilia in elderly patients assessing emerging infectious threats to blood safety for the blood disorders community blood processing who technical report: guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products pathogen safety of long-term treatments for bleeding disorders: still relevant to current practice factor viii safety: plasma-derived versus recombinant products pathogen safety of long-term treatments for bleeding disorders: (un)predictable risks and evolving threats transfusion-transmitted emerging infectious diseases: 30 years of challenges and progress experience of german red cross blood donor services with nucleic acid testing: results of screening more than 30 million blood donations for human immunodeficiency virus-1, hepatitis c virus, and hepatitis b virus recommendations: screening donated blood for transfusion-transmissible infections registry of clotting factor concentrates medical virology of hepatitis b: how it began and where we are now a novel acute hiv infection staging system based on 4th generation immunoassay pathogen inactivation and removal methods for plasma-derived clotting factor concentrates how safe is safe: new human immunodeficiency virus type 1 variants missed by nucleic acid testing prevalence of human immunodeficiency virus rna and antibody in first-time, lapsed, and repeat blood donations across five international regions and relative efficacy of alternative screening scenarios recent advances in molecular diagnostics of hepatitis b virus surface antigen-negative hepatitis b virus infection in dutch blood donors potential threat of drug-resistant and vaccine-escape hbv mutants to public health hepatitis c virus: screening, diagnosis, and interpretation of laboratory assays the incidence/window period model and its use to assess the risk of transfusion-transmitted human immunodeficiency virus and hepatitis c virus infection comparative analysis of triplex nucleic acid test assays in united states blood donors novel treponema pallidum serologic tests: a paradigm shift in syphilis screening for the 21st century transmission of west nile virus through blood transfusion in the united states in 2002 emerging infectious agents and the nation's blood supply: responding to potential threats in the 21st century west nile virus in the transfusion setting with a special focus on italian preventive measures adopted in 2008-2012 and their impact on blood safety novel chikungunya virus variant in travelers returning from indian ocean islands the chikungunya epidemic in italy and its repercussion on the blood system chikungunya virus: new risk to transfusion safety in the americas a single mutation in chikungunya virus affects vector specificity and epidemic potential parvovirus transmission by blood products -a cause for concern? prevalence of nucleic acid sequences specific for human parvoviruses, hepatitis a and hepatitis e viruses in coagulation factor concentrates evidence for the continued transmission of parvovirus b19 in patients with bleeding disorders treated with plasma-derived factor concentrates human parvovirus b19 viral pathogens hepatitis e screening for blood donations: an urgent need? transfusiontransmitted hepatitis e in a 'nonhyperendemic' country transfusion-associated hepatitis e, france transfusion-transmitted hepatitis e in germany an assessment of hepatitis e virus (hev) in us blood donors and recipients: no detectable hev rna in 1939 donors tested and no evidence for hev transmission to 362 prospectively followed recipients autochthonous hepatitis e virus infections: a new transfusion-associated risk a nationwide survey for prevalence of hepatitis e virus antibody in qualified blood donors in japan hepatitis e virus antibodies in blood donors hepatitis e virus in blood components: a prevalence and transmission study in southeast england new generation quic assays for prion seeding activity protein misfolding cyclic amplification of infectious prions plasminogen-based capture combined with amplification technology for the detection of prp(tse) in the pre-clinical phase of infection evaluation of a test for its suitability in the diagnosis of variant creutzfeldt-jakob disease population screening for variant creutzfeldt-jakob disease using a novel blood test: diagnostic accuracy and feasibility study preclinical detection of variant cjd and bse prions in blood transfusion transmitted virus: a review on its molecular characteristics and role in medicine hepatitis b virus testing by minipool nucleic acid testing: does it improve blood safety? human immunodeficiency virus transfusion transmission despite nucleic acid testing first transmission of human immunodeficiency virus type 1 by a cellular blood product after mandatory nucleic acid screening in germany blood screening nucleic acid amplification tests for human immunodeficiency virus type 1 may require two different amplification targets instruction to implement measures for risk minimisation in using hiv-1 nat test systems erythroid crisis caused by parvovirus b19 transmitted via red blood cell transfusion relative distribution of west nile virus rna in blood compartments: implications for blood donor nucleic acid amplification technology screening epidemiology, treatment, and prevention of human t-cell leukemia virus type 1-associated diseases approaches to minimize infection risk in blood banking and transfusion practice minimum infective dose of the major human respiratory and enteric viruses transmitted through food and the environment mechanisms of pathogenesis, infective dose and virulence in human parasites cytomegalovirus: pathogen, paradigm, and puzzle seroprevalence of cytomegalovirus antibodies in blood donors in southern hepatitis b virus vaccination of blood donors-what costs may be expected a cost-benefit analysis of blood donor vaccination as an alternative to additional dna testing for reducing transfusion transmission of hepatitis b virus guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products quarantine plasma: quo vadis pathogen inactivation technology: cleansing the blood supply eis-hubinger am. contamination of coagulation factor concentrates with human parvovirus b19 genotype 1 and 2 removal of tse agent from plasma products manufactured in the united kingdom characterization of thrombate iii(r), a pasteurized and nanofiltered therapeutic human antithrombin concentrate comparison of nanofiltration efficacy in reducing infectivity of centrifuged versus ultracentrifuged 263 k scrapie-infected brain homogenates in "spiked" albumin solutions pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and uv light uvc irradiation for pathogen reduction of platelet concentrates and plasma development of a riboflavin and ultraviolet light-based device to treat whole blood guideline on the selection and use of therapeutic products to treat haemophilia and other hereditary bleeding disorders. a united kingdom haemophilia center doctors' organisation (ukhcdo) guideline approved by the british committee for standards in haematology incidence of transfusion-related adverse reactions per patient reflects the potential risk of transfusion therapy in japan setting standards of quality and safety for the collection, testing, processing, storage and distribution of human blood and blood components and amending directive 2001/83/ec emerging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells is the incidence and prevalence of inhibitors greater with recombinant products? yes influence of the type of factor viii concentrate on the incidence of factor viii inhibitors in previously untreated patients with severe hemophilia a intensity of factor viii treatment and inhibitor development in children with severe hemophilia a: the rodin study clinical perspectives of emerging pathogens in bleeding disorders hemophilia therapy and blood-borne pathogen risk creutzfeldt-jakob disease: reflections on the risk from blood product therapy the european haemophilia safety surveillance system variant cjd infection in the spleen of a neurologically asymptomatic uk adult patient with haemophilia detection of prion infection in variant creutzfeldt-jakob disease: a blood-based assay world health organisation (who) development of a sensitive real-time reverse transcriptase pcr assay with an internal control to detect and quantify chikungunya virus development and evaluation of sybr green i-based one-step real-time rt-pcr assay for detection and quantification of chikungunya virus poor diagnostic accuracy of commercial antibody-based assays for the diagnosis of acute chikungunya infection cmv hhv6,7,8 r-gene® real-time pcr kit technicial specifications. 2015. available from detection of cytomegalovirus (cmv) dna in edta whole-blood samples: evaluation of the quantitative artus cmv lightcycler pcr kit in conjunction with automated sample preparation detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification simplexa™ dengue kit technical specifications development of multiplex real-time reverse transcriptase pcr assays for detecting eight medically important flaviviruses in mosquitoes evaluation of commercially available diagnostic tests for the detection of dengue virus ns1 antigen and anti-dengue virus igm antibody world health organisation (who) procleix® ultrio® assay hepatitis b virus serology: use and interpretation the reliability of hbv core antibody in serological screening for hepatitis b virus world health organisation (who) laboratory diagnostics for hepatitis c virus infection hepatitis c virus core antigen testing: role in diagnosis, disease monitoring and treatment hepatitis c diagnostics: clinical evaluation of the hcvcore antigen determination transfusion-transmitted hepatitis e: is screening warranted? comparison of real-time pcr and antigen assays for detection of hepatitis e virus in blood donors genotype 3 diversity and quantification of hepatitis e virus rna novel approach for detection of hepatitis e virus infection in german blood donors serologic assays specific to immunoglobulin m antibodies against hepatitis e virus: pangenotypic evaluation of performances transmission and disease association of kaposi's sarcoma-associated herpesvirus: recent developments quantitative real-time pcr detection of antibodies to kaposi's sarcoma-associated herpesvirus: a new approach using k8.1 elisa and a newly developed recombinant lana elisa global report: unaids report on the global aids epidemic comparison of hiv-1 viral load assay performance in immunological stable patients with low or undetectable viremia laboratory diagnostics for hiv infection molecular determinants of human t-lymphotropic virus type 1 transmission and spread abbott prism human t-lymphotrophic virus types i and ii assay development and validation of a multiplex real-time pcr for htlv-1/2 confirmatory diagnosis evaluation of a new screening assay for htlv-1 and -2 antibodies for large-scale use evaluation of the use of real-time pcr for human t cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors keeping pace with parvovirus b19 genetic variability: a multiplex genotype-specific qpcr assay quantitative real-time detection of parvovirus b19 dna in plasma detection of human parvovirus b19, human bocavirus and human parvovirus 4 infections in blood samples among 95 patients with liver disease in nanjing by nested pcr detection of parvovirus b19 igm by antibody capture enzyme immunoassay: receiver operating characteristic analysis placental transmission of human parvovirus 4 in newborns with hydrops serodiagnosis of primary infections with human parvovirus 4, finland human parvovirus 4 infection human parvovirus 4 viremia in young children novel parvovirus and related variant in human plasma detection of a novel dna virus (ttv) in blood donors and blood products validation of sybr green based quantification assay for the detection of human torque teno virus titers from plasma west nile virus: detection with serologic and real-time pcr assays diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies second international diagnostic accuracy study for the serological detection of west nile virus infection global incidence and prevalence of selected curable sexually transmitted infections use of pcr in the diagnosis of early syphilis in the united kingdom evaluation of a pcr test for detection of treponema pallidum in swabs and blood the laboratory diagnosis of syphilis current advances in detection and treatment of babesiosis a new real-time pcr assay for improved detection of the parasite babesia microti multiplex assay detection of immunoglobulin g antibodies that recognize babesia microti antigens world health organisation (who) leishmaniasis: prevention, parasite detection and treatment evaluation of pcr procedures for detecting and quantifying leishmania donovani dna in large numbers of dried human blood samples from a visceral leishmaniasis focus in northern ethiopia comparison of pcr assays for diagnosis of cutaneous leishmaniasis world health organisation (who) emerging nucleic acid-based tests for point-ofcare detection of malaria serological detection of plasmodium vivax malaria using recombinant proteins corresponding to the 19-kda c-terminal region of the merozoite surface protein-1 factsheet no 259 -trypanosomiasis options for field diagnosis of human african trypanosomiasis nucleic acid sequence-based amplification with oligochromatography for detection of trypanosoma brucei in clinical samples diagnostic accuracy of pcr in gambiense sleeping sickness diagnosis, staging and post-treatment follow-up: a 2-year longitudinal study factsheet no 340 -chagas disease international study to evaluate pcr methods for detection of trypanosoma cruzi dna in blood samples from chagas disease patients evaluation of serological tests to identify trypanosoma cruzi infection in humans and determine cross-reactivity with trypanosoma rangeli and leishmania spp a novel rhabdovirus associated with acute hemorrhagic fever in central africa a novel bunyavirus in china hemorrhagic fever caused by a novel bunyavirus in china: pathogenesis and correlates of fatal outcome what we are watching-five top global infectious disease threats, 2012: a perspective from cdc's global disease detection operations center surveillance on a/h5n1 virus in domestic poultry and wild birds in egypt comparative epidemiology of human infections with avian influenza a h7n9 and h5n1 viruses in china: a population-based study of laboratory-confirmed cases viral lung infections: epidemiology, virology, clinical features, and management of avian influenza a(h7n9) genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa nosocomial outbreak of novel arenavirus infection, southern africa marseillevirus-like virus recovered from blood donated by asymptomatic humans marseillevirus prevalence in multitransfused patients suggests blood transmission transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk severe acute respiratory syndrome (sars) -frequently asked questions current status of severe acute respiratory syndrome in china confronting sars: a view from hong kong identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy no evidence of marseillevirus-like virus presence in blood donors and recipients of multiple blood transfusions the detection of acute hiv infection improving hiv-2 detection by a combination of serological and nucleic acid amplification test assays including source plasma, to reduce the risk of transmission of hepatitis b virus prevalence and incidence of hiv and hepatitis b among blood donors and estimated residual risk of transmission of hiv and hbv virus by blood transfusion. a study at the provincial general referee hospital bukavu, democratic republic of the congo risk factors for human immunodeficiency virus infection among brazilian blood donors: a multicentre case-control study using audio computer-assisted structured interviews the diversity of chronic hepatitis b virus infections within blood donors in england and north wales a method for estimating the residual risk of transfusion-transmitted hbv infection associated with occult hepatitis b virus infection in a donor population without universal anti-hbc screening hiv, hcv, hbv and syphilis surveillance among blood donors in evolution of residual risk for hiv, hcv and hbv hepatitis e virus in scottish blood donors occurrence of hepatitis e virus rna in plasma donations from sweden, germany and the united states world federation of hemophilia (wfh) annual global surveys we wish to thank professor hermann eichler for his appraisal of this manuscript and helpful suggestions. medical writing assistance was provided by hanna mourad-agha of inscience communications, springer healthcare. this assistance was funded by pfizer. a.t. has received grants and personal fees from bayer, grants and personal fees from baxter, grants and personal fees from biotest, grants and personal fees from csl behring, grants and personal fees from novo nordisk, grants and personal fees from pfizer, and grants and personal fees from octapharma, during the conduct of the study.c.f.p. has received grants as bureau speaker, consultant, or advisor, from gilead, merck sharp and dohme, roche, pfizer, abbott, bristol-myers squibb, viiv, and boehringer-ingelheim. none of these personal activities is in conflict with the opinions he expressed in this manuscript. d.n. has received honoraria for conferences from pfizer, roche pharma, roche diagnostics, abbott, msd, and astellas.g.d.m. has disclosed the following financial relationshipsspeaker or a member of a speaker bureau for: boehringer-ingelheim, sanofi-aventis, bayer, novo nordisk, pfizer, biotest, and grifols. consultant or ad hoc speaker/consultant for: boehringer-ingelheim, eli-lilly, sanofi-aventis, bayer, csl behring, novo nordisk, pfizer, biotest, and grifols.j.w.i. has received personal fees from piramal and grants from the department of health, uk, outside the submitted work.l.g. reports no potential conflicts of interest. m.c. has received research grants, lecture fees, and honoraria for consultancy from baxter, bayer, and pfizer. funding for medical writing assistance and two author meetings were provided by pfizer. pfizer had no editorial influence regarding the contents of this manuscript and did not comment on the outline or any drafts prior to journal submission. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.blre.2015.07.004. key: cord-002687-ql6zo8ka authors: li, dan; he, wenhui; liu, ximing; zheng, sanduo; qi, yonghe; li, huiyu; mao, fengfeng; liu, juan; sun, yinyan; pan, lijing; du, kaixin; ye, keqiong; li, wenhui; sui, jianhua title: a potent human neutralizing antibody fc-dependently reduces established hbv infections date: 2017-09-26 journal: nan doi: 10.7554/elife.26738 sha: doc_id: 2687 cord_uid: ql6zo8ka hepatitis b virus (hbv) infection is a major global health problem. currently-available therapies are ineffective in curing chronic hbv infection. hbv and its satellite hepatitis d virus (hdv) infect hepatocytes via binding of the pres1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (ntcp). here, we developed novel human monoclonal antibodies that block the engagement of pres1 with ntcp and neutralize hbv and hdv with high potency. one antibody, 2h5-a14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. it also acts therapeutically by eliciting antibody-fc-dependent immunological effector functions that impose durable suppression of viral infection in hbv-infected mice, resulting in reductions in the levels of the small envelope antigen and viral dna, with no emergence of escape mutants. our results illustrate a novel antibody-fc-dependent approach for hbv treatment and suggest 2h5-a14 as a novel clinical candidate for hbv prevention and treatment of chronic hbv infection. human hepatitis b virus (hbv) infects more people than does hepatitis c virus (hcv) and human immunodeficiency virus (hiv) combined (world health organization, 2016) . despite the fact that there is an effective prophylactic hbv vaccine, 240 million people are chronically infected. hbv carriers are at high risk of developing severe liver diseases ranging from chronic hepatic insufficiency to cirrhosis and hepatocellular carcinoma (hcc) (world health organization, 2016; schweitzer et al., 2015) . current therapies are inadequate for treating chronic hbv infection. for example, the immune-modulatory interferon alpha therapies have a curative effect in a small portion of patients; nucleos(t)ide analog (nuc) therapies can suppress viral replication but hardly achieve sustained virological control after treatment withdrawal and are non-curative (lok et al., 2016) . an additional problem involves co-infection with hepatitis d virus (hdv), which is a satellite virus that propagates only in the presence of hbv. approximately 15 million of hbv carriers are co-infected with hdv, which accelerates disease progression and exacerbates disease severity. no effective therapies are currently available for hbv patients co-infected with hdv, and there are no anti-viral therapies that specifically target hdv (rizzetto, 2015; hughes et al., 2011; thomas et al., 2015) . hbv is a small enveloped dna virus with a relaxed circular genome. upon entering hepatocytes, hbv covalent closed circular dna (cccdna) is generated in the nucleus of infected cells, and this cccdna serves as both the intermediate for viral replication and as a viral persistence reservoir. it is believed that curing chronic hepatitis b (chb) would require either elimination or permanently silencing of the cccdna from hepatocytes and/or elimination of the infected hepatocytes carrying cccdna (zeisel et al., 2015; seeger and mason, 2016; liang et al., 2015; block et al., 2013; gane, 2017) . immunotherapies based on human monoclonal antibodies (mab) have achieved remarkable clinical success in treating multiple cancers and various autoimmune and inflammatory diseases. more recent efforts have shown that chronic viral infectious diseases can be treated via mab-based immunotherapy; studies have reported promising clinical outcomes using a mab with broad and potent activity in neutralizing hiv scheid et al., 2016; caskey et al., 2015) . neutralizing antibodies (nabs), in addition to their capacity to specifically block viral entry via fab (fragment of antigen binding) recognition of virus, have been found to exert a variety of immunological 'effector' functions, including clearance of circulatory viruses as well as by mediating cytotoxic killing or phagocytosis of infected cells (dilillo et al., 2014; corti et al., 2011; bournazos et al., 2014; bruel et al., 2016; lu et al., 2016; hessell et al., 2007) , or even possibly triggering sustained host immune responses in vivo pelegrin et al., 2015) . for example, just as with cancer cells, virus-infected cells can be eliminated by antibody dependent cell-mediated cytotoxicity (adcc) and phagocytosis (adcp) through interaction of the fc (fragment crystallizable domain) of an antibody with its cognate fcg receptors (fcgrs) expressed on immune cells. therefore, although the results from other immune-based therapies in treating hbv have so far been disappointing , novel nab-based immunotherapy may represent a new modality for curing chronic hbv infection. infection of hbv is mediated by hbv envelop proteins, which are the main target of neutralizing antibodies. no structural data are available for these multi-transmembrane proteins. the large (l) envelope protein of hbv has a pres1 domain at the n-terminal end of s domain and plays a pivotal role in hbv and hdv infections (le seyec et al., 1999; blanchet and sureau, 2007) (figure 1-figure supplement 1) . we discovered that the pres1 domain of the l protein specifically binds to the liver bile acid transporter sodium taurocholate cotransporting polypeptide (ntcp) in hepatocytes and thusly mediates viral entry of hbv and hdv (yan et al., 2012; li, 2015) . given this key functional role, the pres1 domain containing the ntcp-binding site is an attractive target for developing nabs against hbv and hdv infection. in the present study, we took advantage of a large non-immune phage display human antibody library and our human ntcp-enabled hbv cell culture system (yan et al., 2012; sun et al., 2016) to identify a panel of nabs that specifically target the pres1 domain. one of the lead nabs, 2h5, showed strong neutralizing ability against both hdv and hbv. we used crystallographic studies to identify the 2h5 epitope, and sequence analysis predicted that this epitope is highly conserved in eight of the ten hbv genotypes. neutralization-oriented optimization of the lead nab by antibody engineering resulted in a more potent anti-pres1 nab, 2h5-a14, with picomolar neutralizing activity against the major epidemic genotypes of hbv (b, c, and d) . importantly, 2h5-a14 demonstrated adcc activity through natural killer (nk) cells and through macrophages, and adcp activity through macrophages in vitro. in animal studies, the prophylactic use of 2h5-a14 resulted in full protection of humanized mice from hdv and hbv infection. moreover, 2h5-a14 greatly reduced hbv infection in a treatment mouse model. studies with 2h5-a14 fc mutant in the animals revealed that fc-mediated immune effector functions are largely responsible for the therapeutic effect of 2h5-a14. identification of a human nab against the pres1 domain of the hbv l protein and structural analysis of the antigen-antibody complex to identify nabs that block viral infection, we used pres1 peptides derived from the pres1 domain of the hbv l protein (figure 1-figure supplement 1 ) as targets to select against a large nonimmune phage display antibody library. by employing hepg2-hntcp cell-based in vitro hbv infection systems , we identified six nabs that were effective in neutralizaling hbv, but each differed in its potency ( figure 1a) . epitope mapping by direct binding and competition elisa assays showed that these nabs recognized four unique epitopes, represented by #71, 2h5, m1q (mge) in the presence of the tested abs. the secreted hbeag and hbsag levels in sample supernatants were measured at 7 days post infection (dpi). the hbv neutralization activity is here presented as the percentage of inhibition of secreted hbeag and hbsag, which was calculated by normalizing the 'infection only' reading to 0% inhibition. (b) neutralization of hdv infection in hepg2-hntcp cells by 2h5 and m1q. the cells were infected with 500 mge of hdv in the presence of the test nabs or matched isotype control ab. at dpi 7, hdv delta antigens were stained with fitc-conjugated mab, 4g5; the cell nuclei were stained with dapi. (c) crystal structure of the scfv of 2h5 in complex with the pres1 peptide. 2h5 is shown in ribbon and residues 20-27 of pres1 (genotype c) are shown in sticks. carbon atoms are colored in cyan for vh of 2h5, magenta for vl of 2h5 and yellow for the pres1 peptide. oxygen atoms are colored in red and nitrogen atoms are colored in blue. (d) interaction between 2h5 and pres1. 2h5 is shown in ribbon and the pres1 epitope and selected cdr side chains are shown in sticks. this view is perpendicular to panel c. dotted lines denote hydrogen bonds. (e) surface view of the peptide binding pocket of 2h5. same color-coding and orientation as panel d. (f) 2h5 protected hntcp-tg mice from hdv infection. human ntcp-tg homozygotes (c57bl/6 background) were ip administered 2h5 (20 mg/kg; n = 5) or pbs control (n = 2) at 8 days after birth. one hour later, each mouse was challenged with 1.47 â 10 10 genome equivalents (ge) of hdv. the mice were sacrificed at dpi 6. hdv total rna titers (on the left y-axis) and hntcp transgene expression (on the right y-axis) in liver tissues were measured by qpcr. the horizontal dotted line indicates the reliable figure 1 continued on next page and #15, respectively (figure 1-figure supplements 1 and 2a-c). the neutralization potency of these nabs correlated with the location of their epitopes in relation to the receptor binding site (rbs) in pres1. the epitopes of 2h5 and m1q are more closer to the c-terminus of rbs, and showed more potent neutralization activity compared to the other two nabs, including #71, with its epitope close to the n-terminus of rbs that only recognizes non-myristoylated pres1 peptides, and #15, with its epitope distant from the c-terminus of rbs, although all four of these nabs had similar pres1 binding activity. 2h5 and m1q were also able to neutralize hdv infection in hepg2-hntcp cells ( figure 1b ). as 2h5 was slightly more potent in neutralizing hbv and hdv than m1q, we selected 2h5 for further characterization and optimization. to better understand the structural basis of 2h5-pres1 binding, we solved the crystal structure of 2h5 (in scfv form (single-chain fragment of variable domain): vh-linker-vl) in complex with a pres1 peptide ('59c') corresponding to residues (à10~48) of pre-s1 (genotype c) (figure 1-figure supplement 1). the 2h5 scfv and the 59c peptide were co-expressed in e. coli and were then co-purified and co-crystallized. the complex structure was determined by molecular replacement and refined at 2.5 å resolution to an r-factor of 0.23 and an r-free value of 0.28 (table 1 ). in the complex structure, residues 20-27 of the 59c peptide showing well-ordered electron density were modeled. the 8-residue peptide adopts a u-shaped conformation and is situated in a pocket formed by five complementarity-determining-region (cdr) loops from both the variable heavy (vh) and light chains (vl) of 2h5 ( figure 1c -e). the interface between 2h5 scfv and the pres1 peptide buries a solvent accessible area of about 460.8 å 2 , accounting for 54.6% of the total peptide surface area. the heavy chain and light chain of 2h5 contribute 236.3 å 2 and 224.5 å 2 of the buried surface area respectively. five cdr loops (hcdr1-3, lcdr1, and lcdr3) are involved in direct binding to the 59c peptide. the 2h5-pres1 interaction interface includes contributions from all types of non-covalent interactions ( figure 1d and table 2 ). all of the eight modeled residues except gly 24 of pres1 interact directly with 2h5. on the buried surface of pres1, which is mainly involved in heavy chain interaction, the asp 20 residue has an electrostatic interaction with arg 55 in the hcdr2 loop. additionally, arg 59 of the hcdr2 loop forms a hydrogen bond with the main-chain carbonyl oxygen of ala 22 from pres1. phe 23 in pres1, which has a large hydrophobic benzyl group, inserts deep into a hydrophobic pocket that is formed by ala 38 , arg 55 , tyr 57 and the backbone atoms of hcdr3. phe 23 alone contributes to 23.5% of the buried surface area of the pres1 peptide. ala 22 and ala 25 in pres1 form weak hydrophobic interactions with, respectively, tyr 57 of hcdr1 and met 109 of hcdr3. on the exposed surface of pres1, which primarily contacts the light chain, pro 21 forms hydrophobic interactions with tyr 37 of lcdr1 and tyr 107 of lcdr3. additionally, asn 26 and ser 27 make several van der waals contacts with both tyr 37 and tyr 38 of lcdr1. we next verified the key binding residues deduced from the crystal structure of the complex. a wild-type synthesized 16-mer pres1 peptide could efficiently compete with the full-length pres1 peptide for binding to 2h5. however, three 16-mer variants of the pres1 peptide that had a key residue (asp 20 , pro 21 , or phe 23 ) mutated to alanine failed to compete the full-length pres1 peptide for binding to 2h5 (figure 1-figure supplement 2d) . further, alanine substitution of pres1 leu 19 , which is immediately adjacent to the 8-residue region of the 59c peptide, also reduced binding competition activity, indicating that this residue is involved in pres1-2h5 binding. of note, sequence alignment showed that 2h5 epitope is highly conserved among hbv genotypes (figure 1-figure figure 1 continued detection limit. the copy numbers shown in the y-axes are from 20 ng of total liver rna for each sample. each square represents one mouse; squares of the same color indicate data from the same mouse. doi: https://doi.org/10.7554/elife.26738.002 the following source data and figure supplements are available for figure 1: source data 1. data for figure 1f . doi: https://doi.org/10.7554/elife.26738.005 figure supplement 1. hbv envelope proteins and amino acid sequence alignment of pres1 peptides. doi: https://doi.org/10.7554/elife.26738.003 figure supplement 2. binding of the six anti-pres1 nabs to pres1 peptides and characterization of their epitopes. doi: https://doi.org/10.7554/elife.26738.004 supplement 1), indicating that 2h5 can potentially provide broad protection against the vast majority of hbv genotypes. although residue 24 in the 2h5 epitope is variable among different hbv genotypes (e.g., gly 24 in genotypes a and c; lys 24 or arg 24 in all other genotypes, figure 1 -figure supplement 1), g24r mutation of the pres1 peptide had no effect on binding to 2h5 (figure 1figure supplement 2e), consistent with our observation from the complex structure that gly 24 does not make direct contact with 2h5. generation of a more potent nab, 2h5-a14 to evaluate the neutralization activity of 2h5 in vivo, we employed a de novo hdv infection mouse model that we recently established (he et al., 2015) in which the transgenic expression of human ntcp (hntcp-tg) in c57bl/6 mouse liver supports acute hdv infection mediated by hbv envelope proteins. using this model, prophylactic administration of 2h5 igg1 (1 hr before hdv viral challenge) resulted in a more than one log reduction of the viral titer in mouse livers as compared to untreated controls ( figure 1f ). nevertheless, 2h5 treatment was not able to completely block hdv infection in mouse livers, even at a 20 mg/kg dose. thus, increasing the neutralization activity of 2h5 while preserving its conserved epitope were needed to yield a potent and broadly-neutralizing antibody with improved in vivo efficacy. to this end, we employed various antibody engineering approaches including chain shuffling, structure-guided design, and cdr-focused mutagenesis. the vh chain shuffling approach was most fruitful in identifying improved anti-pres1 nabs. vh chain shuffling by fixing the variable light (vl) chain of 2h5 and pairing it with a repertoire of naturally-occurring variable heavy (vh) genes (~1â10 10 ) led to the identification of 16 abs with higher binding affinity. hbv and hdv neutralizing assays were then performed to rank the neutralizing ability of these 16 abs relative to that of 2h5 ( 2h5-a14 is a potent and broadly-neutralizing nab against hbv infection that acts by blocking the binding of pres1 with the hbv receptor ntcp competition elisa assays showed that 2h5-a14 had almost the same peptide competition pattern as did 2h5, and mutation of g24r of pres1 did not affect 2h5-a14 binding activity, indicating that 2h5-a14 targets the same epitope as does 2h5 (figure 2-figure supplement 4) . this epitope is in close proximity to, but does not overlap, the receptor ntcp-binding site of pres1 (figure 1-figure supplement 1). to test whether the neutralization activity of 2h5-a14 can be attributed to competition with pres1 for binding with ntcp, we used a stable hepg2-hntcp cell clone that expresses a high level of hntcp and supports the binding of a fluorescent-labeled pres1 peptide. an immunofluorescence staining assay showed that 2h5-a14 inhibited the binding of fitc-labeled pres1 to hepg2-hntcp cells in a dose-dependent manner, whereas an isotype-matched control ab did not ( figure 2a ). this result indicates that the neutralization mechanism of 2h5-a14 likely involves blocking of viral entry by interfering with the binding of ntcp and pres1, probably owing to steric hindrance at or near the pres1 receptor binding site. we next examined the potency and breadth of 2h5-a14 in neutralizing hbv and hdv. hbv has 10 known genotypes, all of which differ from each other by at least 8% at dna sequence level (sunbul, 2014) . genotypes b, c, and d represent the major epidemic genotypes, and 2h5-a14 potently neutralized all three of these genotypes ( figure 2b ). we also compared the potency of 2h5-a14 to other agents known to block hbv entry, including commercial hbig (hepatitis b immune globulin), kr127 (a humanized pres1 mouse mab [chi et al., 2007] ), and two prototype peptides of myrcludex-b (bogomolov et al., 2016) (an ntcp-binding pres1-derived synthetic myristoylated lipopeptide): m47-d (genotype d) and m47 (genotype c). assays using a hepg2-hntcp cell-based hbv infection system showed that 2h5-a14 had more than 1000-fold stronger neutralization activity than did hbig against infection with recombinant hbv ( figure 2c ) and hdv ( figure 2d ), as well as against infection with six hbv primary isolates derived from hbv-infected patients (genotype c) ( hbv than did kr127, and 50-fold stronger anti-hbv activity than did either m47-d or m47 ( figure 2c ). consistently, in a primary human hepatocyte (phhs) infection system, 2h5-a14 showed similar neutralization potency against a clinical viral isolate from an hbv-infected patient (genotype b) ( figure 2e ). we examined variations in the epitope-forming amino acid sequences among hbv genotypes to assess the breadth of the neutralization activity of 2h5-a14 against hbv. in addition to the aforementioned variability at position 24 of pres1, position 22 also exhibited heterogeneity; genotypes f and h had leucine at this position while all other genotypes had alanine (figure 1-figure supplement 1). analysis using the hbvdb database revealed that this leucine mutation is present at a very low frequency (about 3.75%) among all reported hbv sequences (total 7407 sequences) (hayer et al., 2013) , and is almost completely limited to genotypes f and h (figure 2-figure supplement 5b). elisa binding assays showed that 2h5-a14 bound weakly to a pres1 peptide variant containing the epitope-forming sequnces of both the f and h genotypes of hbv (figure 2-figure supplement 5c), suggesting that 2h5-a14 likely has limited efficacy against these rare genotypes. even so, our sequence analysis showed that the epitope 2h5-a14 is highly conserved and our neutralization assay results clearly showed that 2h5-a14 neutralizes the three major epidemic genotypes of hbv with remarkable potency. given this, it is reasonable to extrapolate that 2h5-a14 can likely neutralize the vast majority (>95%) of hbv virus strains. we evaluated the prophylactic efficacy of 2h5-a14 against hdv infection in a recently established gene-edited mouse model in which residues 84-87 of mntcp have been replaced by their hntcp counterparts . 2h5-a14, hbig or an isotype-matched control antibody was administered at indicated doses to ntcp gene-edited homozygous fvb mice 1 hr prior to hdv challenge. a 15 mg/kg dose of 2h5-a14 completely blocked hdv infection, reducing hdv titers in liver to below their limit of detection at day six after hdv challenge. notably, 3 mg/kg and 0.6 mg/kg doses reduced hdv titers by 99.4% and 98.5%, respectively. in contrast, even a high dose of hbig (100 iu/ kg) only reduced hdv titers by 90%, which was still insufficient to prevent hdv infection ( figure 3a ). we next examined prophylactic efficacy of 2h5-a14 against hbv infection in human liver chimeric mice. chimeric livers generated by transplanting human hepatocytes into fah -/-rag2 -/-/il2rg -/-(frg) triple knock-out mice ('hfrg mice') are known to be highly susceptible to hbv infection (strom et al., 2010; bissig et al., 2010a; azuma et al., 2007) . we tested the in vivo efficacy of 2h5-a14 in neutralizing viral challenge both before (prophylactically) and after (therapeutically) administration of patient-derived viruses to these chimeric mice. the hfrg mice used in this were ip administered 2h5-a14, hbig, or matched isotype control ab at the indicated concentrations at 9 days after birth. one hour later, each mouse was challenged with 1 â 10 10 ge of hdv and was then sacrificed on post infection day 6. the liver tissues were collected from each mouse. hdv total rna levels and the edited ntcp gene expression were measured by qpcr. the data were presented similarly as in figure 1f . (b) schematic diagram illustrating hbv challenging, bleeding and antibody administration schedules in the mouse study. five animals were used in each group. recipient hfrg mice were challenged with 1 â 10 9 ge hbv (genotype b) on day 0. a single dose of 15 mg/kg 2h5-a14 was ip administered one day before viral challenge (prophylaxis), or biweekly doses of 20 mg/kg 2h5-a14 were ip administered starting from day 5 to day 21 after the challenge (therapeutic). hbig (40 iu/kg) treatment was used as a control. experiment had high level of humanization with an average human serum albumin level of about 10 mg/ml. this experiment also included virus infection only (vehicle ctrl.) and hbig control group. all of the treatments began at five dpi and lasted until 21 dpi ( figure 3b ). secreted hbsag, as well as hbv dna, and rna levels ('virological markers') were measured periodically from serum samples. hbv rapidly and robustly replicated in the hfrg mice ( figure 3c ). in the infection-only control group, the serum hbv dna and rna levels reached about 1 â 10 11 and 1 â 10 10 copies/ml, respectively, and hbsag level exceeded 1 â 10 5 iu/ml. a single administration of 2h5-a14 (15 mg/ kg) one day prior to hbv challenge completely protected mice from hbv infection, as indicated by the levels of all of the virological markers in serum samples. therapeutically, biweekly 2h5-a14 treatment (20 mg/kg) greatly reduced all of the virological markers in sera as compared to infection-only control and to the hbig treatment groups. remarkably, by the end of the experiment (37 dpi), 2h5-a14 treatment resulted in three log reductions in the hbv dna and rna levels relative to the two control groups ( figure 3c ). in addition, human albumin levels and alanine aminotransferase (alt) activities in mouse serum samples were monitored throughout the study period. there were no significant differences between 2h5-a14 treated group and other groups including vehicle control, 2h5-a14 prophylaxis and hbig control groups (figure 3-figure supplement 1). liver tissues collected at the end of the experiment were used for immunohistochemical staining (ihc) of hbsag and hbcag, southern blotting of viral dna and cccdna, and northern blotting of viral rna. consistent with our sera-based measurements, liver tissues from 2h5-a14 prophylaxis group animals had no detectable hbsag and hbcag or hbv dna, cccdna, or rna; while both the infection vehicle control and hbig treatment groups had strong signals for these markers; liver tissues from 2h5-a14 treatment had greatly reduced hbsag and hbcag, and had almost undetectable levels of hbv dna and rna ( figure 3d and encouraged by the therapeutic effects observed for 2h5-a14 in the hfrg mouse model, we further dissected the mechanism-of-action of 2h5-a14. to determine whether viral neutralization mediated by fab of 2h5-a14 is the sole anti-viral mechanism or if, additionally, effector functions mediated by the fc region may be involved, we first examined the effector functions of 2h5-a14 using in vitro assays. human nk cells and macrophages are two types of immune effector cells capable of mediating adcc and adcp. nk cells primarily express fcgriiia (cd16) and mediate adcc, whereas macrophages express all classes of fcgrs and can perform both adcc and adcp (braster et al., 2014; herter et al., 2014; bournazos et al., 2015) . we initially tested whether 2h5-a14 can activate nk cells to induce adcc. these experiments used an in vitro adcc assay that employed human natural killer cells expressing both the v158 allele of fcgriiia and the g chain of the fc receptor (nk92-mi hcd16 line) as effectors (klingemann et al., 2016) . two stable cho cell lines expressing either the wild type (wt) epitope or a mutant epitope of 2h5-a14 were established: cho-59c cells expressing a recombinant single-transmembrane protein (wt pres1 fused with the transmembrane domain of vamp2, vesicle-associated membrane proteins), to which 2h5-a14 efficiently binds, was used as the target cell line; cho-59c-mut cells carrying a d20a mutation in pres1 that abolishes 2h5-a14 binding was used as the control cell line (figure 4-figure supplement 1a) . 2h5-a14 but not an isotype-matched control antibody treatment resulted in a dose-dependent cytotoxic effect against cho-59c cells; no such effect was observed for the cho-59c-mut control cells, demonstrating that nk cell-mediated adcc requires the presence of the 2h5-a14 epitope on target cells ( figure 4a ). to further confirm the adcc activity of 2h5-a14, a process that invariably requires fc-fcgr interaction, we generated a 2h5-a14 variant (2h5-a14-dana) with two mutations in its igg1 fc region t ratio was about 1:1. the adcc activity (left) was measured as described above. for the adcp assay (right), the target cells were labeled with cfse fluorescent dye prior to mixing with macrophages. adcp activity was monitored by fluorescence microscopy. the phagocytosis index was determined as the number of cfse-positive target cells per 100 macrophages. the means and error bars shown were from two independent experiments. (d) cdc activity of 2h5-a14 and other control antibodies. rabbit complement sera (10%) (sigma-aldrich) were used. all the samples were tested in triplicates. rituximab (anti-cd20) and gc33 (anti-gpc3) mabs were positive control mabs for the assay. they have the same fc region of human igg1 as 2h5-a14. all data shown in panel a, b and d represent at least two independent experiments. all the antibodies were tested at 10 mg/ml in panel c-d. doi: https://doi.org/10.7554/elife.26738.020 the following source data and figure supplements are available for figure 4: (d265a and n297a) that are known to abolish the binding of fc with all classes of fcgrs (shields et al., 2001; wilson et al., 2011) . as expected, the 2h5-a14-dana variant could still bind to cho-59c cells and pres1 peptides, and neutralize hbv infection to the same extent as 2h5-a14 (figure 4-figure supplement 1a-c), but could not bind to fc receptors as revealed by facs binding assays using nk92-mi hcd16 cells and spr analysis (biacore) using soluble human and mouse fcgrs (figure 4 -figure supplement 1d and figure 4-figure supplement 2) . consistently, the 2h5-a14-dana variant could not induce adcc activity ( figure 4a ). using the same adcc assay, we also demonstrated that 2h5-a14, but not the 2h5-a14-dana variant, caused adcc against hbv-infected cells. the hepg2-hntcp cells infected by hbv were specifically killed by nk cells, whereas the hepg2-hntcp cells uninfected by hbv were spared from nk cell killing ( figure 4b ). we next examined if 2h5-a14 can mediate adcc and adcp via macrophages. when fully differentiated human macrophages were mixed with target cells (293f-59c, see materials and methods) expressing the 2h5-a14 epitope on their surface, both adcp and adcc were observed following treatment with wild type 2h5-a14 but not with the 2h5-a14-dana variant ( figure 4c, figure 4 figure supplement 3a). facs analysis confirmed that 2h5-a14 and 2h5-a14-dana bound to these target cells to a similar extent (figure 4-figure supplement 3b ). when the target cells were expressing a mutant epitope (293f-59c-mut), no specific adcc and adcp were observed ( figure 4c ). similar results were obtained using mouse bone marrow-derived macrophages (bmdms) as effector cells (figure 4-figure supplement 3c). taken together, the results of these in vitro assays suggest that 2h5-a14 can kill target cells via fc-fgr interaction-dependent effector functions such as adcc and adcp. we further analyzed if 2h5-a14 can activate complement and induce cdc. in the presence of complement sera (from rabbit or human), 2h5-a14 did not cause cell lysis of cho-59c cells. under the same testing conditions, anti-cd20 (rituximab) and an anti-gpc3 chimeric antibody (gc33) that are known to cause cdc all induced target-specific cdc ( figure 4d and (nakano et al., 2009; van meerten et al., 2006) . thus, 2h5-a14 is not competent to induce cdc against target cells. consistently, 2h5-a14 has very weak binding activity to both human-and mouse c1q (figure 4-figure supplement 4b ). to assess whether the effector functions of 2h5-a14 contribute to its in vivo efficacy, we compared the anti-viral activities of 2h5-a14 and 2h5-a14-dana in the aforementioned hdv and hbv infection mouse models. the fc of 2h5-a14 is from the human igg1 isotype, which is able to engage mouse fcgrs on mouse effector cells (figure 4-figure supplement 2) (overdijk et al., 2012) . in the ntcp gene-edited homozygous fvb mice, 2h5-a14-dana and 2h5-a14 showed similar prophylactic efficacy: both offered complete protection from hdv infection at a 5 mg/kg dose ( figure 5a ). this result suggests that fab-mediated neutralization, rather than the fc-mediated effector function, is primarily responsible for preventing viral infection. this mouse model only supports transientlyacute hdv infection and is thus only suitable to assess prophylactic efficacy. we therefore used the hfrg mouse model, which supports sustained hbv infection, to determine if the therapeutic effect of 2h5-a14 requires fc-fgr interaction-dependent effector functions. hfrg mice with a medium level of humanization (an average human serum albumin level of about 3 mg/ml) were challenged with hbv on day 0. at 33 dpi, nine mice with similar level of stably-established hbv infection were divided into three groups and treated with 2h5-a14 (5 mg/kg), 2h5-a14-dana (5 mg/kg), or pbs (vehicle control). treatment was administered twice weekly for a total of 4 weeks ( figure 5b ). blood was collected periodically for monitoring virological markers, including dna titer and the hbsag level until the end of the experiment (89 dpi). the hbv dna titer and the hbsag level increased over time in the control group, with an average dna titer of~2.0â10 9 /ml and an average hbsag level of~3â10 6 iu/ml at 89 dpi. in the 2h5-a14-dana group, the average hbv dna titer and the hbsag levels were~3.8â10 8 /ml and~4.0â10 4 iu/ml, respectively, at 89 dpi. in contrast, compared to the control group, the 2h5-a14 treatment group had an about 200-fold reduction in dna titer (~1.0â10 7 /ml) and a~40000 fold reduction in the hbsag level (~70 iu/ml) at 89 dpi. importantly, in contrast to control pbs or 2h5-a14-dana, treatment with 2h5-a14 resulted in reductions in both hbv dna titer and hbsag levels over time, starting from the beginning of the treatment and continuing through the treatment window and 3 weeks after treatment withdrawal, with only a slight the experiment was performed similarly to that described in figure 3a . both 2h5-a14 and fc mutant 2h5-a14-dana were tested at 5 mg/kg. the copy numbers shown in the y-axes are from 20 ng of total liver rna for each sample. (b) schematic diagram illustrating hbv challenge, bleeding and antibody treatment schedules in the mouse study. figure 5 continued on next page rebound at the end of the experiment (4 weeks after treatment withdrawal) ( figure 5c ). these results showed that 2h5-a14 treatment not only efficiently suppressed the spread of hbv in mice, but could also gradually reduce the extent of an established hbv infection over time. in contrast, 2h5-a14-dana was able to suppress the spread of hbv as compared to the control group, but it did not reduce the extent of an established hbv infection: the levels of hbv dna and hbsag at all tested time points after the treatment were all higher than those at the starting point of treatment. note that both 2h5-a14 and 2h5-a14-dana treated mice had comparable serum antibody concentrations at all time points tested ( figure 5d ) and had comparable human albumin levels both before viral challenge and at 80 dpi ( figure 5-figure supplement 1a) . liver tissues collected at the end of the experiment were used for ihc staining of hbsag and southern blotting of viral dna and cccdna. consistent with our sera-based measurements, liver tissues from 2h5-a14-treated but not 2h5-a14-dana-treated mice had greatly reduced levels of hbsag (ihc assay) comparing to the control mice ( figure 5e and figure 5-figure supplement 1b) . no significant histopathological changes related to the treatment were observed. southern blotting showed that the hbv total dna and cccdna were undetectable for the 2h5-a14-treatment group, whereas strong total dna and cccdna signals were observed for the 2h5-a14-dana and control groups ( figure 5f ). quantification of the cccdna level in the liver tissues by qpcr further independently confirmed that 2h5-a14-treated mice indeed had much lower cccdna copy numbers than the other two groups ( figure 5g ). these results demonstrate the potent in vivo anti-viral effect of 2h5-a14. moreover, the comparison of the effects of 2h5-a14 and 2h5-a14-dana treatments side by side clearly proves that the anti-viral effect of 2h5-a14 results only in part from fab-mediated neutralization, while the fc-dependent effector functions of 2h5-a14 are particularly critical in conferring its overall therapeutic effect. to examine if hbv escape mutants will arise under selection pressure from 2h5-a14 treatment, we sequenced the hbv l gene amplified from the mouse liver tissues collected at the end of the two figure 5 continued three animals were used in each group. recipient hfrg mice were challenged with hbv viruses (genotype d) at 2 â 10 9 ge. twice weekly treatment with pbs, 2h5-a14 (5 mg/kg), or 2h5-a14-dana (5 mg/kg) began at 33 dpi and lasted for 4 weeks. (c) hbv dna titers and hbsag levels in serum. blood samples were collected at the indicated time points for measuring hbv dna titers and/or hbsag levels. (d) antibody concentrations. the antibody concentrations in serum samples were measured by elisa using antibody standards with known concentrations. note, for dpi 40 and dpi 54, the blood samples were collected at three days after ab administration; for dpi 60, the blood samples were collected 1 hr after ab administration. the horizontal dotted lines indicate the lowest detection limits; the vertical dotted lines and the grey-shaded areas indicate the treatment window (c-d). (e) ihc staining of human cytokeratin-18 (hck18) and hbsag in serial sections of liver tissues from sacrificed hfrg mice at the end of the experiment (dpi 89). intrahepatic hbsag was detected by a specific anti-hbsag mouse mab, followed by hrp-anti-mouse secondary ab and stained brown using dab substrate, nuclei were stained blue by hematoxylin. human hepatocytes in consecutive tissue sections were visualized by staining with a human-specific hck18 mab and dab substrate in blue violet, nuclei were stained with nuclear fast red. each image shown represents the staining result for one mouse of each group. (f) southern blot analysis of intrahepatic viral dna. total hbv dna was extracted from mouse liver tissues at dpi 89. the extracted dna samples were analyzed by southern blotting with an [a-32p] dctp-labeled full-length hbv dna probe. dna samples prepared from normal mouse livers were used as a negative control. 100 pg each of 3.2 kb, 2.1 kb, and 1.7 kb hbv dna fragments were used as dna markers. rcdna (relaxed circular dna), cccdna, and ssdna (single-strand dna) intermediates are labeled. (g) qpcr quantification of intrahepatic viral cccdna level. 500 ng of total dna prepared from the liver tissues collected at dpi 89 was digested by psad and 1/10 of the digested samples were used to quantify hbv cccdna by qpcr using specific primers (see materials and methods). the hntcp gene copy numbers, which represent the amount of human hepatocytes in the liver tissues of chimeric mice, were used to normalize the cccdna level in the human hepatocytes for each sample. the cccdna copy number shown in the y-axis is the relative value normalized to 1000 copies of hntcp gene in~50 ng total dna samples. each square represents one mouse. the horizontal dotted line indicates the reliable detection limit. aforementioned studies in hfrg mice. few random mutations were observed within the l genes in both mouse studies, and it appeared that more mutations were in the 2h5-a14 treatment group than that in the control group, but no mutation was specifically associated with 2h5-a14 treatment and no mutations were found within the antibody's epitope ( figure 5-figure supplement 2) . although we cannot completely rule out the possibility that 2h5-a14 treatment may induce escape mutants, these results indicate that the epitope of 2h5-a14, a receptor-binding proximal and highly conservative region centered by phe 23 , is likely unsusceptible to escape mutation. highly potent direct anti-viral agents for chronic hepatitis c (hcv) can now cure most hcv-infected patients (schreiber et al., 2016; feld et al., 2015) . curing hbv has thus become the next major goal in fighting viral hepatitis. monoclonal antibodies have rapidly become an important drug class, especially for treating cancer and autoimmune diseases. however the potential of using mabs to treat chronic viral infections is only beginning to be explored. in hbv, nabs against the hbv small (s) envelop protein (hbsag) constitute the major protective components of both the widely-used recombinant hbv vaccine and of the post-exposure prophylactic blood-derived hbig. however, neither the vaccine nor hbig are effective in treating persistent hbv infection (tsuge et al., 2016) . a previous study reported that a combination of two monoclonal nabs against hbsag (hepex-b) only exhibited short-term anti-viral effects in animal models and an early phase i human study (eren et al., 2000; neumann et al., 2010) . a humanized anti-hbsag nab was recently shown to have longer anti-viral effects in mouse models mainly through clearance of the ab-hbsag immune complexes by phagocytosis . high levels of circulating hbsag-containing non-infectious subviral particles are commonly present in the blood of chronicallyinfected hbv patients (seeger et al., 2007) . it has been assumed that hbsag can act as a decoy that prevents anti-hbsag nabs from effective targeting of infectious virions and infected cells, the formation of circulating immune complexes that may lead to immune complex disease also remains a potential risk. compared to the hbsag, the l protein is much less abundant in circulation and preferentially present on infectious hbv particles (seeger et al., 2007) . moreover it is now known that the pres1 domain of the l protein is responsible for receptor ntcp binding (yan et al., 2012) . to date, two anti-pres1 nabs, the murine bx-182 nab and humanized kr127 nab, have been shown to be able to prevent hbv infection when injected in chimpanzees prior to hbv challenge (chi et al., 2007; zhang et al., 2006) . however, neither these nor any other anti-pres1 nabs have been tested as a treatment against established hbv infection and no mechanistic studies have been conducted with these nabs. bx-182 is an hbv subtype-specific nab owing to sequence variations in its epitope (residues 6-10 of pres1, svpnp). kr127 recognizes residues 37-45 (nsnnpdwdf) of pres1 and has broader neutralizing activity than bx-182, but its potency is limited, diminishing its promise for therapy. 2h5-a14 has potent viral neutralization activity with low picomolar ic 50 values in cell cultures. consistent with this in vitro potency, it is extremely effective in protecting mice from de novo hdv and hbv infection. remarkably, 2h5-a14 demonstrated impressive therapeutic efficacy against hbv infection in two independent mouse studies using liver-humanized hfrg mice. at present, there is no immune-competent small animal model that supports natural and long-lasting hbv infection. although the hfrg mice are immunodeficient (azuma et al., 2007) , this model recapitulates the complete life cycle of hbv and is a valuable model for evaluating the efficacy of an antibody against hbv infection in vivo. our initial animal study in hfrg mice with high-level of humanized chimeric liver was ended at 37 days due to the rampant viral propagation in the untreated control group, which had an adverse effect on the general health of these mice. the second study had slower viral propagation rates (likely due to the medium-level of liver humanization and/or the relatively lower propagation rate of the challenging virus) and lasted for 89 days, allowing us to assess the antiviral effect of 2h5-a14 over a longer period. monitoring infection during this longer experimental period revealed a marked reduction of the extent of an established hbv infection in 2h5-a14-treated mice. mechanistically, we found that viral neutralization by 2h5-a14 is the dominant mechanism contributing to the protection of mice from hdv infection. in contrast, in addition to protecting hepatocytes from viral entry, fc-mediated effector functions contribute substantially to the overall antiviral effect of 2h5-a14 against hbv infection in hfrg mice. our experiments in the hfrg model revealed that wildtype 2h5-a14, but not the fc-dana mutant of 2h5-a14 (a variant incapable of mediating antibody-dependent effector functions), elicited remarkable anti-viral effects against an established hbv infection. hfrg mice lack t and b lymphocytes, and nk cells. nk cells are generally considered to be the main effector cells in mediating adcc to induce apoptosis in target cells . the observed effector functions in hfrg model thus cannot be attributed to nk cells but to the remaining immune effector cells in these mice; for example fcgr-expressing macrophages or other meloid cells (e.g. neutraphils) that are capable of killing infected hepatocytes by adcc and/or adcp or antibody-dependent scretion of antiviral cytokines or by direct clearance of the antibodyopsonized viruses via phagocytosis. considering that primary hepatocytes and hbv-infected hepatocytes may be resistant to cdc (halme et al., 2009; koch et al., 2005; liu et al., 2013; zhang et al., 2013) , and 2h5-a14 lacked complement c1q binding activity and cdc activity in vitro, cdc is unlikely involved in the anti-viral effect of 2h5-a14 in the hbv-infected hfrg mice. macrophages have previosuly been shown to be the major effector cells invovled in anti-tumor activity of several mabs in mice (clynes et al., 2000; hamaguchi et al., 2006; minard-colin et al., 2008) . macrophages exert their cytotoxic functions via diverse mechanisms such as adcc, adcp, etc. (braster et al., 2014) . neutrophils have also been reported to be involved in the anti-tumor efficacy of two mabs in tumor models in mice (siders et al., 2010; golay et al., 2013) . it will be interesting to identify which immune effector cells are involved in mediating the in vivo antiviral effect of 2h5-a14 in future studies. the cells involved could be identified via the selective depletion of macropahges (van rooijen and hendrikx, 2010) or neutraphils (daley et al., 2008) using specific reagents targeting each of them in hfrg mice. in humans, the mechanisms underlying hbv control is only partially understood, and harnessing immune control to combat chronic hbv infection is promising but remains a challenge (liang et al., 2015; block et al., 2013; zhang et al., 2015; guidotti et al., 2015; isorce et al., 2015; lucifora et al., 2014; bertoletti and rivino, 2014; bertoletti and ferrari, 2012) . during chronic hbv infection, it appears that nk cells have impaired secretion of antiviral cytokines (e.g. ifn-g) yet are able to maintain their cytotoxicity (maini and peppa, 2013; sun et al., 2015; shabani et al., 2014) . the proportion of fcgr-expressing nk cells in liver (27-75% of total nk cells in liver) is lower than that in blood (burt et al., 2009) . nontheless, given that they retain their cytotoxicity, these fcgr-expressing nk cells can be assumed to have adcc activity. hepatic macropahges include both infiltrating macrophages and liver-resident macrophages (kupffer cells, accounting for 80-90% of all tissue macrophages in entire body [jenne and kubes, 2013] ). the roles of these cells in hbv infection appear to be complicated (ju and tacke, 2016) . similar to human tumor-associated macrophages, hepatic macropahges likely retain antibody-dependent effector fuctions via their expressed fcgrs (herter et al., 2014; grugan et al., 2012) . future human clinical trials will allow us to determine if, as we found in hfrg mice, 2h5-a14 elicits its fc-dependent effect functions via activating immune cells in liver to exert its anti-viral effect. two aspects of the anti-hbv effect of 2h5-a14 are worth noting. first, 2h5-a14 treatment resulted in substantial decreases in hbsag levels in the blood of hbv-infected hfrg mice over time. serological hbsag reduction, and eventually hbsag loss, is considered as a critical step toward a 'functional cure'. current nucs therapy rarely reduces hbsag level in chb patients. 2h5-a14 specifically targets pres1 but not the vast majority of hbsag particles, therefore, in contrast to nabs targeting hbsag, the decline of hbsag by 2h5-a14 cannot be attributed to the ab-mediated clearance of hbsag from the blood, but to a gradual reduction of established hbv infection. second, 2h5-a14 treatment at least effectively suppressed cccdna accumation and more likely reduced cccdna in liver tissues. due to techniqucal difficulties, we cannot quantify the intrahepatic cccdna when treatment began, however the continuing reduction of the levels of dna titers and hbsag levels after treatment clearly demonstrates that 2h5-a14 does reduce the extent of an established hbv infection. our data showed that 2h5-a14 can mediate immune effector cells to kill hbv-infected cells in vitro via adcc and adcp, although more evdience is needed, it is conceivable to speculate that the clearance of cccdna-carrying infected cells or decrease their in vivo half-lives by fc-dependent effector functions via fcgr engagement, including cytotolytic and/or noncytolytic mechanisms, may have contributed to the anti-hbv effect of 2h5-a14 in a manner beyond fab-mediated blockage of new infections. it will be interesting to further determine the detailed mechanism of the antiviral effect of 2h5-a14, in particular the relative contribution of cytolytic versus noncytolytic effects in this process. in summary, we identified six anti-pres1 human nabs from a phage library and then improved the lead hit into 2h5-a14, a high-affinity and broad-spectrum nab that is extremely potent in vitro and in vivo against both hdv and hbv. 2h5-a14 inhibits ntcp binding by recognizing a structurallydefined and highly-conserved epitope located in close proximity to the rbs of pres1. 2h5-a14 is also capable of triggering epitope-specific and fc-dependent effector functions. importantly, the effector functions contribute substantially to the anti-viral effect of 2h5-a14 against hbv infection in vivo. these results for the first time demonstrate a nab targeting the rbs adjacent region on pres1 can exert anti-hbv effect through fc-dependent effector functions by engaging fcgrs, pointing toward a novel antibody-fc-dependent strategy for hbv treatment. human embryonic kidney cell lines 293t (rrid:cvcl_0063) and human hepatocellular carcinoma cell line hepg2 (rrid:cvcl_0027) were from american type culture collection (atcc, manassas, va); human hepatocellular carcinoma cell line huh-7 (rrid:cvcl_0336), raji (rrid:cvcl_0511) and chinese hamster ovary cell line cho (rrid:cvcl_0213) were from the cell bank of type culture collection, chinese academy of sciences. hepg1-hntcp cell line used for hbv and hdv in vitro neutralization assays were constructed as previously described . hepg2, huh-7 and cho cell lines were cultured with dulbecco's modification of eagle's medium (dmem) supplemented with 10% fetal bovine serum, 100 u/ml penicillin and 100 mg/ml streptomycin. raji cells were cultured with rpmi 1640 medium supplemented with 10% fetal bovine serum, 100 u/ml penicillin and 100 mg/ml streptomycin. these cells were cultured at 37˚c in 5% co2 humidified incubator. freestyle 293f cell line (rrid:cvcl_d603) was from life technologies, and the cells were cultured following manufacturer's instruction. the nk92-mi hcd16 cell line was generously provided by drs. zhang and li (beigene) . it was established by stably expressing fcgriiia (cd16, v158 allele) and fcrg chain in the parental nk92-mi cell line (atcc). all the cell lines used in this study had been tested for mycoplasma and that the tests were negative. two peptides derived from the pre-s1 domain of hbv genotype c were synthesized by scilight-peptide (beijing, china) with purity greater than 95%. nc36b, a peptide comprised of residues 4-36 of the pre-s1 domain of the hbv l protein with a biotin modification at its c-terminus. m47b, a lipopeptide comprised of amino acids 2-48 of the pre-s1 domain with a biotin modification at the c-terminus and a myristoylation modification at the n-terminus. a human non-immune scfv (single-chain fragment of variable domain: vh-linker-vl) antibody library (1.1 â 10 10 ) constructed from peripheral blood mononuclear cells of 93 healthy donors was used for selection against the synthesized nc36b or m47b pres1 peptides. briefly, the peptides were captured on streptavidin-conjugated magnetic m-280 dynabeads (thermo fisher, waltham, ma) and then incubated with 5 â 10 12 phage-scfv particles prepared from the library. for each peptide, two rounds of selection were performed. to recover high-affinity binders from the magnetic beads and to increase the diversity of the recovered phage-scfvs, two elution methods were used: peptide competition elution and conventional basic triethanolamine solution elution. subsequently, a total of about 2000 single clones were randomly picked and rescued to produce phage-scfvs in the bacterial culture supernatant. these clones were screened for specific binding to nc36b and/or m47b by elisa. more than 90% of the clones screened were positive for one or both peptides. clones that bound to nc36b and/or m47b with an optical density at 450 nm >1.0 were selected for further antibody gene sequencing. the genes of the variable heavy chain (vh) and light chain (vl) of the selected clones were sequenced. a total of 109 clones with unique sequences were identified. these clones were either produced as purified phage-scfv particles or converted to full-length human igg1s and then analyzed for binding to the pres1 peptides by elisa again, and evaluated in hbv and/or hdv neutralization assays. at the end, top six clones were selected based on their binding and hbv/hdv neutralization activities. preparation and purification of phage-scfvs for elisa or neutralization assays the phage-scfvs in the supernatant of 10-30 ml bacterial cultures were precipitated and concentrated with peg/nacl (sui et al., 2008) . the physical particle concentrations of the peg-precipitated phage-scfvs were then quantified by spectrophotometry based on the uv absorption spectra of purified filamentous phages. the binding or viral neutralization activities of different phage-scfvs were then evaluated in elisa assays or in cell-based viral neutralization assays, in a manner similar to the assays used for their full-length igg1 form. the coding sequences of the vh and vl of a scfv were subcloned, respectively, into a human igg1 heavy chain (hc) expression vector and a light chain (lc) expression vector. to generate full-length higg1 antibodies, 293f or 293 t cells were co-transfected transiently with the two expression plasmids (hc +lc plasmids) at a 1:1 ratio. 3-5 days after transfection, the cell culture supernatant was harvested for purification of igg1 via protein a bead affinity chromatography. elisa binding assay 1 mg/ml of streptavidin (sigma aldrich, st louis, mo) in phosphate buffered saline (pbs) was coated onto u-bottom 96-well plates (nunc, maxisorp, denmark) at 100 ml per well, then incubated at 4˚c overnight or 37˚c for 1 hr. biotin-labeled peptides (nc36b or m47b) at various concentrations in pbs (100 ml per well) were then captured onto the plates by incubating at 30˚c for 1 hr. for phage-scfv based elisa, serially diluted phage-scfvs in pbs containing 2% nonfat milk were added to each well at 100 ml per well. specifically bound phage-scfvs were then detected by adding hrp-conjugated mouse anti-m13 antibody (ge healthcare, chicago, il) and incubating for 1 hr at 30˚c. after each incubation step, the elisa plate was washed six times with pbs-t solution (0.05% tween 20 containing pbs) at a volume of 300 ml of wash solution per well per wash. following hrp-conjugated antibody incubation, the elisa signal was developed with tmb substrate (sigma) for 5-10 min at room temperature. the reaction was then stopped by adding 50 ml of 2m h 2 so 4 per well. the colorimetric absorbance was measured at 450 nm, with automatic subtraction of the reference absorbance at 630 nm by use of a microplate reader (bio-rad, hercules, ca). for full-length higg1-based elisa, the method was basically the same as described above for the phage-scfvs, with the exception that the bound antibodies were detected by hrp-conjugated mouse anti-human igg fc antibody (pierce-thermo fisher scientific, waltham, ma). the competition elisa assays were performed in a manner similar to the aforementioned binding elisa assays, except that the tested abs were incubated with captured peptide antigen in the presence of competition peptides. briefly, different short peptides at serially diluted concentrations were mixed with 0.5 nm (75 ng/ml) of igg1 antibody and added to the elisa plates to compete for the binding of abs to the captured peptides on the elisa plates. the amino acid sequence of 59c corresponds to aa (à10~48) of pres1 of genotype c. 2h5-scfv and 59c were co-expressed in e. coli. the complex was purified with immobilized metal ion affinity chromatography using ni-nta agarose beads (qiagen, germantown, md), followed by size exclusion chromatography with a superdex s200 10/300 column (ge healthcare). the purified 2h5-scfv/ 59c complex was then concentrated and crystallized at 20˚c using the hanging drop vapor diffusion method by mixing 1 ml of protein (29 mg/ml in 10 mm tris-hcl ph 8.0 and 100 mm nacl) and 1 ml of reservoir solution containing 2.8 m sodium acetate, ph 7.0. needle-shaped crystals appeared after 10 days. the crystals were flash cooled in liquid nitrogen. the x-ray diffraction data were collected at beamline bl17u of the shanghai synchrotron radiation facility and processed by hkl2000 (otwinowski and minor, 1997) . the structure was determined at 2.5 å resolution by molecular replacement in phaser mccoy, 2007) using vh and vl derived from the structure of the herceptin-fab complex (pdb 3h0t) (jordan et al., 2009 ) as a starting model. the initial model from molecular replacement was further refined in phenix (adams et al., 2010) and manually rebuilt with coot (emsley and cowtan, 2004 ). the final model includes 220 residues of 2h5 scfv and residues 20-27 of the 59c peptide. rampage analysis showed that 94.7% of the residues are in the favored region and 5.3% of residues are in the allowed region (lovell et al., 2003) . recombinant hbv viruses of genotype d and hdv viruses with genotype d envelopes were produced by plasmid transfection of huh-7 cells (yan et al., 2012; sun et al., 2016) . the serum derived hbv viruses (genotype b and c) were from de-identified blood remnant samples of hbv patients. the neutralization assays were performed as previously described (yan et al., 2012; yan et al., 2014) , with minor modifications. for hbv infection of hepg2-hntcp cells, the cells were cultured in pmm medium (yan et al., 2012) for 24 hr in 48-well plates (~5â10 4 cells/well) or in 96-well plates (~2â10 4 cells/well) prior to viral infection. about 200 multiplicities of genome equivalents (mge) of hbv mixed with abs were inoculated with hepg2-hntcp cells in the presence of 5% peg8000 and incubated for 16 hr. cells were then washed three times with media and maintained in pmm. the cell culture medium was changed with fresh pmm medium every 2 days after the first medium change. at 3, 5, and seven dpi, the culture supernatants were collected and tested for hbv-secreted viral antigen hbsag and/ or hbeag with commercial elisa kits (wantai, china). the levels of hbeag and/or hbsag were used to evaluate the hbv neutralization activity of the antibodies. data were normalized to the virus infection control and ic 50 values calculated from nonlinear regression curve fitting using graphpad prism six software. for hbv neutralization assays using phh cells, plasma-derived hbv (genotype b) were used. the neutralization assay was performed in 48-well plates with phh cells harvested from humanized frg mice. the phh cells were infected with 200 mge of hbv in the presence of 2h5-a14, an isotypematched control ab, or hbig at various concentrations. 16 hr after infection, the medium was changed to maintenance culture medium. cell culture supernatants were harvested every 2 days for measuring hbsag levels. the hbsag level in serum samples was determined using a commercial kit (autobio-cl0310, china). for hdv infection, the hepg2-hntcp cells were infected similarly as with the hbv infection, but with about 500 mge of hdv viruses. abs were mixed with viruses and then inoculated with the cells. at seven dpi, hdv-infected cells were fixed with 100% methanol at room temperature for 10 min. intracellular delta antigen was stained with 5 mg/ml of fitc-conjugated 4g5 (a mouse anti-hdv delta antigen monoclonal antibody) and nuclear delta antigen was stained with dapi. images were captured with a nikon eclipse ti fluorescence microscope or a zeiss lsm 510 meta confocal microscope. the neutralization activity against hdv was determined based on the stained delta antigen amount (number of infected cells) and staining intensity. the vl gene of 2h5 was cloned into a phagemid vector containing a repertoire of non-immune vh genes (~1â10 10 ) derived from 93 healthy donors. the constructed library had a size of 1.1 â 10 9 . the library selection was done similarly as described above for antibody library selection of anti-pres1 antibodies. only one round of selection was performed. a total of 576 individual clones were then randomly picked and screened by elisa. positive clones were sequenced to identify abs with unique sequences. kinetic analyses of 2h5-a14 or 2h5-a14-dana mutant binding to fcgrs were performed on a biacore t200 (biacore, ge healthcare) at 25˚c similarly as described above. the fcgrs were expressed in 293f suspensions cells and purified by immobilized metal affinity chromatography using the his 6 tag fused to the c-terminus of fcgrs. proteina/g (pierce, thermo fisher) was covalently attached to individual flow cell surfaces of a cm5 sensor chip by amine coupling using an amine coupling kit (biacore). antibodies at optimal concentrations were then captured on the chip to ensure the binding occurred as a homogenous 1:1 langmuir interaction. the analyte fcgrs were then injected over each flow cell at serial diluted (2-fold) concentrations. a buffer injection served as a negative control. upon completion of each association and dissociation cycle, surfaces were regenerated with 10 mm ph2.0 glycine-hcl solution. the association rates (ka), dissociation rates (kd), and affinity constants (kd) were calculated using biacoret200 evaluation software. for low affinity interactions, the starting fcgr concentration was from 4000 to 8000 nm, whereas for high-affinity interactions that was from 100 nm. the fitc-labeled pres1 peptide, m59-fitc, was mixed with 2h5-a14 or control ab at the indicated concentrations and then added to hepg2-hntcp cells. the final concentration of m59-ftic in the mixture was 100 nm. after incubation at 37˚c for 15 min, the cells were washed five times with william's e medium. the dapi-containing mounting solution was then added to the cells. the images were captured with a zeiss lsm 510 meta confocal microscope. the nk92-mi hcd16 cells were used as effector cells in these assays; these cells express fcgriiia (cd16, v158 allele) and fcrgchain. two types of cells were used as target cells: a cho cell line (cho-59c) stably expressing the epitope of 2h5-a14 (59c) and hbv-infected hepg2-hntcp cells. construction of cho-59c cell line: an expression plasmid was first constructed by inserting the pres1-59c coding dna into the c-terminal (ct) extracellular domain of the vamp2 gene that encodes a single-pass type iv transmembrane protein. the nt of vamp2 is on the cytoplasmic side of the membrane. its ct extracellular domain only contains two amino acids. the 59c was added to the ct end of vamp2; there was a 12aa long linker between 59c and vamp2. the expression plasmid was then transfected into cho cells, followed by facs sorting of 2h5-a14-staining positive populations to obtain the stable cell line, cho-59c. the cho-59c-mut (d20a mutant in pres1-59c) stable cell line was similarly established and was used as a negative control cell line in the adcc or adcp assays. d20a mutant in pres1 knocked out the binding to 2h5-a14. m1q mab, which recognizes the d20a mutant form of 59c, was used for the facs sorting of d20a mutant expressing cells. the hbv-infected cells were hepg2-hntcp cells infected with hbv for 5 days before use in the adcc assays. for the adcc assays, target cells were washed and resuspended in rpmi +5% fbs medium, and plated at 10000 cells/well in u-bottom 96-well plates and incubated briefly with various concentrations of 2h5-a14 or 2h5-a14-dana. the effector cells were then added (60000 cells/well resulting in a raito of e:t = 6:1) to the wells containing the target cells and antibodies, and incubated for 6 hr at 37˚c. cytolysis was determined by lactate dehydrogenase (ldh) release following the instructions of a cytotox 96 non-radioactive cytotoxicity assay kit (promega, fitchburg, wi). percentage cytotoxicity was calculated following the manufacturer's instruction. adcc and adcp by human macrophages. human pbmcs were differentiated by 20 ng/ml macrophage colony-stimulating factor (m-csf) (peprotech, rocky hill, nj) for 9 days before use as effector macrophages. suspension 293f-59c and 293f-59c-mut cells were used as target cells for both adcc and adcp assays. these cells were similarly established as cho-59c and cho-59c-mut, except they were not stable transfectants but transiently-transfected cells. they were harvested and used as target cells at 30-48 hr after transient transfections. for both assays, the target cells (2 â 10 5 cells/well) were incubated with 10 mg/ml 2h5-a14-wt or 2h5-a14-dana at rt for 10-30 mins and then added to the differentiated macrophages (~2â10 5 cells/well, resulting in a e:t ratio of 1:1) at 37˚c for 2 hr. for adcc, the cytolysis was determined by lactate dehydrogenase (ldh) release following the instructions of a cytotox 96 non-radioactive cytotoxicity assay kit. for adcp, the target cells were labeled with cfse according to the manufacturer's protocol (life technologies) prior to incubation with antibodies. phagocytosis of cfse-labeled target cells by macrophages was recorded with a zeiss pascal confocal system, and the phagocytic index was determined as the number of cfse-positive cells per 100 macrophages. about 200 macrophages were counted per sample. to prepare mouse bone marrow-derived macrophages (bmdms), mouse bone marrow cells were flushed with a syringe from the tibia and femurs of c57bl/6 mice into dmem medium. cells were collected and washed by pbs and filtered through a 40 mm cell strainer. to differentiate the cells into bmdms, they were resuspended in dmem medium supplemented with 15% l929 (secreting granulocyte/macrophage colony-stimulating factor, gm-csf [englen et al., 1995] ) cell culture medium and cultured for 3 days without replenishing or changing medium. suspension 293f-59c or 293f-59c-mut cells were used as target cells and labeled with cfse. the differentiated bmdms were labeled with a 1:200 dilution of anti-mouse f4/80-alex fluor647 (thermo fisher, clone bm8) prior to incubation with target cells. the cfse labeled target cells were plated at a density of 2 â 10 5 cells/well and incubated with 10 mg/ml 2h5-a14-wt or 2h5-a14-dana at rt for 15 mins and then added to the differentiated and labeled bmdms (~2â10 5 cells/well, resulting in a e:t ratio of 1:1) at 37˚c for 2 hr. phagocytosis of cfse-labeled target cells by macrophages was recorded with a nikon a1r confocal microscope. target cells were washed and resuspended in rpmi medium (without fbs) containing testing antibodies (10 mg/ml) and plated in a 96-well u-bottom plate at 2.5 â 10 4 cells/well. complement sera derived from rabbit or human (sigma-aldrich) were then added to the wells containing the target cells and antibodies, the final sera concentration was 10%. after 2 hr (for rabbit serum) or 4 hr (for human serum) incubation at 37˚c, the supernatants in each well were recovered and detected ldh release using a cytotox 96 ò non-radioactive cytotoxicity assay kit (promega). the cytolysis was calculated following the instructions of the kit. rituximab or gc33 were used as systemic positive controls. rituximab (anti-cd20) is commercial antibody used in clinic for treating lymphoma. gc33, a chimeric antibody against gpc3, was expressed and purified using the same method as described for full-length human igg1 antibody. they both are capable of inducing cdc against their target cells (nakano et al., 2009; van meerten et al., 2006) . 2h5-a14 has the same fc region of human igg1 as rituximab and gc33. prophylactic efficacy studies of 2h5, 2h5-a14 and 2h5-a14-dana mutant against hdv infection in mouse models with humanized ntcp. human ntcp transgenic c57bl/6 mice (hntcp-tg) were used for evaluating 2h5 ab ( figure 1f ). the ntcp-edited fvb mice (residues 84-87 of mntcp replaced by their human ntcp counterparts) were used for evaluating 2h5-a14 ( figure 3a) and for comparing the effect of 2h5-a14 and 2h5-a14-dana ( figure 5a ). for all experiments, homozygotes at 8-9 days after birth were used. animals were hosted in the animal facility of nibs, beijing. the experiments were conducted following the national guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after approval by the iacuc at nibs. antibodies at the indicated concentrations were administrated 1-2 hr prior to hdv viral challenge. at day six after hdv challenge, mice were sacrificed and liver tissues were harvested and frozen liquid nitrogen immediately. the liver tissues were then homogenized and lysed. trizol reagent was used to extract total rna. the rna samples were reverse transcribed into cdna with a prime script rt-pcr kit (takara, japan). cdna obtained from 20 ng of rna from each sample was used as the template for qpcr to quantify both the number of total hdv rna and hntcp mrna copies with abi fast 7500 real time system instrument (applied biosystems, waltham, ma) (he et al., 2015) . the gapdh mrna was also quantified and used as an internal reference control for the qpcr assays. the hdv total rna qpcr primers are: hdv-1184f: 5 0 -tcttcctcggtcaacc tctt-3 0 ; hdv-1307r: 5 0 -acaaggagaggcaggatcac-3 0 . the hntcp transgene qpcr primers are: forward primer: 5 0 -gcttctcctcattgccatatttt-3 0 ; backward primer: 5 0 -gggagcagtcc tcccct-3 0 . the edited chimeric m-hntcp qpcr primers are: forward primer: 5 0 -ggtctttcggc tgaagaac-3 0 ; backward primer: 5 0 -catggccagggtgaagagg-3 0 . the hdv rna or ntcp mrna copies were calculated with standard curves generated from samples with known copy numbers. a mouse hbv infection model was previously established using frg (fah -/-rag2 -/-/il2rg -/-) triple knock-out mice transplanted with human hepatocytes (bissig et al., 2010b ) (hfrg). the hfrg mice were generated commercially (yecuris, tualatin, or) as described previously (bissig et al., 2010b) . figure 3b -d and figure 3 -figure supplements 1-2, the study was carried out at the bsl2 facility of wuxi apptec using approved iacuc protocols. human hepatocyte-repopulated frg mice with serum human albumin levels around 10.0 mg/ml were used for hbv challenge. the hbv virus (genotype b, hbeag negative) was obtained from an hbv-infected patient plasma sample with written consent. a total of 20 hfrg mice were challenged with high viral dose at 10 9 ge hbv viruses in 200 ml per mouse by tail vein injection on day 0. all the animals were monitored on a daily basis for body weight changes and for clinical signs of viral infection for the duration of the in vivo study. for the 2h5-a14 prophylaxis group, five mice were injected with 2h5-a14 at 15 mg/kg by a single ip administration one day prior to hbv virus challenge. for the 2h5-a14 and hbig treatment groups, the treatment started on day five post infection and lasted until day 21. 2h5-a14 or hbig were administrated every three days by ip injection at 20 mg/kg and 40 iu/kg (72 mg/kg), respectively. for both the prophylaxis and the treatment models, blood samples were collected at different time points. these samples were used for quantifying viral hbsag, hbv dna, and rna copies in sera. the mice were sacrificed at the end of the experiment at 37 dpi, and the liver tissues from all mice were harvested. one portion of the liver tissues was frozen in liquid nitrogen immediately upon harvest to be used for analysis of hbv dna, rna, and cccdna. the remaining portion of the liver sample was fixed with 4% pfa in neutral pbs and paraffin-embedded for ihc analysis of hbsag and hbcag. for comparing the effect of 2h5-a14 and 2h5-a14-dana in hbv-infected hfrg mice ( figure 5b -g, and figure 5 -figure supplement 1), the study was carried out at nibs's bsl2 animal facility according to approved iacuc protocols. the hfrg mice used this experiment were purchased from yecuris. these mice had serum human albumin levels around 2.0-3.0 mg/ml. the hbv virus (genotype d) was prepared from hepde19 cells (sells et al., 1988) . a total of nine hfrg mice were challenged with high viral doses (2 â 10 9 ) mge hbv per mouse by tail vein injection on day 0. all the mice were monitored on a daily basis for body weight changes and for clinical signs of viral infection for the duration of the in vivo study. pbs, 2h5-a14, or 2h5-a14-dana was administrated by ip starting on day 33 post infection, twice a week until day 60, n = 3 for each group. antibodies were given at 5 mg/kg. blood samples were collected periodically until dpi 89 and used for quantifying viral hbsag as described above and hbv dna copies in sera. one mouse in the pbs control group was found dead with an undetermined cause at dpi 83 and excluded for blood collection and liver tissue collection at the end of the experiment (dpi 89). the rest eight mice were euthanized at dpi 89, and the liver tissues were harvested. one portion of the liver tissues was frozen in liquid nitrogen immediately upon harvest to be used for analysis of hbv viral dna. the remaining portion of the liver sample was fixed with 4% pfa in neutral pbs and paraffin-embedded for ihc analysis of hbsag. hbsag levels in mouse serum samples were measured using commercially available kits (autobio diagnostic co). serum samples were diluted 100 to 5000 times depending on hbsag levels in the samples. quantification of hbv dna copies in serum samples 1 ml serum was diluted with 8.5 ml ddh 2 o, to which was added 0.5 ml1m naoh. samples were mixed well and incubated 100˚c for 10 min. then, 2 ml was taken to use as a qpcr template. the qpcr was performed with a sybr premix ex taq kit (takara) using hbv dna specific primers: 5'-gagtgtggattcgcactcc-3' (forward) and 5'-gaggcgagggagttcttct-3' (reverse) using an abi 7500 fast real-time system instrument (applied biosystems). the viral dna copies were calculated based on a standard curve generated from samples with known copy numbers. total rna were extracted from 1 ml of each serum sample diluted in 199 ml of 0.9% nacl solution using a tianamp virus dna/rna kit (tiangen). the extracted rna were treated with dnase i and then reverse-transcribed into cdna with a primescript rt kit (takara) in a 20 ml reaction. 2 ml of cdna was used as a template for qpcr as described previously (yan et al., 2012) . primers hbv1805f: 5'-tcaccagcaccatgcaac-3' and hbv1896r: 5'-aagccacccaaggcacag-3' were for hbv-specific rna. chimeric mouse livers fixed with 4% pfa in neutral pbs and paraffin-embedded were processed for ihc analysis of hbsag and hbcag using a standard ihc protocol. for figure 3d and figure 3 -figure supplement 2, intrahepatic hbsag and hbcag were detected using the specific goat polyclonal anti-hepatitis b virus surface antigen (abcam, uk), rabbit polyclonal anti-hepatitis b virus core antigen (dako, denmark), donkey polyclonal secondary antibody to goat igg-h and l(ap) (abcam), and donkey polyclonal secondary antibody to rabbit igg-h and l(hrp) (abcam), respectively. an ap-red substrate kit (zhongshan jinqiao company, china) and immpact dab peroxidase substrate (vector laboratories,burlingame, ca) were used for staining. for figure 5e and figure 5 -figure supplement 1, intrahepatic hbsag were detected using a mouse mab against hepatitis b virus surface antigen (clone: 56a1), followed by hrp conjugated anti-mouse igg1 (boster biological technology,pleasanton, ca). dab substrate (brown) kit (boster) was used for developing the hbsag staining signals, nuclei were stained by hematoxylin. consecutive tissue sections were immunostained using a human-specific cytokeratin-18 (hck18) mouse monoclonal antibody, clone dc10 (dako) to visualize human hepatocytes. dab substrate (blue violet) kit (boster) was used for developing the hck18 staining signals, nuclei were stained using nuclear fast red (boster). approximately 100 mg of frozen liver tissue were ground in a mortar and lysed in a lysis buffer (20 mm tris, 0.4 m nacl, 5 mm edta, 1% sds, ph = 8.0) in the presence of proteinase k (qiagen) at 56˚c overnight. after rnase a digestion, the cell lysate was extracted twice with phenol:chloroform: isoamyl alcohol (25:24:1, ph = 8.0). the extracted genomic dna was precipitated with equal volumes of isopropanol at à20˚c overnight. the dna pellet was washed with 70% ethanol and dissolved in te buffer (10 mm tris-hcl, 1 mm edta, ph = 8.0), and digested with hindiii (neb, ipswich, ma) before being analyzed by southern blotting. the dna samples of normal mouse livers were prepared as well and used as negative controls in the southern blotting. for figure 3 figure supplement 1, about 500 ng of the prepared dna samples were loaded one sample per lane and separated on 1.2% agarose gels and then transferred to a positively charged nylon membrane. the membranes were probed with dig-labeled full-length hbv dna to detect the hbv sequence, and membrane hybridization was detected using x-ray film. for figure 5f , about 20 mg of the prepared dna was separated on 1.2% agarose gel electrophoresis and transferred onto amersham hybond-n + membrane (ge healthcare). the loading amount for each sample was normalized to the copy number of hntcp dna quantified by qpcr in the same dna sample (see details in the method section of 'quantification of hbv cccdna in mouse liver tissues by qpcr'). for southern blot, the hybond-n + membrane was crosslinked and subsequently probed with [a-32p] dctp (250 mci, perkin elmer)-labeled hbv genotype d (accession number: u95551.1) linear full-length genomic dna. after overnight hybridization in perfecthyb plus hybridization buffer (sigma) at 65˚c, the membrane was washed and exposed to carestream x-omat bt film (xbt-1). 100 pg each of 3.2 kb, 2.1 kb and 1.7 kb hbv dna fragments prepared by pcr amplification of a plasmid containing 1.0 copies linear hbv genotype d genome was used as dna marker. the cccdna was selectively extracted using a protein-free hirt method (hirt, 1967) as previously described, with modifications. briefly, to selectively extract hbv cccdna, 100 mg of frozen liver tissue was lysed with 7.3 ml lysis buffer at room temperature for 30 mins, followed by addition of 2 ml of 2.5 m kcl and incubation at room temperature overnight. the lysis buffer was not supplemented with proteinase k, containing 1 mm tris-hcl, ph 7.5, 10 mm edta, 150 mm nacl, 10% sds. the lysate was then clarified by centrifugation at 12,000 g for 30 min at 4˚c and extracted with phenol and phenol:chloroform. dna was precipitated with equal volume of isopropanol and finally dissolved in te buffer (10 mm tris, 1 mm edta, ph8.0). for southern blotting, the dna samples were separated on a 1.2% agarose gel and then transferred to a nylon membrane (hybond-n+; ge-rpn2250b) using a standard neutral transfer procedure. 3.2 kb, 2.0 kb, and 1.3 kb hbv fragment dna was also run on the same agarose gel to serve as the molecular marker. the membrane was probed with the dig-labeled hbv dna probe. hybridization was carried out in 10 ml of hybridization buffer with a 1 hr prehybridization at 60˚c and overnight hybridization at 60˚c, followed by 2 â 5 min wash with 2 â ssc, 0.1% sds at room temperature and 2 â 5 min wash with 0.2 â ssc, 0.1% sds at 60˚c. the membrane was incubated with blocking buffer for 30 min, followed by 30 min incubation with antibody solution. after equilibration with detection buffer for 5 min, the membrane was rinsed with cdp-star and then exposed to x-ray film at room temperature. quantification of hbv cccdna in mouse liver tissues by qpcr hbv cccdna was quantified by qpcr using a protocol as previously described , with modifications. briefly, the frozen liver tissues were homogenized and then lysed in a lysis buffer (20 mm tris, 0.4 m nacl, 5 mm edta, 1% sds, ph = 8.0) in the presence of proteinase k (qiagen). the total dna was extracted according to a standard phenol-chloroform extraction protocol. 5u (0.5 ml) plasmid-safe adenosine triphosphate (atp)-dependent deoxyribonuclease dnase (psad) (epicentre technologies, madison, wi) was used to digest 500 ng of total dna in 20 ml reaction volume, and incubated at 37˚c for 8 hr to remove the linear genomic and hbv replication intermediate dnas. the reaction was then incubated at 70˚c for 30 mins to inactivate the dnase. then, 2 ml of the digested dna for each sample was used as a qpcr template for quantification of intrahepatic hbv cccdna copies. hbv cccdna specific qpcr primers are: 5'-tgcacttcgcttcacct-3' (forward) and 5'-aggggcatttggtggtc-3' (reverse). the hntcp gene copy numbers, which represent the amount of human hepatocytes in the liver tissues of chimeric mice, were used to normalize the relative cccdna amount in the infected cells for each sample. for quantification of human ntcp gene copies, 50 ng of the undigested dna samples were used. human ntcp specific primers (annealing to the second intron of hntcp gene) are: 5'-tccaggagccactttcaccataa-3' (forward) and 5'-agcagggacaagtgtcagaacaga-3' (reverse). the amount of hbv cccdna or hntcp gene copies were calculated using the standard curves generated from the standard plasmids with known copy numbers. total rna was isolated from frozen liver tissues using trizol reagent. about 2 mg of total rna per sample was separated on a 1.2% formaldehyde-agarose gel and blotted onto positively charged nylon membrane. dig-labeled probes were prepared with a dig rna labeling kit (roche, switzerland). after hybridization, the membrane was washed and exposed to x-ray film. cloning and sequencing the l gene of hbv total genomic dna was isolated from frozen homogenized liver tissues collected from each individual mouse in the 2h5-a14 treatment group and the control group. l gene was amplified from the extracted dna by pcr using a set of specific primers. two pcrs were performed for each sample. the pcr products were then cloned by ta cloning. for each ta cloning, clones were randomly picked for sequencing the l gene. the coordinates and structural factors have been deposited into the protein data bank with accession code 5yax. phenix: a comprehensive python-based system for macromolecular structure solution robust expansion of human hepatocytes in fah-/-/rag2-/-/il2rg-/-mice innate and adaptive immune responses in chronic hepatitis b virus infections: towards restoration of immune control of viral infection hepatitis b: future curative strategies human liver chimeric mice provide a model for hepatitis b and c virus infection and treatment human liver chimeric mice provide a model for hepatitis b and c virus infection and treatment infectivity determinants of the hepatitis b virus pre-s domain are confined to the n-terminal 75 amino acid residues chronic hepatitis b: what should be the goal for new therapies? treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study the role of fc-fcgr interactions in igg-mediated microbial neutralization broadly neutralizing anti-hiv-1 antibodies require fc effector functions for in vivo activity myeloid cells as effector cells for monoclonal antibody therapy of cancer elimination of hiv-1-infected cells by broadly neutralizing antibodies the lytic potential of human liver nk cells is restricted by their limited expression of inhibitory killer ig-like receptors viraemia suppressed in hiv-1-infected humans by broadly neutralizing antibody 3bnc117 broadly neutralizing antihepatitis b virus antibody reveals a complementarity determining region h3 lid-opening mechanism inhibitory fc receptors modulate in vivo cytotoxicity against tumor targets a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins use of ly6g-specific monoclonal antibody to deplete neutrophils in mice broadly neutralizing hemagglutinin stalk-specific antibodies require fcgr interactions for protection against influenza virus in vivo coot: model-building tools for molecular graphics granulocyte/macrophage colony-stimulating factor is expressed and secreted in cultures of murine l929 cells preclinical evaluation of two human anti-hepatitis b virus (hbv) monoclonal antibodies in the hbv-trimera mouse model and in hbv chronic carrier chimpanzees sofosbuvir and velpatasvir for hcv genotype 1, 2, 4, 5, and 6 infection future anti-hbv strategies glycoengineered cd20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through cd16b more efficiently than rituximab tumor-associated macrophages promote invasion while retaining fc-dependent anti-tumor function host-virus interactions in hepatitis b virus infection primary human hepatocytes are protected against complement by multiple regulators antibody isotype-specific engagement of fcgamma receptors regulates b lymphocyte depletion during cd20 immunotherapy hbvdb: a knowledge database for hepatitis b virus modification of three amino acids in sodium taurocholate cotransporting polypeptide renders mice susceptible to infection with hepatitis d virus in vivo hepatitis d virus infection of mice expressing human sodium taurocholate co-transporting polypeptide glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity fc receptor but not complement binding is important in antibody protection against hiv selective extraction of polyoma dna from infected mouse cell cultures hepatitis delta virus immune-modulators to combat hepatitis b virus infection: from ifn-a to novel investigational immunotherapeutic strategies immune surveillance by the liver hepcidin revisited, disulfide connectivity, dynamics, and structure hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies natural killer cells for immunotherapy -advantages of the nk-92 cell line over blood nk cells intrinsic resistance of hepatocytes to complement-mediated injury infection process of the hepatitis b virus depends on the presence of a defined sequence in the pre-s1 domain the hepatitis b virus receptor present and future therapies of hepatitis b: from discovery to cure hepatitis b virus core protein interacts with cd59 to promote complement-mediated liver inflammation during chronic hepatitis b virus infection antiviral therapy for chronic hepatitis b viral infection in adults: a systematic review and meta-analysis structure validation by calpha geometry: phi,psi and cbeta deviation enhanced clearance of hiv-1-infected cells by broadly neutralizing antibodies against hiv-1 in vivo specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna nk cells: a double-edged sword in chronic hepatitis b virus infection phaser crystallographic software solving structures of protein complexes by molecular replacement with phaser lymphoma depletion during cd20 immunotherapy in mice is mediated by macrophage fcgammari, fcgammariii, and fcgammariv anti-glypican 3 antibodies cause adcc against human hepatocellular carcinoma cells novel mechanism of antibodies to hepatitis b virus in blocking viral particle release from cells processing of x-ray diffraction data collected in oscillation mode crosstalk between human igg isotypes and murine effector cells antiviral monoclonal antibodies: can they be more than simple neutralizing agents? dna polymerase k is a key cellular factor for the formation of covalently closed circular dna of hepatitis b virus hepatitis d virus: introduction and epidemiology hiv-1 antibody 3bnc117 suppresses viral rebound in humans during treatment interruption hiv-1 therapy with monoclonal antibody 3bnc117 elicits host immune responses against hiv-1 treatment of a patient with genotype 7 hepatitis c virus infection with sofosbuvir and velpatasvir estimations of worldwide prevalence of chronic hepatitis b virus infection: a systematic review of data published between hbv replication, pathobiology and therapy: unanswered questions hepadnaviruses. in: virology f. ields (ed). dm knipe. philadelphia: lippincott: pm howleywilliams & wilkins replicative intermediates of hepatitis b virus in hepg2 cells that produce infectious virions nk cells in hepatitis b virus infection: a potent target for immunotherapy high resolution mapping of the binding site on human igg1 for fc gamma ri, fc gamma rii, fc gamma riii, and fcrn and design of igg1 variants with improved binding to the fc gamma r involvement of neutrophils and natural killer cells in the anti-tumor activity of alemtuzumab in xenograft tumor models chimeric mice with humanized liver: tools for the study of drug metabolism, excretion, and toxicity broadening of neutralization activity to directly block a dominant antibody-driven sars-coronavirus evolution pathway structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses natural killer cell dysfunction in hepatocellular carcinoma and nk cell-based immunotherapy ntcp-reconstituted in vitro hbv infection system hepatitis b virus genotypes: global distribution and clinical importance viral hepatitis: past and future of hbv and hdv. cold spring harbor perspectives in medicine 5:a021345 complement-induced cell death by rituximab depends on cd20 expression level and acts complementary to antibody-dependent cellular cytotoxicity liposomes for specific depletion of macrophages from organs and tissues world health organization nk cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy the macromolecular crystallography beamline of ssrf an fcg receptordependent mechanism drives antibody-mediated target-receptor signaling in cancer cells viral entry of hepatitis b and d viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus towards an hbv cure: state-of-the-art and unresolved questions-report of the anrs workshop on hbv cure current status of immunomodulatory therapy in chronic hepatitis b, fifty years after discovery of the virus: search for the "magic bullet" to kill cccdna neutralization epitope responsible for the hepatitis b virus subtype-specific protection in chimpanzees hepatitis b virus x protein protects hepatoma and hepatic cells from complement-dependent cytotoxicity by up-regulation of cd46 prolonged suppression of hbv in mice by a novel antibody that targets a unique epitope on hepatitis b surface antigen we thank w chen, z cao, y li and x tian and all other members in the sui and li labs for their technical assistance and discussions. we also thank the nibs animal facility for their help in handling and care of mice, the nibs biological resource centre for dna sequencing, and the staff at the shanghai synchrotron radiation facility beamline bl17u for assistance in data collection of crystals. this work was supported by the ministry of science and technology, china (973 program: #2012cb837600 to js and #2014cb849600 to wl); the thousand young talents plan (china) to js; national natural science foundation of china (nsfc81525018 to wl, 31325007 to ky), and national science and technology major project (china) to wl (2013z â 09509102). this work was also supported by beijing municipal commission of science and technology. competing interests dan li: dl is a co-inventor of patent applications of antibodies reported in this study. wenhui li: wl is a co-inventor of patent applications of antibodies reported in this study. jianhua sui: js is a coinventor of patent applications of antibodies reported in this study. the other authors declare that no competing interests exist. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-318853-mxyxwkhx authors: sallie, richard title: replicative homeostasis ii: influence of polymerase fidelity on rna virus quasispecies biology: implications for immune recognition, viral autoimmunity and other "virus receptor" diseases date: 2005-08-22 journal: virol j doi: 10.1186/1743-422x-2-70 sha: doc_id: 318853 cord_uid: mxyxwkhx much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. rna polymerases (rna(pol)) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. thus, rna(pol )causes more morbidity and premature mortality than any other molecule. the extraordinary genetic heterogeneity defining viral quasispecies results from rna(pol )infidelity causing rapid cumulative genomic rna mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking rnapol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral rna is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. this mechanism – "viral receptor disease (vrd)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type ii diabetes mellitus, and some forms of obesity. viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations. many of the world's population suffer from acute and chronic viral infection. the two common types of chronic viral hepatitis (cvh), hepatitis b (hbv) and c (hcv) are major causes of death and morbidity; conservative esti-mates suggest 400 million people are persistently infected with hbv, while hcv may infect a further 200 million. annually, in excess of two million people will die from cirrhosis or liver cancer caused by cvh, and many more suffer chronic ill health as result. during the 20 years since the human immunodeficiency virus (hiv) was identified, perhaps 40 million people have become infected worldwide and each year about a million die from resulting immunodeficiency and consequent opportunistic infections, particularly tuberculosis, and other complications. poor countries bear a disproportionate burden of disease caused by these viruses that further exacerbate poverty through pervasive economic disruption and diversion of limited resources to healthcare and disease control. emerging viral pathogens including west nile virus (wnv), the sars coronavirus, endemic viruses like murray valley, japanese, and other encephalitis viruses, dengue and yellow fever, and seasonal influenza, hepatitis a (hav) and e (hev) cause enormous further morbidity and mortality, while pandemic outbreaks of virulent influenza strains remain a constant threat. together, these viruses probably kill more people every ten days than the boxing day tsunami. rna viral infections, including foot and mouth, bovine viral diarrhea virus (bvdv) and hog cholera virus (hchv), cause similar devastation of animal populations with enormous economic consequences. rna polymerases generate massive genetic variability of rna viruses and retroviruses that circulate within infected hosts as vast populations of closely related, but genetically distinct, molecules known as quasispecies. after translation, this genetic variability causes near-infinite antigenic heterogeneity, facilitating viral evasion of host defences. tuberculosis, malaria and other cellular pathogens also express broad cell-surface antigenic heterogeneity, generated by dna-dependent rna pol . thus, rna polymerases probably cause more morbidity and premature mortality in man, and other animals, and greater economic loss, than any other molecule. despite a depressing global epidemiology that strongly suggests otherwise, the immune system is thought to "control" viruses. what practical meaning does "immune control" have for the individual? there is no argument for hbv, and other viruses, high affinity antibody, generated by prior vaccination or other exposures and directed against neutralizing epitopes, will prevent hbv infection (excepting vaccine escape mutations [1, 2] ), in part by blocking viral ligand interaction with cell receptors, or that most patients exposed to hbv develop neutralizing antibodies (hbsab), clear hbsag from serum, and will normalize liver function long term. however, even patients who develop robust immune responses to hbv, defined by high-affinity antihbsab and specific antiviral cytotoxic t cell (ctl) responses, will have both "traces of hbv [3] ... many years after recovery from acute hepatitis" [3] and transcriptionally active hbv demonstrable in peripheral blood mononuclear cells (pbmcs) [4] . furthermore, occult hbv is detected in liver tissue of patients with isolated antihbc (i.e. hbsag/hbsab negative) [5] and in patients with hbsag-negative hepatocellular carcinoma [6] suggesting, at least some patients, hbv in may persist irrespective of any immune responses, implying long term latency and low level basal replication may be a survival/reproductive strategy for hbv. for most patients, acute hcv or hiv infection results in life-long viral persistence. although many patients develop immunological responses, including specific antibody and ctl reactivity to various viral antigens, these responses have little discernible impact on either hcv or hiv replication that occurs essentially unchecked at rates estimated between 10 10 and 10 12 virions per day [7, 8] , indefinitely, while progressive destruction of liver or immune cells proceeds, commonly resulting in cirrhosis or liver cancer (for hcv) or death from immune deficiency (for hiv). evidence that prior hcv infection confers no protective immunity against heterologous hcv infection in humans [9] or chimpanzees [10] or against either homotypic [11] or heterotypic [12] human reinfection, confirmation that active hcv infection persists long after either apparent spontaneous [13] or treatmentinduced [14] viral clearance, or that vaccines causing specific antiviral b and t cell responses fail to protect against infection in animals [15] , and that antibodies to hcv envelope protein e2 are only detected in animals with persistent infection [16, 17] , further undermines the potency of "immune control" and suggests, at least for patients with hcv, the definition of "control" may need to broadened significantly. based on observations that stronger specific cd4/cd8 immune responses with t-helper (th1) cytokine profiles are found more frequently in patients with self limiting viral infections than those who develop chronic viral carriage [18, 19] it is thought ability to mount robust adaptive immune responses predicts viral clearance while failure to do so results in chronic viral carriage [20] . however, detailed and very painstaking studies, albeit in small numbers of chimpanzees [21] and patients following antiviral therapy [22] , have failed to demonstrate any relationship between t cell responses and viral clearance. although development of th1and other immune responses are certainly temporally and, probably, causally related to reduced viral replication and viral clearance the assumed direction of causality (immune response -> reduced viral replication), is not proved by the fact those responses develop, post hoc ergo propter hoc, as comforting a conclusion as it may be to reach. the first part of this paper explores the impact of rna pol fidelity on quasispecies behaviour, specifically in mediating immune avoidance during acute hcv infection. we suggest the primary event causing reduction in viral replication is inhibition of rna pol processivity by variant viral proteins, specifically envelope and envelope-related proteins. we also suggest that immune responses to viruses are thwarted initially by broad antigenic diversity generated by low rna pol fidelity but develop, when they do, after viral replication falls (because of reduced rna pol processivity) and polymerase fidelity increases -linked events that occur because of replicative homeostasisthus restricting antigenic diversity sufficiently to permit focused immune recognition. we further suggest immune responses strategically exploit replicative homeostasis to force viruses to reveal critical dominant antigenic epitopes, facilitating progressively more focused immune responses. the second part explores the ineluctable consequence of viral rna quasispecies: that is, translation of rnas into protein quasispecies with a spectrum of phenotypes and unpredictable properties, among which may be disruption of the cell surface receptors that viruses co-opt for cell entry. this innate property of viral quasispecies may explain a wide variety of diseases apart from viral autoimmunity. acute hcv and hbv infection have characteristic kinetics of viral replication, adaptive immune responses, and cause predictable tissue injury, reflected in elevated serum aminotransferases. these kinetic and transaminase responses are summarized schematically for patients with persistent infection (figure 1) [23]. initial hcv replication is very rapid and viral load increases exponentially until about week 4, at which point viraemia increases more slowly, and asymptotically, towards ~10 7 genome equivalents (geq)/ml by weeks 7-8 (these kinetics alone suggesting competitive inhibition of rna pol ). this exponential increase of viral rna in serum reflects explosive dissemination of virus in tissues, detectable by in-situ hybridisation throughout hepatocytes, including the nuclei, within days of infection [24] . viral replication declines rapidly from weeks 10-11 to weeks 14-16 falling by 10 2-3 geq/ml but lower level (~10 5 geq/ml) fluctuating replication persists, generally indefinitely, thereafter. by contrast, neither hbv dna nor hbv antigens are detectable in either viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection figure 1 viral replication, immunological and tissue injury kinetics following acute hcv and hbv infection. data summated from figure 1 [29] and modified to represent typical patients with chronic viral persistence. note: a) high level hcv replication for 6-8 weeks prior to any immune responses, b) onset of humoral immune response well after down-regulation of viral replication [34] , and c) transaminase peaks occurs ~ 2weeks later. both hbv and hcv are non-cytolytic and viral clearance from hepatocytes, as well as hepatocyte injury, thought to be immune mediated. however, for both hbv and hcv the brisk fall in viral replication following acute infection paradoxical hcv replication kinetics figure 2 paradoxical hcv replication kinetics. if host immune clearance forces (i c , black arrows) reduce viral replication acutely (point a), then they must exceed viral expansive forces (v e , grey arrows) at that point. at equilibrium (e.g. points b through d), viral concentrations (-) and, therefore, viral forces, have fallen by 10 2-3 hence, immune forces i c must fall by >10 2-3 from a to b for equilibrium to develop. there is no evidence this happens. http://www.virologyj.com/content/2/1/70 precedes the peak of transaminase rise by at least two weeks (figure 1). if falling viral replication is due to adaptive immune responses causing hepatocyte lysis the transaminase peak should either precede or be coincident with falling replication. this temporal relationship is also inconsistent with the belief immune factors cause the falling replication seen during acute hcv or hbv, and is analagous to non-cytolytic reductions of viral replication observed for both hbv and lymphocytic choriomeningitis virus (lcv) experimentally, that suggested either [unspecified] antiviral mechanisms are operative [30,31], or that auto-inhibition of rna pol by viral mechanisms (replicative homeostasis) occurs [28] . however, if other non-cytopathic host anti-viral mechanism(s) are responsible, the kinetic paradox implies their potency falls significantly between points a and b. hepatitis c replication kinetics and their relationship to immune responses are well documented [32,33] but reveal an unexplained paradox. despite high level viral replication, adaptive cellular immune responses to hcv are completely undetectable for at least 7-10 weeks [33] after infection, while humoral responses are rarely detected before 12-14 weeks [34] , and in some patients [35] , and some chimpanzees [36] , are never detected at all. an exhaustive and very careful review of the clinical and experimental data relating adaptive immune response and hcv replication kinetics has been published recently [29] . seeking to rationalize the enigma posed by a complete lack of immune responses to hcv replication of ~10 6-7 geq/ml at week 6 but [variable] immune responses to replication at ~10 5 geq/ml after week 14, the authors conclude "..[the data]...appear[s] to be consistent with the interpretation that hbv and hcv are ignored by the adaptive immune system for about 2 months after primary infection" and "[in hcv].. the adaptive response seems to really ignore for several weeks a substantial quantity of virus (at least 10 6 copies/ml)..". this is certainly an accurate synthesis of an extensive and highly complex literature but does it make any sense? if adaptive immune responses really ignore high level hcv replication for two months, as suggested, then the following mechanism(s) are implied: a) an accurate mechanism for prompt detection of infection; b) a timing mechanism; c) a trigger mechanism for immune responses independent of any viral factor (given levels of virus are greater before immune recognition than afterwards the trigger for immune response must be either non-viral or falling (!) viraemia); and, as cytomegalovirus (cmv)-specific cd4(+) t cell responses arise within 7 days of cmv infection [37] ; d) a mechanism allowing the immune system to differentiate hcv from cmv and other viruses (and reasons to do so). while possible, this seems unusually inelegant and pointlessly counterproductive, especially as events soon after infection probably determine whether virus is cleared or chronic infection develops. it is much more likely that adaptive cellular or humoral immune responses do not develop in the first 6-7 weeks of hcv infection simply because the virus isn't "seen". why should hcv replicating at 10 6-7 geq/ml at week 6 be invisible to the immune system but visible when replicating at 10 5 geq/ml long term? dissection of this problem requires explicit analysis of what is being measured and how. assay of hcv rna and detection of hcv by immune responses measure two quite different things. quantitation of hcv is typically performed by branch-chain cdna assay (bdna) or quantitative pcr (qpcr) using probes or primers complementary to conserved 5'untranslated (5'utr) hcv rna sequences. immune responses to hcv typically "measures" envelope proteins translated from envelope-encoding rna (eerna) sequences and are directed at specific antigenic amino acid sequences and polypeptide conformations, not total viral envelope protein concentrations. while concentrations of 5'utr rna will be proportional to eerna concentrations in any given sample, they may not be identical for two reasons; i) rna transcription may prematurely terminate making 5'utr rnas relatively more prevalent than eernas and ii) hcv 5'utr is highly conserved, while eerna s are less constrained, making hybridization efficiencies of pcr primers or bdna probes greater for 5'utr rnas than for the population of eernas, causing relative under-estimation of true envelope rna concentration 1 . nonetheless, as 5'utr hcv rna concentrations will be proportional to eerna concentration, the question remains; why should envelope proteins translated from eerna sequences present at concentrations corresponding to ~10 5 5'utr geq/ml at 16 weeks be visible immunologically, but envelope proteins derived from eerna sequences corresponding to ~10 6-7 5'utr geq/ml at 4-6 weeks remain unseen? quasispecies biology, specifically variable rna pol fidelity, replicative homeostasis, and sequence-specific requirements for both genetic and immunological detection suggest an answer. rna viruses replicate by copying antigenomic templates, a process catalysed by rna pol , an enzyme lacking fidelity or proof reading function [38] [39] [40] [41] . theoretically, an rna viral genome like hcv (about 9200 bases) could assume any of 4 9200 (about 8.95 × 10 5538 ) possible sequence combinations exceeding, by some margin, population estimates of protons in the known universe (about 10 80 ), meaning the potential complexity of rna viral quasispecies is infinite, for all practical purposes. an rna pol fidelity rate of 10 -5 errors per base copied predicts at least one and as many as 10 (estimated for hiv) [39] genomic mutations will be introduced during each cycle of replication. furthermore, as hcv replication results in synthesis of ~10 12 virions per person per day [8] , on average, mutations will develop at each genomic locus ~10 7 times/day, while the probability any two genomes synthesized consecutively will be identical is about 10 -6 . the sum effect is inexorable accumulation of genomic mutationsthat, by itself, should threaten replicative fitness because of muller's ratchet [42] -and progressive dilution of wildtype genomes (figure 3), processes that make long-term stability of rna virus quasispecies highly paradoxical [43] . as argued previously, a combination of selective genomic replication and variable rna pol fidelity, both mediated by replicative homeostasis, act together to prevent rna quasispecies extinction [28] . the phenotypic consequences of viral quasispecies biology may be more important. progressive divergence of genomic rna sequences away from wild-type sequences caused by rna pol infidelity generates a massive population of closely related, but genetically distinct, rna molecules (figure 3), an effect operative at all scales from each open reading frame (orf) to whole virus species. a quasispecies of orf rnas has but one inevitable outcome; translation of a quasispecies of viral proteins with a vast and highly variable spectrum of phenotypes, some subtly nuanced, others grossly defective. furthermore, mutations simplified, two dimensional clade diagram of hyperdimensional viral rna and protein sequence-space figure 3 simplified, two dimensional clade diagram of hyperdimensional viral rna protein sequence-space. because of rna pol (p) infidelity and müller's ratchet, mutations ( ) are introduced into each rna template synthesized, and progressively accumulate, resulting in an rna quasispecies with sequence progressively divergent from consensus sequence. translation results in a spectrum of proteins ( , , , etc.) with properties that also vary progressively from wild-type sequence ( ) to highly variant proteins ( , , etc.). some rnas will be so abnormal that translation or replication fails or is truncated ( ), while others will code for grossly defective proteins ( , etc.). that create new, or obliterate pre-existing, start or stop codons in a significant proportion of rnas, will cause translation of highly unusual and heterogeneous proteins, particularly during high-level viral replication, a phenomenon that may explain hbeag. viral quasispecies cannot, and will not, produce homogeneous proteins with predictable and consistent phenotypic and antigenic properties. while rna pol infidelity will cause progressive divergence of copied sequences away from wild-type or consensus sequences, the probability of any particular sequence arising will fall dramatically with increasing genetic distance from that consensus sequence (figure 4), allowing conceptual representation of the resulting genomic (and consequent phenotypic) diversity as a frequency distribution curve, with increasingly variant sequences surrounding a 'centre of gravity of replication', formed by wild-type sequences. viral quasispecies occupy hyperdimensional sequence-spaces, hence any physical representation is necessarily simplified, but because mutation away from wildtype sequences is equally probable in all directions, variant rna and protein frequencies will be normally distributed and the standard deviation (sd, σ) -insofar as 'normal' or 'standard' can be applied to a hyperdimensional space -of that distribution will be a function of rna pol fidelity; if rna pol is completely faithful, the rnas and proteins will be monoclonal and σ = 0; if rna pol has no fidelity, rna will be synthesised randomly, and all rna and consequent protein sequences will arise with equally probability, therefore σ = ∞. while viral rna and related protein sequences are theoretically unconstrained (at least before any consideration of functionality), the sequence specificities of any reagents used in their detection (bdna probes, pcr primers, mabs etc) are not, by definition, and their specificity and the efficiency with which they detect variant molecules will fall progressively the further those variant sequences are from the consensus sequence. a zone of 'reagent specificity' may therefore be defined probably encompassing wild type and some variant sequences, but there will exist some rna sequences and corresponding proteins of any quasispecies that are undetectable with these sequence-specific reagents. a threshold of detection of any assay (including immune detection) may similarly be defined; rna or protein sequences present at concentrations below this conceptual level being undetectable by that particular assay. the hcv "early replication" paradox now partially resolves; the 5'utr sequences are both highly conserved and common to virtually all rnas in the quasispecies, therefore, the 5'utr concentration -that is, the common measure of hcv viraemia -corresponds to the area under the frequency distribution curve. by contrast, envelope rna sequences (and related envelope proteins) are not so constrained and their relevant concentrations (i.e. whether or not that rna or protein sequence is detectable) corresponds to the frequency of that specific sequence in the quasispecies and that, in turn, depends on rna pol fidelity; if rna pol fidelity is low, the frequency or concentration of any particular rna or protein sequence will also be low and may be below the detection threshold, while increasing rna pol fidelity may increase sequence frequency [i.e. the concentration of specific proteins] above detection threshold. but why should specific eerna sequence frequencies -in other words, hcv rna pol fidelity -increase after week 8, facilitating adaptive immune responses? viral autoregulation, specifically replicative homeostasis, provides an answer. interactions among species, whether between humming birds and flowering plants, primitive viroids and prokaryotic cells or hcv and man, results in an unremitting process of adaptation and responsive counter-adaptation -in effect, a molecular arms race -for each species just to maintain ecological parity. the price of survival for a species is continual evolution. survival, for viruses, requires cell entry, a precondition long antedating necessity to evade more complex host defenses, including interferons and other cytokines and adaptive immune responses, while for cells, and complex cellular organisms, cell wall defenses, including receptor polymorphisms, form a principal barrier against viral invasion. viral survival -effectively meaning rna pol survival -on an evolutionary timescale, as argued previously [28, 44] , requires control of mutation and replication rates in a manner adaptively responsive to constantly changing biota and this implies dynamic linkage of rna pol fidelity and processivity with quasispecies phenotypic and antigenic diversity, meaning an autoregulatory linkage -replicative homeostasis -between rna pol fidelity and processivity and envelope proteins, as argued previously [28] . by definition, evolutionary co-adaptation occurs in response to adaptations in locally prevalent interacting species. natural selection for beak variation(s) in darwin's finches occurs as a consequence of concrete survival benefits these variations -mediating, for example, enhanced food harvesting interactions with other variable plant or animal species -confer to individual galapagos island birds, rather than any inexorable hypothetical 'improvement' in beak function for finches in general. if a species is widely distributed in space, but population mixing is slow or incomplete, locally prevalent interactions with other species will vary and regional genetic variations will arise and be maintained, hence progressive divergence from the original genotype (speciation) may result. for viruses, and their hosts, genetic variations -reflected in viral genotype and cell surface polymorphisms and resulting disease susceptibilities -would be predicted, and are observed [45] [46] [47] [48] [49] [50] , to have frequencies that vary geographically. consider the following; an enzyme (e) functioning in a closed system synthesizes either product a or b that both interact with e to influence output such that a:e interactions cause production of b, while b:e interactions pro-duce a. irrespective of starting conditions (excluding substrate exhaustion and product inhibition), an equilibrium will eventually develop ( figure 5 ) with the relative concentrations of a:b determined by the relative association constants (k) of a:e (k a:e ) and b:e (k a:b ) and the velocity (ν) of production of a from b:e (ν a ) and b from a:e (ν b ). removal or addition of either a or b will alter equilibrium conditions but not the fact equilibrium is reached; if a is removed, for example, the increased two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent spe-cificity (red shading) and threshold of detection (tod) of any assay figure 4 two-dimensional representation of hyperdimensional rna (or corresponding protein) frequency distribution curve (scale arbitrary) with conceptual centre of gravity of replication (wild type, green) and variant sequences (blue), zone of reagent specificity (red shading) and threshold of detection (tod) of any assay. as mutations ( , ) accumulate and rna sequence progressively diverges from consensus sequence (0) the probability of that rna sequence and corresponding protein (e.g. envelope, env.) arising falls rapidly. standard deviation (σ) of frequency distribution is proportional to rna pol fidelity. frequency genetic distance 0 threshhold of detection (tod) reagent specificity ♦ ♦ frequency of b:e interactions will cause compensatory increased a synthesis; in this sense enzymatic autoregulation occurs. intuitive analysis suggests that enzymes acting in a milieu of increasing concentrations of inhibitory molecules become progressively less processive until reduced enzyme output is insufficient to further inhibit enzyme activity, and an equilibrium state is reached. considering viral replication, if alteration of rna pol fidelity causes synthesis of either wild-type or variant rna sequences (simplified, as a continuum between these two must exist) that are subsequently translated into either wild-type or variant polypeptides that then interact with rna pol such that wild-type: rna pol are high affinity interactions that induce rapid, low fidelity rna pol replication while variant protein: rna pol interactions are low affinity and cause high fidelity rna pol replication at low rate then an equilibrium will eventually develop. hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of rna pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. stable, highly reactive equilibria not only develop as a result of rna pol /envelope interactions and viral autoregulation, there is no option but for this to occur. generation and maintenance of polymorphisms, that is, replacement of existing genes -that, by operational dar-winian definition, have proved their functionality and evolutionary fitness by surviving to reproduce -with variant genes (polymorphisms) of uncertain functionality, fitness or overall compatibility within an organism, is an evolutionary strategy that will only be sustained on a geological timescale if new polymorphisms confer survival benefits to organisms that exceeds the risks and metabolic costs of generating and sustaining those polymorphisms. for primitive cells, lacking functional humoral, cellular or cytokine defense mechanisms, development of cell-surface protein polymorphisms is an obvious adaptive strategy to thwart invasion by primitive viruses. like other adaptive strategies, cell-surface polymorphisms are strongly selected for, and have been highly conserved over deep time, and are found in all organisms from primitive prokaryotic cells [51] and thermophilic bacteria [52] through to plants [53] as well as mammalian cells, strongly suggesting a critical evolutionary function. the lock and key hypothesis, for which there is very considerable evidence [54] [55] [56] [57] , first proposed by jbs haldane [58] , contends polymorphisms arise, and are maintained, as protection against cellular parasitism, particularly by viruses 2 . while dna-encoded protein polymorphisms form necessary defenses against viral access, they may not be sufficient; a quasispecies of cells (e.g. the liver) expressing similar and static receptor variations renders those cells vulnerable to sustained attack from any virus that successfully invades any one cell, and further dynamic modification of cell receptors, triggered by viral infection and mediated at the transcriptional level by modulation of dna dependent rna polymerase fidelity in nearby uninfected cells, by a mechanism similar to replicative homeostasis would seem possible. a clear, unambiguous band at the "c" position on a sequencing gel, causes "cytosine" to be assigned to that genetic locus. but does this certitude reflect reality, at least for viral rna quasispecies? direct pcr sequencing is an "averaging" procedure revealing the most frequent nucleotide at any particular locus. however, nucleic acids and proteins cannot express 'an average', and discrete quanta of specific nucleotides or amino acids are present at every locus. a typical clinical serum sample, containing 4 × 10 5 geq/ml hcv and mutating at 10 -5 substitutions/base, will contain examples of each possible nucleotide at every locus, but most variations will remain undetected during sequencing or any other method of quasispecies analysis. analysis of cloned dna gives cleaner data than pcr sequencing but if 100 clones (and multiple hcv quasispecies clones are highly unlikely to be identical) provides definitive sequence, would we process the 101 st to reveal different and, potentially, critical sequence variations? and if we did, how would we recognise its importance? is important sequence likely to be present at frequencies of http://www.virologyj.com/content/2/1/70 < 1%? infectious virions containing, presumably, fulllength functional genome and corresponding wild-type proteins, are often outnumbered by ~6 × 10 4 :1 in serum by defective and non-infectious particles [53] that presumably do not, suggesting that important genetic sequence and associated phenotype may occasionally be extremely rare. how the immune system recognizes uncommon, nondescript, but important protein sequences in a featureless background of similar molecules is a non-trivial problem for which replicative homeostasis may suggest a solution. replicative homeostasis, described in detail elsewhere [28, 44] , is an epicyclic mechanism of viral autoregulation that results when viral proteins, notably envelope (env), influence rna pol fidelity and processivity. the predicted consequences of replicative homeostasis for rates of intracellular viral replication and mutation, cellular expression of viral proteins and immunological responses occurring because of replicative homeostasis is represented schematically (figures 6, 7). during early viral replication in a naive cell devoid of inhibitory molecules (panel a, a), high affinity wild-type envelope:polymerase interactions predominate, causing rapid low-fidelity polymerase activity resulting in rapid synthesis of variant viral rnas and subsequently proteins, hence causing a broad spectrum of viral proteins to be expressed on the cell surface, each at concentrations below the threshold of immune detection (tod). rna pol infidelity ensures synthesis of variant viral rnas and proteins predominates early, hence variant protein molecules progressively accumulate within cells relative to wild-type viral molecules (panels b-d) and increasing the probability of variant viral envelope:rna pol interactions. variant viral envelope:rna pol interactions causing progressive inhibition of rna polymerase processivity and increasing rna pol fidelity, reducing diversity of viral rnas synthesized and progressively restricting dynamic progression of rna pol functional properties, processivity () and fidelity () predicted by replicative homeostasis figure 6 dynamic progression of rna pol functional properties, processivity ( ) and fidelity ( ) predicted by replicative homeostasis. initial state (a, corresponding to panel a, figure 7 ): in a newly infected cell, high-affinity wild-type:rna pol interactions will predominate resulting in high rna pol processivity but low fidelity causing high-level viraemia with broad virus phenotypic spectrum, maximizing cell tropism. intracellular accumulation of variant viral proteins (b, c.f. panel b, figure 7 ) reduces rna pol processivity but increases fidelity reducing viral rna synthesis and consequently, viraemia before a dynamic, fluctuating equilibrium (c, c.f. panel c or d, figure 7 ) develops in which inhibition of rna pol by variant viral proteins is balanced by increases in rna pol fidelity (with consequent synthesis of wild-type viral products tending to cause high rna pol processivity). conceptual progression of intracellular viral replication events, including variable rna pol fidelity and processivity, restriction of antigenic diversity and immune recognition under influence of replicative homeostasis env viral protein diversity expressed on the cell surface (panels b to d), increasing cell-surface concentrations of individual viral proteins above the threshold of detection (panels c, c) at which point a polyclonal immune response develops. development of low-affinity polyclonal blocking antibodies, restricting cellular egress of viral proteins, further increasing intracellular concentrations of variant envelope proteins, still further increasing the probability of variant viral envelope:rna pol interactions and inexorably further restricting antigenic diversity increasing relative expression of wild-type proteins thus further exposing these epitopes to immune surveillance and facilitating specific high-affinity immune responses, including cytotoxic t cell responses, (d,d) to wild-type proteins. thus, the immune responses can strategically utilize replicative homeostasis to force viruses to reveal important and dominant wild-type epitopes, but those responses develop initially as a consequence of restriction of rna pol fidelity that occur because of replicative homeostasis. high-affinity responses further deplete intracellular concentrations of wild-type proteins, progressively reducing wild-type envelope:rna pol interactions, greatly reducing rna pol processivity to the point of viral latency (e,e), caused by variant viral envelope:rna pol interactions. the hepatitis c "early replication" paradox now resolves completely when considered in the context of replicative homeostasis; initial high level hcv replication (due to high rna pol processivity) remains immunologically undetectable for 6-8 weeks, or more, because of low rna pol fidelity causing a broad spectrum of hcv envelope proteins each expressed on cell surfaces at concentrations below the threshold of detection even while viraemia, reflected in concentrations of 5'utr rna common to each rna species, are present at 10 6-7 geq/ml. as replication progresses, intracellular accumulation of variant viral proteins increase rna pol fidelity but decrease processivity (replicative homeostasis), downregulating hcv replication and reducing viraemia but restricting antigenic diversity and increasing expression of hcv envelope proteins to beyond the threshold of immune detection. furthermore, the temporal tissue injury (aminotransferase) paradox also resolves in this light: focussed immune recognition (including cytotoxic t cell responses) doesn't develop until after viral antigenic diversity is restricted by replictive homeostasis the transaminase peak would not be expected until after viral replication falls due to autoinhibition of rna pol processivity. varying expression of viral proteins by modulating rna pol fidelity to facilitate immune escape would seem a useful evolutionary adaptation that might be retained by more complex organisms, including cellular pathogens like tuberculosis and malaria, to optimize their stability within hosts. this mechanism of immune avoidance might also explain maternal-foetal tolerance. the human foetus maintains a stable parasitic existence during gestation (and, i expect, to university age and beyond) that is tolerated despite normal maternal immune responsiveness in general and lack of specific tolerance to paternal antigens in particular, a situation made more problematic as expressed foetal antigens are predominantly of paternal origin [54] . while immunological isolation of foetal tissue by the placental trophoblastic layer [55] , and placental display of hla-g [56] , probably contribute to foetal stability in the face of a potentially robust immune attack, neither mechanism would explain persistence of viable foetal nucleated red blood cells within the maternal circulation [57] in quantities sufficient to permit clinical prenatal diagnosis [58] . is it possible foetal tolerance is mediated by regulating the fidelity of foetal dna dependent rna transcriptases to ensure any cell-surface antigens are expressed heterogeneously and at levels below the threshold of maternal immune responsiveness? for many classical autoimmune disorders, including primary biliary cirrhosis [59] , multiple sclerosis, and rheumatoid arthritis, convincing epidemiological evidence [60] , including cases clustering [61, 62] , strongly suggests these diseases are triggered by infectious agents in genetically predisposed individuals. in others, such as diabetes mellitus, tantalizing epidemiological [63] , clinical [64] and laboratory [65] evidence has implicated enteroviruses, but has suggested viral-triggered autoimmune processes, rather than cytolytic destruction of pancreatic betacells [66] . similar circumstantial evidence exists for myocarditis, demyelinating diseases, myositis and other post infectious inflammatory disorders. when macfarlane burnet wrote autoimmunity arises from "inability to distinguish self from non-self" hbv, hcv, hiv and other viruses, now established to cause diseases with clear autoimmune features were unknown. viral infections, particularly hepatitis c -and its treatment with interferon -are associated with many varied autoimmune phenomena [67] , and thyroid disease [68] [69] [70] , diabetes mellitus [71, 72] , membranous, membranoproliferative and cryoglobulinemic glomerulonephritis, vasculitis and peripheral neuropathy [73] , and autoimmune gastritis [74] are all very well documented, although the mechanism(s) are unknown and causality is certain. classical serological markers of autoimmunity, including rheumatoid factor, antinuclear antibodies (ana), anticardiolipin, antithyroid, anti-liver/kidney/microsomal antibodies (anti-lkm), as well as hcv/anti-hcv immune complex formation and mixed essential cryoglobulinemia are common accompaniments of chronic hcv infection [73] , raising the obvious question of whether all "autoimmunity" has a viral basis. indeed, zinkernagel's pragmatic and subtly anticipatory; "if we know the infection, we call the disease immunopathologically mediated; if we do not recognize or know it, we call the disease autoimmune [75] " fully reflects recent explosive growth of information and the deeper questions this information poses. rna virus quasispecies biology, specifically the generation of rna quasispecies by rna pol , and translation of these immensely variable rnas into protein quasispecies, suggests an immediate solution to the problem of viral autoimmunity and, by extension, to autoimmunity in general, as well as suggesting a unifying hypothesis to explain other diseases known to have multi-factorial aetiologies that include inflammatory components -such as coronary artery disease -in addition to other diseasesincluding schizophrenia and some forms of depressionthat currently lack rational and coherent pathogenic explanations. viruses are known to co-opt cell surface molecules, including lectins, hormone receptors and cell signaling molecules, to access cells. receptors, and other cell surface molecules, identified as "viral receptors"or to specifically interact with viral proteins include prostaglandins, catecholamines and acetylcholine receptors [76] , serotonergic neurotransmitters (5ht) [77] , endothelial cell glycoproteins [78] , insulin-like growth factor (igf-ir) and its major signaling molecules insulin receptor substrates irs-1 [79] and irs-2 [80] , epidermal growth factor (egf) [81] , neurotrophin receptor [82] , thyroid hormone receptor tralpha1 [83] , an immunoglobulin protein superfamily [84] , low density lipoprotein (ldl) receptors [85, 86] , transferrin receptor (tfr) [87] , asialoglycoprotein receptor (asgp-r) [88, 89] , and angiotensin-converting enzyme 2 [90] , to cite biologically diverse examples. of necessity, some receptor affinity studies have used cloned viral protein ligands, an artificial situation that cannot approach the phenotypic complexity of rna viral protein quasispecies. nonetheless, variable virus receptor affinities [91, 92] , evolutionary adaptation of receptor affinity [93] , emergence of escape variants with altered receptor affinities [94] , temporal alteration of receptor usage [92] and capacity to exploit alternative entry pathways [95] have all been confirmed, suggesting viruses are capable of generating highly plastic ligands with very broad receptor affinities. if a virus co-opts a receptor for cell entry, then wild-type envelope (consensus sequence) epitopes, coded for by wild-type rna sequences, will probably form the common viral ligand. however, any viral rna quasispecies also contain a vast spectrum of rnas derived from, and similar to, envelope open reading frame (orf) consensus sequence, but variant from it. as the envelope orf quasis-pecies sequences progressively diverge from wild-type, the quasispecies of envelope proteins translated from these variant orfs will also, and inexorably, diverge in sequence, structure and biological function from wildtype envelope sequence proteins. some of these envelope proteins will be functionally identical, but others, and probably the vast majority, will range from subtly different to grossly abnormal, either due to major differences of sequence and/or chemical or steric amino acid incompatibility, or because of premature introduction of stop codons. even minor amino acid differences, as sickle cell anaemia illustrates, and has been confirmed specifically for viral receptor usage [96, 97] , may catastrophically alter a proteins' function with respect to co-opted viral receptors, with some having no binding affinity, while others will bind strongly and act as agonists, antagonists or competitive inhibitors of normal receptor function. variant and defective viruses, and their polypeptides, will be in vast molar excess compared to wild-type [53] but will exhibit similarly high antigenic variability, permitting escape from immune and other scavenger mechanisms. as many variant viral polypeptides will bind tightly to "self" receptors, but contain immunogenic non-self motifs, a polymorphic (because variant viral proteins will themselves be highly polymorphic due to the quasispecies process) immune response, apparently directed against "self" antigens, but actually targeting virus protein-receptor complexes virtually indistinguishable from normal cell receptors, will result causing apparent 'autoimmune' tissue damage. this mechanism suggests an explanation for common autoimmune phenomena. if a virus enters cells because wild-type envelope motifs interact with insulin, insulin receptor substrate [79, 80] , tsh or related molecules [83] , or acetylcholine [76] receptors, many variant envelope polypeptides, generated by envelope orf quasispecies rnas, would have similar receptor binding affinity, but may effectively disrupt receptor function, predictably causing impaired glucose tolerance or diabetes mellitus, thyroid dysfunction, or myasthenia gravis with secondary resistance to, and elevation of, the normal hormone ligand (insulin, tsh etc.). the expected consequences disruption of receptor function by variant viral proteins might explain many common biochemical pathologies; for example, what effect would chronic blockade of parathyroid (pth) receptors by viral proteins have on pth levels, the parathyroid glands, or bone? leptin is a 16kda protein hormone secreted by adipocytes and carried across the blood-brain barrier by a rate-limiting transporter to act on hypothalamic receptors [98] where, among other functions, it regulates thyrotropinreleasing hormone (trh) genes and upregulates alphamelanocyte-stimulating hormone and other anorexigenic neuropeptides [99] important to appetite-regulation and energy balance [100] . leptin also regulates a broad spectrum of other processes and behaviours including thermogenesis, blood pressure and immune function. s=serum leptin concentrations and leptin resistance, are independent markers of obesity, weight gain, systemic hypertension [101] , diabetes mellitus [102] , obstructive sleep apnoea [103] and myocardial infarction [104] , while polymorphisms of the leptin gene are associated with insulin resistance [105] and long-term risk of developing diabetes mellitus [102] . predictably, variant envelope proteins generated by envelope orf rna quasispecies from viruses utilizing leptin receptors for cell access would have similar receptor affinity, but exhibit non-physiological leptin antagonist or agonist properties, thus disrupting leptin receptor function, altering energy regulation, and causing either excess caloric intake unrestrained by satiety responses, or inappropriate satiety signals with pathologically reduced caloric intake. as clear evidence exists for viral disruption of leptin function [106] and virus-associated weight gain in humans [107] and monkeys [108] , is it possible the global epidemics of type ii diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [109] [110] [111] [112] [113] [114] [115] [116] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function? could it also be that ethnically based epidemics of obesity, diabetes mellitus, hypertension and reno-vascular disease (the 'metabolic syndrome'), as seen in pima indians, nauruans and australian aborigines [115] have developed not primarily because of exposure to "western" foods and lifestyles -that, after all, are all-pervasive without necessarily having so dramatic an effect on other groups -but because of chronic or recurrent exposure to viruses, or genotypes of viruses to which their particular repertoire of receptor polymorphisms confer no protection? or that anorexia nervosa develops, in some patients, when variant viral proteins with aberrant leptin-agonist function arise during the course of viral infection, as the temporal relationship between infection and disease onset, very clearly documented in one study [117] , suggests. cardiovascular disease, the leading cause of premature death and disability in most western countries, has a wellestablished multi-factorial basis involving a complex interplay between genetic predisposition, environmental and personal risk factors -including systemic hypertension, diabetes mellitus, hyperlipidaemia, obesity and cigarette smoking -and more recently recognized mechanisms, including endothelial dysfunction [118] , vascular inflammation [119] and leptin levels [104] . systemic hypertension, diabetes mellitus and hyperlipidaemia have long-established, but complex, patterns of inheritance, a situation further compounded by evidence receptor polymorphisms -including those of angiotensin ii type 1 receptor [120] , irs-1 gene [121] and low density lipoprotein receptor (ldlr) [122] -both confer disease susceptibility and have regionally variable prevalences [123, 124] . the flaviviradae -including hcv -as a family, and the rous sarcoma virus, utilize low density lipoprotein receptors to enter cells [85, 125] , while angiotensin ii [90] , insulin receptor substrates (irs1 and irs 2) [79] , and endothelial cell glycoproteins [78] and other receptors widely distributed in vascular tissues are known to be permissive for virus cell entry establishing, in principle and in fact, viral-protein receptor affinity relevant to cardiovascular diseases. viruses accessing cells through these receptors will generate a quasispecies of variant proteins capable of disrupting receptor function potentially causing hyperlipidaemia, hypertension, hyperglycaemia and endothelial dysfunction, as well as immune-mediated endothelial cell damage, thus establishing the necessary and sufficient conditions and a chain of events that potentially link viruses and vascular diseases, including myocardial infarction. this hypothesis exists at the confluence of established risk factors for coronary artery disease, including genetic susceptibility, polymorphisms predisposing to hypertension [126] [127] [128] , diabetes [126] and hypercholesterolaemia and substantial new data implicating vascular inflammation [119, 129] , endothelial dysfunction [119, 130] , leptin dysregulation [104] and viral infection [131, 132] in the pathogenesis of vascular disease. furthermore, this final common pathway can account for that small, but significant, group of patients with vascular diseases but no clinically identifiable risk factors, as well as the non-random co-incidence of depression and coronary artery disease [133] (as discussed below) in addition to the anti-inflammatory action of hmg-coa reductase inhibitors (statins) [134] , and their effect in lowering cardiovascular mortality independent of cholesterol reduction [135] ; if statins compete with variant viral proteins for hmg-coa reductase receptor binding, and displace immunologically attractive molecules, inflammatory responses directed at viral product, but involving endothelial cell receptors, will be ameliorated (figure 8). human immunodeficiency virus hiv-associated dementia (hivd) occurs in 15% of hiv-infected adult patients, and as a major cause of dementia in the young represents "proof of principle" of virus-caused dementia, raising the possibility other forms of virus related dementia exist. although highly active antiretroviral therapy (haart) has reduced the incidence of hiv-d by 40-50% [136] , it remains a major cause of morbidity and the pathogenesis poorly understood. direct cytopathic effects of hiv or other viruses are unlikely, while active replication of virus, high-level viral protein expression [137] , and increased viral envelope sequence-diversity in blood and brain [138] are all important, clearly indicating viral proteins are pathogenically important. the clinical features of hivd, including psychomotor slowing, apathy, and altered gait and posture, strongly suggest a subcortical dementia with involvement of the basal ganglia and striatal dopamine receptor pathways. schizophrenia, depression and bipolar affective disorder, and anorexia nervosa are highly prevalent, chronic conditions of unknown aetiology that cause enormous morbidity and generate significant health care costs. each of these disorders have well documented, albeit regionally variable, associations with receptor -including dopamine -polymorphisms [124, [139] [140] [141] [142] [143] , as well as epidemiological evidence that viral infections are aetiologically important, either directly or as precipitating events [117, [144] [145] [146] [147] , although other sero-epidemiological studies [148] and work directly seeking viral nucleic acids in patients with schizophrenia have proved negative [149] . if a virus, or viruses, use dopamine, acetylcholine [76] , neurotrophin [82] , serotonergic (5-ht) [77] , or other neuro-transmitter receptors to access cells (and, given rna virus quasispecies biology, it would be surprising if some didn't), then the rna quasispecies will generate a quasispecies of variant polypeptides potentially reactive to these receptors. while it is difficult to imagine what effect perfusing a functional human brain with a solution of antigenic, inflammatory polypeptides that bind to, and are variably disruptive of, critical neurotransmitter receptor function, might have on cognition, perception, behaviour, attention span, abstract thought, fine motor or emotional control, it is unlikely to be beneficial. in this context, the welldocumented cognitive abnormalities -unrelated to depression -found in patients with early hcv and hiv infection [150] [151] [152] are unsurprising. virus receptor disease (vrd) is quite distinct from either immune complex deposition disease due to deposition of macromolecules in tight vascular arcades, or from disease related to altered cell tropisms and is also completely independent of the primary site of viral replication; both non-inflammatory receptor blockade and immune-mediated inflammation directed at viral protein-receptor complexes could cause pathology of tissues non-permissive for and remote from the primary site(s) of viral replication with "autoimmune" damage to the liver, pancreas, brain, skin or lungs arising, for example, from chronic small intestinal virus infection. viral quasispecies biology predicts vrd will have other characteristics. first, due to replicative homeostasis, the ratio of wild type to variant viral proteins of the quasispecies will both fluctuate with time and will alter dramatically after initial infection; if wildtype proteins are dominantly agonist in function with respect to their receptor, variant proteins, most likely, will predominantly exhibit antagonist function (and vice versa). furthermore, the net effect of viral proteins (because of viral autoregulation) will fluctuate initially between receptor agonist and antagonist function, before becoming predominantly antagonistic, thus providing a possible explanation for transient thyrotoxicosis during early thyroiditis (before hypothyroidism supervenes), for hypoglycaemia seen during early insulin-receptor antibody-mediated insulin resistance [153] , and for the contradictory functions ascribed to hiv nef [154] . a corollary of fluctuating phenotypic dominance of viral protein quasispecies is that receptor affinity of these proteins will also fluctuate, and any resulting inflammation may vary in both intensity and anatomical distribution over time. second, because viruses utilize alternate receptors for cell access, apparently homogeneous disease processes could result from multiple different viruses. similarly, because cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship (1; unbound, 2; activated), receptor permis-sive for virus cell entry (3) or blocked by polymorphism (rp, 4) figure 8 cell receptor (r) and normal ligand (l; insulin, pth, leptin etc.) relationship (1; unbound, 2; activated), receptor permissive for virus cell entry (3) or blocked by polymorphism (rp, 4). receptor blockade by variant viral envelope proteins (green e, 5), blockade by antigenic envelope proteins stimulating "autoimmune"response apparently directed against self receptors (e, 6), competitive displacement of antigenic proteins by drug (d, e.g. statin, aspirin) abrogating immune response (7). virus quasispecies produce a broad spectrum of protein phenotypes, and the receptor polymorphisms permissive for cell entry for specific viruses will be variably distributed in host populations, pathology of widely variable tissues in different individuals could result from the same virus. third, as evolutionary co-adaptation results in progressive genetic co-divergence of interacting species, the receptor polymorphisms predisposing to (or protecting against) infection by any particular virus, and resulting vrd, and the common viruses causing them, would be predicted to vary geographically, an expectation multiply confirmed for disease associated polymorphisms. as a corollary this suggests individuals migrating from regions where hosts and virus strains are stably co-adaptated to other areas, where different viruses are prevalent, might experience increased rates of vrd -beaks optimally adapted for finch survival on the galapagos may be a liability elsewhere -a prediction again amply confirmed [155] [156] [157] ;. finally, if immune mechanisms are unable to clear rna viruses like hcv and do not cause the reduced viral replication seen during acute infection, are they any more likely to be effective against other rna viruses? is it possible that self-limiting infections like influenza and sars also autoregulate their replication, and, like hcv or hbv, become partially dormant, yet remain transcriptionally active, in the face of an active and powerful immune response? pcr amplification of influenza rna from convalescent samples makes this readily testable, while the documented relationship of influenza to myocardial infarction [132] and juvenile rheumatoid arthritis [61] makes the question important. if confirmed, the well-documented seasonality of some depressive illnesses [158] and schizophrenia, [146] and increased rates of schizophrenia during influenza epidemics [144] , and the increased incidence of both depression [146] and schizophrenia [144, 145] following in-utero exposure to influenza may be more rationally explained. if quantitative pcr (qpcr) assays of both 5'utr and envelope rnas are performed serially, and data expressed as [5' utr rna]/[env rna] for each sample, then a numerical expression describing changing quasispecies complexity over time may be obtained. in case prescient genius is unappreciated, haldane formulated the "lock and key" hypothesis on the basis of protein polymorphisms, defined by gel electrophoresis, and some general musing about predation and evolutionary struggle, two decades before the nature of dna was elucidated. i have no pecuniary interests, whatever, in this work and do not stand to gain financially or otherwise from it. publish with bio med central and every scientist can read your work free of charge a new immune escape mutant of hepatitis b virus with an asp to ala substitution in aa144 of the envelope major protein emergence of vaccine-induced escape mutant of hepatitis b virus with multiple surface gene mutations in a korean child the hepatitis b virus persists for decades after patients' recovery from acute viral hepatitis despite active maintenance of a cytotoxic tlymphocyte response hepatitis b virus persistence after recovery from acute viral hepatitis hepatitis b virus occult infection in subjects with persistent isolated anti-hbc reactivity occult hepatitis b virus infection in hbs antigen-negative hepatocellular carcinoma in a japanese population: involvement of hbx and p53 hiv-1 dynamics in vivo: virion clearance rate, infected cell lifespan, and viral generation time hepatitis c viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy hepatitis c virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children lack of protective immunity against reinfection with hepatitis c virus superinfection by homotypic virus in hepatitis c virus carriers: studies on patients with post-transfusion hepatitis superinfection of heterologous hepatitis c virus in a patient with chronic type c hepatitis hepatitis c virus persistence after spontaneous or treatment-induced resolution of hepatitis c persistence of hepatitis c virus in patients successfully treated for chronic hepatitis c vaccina analysis of hepatitis c virusinoculated chimpanzees reveals unexpected clinical profiles development of virus-specific cd4(+) t cells during primary cytomegalovirus infection fidelity of hepatitis b virus polymerase fidelity of hiv-1 reverse transcriptase high fidelity of yellow fever virus rna polymerase lack of evidence for proofreading mechanisms associated with an rna virus polymerase rapid fitness losses in mammalian rna virus clones due to muller's ratchet eigen m: viral quasispecies replicative homeostasis: a mechanism of viral persistence the molecular epidemiology of mumps virus interleukin 6-174 g/c promoter gene polymorphism and sporadic alzheimer's disease: geographic allele and genotype variations in europe biological divergences in south-central bougainville: an analysis of blood polymorphism gene frequencies and anthropometric measurements utilizing tree models, and a comparison of these variables with linguistic, geographic, and migrational "distances classification of hepatitis c virus into six major genotypes and a series of subtypes by phylogenetic analysis of the ns-5 region a novel mechanism to explain protein abnormalities in schizophrenia based on the flavivirus resistance gene ethnic heterogeneity in allele variation in the drd4 gene in schizophrenia characterization of microbial diversity in hypersaline environments by melting profiles and reassociation kinetics in combination with terminal restriction fragment length polymorphism (t-rflp) structural polymorphism of the reca protein from the thermophilic bacterium thermus aquaticus single nucleotide polymorphism, haplotype diversity and recombination in the isa gene of barley polymorphism of the il-10 gene is associated with susceptibility to herpes zoster polymorphism of the interleukin-10 gene is associated with susceptibility to epstein-barr virus infection hla-dr antigen frequencies in mexican patients with dengue virus infection: hla-dr4 as a possible genetic resistance factor for dengue hemorrhagic fever polymorphism of fc receptor iia for igg in infants is associated with susceptibility to perinatal hiv-1 infection symposium sui fattori ecologi e genetici dela specilazione negli animali. supplemnto a la la ricerca scientifica high levels of hiv-1 in plasma during all stages of infection determined by competitive pcr differential imprinting and expression of maternal and paternal genomes maternal immune response to pregnancy maternal and fetal immune responses during pregnancy fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age prenatal diagnosis using fetal cells isolated from maternal peripheral blood: a review gershwin me: lack of immunological or molecular evidence for a role of mouse mammary tumor retrovirus in primary biliary cirrhosis multiple sclerosis in australia antibodies to influenza a in a cluster of children with juvenile chronic arthritis the geographical distribution of primary biliary cirrhosis in a well-defined cohort indications that maternal coxsackie b virus infection during pregnancy is a risk factor for childhood-onset iddm. diabetologia coxsackie b virus infection and beta cell autoantibodies in newly diagnosed iddm adult patients coxsackievirus b4-induced development of antibodies to 64,000-mr islet autoantigen and hyperglycemia in mice the role of enteroviral infections in the development of iddm: limitations of current approaches hepatitis c infection and autoimmunity interferon-alpha and autoimmune thyroid disease hepatitis c and d, retroviruses and autoimmune manifestations editorial: autoimmune thyroid disease in interferon-treated patients further evidence for an association between non-insulin-dependent diabetes mellitus and chronic hepatitis c virus infection fulminant autoimmune type 1 diabetes during interferon-alpha therapy: a case of th1-mediated disease hepatitis c virus infection and autoimmunity gastric autoimmune disorders in patients with chronic hepatitis c before, during and after interferon-alpha therapy rabies virus infection selectively impairs membrane receptor functions in neuronal model cells rabies virus selectively alters 5-ht1 receptor subtypes in rat brain role of virus receptor-bearing endothelial cells of the blood-brain barrier in preventing the spread of mouse hepatitis virus-a59 into the central nervous system insulin-like growth factor i receptor signaling system in jc virus t antigen-induced primitive neuroectodermal tumors -medulloblastomas hepatitis c virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3 vaccinia virus and the egf receptor: a portal for infectivity? alterations in neurotrophin and neurotrophin receptor gene expression patterns in the rat central nervous system following perinatal borna disease virus infection direct modulation of simian virus 40 late gene expression by thyroid hormone and its receptor receptor for the group b coxsackieviruses and adenoviruses: car hepatitis c virus and other flaviviridae viruses enter cells via low density lipoprotein receptor low density lipoprotein receptor as a candidate receptor for hepatitis c virus the natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor sendai virus efficiently infects cells via the asialoglycoprotein receptor and requires the presence of cleaved f0 precursor proteins for this alternative route of cell entry role of the asialoglycoprotein receptor in binding and entry of hepatitis c virus structural proteins in cultured human hepatocytes angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus clinical coxsackievirus b isolates differ from laboratory strains in their interaction with two cell surface receptors early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals adaptation of wild-type measles virus to cd46 receptor usage evolutionary pressure of a receptor competitor selects different subgroup a avian leukosis virus escape variants with altered receptor interactions evolution of cell recognition by viruses a single amino acid alteration in the human parainfluenza virus type 3 hemagglutinin-neuraminidase glycoprotein confers resistance to the inhibitory effects of zanamivir on receptor binding and neuraminidase activity single amino acid substitutions in influenza haemagglutinin change receptor binding specificity the many lives of leptin leptin signaling, adiposity, and energy balance leptin signaling in the central nervous system and the periphery leptin gene polymorphism is associated with hypertension independent of obesity association between baseline plasma leptin levels and subsequent development of diabetes in japanese americans increases in leptin levels, sympathetic drive, and weight gain in obstructive sleep apnea leptin is associated with increased risk of myocardial infarction association of leptin receptor polymorphism with insulin resistance alteration of the leptin network in late morbid obesity induced in mice by brain infection with canine distemper virus human adenovirus-36 is associated with increased body weight and paradoxical reduction of serum lipids human adenovirus ad-36 promotes weight gain in male rhesus and marmoset monkeys obesity, insulin resistance and diabetes -a worldwide epidemic the worldwide epidemic of obesity in adolescents the obesity epidemic development of the obesity epidemic in denmark: cohort, time and age effects among boys born 1930-1975 the spread of the obesity epidemic in the united states global and societal implications of the diabetes epidemic etiology of the metabolic syndrome: potential role of insulin resistance, leptin resistance, and other players the obesity epidemic as harbinger of a metabolic disorder epidemic: trends in overweight, hypercholesterolemia, and diabetes treatment in geneva post-viral onset of anorexia nervosa endothelial dysfunction of the non-infarct related, angiographically normal, coronary artery in patients with an acute myocardial infarction persistent endothelial dysfunction is related to elevated c-reactive protein (crp) levels in type ii diabetic patients after acute myocardial infarction association of angiotensin ii type 1 receptor gene polymorphism with essential hypertension association between vitamin d receptor polymorphism and type 2 diabetes or metabolic syndrome in community-dwelling older adults: the rancho bernardo study normal dna polymorphism at the low density lipoprotein receptor (ldlr) locus associated with serum cholesterol level varmus he: a receptor for subgroup a rous sarcoma virus is related to the low density lipoprotein receptor a common polymorphism of uncoupling protein 2 gene is associated with hypertension epidemiology of essential hypertension: the role of genetic polymorphism genetic polymorphism of the renin-angiotensin-aldosterone system and arterial hypertension in the italian population: the geniper project low grade inflammation and coronary heart disease: prospective study and updated meta-analyses impaired endothelial function in never-treated hypertensive subjects carrying the arg972 polymorphism in the insulin receptor substrate-1 gene influenza infection exerts prominent inflammatory and thrombotic effects on the atherosclerotic plaques of apolipoprotein e-deficient mice association of influenza vaccination and reduced risk of recurrent myocardial infarction depression and cardiovascular disease: mechanisms of interaction crisby m: modulation of the inflammatory process by statins. drugs today (barc) 2003 statins and endothelial dysfunction hiv dementia: an evolving disease circular forms of unintegrated human immunodeficiency virus type 1 dna and high levels of viral protein expression: association with dementia and multinucleated giant cells in the brains of patients with aids hiv dementia patients exhibit reduced viral neutralization and increased envelope sequence diversity in blood and brain association of egf polymorphism with schizophrenia in finnish men meta-analysis identifies an association between the dopamine d2 receptor gene and schizophrenia dias neto e: association of a new polymorphism in alox12 gene with bipolar disorder anorexia nervosa (restrictive subtype) is associated with a polymorphism in the novel norepinephrine transporter gene promoter polymorphic region serologic evidence of prenatal influenza in the etiology of schizophrenia relationship between in utero exposure to influenza epidemics and risk of schizophrenia in denmark adult major affective disorder after prenatal exposure to an influenza epidemic detection of serum antibodies to borna disease virus in patients with psychiatric disorders immunoglobulin studies in patients with psychiatric diseases polymerase chain reaction (pcr) search for viral nucleic acid sequences in schizophrenia subclinical impairment of brain function in chronic hepatitis c infection hepatitis c and cognitive impairment in a cohort of patients with mild liver disease cognitive changes as early signs of hiv infection the evolving clinical course of patients with insulin receptor autoantibodies: spontaneous remission or receptor proliferation with hypoglycemia the paradox of hiv nef function: resolution of the contradictions childhood migration and cardiovascular risk migration as a risk factor for schizophrenia: a danish population-based cohort study impact of migration on coronary heart disease risk factors: comparison of gujaratis in britain and their contemporaries in villages of origin in india variability in the 5-ht(2a) receptor gene is associated with seasonal pattern in major depression i thank my wife sophie j coleman, and sons matt and tim, for everything important, my parents dick and janet for extraordinary opportunity, and some great physician-teachers -that most noble vocation -of the university of western australia medical school; professors mike mccall, dick joske, bill reed, bill musk, peter pullan, michael quinlan, dick lefroy and ted haywood. special thanks to karl ruckriegel for turning back-of-envelope sketches into first-class graphics. any remaining lack of clarity is my fault. key: cord-286334-d9v5xtx7 authors: li, rui; qiao, songlin; zhang, gaiping title: analysis of angiotensin-converting enzyme 2 (ace2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov date: 2020-04-30 journal: journal of infection doi: 10.1016/j.jinf.2020.02.013 sha: doc_id: 286334 cord_uid: d9v5xtx7 nan a novel coronavirus from wuhan in central china, named 2019-ncov, has recently caused an epidemic of pneumonia in humans and posed a huge threat to global public health. 1 , 2 to the date 06/02/2020, 2019-ncov has led to more than 31,0 0 0 confirmed cases and 637 deaths in china according to national health commission of the people's republic of china ( http://en.nhc.gov. cn/index.html ). cases have also been documented in a growing number of other international locations, including the united states ( https://www.cdc.gov/coronavirus/2019-ncov/index.html ). as a consequence, it is urgent to develop effective measures to control this novel coronavirus on the basis of its pathogenesis. host receptor recognition is a determinant for virus infection. during the time of this letter preparation, three works have just been published to explore the receptor usage of 2019-ncov. a work by zheng-li shi et al. has shown that angiotensin-converting enzyme 2 (ace2), the receptor for severe acute respiratory syndrome coronavirus (sars-cov), 3 from human, rhinolophus sinicus bat, civet, swine but not mouse mediate 2019-ncov infection in vitro , while the detailed mechanisms are not yet determined. 4 the other two works have reported or predicted human ace2 usage of 2019-ncov in a similar way to sars-cov mainly based on the coronavirus spike (s) glycoproteins. 5 , 6 considering the fact that the s proteins mutate and gain capability to recognize host receptors among species, 7 , 8 there is still a lack of analyses on receptor usage of 2019-ncov from the receptor perspective, which does not evolve as quickly as viruses. here, we firstly performed amino acid sequence alignment of ace2 from different species, including human, five non-human primates (gibbon, green monkey, macaque, orangutan and chimpanzee), two companion animals (cat and dog), six domestic animals (bovine, sheep, goat, swine, horse and chicken), three wild animals (ferret, civet and chinese horseshoe bat) and two rodents (mouse and rat). the alignment by clustal w 2.1 shows that they share a high sequence similarity except chicken (data not shown). the result suggests that 2019-ncov of probable bat origin may not interact with chicken ace2 and subsequently infect them, which were not considered in the following analyses. in ace2, the regions at position 30-41, 82-84 and 353-357 are demonstrated to be involved in the interaction with sars-cov s protein, where the residues at positions 31, 35, 38, 82 and 353 are critical. 9 therefore, we took a close comparison in these regions and residues. as shown in fig. 1 , human and non-human primates share the identity sequences in the regions and residues, implying that ace2 from non-human primates may recognize 2019-ncov and medi-ate its infection. as a result, non-human primates may be susceptible to 2019-ncov and serve as animal models for antiviral research or intermediate hosts for cross-species transmission. in fig. 1 , the residues of most companion, domestic and wild animals are conserved, especially for the critical ones stated above, while certain ones are variable. for example, lys31, glu35, asp/glu38 and lys353 are conserved, which probably form salt bridges. interestingly, the changes at positions 31, 38 and 82 are observed. these changes suggest steric hindrance and electrostatic interference for host-virus interaction. taking civet ace2 as an example, the change of lys31 to thr31 is likely to form a hydrogen bond instead of a salt bridge. in addition, the polar side chain of thr82 may influence the hydrophobic interaction of the original met82. all these changes may result in a lower binding affinity. however, an additional region covering residues 90-93 has been shown to be involved in civet ace2 binding to sars-cov and enhance their interaction. 10 consequently, we can't preclude the existence of other regions to compensate for the residue changes. with most residues in human ace2, the ones from these compaion, domestic and wild animals may be favorable for 2019-ncov recognition, which is in consistent with the recent work by zheng-li shi et al. in case cross-species transmission, close contact with sick or asymptomatic companion, domestic and wild animals should be cautious, such as for workers in livestock farming and travellers in the wild. in contrast, certain significant changes occur in the mouse and/or rat ace2 compared to the human one ( fig. 1 ) . the asn31 and ser82 in mouse ace2 may not form favorable interactions with 2019-ncov due to their electrostatic or hydrophilic characteristics. importantly, the change into his353 in both mouse and rat ace2 does not form a strong salt bridge as lys353 does. since the structural information for mouse and rat ace2 is unavailable, we carried out homology modeling using human ace2 (pdb code 2ajf) as template on online ( https://swissmodel.expasy.org ) for further analyses. in fig. 2 a, the change into ser82 in mouse ace2 may interfere with the hydrophobic interaction of the original met82. additionally, the changes into asn31 and his353 definitely affect the salt bridge formation and electrostatic potential. the change into his353 in rat ace2 is similar in the effect on receptor-virus interaction ( fig. 2 b) . these analyses partially explain why mouse ace2 does not mediate 2019-ncov infection reported by zheng-li shi et al. and assume that rodents are not likely to be the susceptible host. in conclusion, we conducted sequence and structural analyses of angiotensin-converting enzyme 2 (ace2) from different species, which sheds some light on cross-species receptor usage of 2019-ncov. all these analyses raise an alert on a potential interspecies transmission of 2019-ncov and propose further surveillance in other animal populations. structural studies on human and other species ace2 in complex with 2019-ncov spike protein will con the authors declare no conflict of interest. very recently, a letter in journal of infection reported the outbreak of the novel cornonavirus from dec. 2019 in china, especially in hubei province. 1 this novel cornonavirus may originate from the bat, 2 is just named as the covid-19 by the world health organization (who). the covid-19 outbroke from wuhan, the capital of hubei province, has spread to other provinces of china and even other countries. 3 strong human-to-human transmission is established. 4 until feb. 11, 2020, there have been 44653 cases of covid-19 infections confirmed in mainland china, including 1113 deaths. to prevent and control the spread of the epidemic, many strategies are needed. 5 predicting the trend of the epidemic are quite important to the allocation of medical resources, the arrangement of production activities, and even the domestic economic development all over china. therefore, it is very urgent to use the latest data to establish an efficient and highly suitable epidemic analysis and prediction model according to the actual situation, and then to give reliable predictions, which could provide an important refer-ence for the government to formulate emergency macroeconomic decisions and medical resources allocation. recently, the susceptible-exposed-infectious-recovered (seir) or other similar models 6,7 are used to forecast the potential domestic and international spread of this covid-19 epidemic with parameters estimated from other sources.the real situation could be much more complicated and changing all the time. especially, with the implementation of the chinese government's multiple epidemic control policies, the control of nationwide epidemic has become obvious. however, the medical supplies in hubei will still affect the implementation of national policies. in this letter, we present the current situation of the epidemic, predict the ongoing trend with data driven analysis, and estimate the outbreak size of the covid-19 in both hubei and other areas in mainland china. the data of the epidemic are listed in table 1 and also graphically shown in fig. 1 , in which "china" is used to denote the mainland china, and "other" mainland china other than hubei province. the data includes the daily confirmed(suspected) infections, totally confirmed(suspected) infections, daily deaths, and total deaths from jan. 20, to feb. 11, 2020, reported by the national health commission of the republic of china (nhc), 8 , and health commission of hubei province (hch). 9 jan. 20, 2020, containing all the cases reported from 0 to 24, is the zeroth day in this letter, and then others are implied. the total number of suspected cases reaches the peak value on the 19th day (feb. 8), and then drops rapidly. notice that, until feb. 11, 2020, almost all the cases of deaths (1068/1113, 96%,) locates in hubei province, which reveals the epidemic in hubei is much more serious than that in the other areas of china. on the hand, it states the strict quarantine and limitation on population mobility have effectively prevented outbreaks in other provinces of china. scribe the data of daily infections and deaths in hubei, where x = (t + 0 . 5 − t t ) with t denoting the day, and t t representing the turning point; a and k are the parameters and determined by the data together with t t . the cumulative data of infections or deaths are obtained by the integration over h ( t ). for the epidemic in the other areas of china, the data of infections shows an asymmetric character, and then will be described as where x = t − t t ; the parameters b, k 1 , and k 2 together with t t , are then determined by fitting to the data. fig. 2 shows the fit and trend predictions to the total infections and deaths in hubei and china other than hubei. the extracted turning point of the infections in hubei is the 17th day, namely, feb. 6, 2020. the epidemic in hubei is predicted to end after mar. 10, 2020. we estimated that the epidemic is to end up with a total of 39, 0 0 0 infections in hubei, not including the clinically diagnosed cases since feb. 12, which may enlarge the prediction by 1.4 times. with considered data, namely, data from jan. 20 to feb. 11, the average errors are bout 166 and 190 for the fits to describe the daily and cumulative infections in hubei, respectively, corresponding to 8.6% and 1.6% for the average relative errors, respectively. fig. 2 (b) and (e) shows the estimations of the total and daily deaths in hubei. the predicted turning point is feb. 12, 2020. the total deaths is estimated to be 2250. notice the distribution of the daily deaths is delayed about 5 ∼6 days compared with the that of the daily infections. the average errors are bout 4 and 22 for the model to describe the daily and cumulative death numbers, respectively, corresponding to the relative errors 8.6% and 6.2%, respectively. the numbers of the daily and total infections in china other than hubei are showed in fig. 2 (c) and 2 (f), respectively. the extracted turning point is feb. 1, 2020 and the epidemic is expected to end on the 45th day, namely, on mar. 5, 2020. the estimated number of cumulative infections is about 12,600 in china other table 1 the data of epidemic caused by the covid-19 pneumonia in the mainland china and hubei, including (a) daily infections, (b) daily deaths, (c) total infections, (d) total deaths, (e) daily and (f) total suspected cases. china hubei a b c d e f a b c d 2020/1/20 77 2 291 6 27 54 72 2 270 6 2020/1/21 149 3 440 9 26 37 105 3 375 9 2020/1/22 131 8 571 17 257 393 69 8 4 4 4 17 2020/1/23 259 8 830 25 680 1072 105 7 549 24 2020/1/24 4 4 4 16 1287 41 1118 1965 180 15 729 39 2020/1/25 688 15 1975 56 1309 2684 323 13 1052 52 2020/1/26 769 24 2744 80 3806 5794 371 24 1423 76 2020/1/27 1771 26 4515 106 2077 6973 1291 24 2714 100 2020/1/28 1459 26 5974 132 3248 9239 840 25 3554 125 2020/1/29 1737 38 7711 170 4148 12,167 1032 37 4586 162 fig. 1 (f)), we did not parameterize this data, and hence did not give a trend prediction. the covid-19 epidemic in china is predicted to end after mar. 20, 2020, and cause 52,0 0 0-68,0 0 0 infections and about 240 0 deaths. however, the data trends show that the quick and active strategies to reduce human exposure taken in china, such as limi-tation on population mobility and interpersonal contact rates, strict quarantine on migrants, have already had good impacts on control of the epidemic. now the outbreak and deaths of the covid-19 epidemic are mainly in hubei province. after this letter has been written, the hubei reported 14,840 confirmed infections (including 13,332 clinically diagnosed cases) on feb. 12, 2020, which is almost 9 times greater than the data of the previous day. the huge fluctuation is due to the changing of diagnostic criteria in hubei. and this clinical criteria taken in hubei is expected to play an active and important role in controlling the outbreak and death rate. the authors declare no conflict of interest. dear editor , recently, several studies in this journal have highlighted the threat of avian influenza virus (aiv) to humans, poultry, and other animals. [1] [2] [3] [4] [5] [6] in equines, there was only one reported influenza outbreak caused by aiv, which occurred in 1989-1990 in china. however, aiv still poses a potential threat to equines. in contrast to aiv, equine influenza virus (eiv) is a commonly known causative pathogen of acute respiratory disease in equines. to date, two subtypes of eivs have been determined in the equine population worldwide: h7n7 and h3n8. 7 h7n7 eiv was initially identified in prague in 1956, and the last outbreak caused by h7n7 eiv occurred in 1979. 7 h3n8 eiv was first isolated in miami in 1963 and is currently responsible for ei outbreaks worldwide. during continuous transmission and evolution in equines, h3n8 eiv strains diverged genetically into three distinct lineages: predivergent, european, and american. 7 historically, there have been four main ei outbreaks in mainland china, occurring in 1974 china, occurring in , 1989 china, occurring in -1990 china, occurring in , 1994 , and 20 07-20 08. in the first, third, and fourth outbreaks, the isolated eiv strains were of the h7n7 subtype, h3n8 subtype european lineage, and h3n8 subtype american lineage, respectively. in the second ei outbreak, the causative pathogen (a/equine/jilin/1/1989) was determined to be h3n8 subtype influenza a virus (iav) by antigenicity characterization. however, after genetic sequencing, all eight segments of a/equine/jilin/1/1989 were genetically closer to those of avian influenza virus than those of eiv, indicating an interspecies transmission event. the 1989-1990 ei outbreaks in china contain two major outbreaks. the first ei outbreak occurred in jilin and heilongjiang provinces between march and june 1989, with a morbidity of 81% and a mortality of up to 20% in some herds. the second ei outbreak occurred in heilongjiang province in april 1990, with a morbidity of 41% and no mortality. the high mortality and unique antigenicity of a/equine/jilin/1/1989 attracted the attention of eiv researchers. however, after the 1989-1990 ei outbreaks in china, the virus disappeared from the equine population for unknown reasons. although no evidence in the epidemiological investigation worldwide supports the continuous circulation of a/equine/jilin/1/1989-like eiv in equines, we should not underestimate the potential of interspecies aiv transmission to equines and the possibility of future ei outbreaks caused by aiv. the haemagglutinin (ha) of iavs recognizes and binds the cell surface sialic acid (sa) receptor of the host respiratory tract, and then the virus enters into cells and replicates. the distribution of the sa receptor influences the host range of iavs. human influenza viruses preferentially bind the saa2,6 gal receptor, while aivs preferentially bind the saa2,3 gal receptor. it has been reported that the saa2,3 gal receptor is abundant in the epithelial cells of the horse trachea, and animal experiments indicate that iavs with an ha recognizing the saa2,3 gal receptor could replicate in horses. this finding provides a prerequisite for cross-species transmission of aiv to equines. in fact, there were several pieces of direct molecular evidence supporting the interspecies transmission of aiv to equines, in addition to the 1989-1990 ei outbreaks in china. in 2010, abdel-moneim et al. reported one h5n1 eiv strain (a/equine/egypt/ av1/2009) isolated from donkeys with cough, fever and serous nasal discharge in egypt. 8 sequencing results indicated that the virus had a close genetic relatedness to h5n1 aiv. in addition, h5 seroconversion was observed in 25.71% (27/105) of the examined donkeys. in 2011, he collected 284 equine lung tissue samples in china and isolated one aiv-derived h9n2 eiv strain (a/equine/guangxi/3/2011) ( https://kns.cnki.net/ kcms/detail/detail.aspx?dbcode=cmfd&dbname=cmfd201301& filename=1012496506.nh&v=mtmxmzc3ck5wrji2sexleedove1x wkviuelsogvymux1efltn0romvqzcvryv00xrnjdvvjmt2vazvpt rknua1u= ). among the tested equine serum samples in china, 0.54% (7/1110) of samples were positive for anti-h9n2 antibody. in the 2015-2016 ei outbreaks in pakistan, khana et al. reported that the isolated eiv strain (a/equine/pakistan/16) was reassorted from aiv. 9 the common characteristic for the reported cross-species transmission events of aiv to equines in china, pakistan, and egypt is that they occur in farming equines. 8 , 9 in the mixed farming system, equines and domestic poultry often live in close proximity. compared with racehorses, farming equines have more opportunities to contact domestic poultry and experience long-term environmental exposure to poultry. aiv infections in poultry increase exposure risks to equines. recently, frequent reports of cross-species transmission events of aiv to farming dogs in china and korea also indicate the potential threat of aiv to farming animals with close contact with poultry. 10 another problem is that the farming equines in china, pakistan, and egypt had no vaccination history. even in some racehorse populations, vaccinations for eiv are not routinely performed as recommended by the world organization for animal health (oie) expert surveillance panel on ei vaccine composition. although h3n8 eiv is antigenically distinct from a/equine/jilin/1/1989, animal experiments indicated that high doses of ei vaccines still provided complete protection against challenge with a/equine/jilin/1/1989. accordingly, routine vaccination with h3n8 ei vaccines in equines might prevent aiv infection to some degree, at least for h3n8 subtype aiv. several strategies may help reduce the threat of aiv to equines, including reducing exposure of equines to poultry, birds, and other hosts of iav, especially animals with clinical signs of influenza virus infection; monitoring aiv prevalence in domestic poultry around equines and routinely vaccinating domestic poultry with aiv vaccines; vaccinating susceptible equines with ei vaccines, especially farming equines in close contact with domestic poultry; and monitoring the prevalence of multiple aiv subtypes in equines, not merely that of those restricted to h3n8 subtype. none. we read with interest recent articles in this journal regarding the utility of next-generation sequencing for the diagnosis bacterial meningitis. 1 , 2 bacterial meningitis causes substantial morbidity and mortality worldwide. 3 rapid identification of the microorganisms is essential for early initiation of appropriate antimicrobial therapy, thereby improving clinical outcome. yet routine diagnostic methods fail to identify the bacteria in the majority of patients. over the last decade, advanced sequencing technologies have greatly improved our capacity to detect the causative agents of infectious diseases in clinical samples. 4 , 5 of these, the single molecule real-time sequencing developed by oxford nanopore technologies (ont) is a promising tool for diagnostic setting because of its short turnaround time. in late april 2019, a 59-year old seller of fish-noodles was referred to our hospital with a 1-day history of headache, fever and vomiting. he had a history of heavy alcohol use and hepatitis c infection, and had cirrhosis and diabetes mellitus. on admission, he was unconsciousness (a glasgow coma scale of 8), with a body temperature of 40 °c, a blood pressure of 140/80 mmhg and neck stiffness. initial gram-stain and microscopy of csf showed grampositive cocci, 8449 white cells/ul with 91% neutrophils, elevated protein and low glucose level, and high lactate concentration ( fig. 1 a) . routine bacterial culture, plus streptococcus pneumoniae and s. suis pcrs were all negative. he was diagnosed with bacterial meningitis, and given a combination of ceftriaxone (2 g/12 h) and dexamethasone (0.4 mg/kg/12 h). his clinical condition steadily improved. his second and third csf samples became negative by gram stain. the other csf parameters also improved, except the glucose, which remained low ( fig. 1 a) . on day 20 of hospitalization, the patient suddenly became unconsciousness with fever. brain magnetic resonance imaging showed bifrontal abscesses ( fig. 1 b) . after consulting a local neurosurgeon, aspiration of the brain abscesses was not advised and the patient was treated empirically with meropenem (2 g/8 h) and vancomycin (1 g/8 h). due to continued diagnostic uncertainty, we performed 16s rrna sequencing of the admission csf, stored as part of an going clinical study (supplementary materials), using an established sangersequencing based 16s rrna method. 6 subsequently, analysis of the obtained sequences revealed evidence of s. agalactiae (supplementary figure 1 ). given this new diagnostic result of the admission csf and because the patient had recovered clinically, the patient was given 24 million units of penicillin g for every 4 h. after day 43 of hospitalization, all csf parameters had normalised ( fig. 1 a) . likewise, on ct scan the brain abscess was now significantly improved ( fig. 1 c) . the patient was discharged with full clinical recovery. additionally, minion sequencing of complete 16s rrna gene was retrospectively carried out on the extracted nucleic acid of the admission csf yielded a total of 14,848 reads after 100 min of sequencing run. of these, 11,556 reads (79%) were successfully aligned to s. agalactiae ( fig. 1 d) . the remaining reads were assigned to other streptococcus species (mostly s. dysgalacticiae ( n = 2.145, 14%)), likely attributed to a combination of the high level of sequence similarities of the 16s rrna region between them and the sequencing errors introduced by the minion systems. analysis of sequencing data generated during the 25, 50 and 75 min of sequencing run time also yielded the same results (supplementary figure 2 ). details about the minion procedure are presented in supplementary materials. to further assess of the utility of csf minion sequencing of 16s rrna gene for the detection of bacterial meningitis pathogens, six csf samples from patients with confirmed bacterial meningitis enrolled in the abovementioned clinical study were tested ( table 1 ) . analysis of the minion reads obtained after two hours of the sequencing run showed that the majority of reads were correctly assigned to the corresponding bacterial species ( s. pneumoniae and s. suis) or genus ( neisseria ) found in the csf samples by diagnostic work up of the clinical study ( fig. 1 e and table 1 ). additional analysis of the obtained reads generated at two earlier time points (20 min and 60 min) of the sequencing run generated the same results ( table 1 ) . collectively, we report the first application of minion sequencing of 16s rrna gene to detect bacterial meningitis causing pathogens in csf samples from a low and middle-income country. the assay was able to detect the bacterial causes in all of the seven tested csf samples. meanwhile, gram stain and culture, the two most commonly used methods in clinical microbiology laboratories worldwide, were negative in 3/7 samples. ( fig. 1 and table 1 ). in addition to csf samples described in the present study and a recent pilot study from korea, 7 successful detections of haemophilus influenzae in sputum and campylobacter fetus in culture materials by minion sequencing of 16s rrna have recently been reported. 8 together, the data suggest that minion sequencing of 16s rrna is a sensitive method for rapid and accurate detection of pan-bacterial pathogens, including unexpected microorganisms, in clinical samples. additionally, the bacterial species information generated by the analysis of 16s rrna sequences can be useful for disease surveillance and vaccine evaluation. thus, the application of the method would be relevant for both patient management and epidemiological research. indeed, to the best of our knowledge the present study represents the first report of s. agalactiae associated meningitis in vietnam. because the incidence of invasive diseases (including meningitis) caused by s. agalactiae has been reported with increased frequency in recent years, 9 s. agalactiae should be considered as an important differential owing to the unavailability of the reagents at the time of patient admission, we were not able to perform real-time diagnosis using minion sequencing on the collected csf samples. however, same day diagnosis is theoretically achievable, because the current workflow takes 5 -6 h to operate. prospective study is urgently needed to assess its translational potential in the diagnosis of bacterial meningitis. since september 2017, a prospective observational study aiming at exploring the utility potential of next-generation sequencing in patients presenting with central nervous system (cns) infections has been conducted in the brain infection ward of the hospital for tropical diseases (htd) in ho chi minh city, vietnam. htd is a tertiary referral hospital for patients with infectious diseases from southern provinces of vietnam, serving a population of > 40 million. any patient ( ≥16 years) with an indication for lumbar puncture was eligible for enrolment. patient was excluded if no written informed consent was obtained. as per the study protocol, csf, plasma and urine samples were collected at presentation alongside demographic, meta-clinical data and results of routine diagnosis. after collection, all clinical specimens were stored at −80 °c until analysis. the clinical study received approvals from the institutional review board of the htd and the oxford tropical research ethics committee of the university of oxford. written informed consent was obtained from each study participant or relative (if the patient was unconsciousness). sequencing of complete 16s rrna gene was retrospectively performed using minion nanopore sequencer (ont), following the manufacturer's instructions. in brief, amplification of the complete 16s rrna gene and library preparation were carried out on extracted nucleic acid using 16s barcoding kit (sqk-rab204, ont) and primers (27f 5 -agagtttgatcctggctcag-3 and 1492r 5 -ggttaccttgttacgactt-3 ), followed by the sequencing of the amplified product using r9.4 flow cells (ont). minion reads were first basecalled using albacore v2.1.7 (ont), followed by demultiplexing using porechop ( https://github.com/rrwick/porechop ). determination of bacterial genus/species composition in the obtained reads was then carried out using epi2me interface (metrichor, oxford, uk), a platform for cloud-based analysis of minion data. overall, the whole procedure of minion sequencing of 16s rrna gene takes 5-6 h to complete (supplementary figure 3) . we, the author of the submitted manuscript declare that we do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership, relevant patents, or research funding). dear editor, several aspects of influenza have been highlighted recently, including its global, comparative seasonality, 1 and issues around rapid point-of-care testing. 2 in addition, the uk has a national surveillance programme, the uk severe influenza surveillance system (usiss) to monitor and investigate severe cases of influenza across the country, 3 including severe cases of influenza admitted to intensive care (icu) and high dependency units (hdu). 4 specifically, the aim of this latter arm was to "monitor and estimate the impact of seasonal influenza on the population" and to "describe the epidemiology of severe disease." this surveillance began in the 2011-2012 influenza season and has continued to the present, with mandatory participation by all nhs trusts. leicester is one of 5 nhs commissioned centres in the uk providing extra-corporeal membrane oxygenation (ecmo) support for severe acute respiratory failure in adults. this process involves draining deoxygenated venous blood from the superior and inferior vena cavae, pumping this blood through a membrane lung, where oxygenation and carbon dioxide elimination take place. oxygenated blood is delivered back into the right atrium, therefore replacing the function of the native lung. in this way, ecmo can be used to support patients with respiratory failure of any cause. acceptance for admission for ecmo support follows referral to the ecmo service via structured questionnaire and discussion with the ecmo consultant on-call. patients are commenced on ecmo where benefits are deemed to outweigh the risks in patients with potentially reversible respiratory failure, who are already on maximal conventional therapy at their referring centre, and who are not achieving lung protective ventilation. 5 here, as part of our national usiss role, we describe severe influenza cases that required ecmo support in whom the predominant indications were severe hypoxia with a pao 2 :fio 2 ratio of < 100 despite maximal conventional therapy, and/or hypercapnoeic respiratory failure with a ph < 7.2 despite ventilation pressures > 30 cm h 2 o. during the 2018-2019 influenza season 33 cases of severe influenza were admitted to glenfield hospital for ecmo from our referring centres. most were male (23/33, 69.7%, 28-62 years, bmi: 17-47; female 10/33, 27-51 years, bmi: 21-38), and of white british ethnicity (30/33, 91.0%; with 1 each of chinese, asian, african ethnicity). comorbidities included, obesity, hypertension, asthma, copd, diabetes, anxiety, depression, epilepsy, a history of smoking and alcohol use (or abuse). all cases except for one influenza a(h3n2) infection (possibly two as subtyping was not performed for another sample) were due to influenza a(h1n1)pdm09. only one case had a history of influenza vaccination. various 'on referral' ecmo-related parameters were extracted, as well as contemporaneous laboratory results. these were statistically compared between patients who died ( n = 8) and those who survived using t -test or mann-whitney test for continuous variables and fisherexact test for categorical variables. correlation between duration on ecmo and laboratory parameters was assessed using spearman's rank correlation coefficient. ( n = 24) ( tables 1 and 2 ) . surprisingly, it was found that on direct comparison, most of the referral parameters for patients starting ecmo were not statistically different between those influenza-infected patients that eventually survived ( n = 24) versus those who died ( n = 8) -a case fatality rate of 25%. one patient was dropped from this analysis due to some missing data. only the respiratory rate (rr, p = 0.041) and the lactate ( p = 0.008) showed statistically significant differences between the two groups, with higher values being found in the patients who ecmo -extra-corporeal membrane oxygenation; sofa -sequential organ failure assessment; peep -positive end expiratory pressure; h 2 o -water; bpm -breaths per minute; pao 2 /paco 2 -partial pressure of arterial oxygen/carbon dioxide; altalanine aminotransferase. * rank correlation coefficient measures the strength and direction of a relationship between two variables. the coefficient ranges from −1 to 1 with 1 indicating a strong positive relationship between two variables and −1 indicating a strong negative relationship. a correlation coefficient close to 0 means the relationship between the two variables is very weak. died ( table 1 ) . however, clinically, these differences are of doubtful significance, as the rr is at the discretion of the parent clinical team prior to referral to ecmo, and the lactate levels are normal in the survivors and only marginally elevated in those who died which will again be dependent on use of cvvh. the higher pao 2 :fio 2 (p:f) ratio was statistically significantly correlated ( p = 0.020) with a shorter duration of ecmo, which suggests those with less severe disease recover quicker ( table 2 ) . many studies have reported on intensive care patient outcomes of the 2009 influenza a(h1n1)pdm09 pandemic, with or without the use of ecmo. in one study from australia, where ecmo was used, of 68 patients with confirmed influenza a (53 a(h1n1)pdm09, 8 un-subtyped) infection, 14 (21%) had died mainly due to intracranial and other forms of haemorrhage ( n = 10), or intractable respiratory failure ( n = 4). 6 another study from the usa, where influenza a(h1n1)pdm09-infected patients had non-ecmo icu admission, of 154 cases, 48 (37%) developed acute respiratory distress syndrome (ards), of whom 37 (24%) died. 7 a more recent study from spain that reviewed influenza a(h1n1)pdm09 cases admitted to icu (non-ecmo) from 2009-2015, found that of a total of 2421 cases, the mortality ranged from 18.8% (for community-acquired influenza) to 32.9% (for hospitalacquired influenza). 8 these ecmo-icu case fatality rates of severe influenza a(h1n1)pdm09 infection are similar to ours of 25% reported here, though compared to the specific ecmo patient cohort, 6 the causes of death in our patients were more variable, including intracranial and other haemorrhage ( n = 3), sepsis and multi-organ failure ( n = 2), respiratory failure ( n = 1), post-cardiopulmonary resuscitation hypoxic brain injury ( n = 1), and ischaemic bowel associated with atrial fibrillation ( n = 1). thus, even after 10 years of experience with this 'new' pandemic influenza a(h1n1)pdm09 virus, across almost the entire adult age-range ( ∼25-70 years in these previous studies), 6 -8 it appears that for severe cases, globally, the survival of such patients appears not to have improved. this may be somewhat surprising as the world's populations have become more immunologically experienced with this virus, which is now considered as a seasonal influenza virus that should be conferring some degree of persisting, cross-reactive individual and herd immunity over consecutive seasons. 9 this may be due to some predisposing genetic or environmental factors in individual patients, which should reinforce the general message that seasonal influenza immunisation is still recommended to minimise the number of people needing icu or ecmo support for severe influenza infection. more detailed monitoring on how these physiological parameters change over time (perhaps including more complex cytokine studies), in these severely ill, influenza a(h1n1)pdm09-infected patients admitted to icu-ecmo units, may eventually yield data to improve their management and clinical outcomes. none. we read with interest the report by stalenhoef and colleagues in this journal who show that biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection (ref). 1 here we report on a prospective observational analysis of the biomarker: cd5 antigen like protein (cd5l), in serum samples collected from 64 patients diagnosed with pneumonia 2 and 44 healthy adults between 2018 and 2019. the demographic and clinical characteristics of the 64 adults (males 70.3%; mean age 66 ± 20 years) with pneumonia were summarized in table 1 . 57 (89.1%) bacterial pneumonia patients (including 34 patients with confirmed bacterial pneumonia 2 and 23 patients with suspected bacterial pneumonia 3 ) and 7(10.9%) viral pneumonia patients were studied. there were significant differences in age, sex, wbc, neu%, crp, pct, cd5l, apache ii scores and length of icu stay between bacterial pneumonia and viral pneumonia. the pathogens responsible for pneumonia were described in supplementary table 1 . globally, gram-negative bacteria infection is more common than gram-positive bacteria infection in pneumonia. among them, acinetobacter baumannii (16, 59.3%) was considered the major pathogen in gram-negative bacteria and staphylococcus aureus (3, 42.9%) was considered the major pathogen in gram-positive bacteria. in viral pneumonia, influenza a (h1n1) was detected as the unique pathogen. in the study of the diagnostic performance of serum cd5l to identify etiology of pneumonia, as we found in supplementary fig. 1 , no matter in total bacterial pneumonia, suspected bacterial pneumonia or confirmed bacterial pneumonia, the serum of cd5l levels on day of pneumonia diagnosis were significantly higher than those in viral pneumonia and healthy control subjects. for evaluating the diagnostic performance of cd5l to differentiate bacterial from viral infection in pneumonia, roc analysis was conducted for the above bacterial pneumonia (supplementary fig. 2 ) and compared with routine laboratory markers ( table 2 ). by horizontal comparison, we found that the best auc was for cd5l (auc = 0.89), better than neu% (auc = 0.79), wbc (auc = 0.79), crp (auc = 0.78) and even pct (auc = 0.85). interestingly, by longitudinal comparison, the auc was observed highest in confirmed bacterial pneumonia (auc = 0.92), intermediate in all bacterial pneumonia (auc = 0.89), and lowest in suspected bacterial pneumonia (auc = 0.84), which may better elucidate the diagnostic performance of cd5l for etiology diagnosis in pneumonia patients. although there are 23 patients with suspected bacterial pneumonia for which no definitive pathogen was found, our result here may still provide a new treatment for bacterial infection identification in pneumonia patients for the current limitations in direct pathogen testing make it difficult to identify the pathogen at the time of diagnosis 4 . besides, as a potential biomarker to distinguish pathogens 5-8 , cd5l levels were also significantly correlated with wbc, neu%, crp and pct ( supplementary fig. 3 ) in patients with pneumonia. in the study of the diagnostic performance of serum cd5l to predict mortality in adults with pneumonia, mortality was defined as death occurring within 30 days after the onset of pneumonia. as we observed in supplementary fig. 4a , serum cd5l levels on day of pneumonia diagnosis were significantly higher in non-survivors ( n = 18) than survivors ( n = 46) ( p < 0.001). to ensure that serum cd5l levels were not influenced by the class of the infection in the specimens, spearman's rank correlation analysis was performed between serum cd5l levels and poor prognosis related scores 9 , 10 (sofa and apache ii scores) in patients with total bacterial pneumonia, suspected bacterial pneumonia, confirmed bacterial pneumonia and viral pneumonia, respectively. our correlation analysis between serum cd5l levels and sofa or apache ii scores shows that no matter for bacterial pneumonia or viral pneumonia, the cd5l levels showed positive correlation with sofa and apache ii scores ( supplementary figs. 5 and 6) . furthermore, the auc of cd5l for identifying 30-day mortality in adult pneumonia patients was 0.79 ( supplementary fig. 4b) , a value means good diagnostic performance, which is consistent with gao's study 9 before. therefore, we found that determining serum cd5l concentrations on day of pneumonia diagnosis was of great value in identifying bacterial infection from viral infection and predicting 30day mortality in adult patients with pneumonia, which suggests that cd5l may work for the etiologic diagnosis and could represent a novel biomarker for identification of a group of patients with pneumonia presenting with higher risk of death. these findings encourage further effort s aimed at exploring the clinical value of circulating cd5l to help early clinical decision-making in human pneumonia. none. high morbidity and mortality (up to 100%). asf is a devastating threat to pig agriculture and is responsible for serious production and economic losses. the asfv genome is 170-193 kilo base pairs in length 2 and has been divided into 24 different genotypes based on their b646l gene sequence, a gene which encodes the capsid protein p72. 3 , 4 our previous study showed that the p72 gene located in a very low genetic diversity region of the genome. 5 ye and colleagues, however, identified two novel genotypes xxv and xxvi, with genotype xxvi being especially divergent from all other genotypes. 1 to examine this unexpected result, in this study, we reanalyzed their data to evaluate the reliability of these two genotypes. we collected 716 available p72 gene sequences from ncbi ( http://www.ncbi.nlm.nih.gov/ ), which we then aligned with mafft software, a fast multiple sequence alignment program. 6 the alignments show that genotype xxv (accession number fr668409) has a single amino acid change at position 506 compared to genotype i, while for genotype xxvi (accession numbers fr668404 and fr668418), the region coding for amino acid residues 509 to 528 differs greatly compared to all other genotypes ( fig. 1 a) . to examine this in greater detail, we calculated genetic distance across the p72 coding sequence in 60 bp sliding windows, with a step size of 10 bp, between these two sequences and the 24 other genotypes ( fig. 1 b) . this sliding window analysis shows these two sequences for genotype xxvi have a region that is very divergent, with genetic distance up to 0.5067 from the other genotypes, while the genetic distances of the remaining regions are less than 0.1 ( fig. 1 b) . genetic distances for asfvs were calculated for 182 coding gene sequences, from 42 available complete genomes of asfvs, to assess the overall divergence. pairwise genetic distances of each gene for all asfv strains were calculated. the mean pairwise genetic distance for asfv genes was 0.041 ( fig. 2 ) , which is much lower than this divergent region of these two sequences for genotype xxvi (0.5067). therefore, it seems highly unlikely that a gene has such a highly divergent region. since recombination frequently occurs in asfvs, 5 we conducted a recombination analysis with the rdp4 program, which used the 3seq, bootscan, chimaera, genecov, lard, maxchi, rdp and siscan detection methods, 7 to determine whether recombination might have occurred in the xxvi genotype. we found reliable evidence for recombination events in both p72 genotype xxvi sequences ( fig. 1 c) . this suggests that the very highly divergent region of this p72 genotype is not homologous with other p72 genotypes. we used blastn, from ncbi ( https://blast.ncbi.nlm.nih.gov/blast.cgi ), to identify a source for this highly divergent region, however, no similarity sequence was found. we then mapped the multiple amino acid changes found in genotype xxvi to the 3d structure of p72 ( fig. 3 ) . the changed sites (marked in red in fig. 3 ) are located in the dec loop, a region which plays an important role in the formation of the trimer spike. 8 the amino acid mutations result in changes of electrostatic potential, hydrophobicity, and steric hindrance. it seems unlikely to have so many changes in such a functionally important region. in our previous studies we noticed that sequences directly submitted by individual laboratories to genbank often contain errors such as misidentification of species, sampling error, contamination, or are pseudogenes, which can lead to sequence analysis problems and erroneous conclusions. 9 , 10 sequences that deviate from the overall intraspecific pairwise divergence are potentially erroneous. 9 , 10 since genotype xxvi deviates from other genotypes not only in genetic distance, but also in protein structure, and mutations occur in the flanking regions of the sequences, we deduced that these reported mutations might be due to low quality sequencing. we identified the original manuscript reporting the three sequences of genotypes xxv and xxvi, 11 where the authors stated that the "alignment and translation of sequences obtained from sardinian isolates revealed that the c-terminal end of p72 gene was completely conserved between the sequences compared". this statement indicates that these sequences did not considerably diverge from previously reported asfv p72 sequences, and thus, suggests that an error in these sequences had been introduced upon submitting them to genbank. further examination of the original samples and sequences is needed. we contacted the corresponding author of this manuscript to check these three sequences, and were told that these three p72 sequences were all genotype i, and had some errors when submitted to genbank. in conclusion, the two novel genotypes of asfvs (xxv and xxvi) identified by ye and colleagues 1 are misled by problematic sequences. as reminded by our previous studies, many problematic sequences are present in genbank, which can lead to problems in downstream analyses. 9 , 10 thus, when published data is used for new analyses, the first set in the process of data analyses should be to filter these sequences for potential errors to reduce the possibility of reaching incorrect conclusions. if conclusions are based on obviously strange sequences, then we should trace back the data to their original source, and manuscript, and contact the corresponding authors before making conclusions based on these sequences. the authors declare no conflict of interest. fig. 1 . the analyses of phylogenetic tree, recombinant, and nucleotide divergence between 6xi and the other known 6 subtypes (6a-6xh) based on full-length hbv genome sequences. (a) the known hcv subtype 6 reference sequences (6a-6xh) from the previous report were used. phylogenetic analysis was performed by the maximum-likelihood method, based on the gtr + g + i substitution model, with 10 0 0 bootstrap replicates using the software mega v6. the sequences of hcv 6xi (ynkh261 and ynkh284) are marked in red dot. (b) bootscan plots were constructed using simplot 3.5.1 software based on 100 replicates with a 300-bp sliding window moving in steps of 50 bases. (c) pairwise comparisons of nucleotides similarities between 3 hcv 6xi strains and 30 reference genotype 6 sequences. 399,0 0 0 deaths per year. 2 currently, daa treatment regimens that target ns3/ns4a protease, ns5a phosphor-protein and the ns5b polymerase have shown high safe and high rates of sustained virologic response in hcv chronically infected patients ( > 90%). 3 however, under selective pressure from these drugs, drug resistance-associated substitutions (ras) can emerge during this therapy and result in treatment failure in 2 −10% of patients. 4 therefore, hcv infection is still a major global health concern. to date, eight confirmed genotypes have been characterized based on > 30% sequence divergence in the complete hcv genome, and genotypes are further classified into > 80 subtypes with a sequence divergence of > 15% to other subtypes of the same geno-type. 5 in the current study, we characterized a new hcv subtypes among chronic hepatitis c patients in yunnan, china, initially designated as 6xi, further analyzed its evolutionary history and investigated its baseline ras by next generation sequencing (ngs) method. plasma samples were collected between january 2018 and october 2018 from 160 chronic hepatitis c patients from kunming city in yunnan, china (fig. s1a) . the samples met the following inclusion criteria: (1) hepatitis c antibody-positive for 6 months with normal serum alanine aminotransferase (alt) levels; (2) subject was residing in yunnan province and was over 18 years old; (3) complete demographic information and clinical data were available; (4) consented to the use of patient information in studies on hcv epidemics; and (5) were treatment-naïve during sampling. there was no epidemiologic link among these individuals. the study was approved by the first people's hospital of yunnan province ethics committee. written informed consent was obtained from all participants prior to the study. out of a total of 160 chronic hepatitis c patients, 152 partial ns5b gene fragments were successfully amplified and sequenced with a success rate of 95.0% (152/160). multiple subtypes were identified in 152 subjects, including subtype 3b (36.2%, 55/152), 3a (23.7%, 36/152), 1b (19.1%, 29/152), 2a (8.5%, 13/152), 6n (7.9%, 12/152), 6a (2.0%, 3/152), and 1a (1.3%, 2/152) (fig. s1b) . interestingly, the remaining two strains (1.3%, 2/152) involving ynkh261 and ynkh284 together with the isolate km35 reported formed a novel separate cluster in the genotype 6 with an 82% bootstrap value, indicating a potential new hcv subtype 6. to confirm that the two strains belong to a novel hcv subtype 6, their complete genome sequences were successfully amplified and sequenced with 12 overlapping fragments. further, phylogenetic analysis was performed along with hcv reference sequences of representative subtypes 6a-6xh. the result showed that the two strains and isolate km35 formed a distinct monophyletic cluster supported by a high bootstrap value of 100%. the three strains were isolated from three hiv-1 infected patients without obvious epidemiological linkage in yunnan and showed no evidence of recombination using bootscan analysis ( fig. 1 (b) ). moreover, the intergroup nucleotide divergence (mean ± sd) % over the fulllength genome sequences of the isolates (ynkh261, ynkh284, and km35) were compared to that of representative subtypes (6a-6xh) ( fig. 1 (c) ). the results revealed that the three strains were different from known hcv subtypes of 6a-6xh by 16.5-28.5%. therefore, the three strains are initially designated 6xi. to better understand the time of emergence of hcv 6xi, we performed bayesian molecular clock analyses using full-length genome sequences to estimate the time to the most recent common ancestor (tmrca). as shown in fig. 2 (a) , the estimated tmr-cas for the genotype 6xi was 1971.1 [95% highest probability density (hpd): 1951. 1, 1991.4 ]. in addition, to further investigate baseline ras of subtype 6xi, naturally occurring resistance-associated substitutions (ras) were analyzed for the ns3, ns5a and ns5b sequences using next generation sequencing (ngs) method. strikingly, hcv 6xi strains contain the substitution 28 v with a 100% frequency of mutations in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor, 6 suggesting that the subtype 6xi maybe basically resistant to ns5a inhibitors ( fig. 2 (b) ). among the hcv eight genotypes, genotype 6 exhibits a high degree of genetic complexity and diversity, and 31 subtypes have been confirmed by the international committee on taxonomy of viruses. 7 in china, hcv genotype 6 is common, and subtype 6a is the most prevalent subtype, primarily distributed in guangdong, 6n is the second most prevalent subtype, mainly found in yunnan, followed by subtypes 6xa, 6 g, 6v, 6 w, 6e, 6b, 6j, 6q,and 6r among genotype 6 isolates. 8 -10 to our knowledge, 6xi is the eighth detection of novel hcv subtypes 6 in china combined with previously identified 6a, 6e, 6n, 6v, 6xa, 6xe and 6xh, resulting in genotype 6 to expand to 32 subtypes in the world. our findings again demonstrated that hcv genotype 6 was more complex and diverse. in summary, we characterized a new hcv subtype 6xi based on the characteristics of a monophyletic cluster, > 15% genetic distances, no significant evidence of recombination, and no epidemiologic link among individuals. in addition, bayesian analyses showed that 6xi may originate around the year 1971, and the strains of hcv 6xi naturally contain the substitution 28 v in the ns5a protein contributing to resistance to velpatasvir of ns5a phosphoprotein inhibitor. the present finding again highlights the genetic characteristics and hcv strains in yunnan, and the urgent need for continuous molecular screening and epidemic surveillance in yunnan to implement effective measures to reduce hcv transmission. the authors declare no competing financial interests. we recently read the article by corma-gómez a. et al. on which the authors described a higher probability of relapses with sofosbuvir/ledipasvir 8 weeks compared with 12 weeks of hcv (hepatitis c virus) among hiv (human immunodeficiency virus)/hcv coinfected patients. 1 in this regard, coinfections of hiv/ hcv also with hepatitis b virus (hbv) is associated with high mortality and comorbidity too. the persistence of hbv dna within the core cell in the absence of hbsag and even after clearance of the infection has been described previously in immunosuppressed patients, where hbv screening and prophylaxis is recommended. 2 interestingly, hbv reactivation has recently emerged during or after hcv treatment with direct-acting antivirals (daa). [3] [4] [5] the us food and drug administration issued a warning about this risk in 2016, 6 until that moment 24 cases were reported, two of which died. although specific mechanisms of this event are not well known, it has been suggested that hcv core proteins could inhibit hbv replication and hbsag production as well as production of envelope proteins, being hbcab the only marker of the presence of hbv. 2 in this context, treatment with daa would produce a drastic and rapidly blocking of hcv replication providing the opportunity to hbv to emerge and produce an immune reconstitution syndrome. the interference hbv-hcv has been outlined in 24% of patients with chronic hbv infection (hbsag-positive serology) and 1.4% of patients with resolved hbv infections (hbsag-negative and hbcabpositive) in a recently published systematic review and metaanalysis of patients with hcv-hbv. 7 in most cases, these reports have focused on coinfection hcv-hbv, but there is still a concern about patients triple-infected with hcv, hbv and hiv. although most cases reported occur in hbsag-positive, the main concern affects to patients with a basal serology showing hbcab-positive, hbsag-negative and hbsab-negative. hbsagpositive was associated with higher rates of hbv reactivations compared with hbsag-negative and hbcab-positive patients. 8 there is no clear information about this issue in hbsag-negative, hbcab-positive, and, when occurs, it is considered an uncommon event. 9 reactivation of hbv is characterized by reappearance or increase in hbv dna levels, and could also be accompanied by symptoms of hepatitis. the cases reported previously did show difference in the time of presentation of clinical symptoms, they used to start either during or after daa treatment. it seems that hbv reactivation usually occurs early after daa initiation treatment (4-8 weeks) while otherwise can occur after treatment completion. in that way, it seems necessary that hbv infection should be monitored early after daa initiation. the most recent european association for the study of the liver (easl) guideline about hbv infection, recommends that patients hbsag-negative and hbcab-positive undergoing daa treatment should be monitored and tested for hbv reactivation only in case of alt elevation. 10 they also recommend performing hbv dna levels only in case of alt increase. at the same time, the american association for the study of liver diseases (aasld) and the infectious disease society of america (idsa) guideline has been recently updated recommendations to monitor hbv dna levels only in patients hbsag-positive, but they emphasize that there is not enough data to monitoring dna among patients hbcabpositive or hbcab-positive and hbsab-positive. they remark that a reactivation should be considered if an unexplained increase in liver enzyme is present. in hiv patients, they provide the same recommendations. 11 in a recent review of the literature, the authors also recommend to perform an hbv dna only in patients with altered alt. 9 a meta-analysis recommended not to perform an hbv dna test in this case due to low rate of incidence and the associated cost which needs to be considered especially in an endemic hbv areas. 7 few data exist in hiv patients. the fact that many triple-infected patients are receiving antiretroviral therapy including (art) nucleoside/nucleotide analogous, as tenofovir disoproxil fumarate (tdf) or tenofovir alafenamide (taf), both active against hbv, suggest that hbv reactivation rate could be underestimated. in a recently published review, chang et al., recommend to perform an hbv dna test at baseline in triple-infected patients. if positive, an hbv-active antiretroviral therapy (art) should be started and hbv dna needs to be monitored every 4 weeks during treatment and until week 12 after completion of treatment. however, if baseline hbv dna is negative they match with aasld/idsa and easl recommendations. 12 one of our patients has a basal serology with hbcab-iggpositive, both hbsag-and hbsab-negatives, and undetectable hbv dna levels, prior to treatment with daas. regarding hiv infection, he has an analogues-free regimen. one month after having finished treatment with daas, the patient consulted because of abdominal pain, nausea and jaundice. he had a total bilirubin level of 11 mg/dl, ast 1025 iu/l, alt 642 iu/l and inr 1.30. a hbv viral load of 6,193,455 iu/ml was detected; hbsag, hbcab-igm and hbeag were positive. there were no reasons to believe that he was re-infected. treatment with entecavir was initiated; however, the clinical evolution was unfavorable and died as a result of an acute liver failure. the hbv viral load was requested from the stored samples drawn during the treatment of hcv showing that hbv viral load was undetectable at the beginning of treatment and in week 2, but progressively increased to 98 iu/ml in week 8 and up to 82,700 iu/ml in week 4 after treatment completion. during the follow-up of daa treatment alt and ast remained normal. this case changed our daily practice, since then all hiv patients and more specifically those not treated with either tdf or taf and presenting a previous hbcab-positive, are followed using dna levels and not only monitoring hepatic enzymes, as recommended by guidelines. we consider that this case may reflect the necessity of change the current guidelines. we recommend to perform a periodic monitoring of hbv reactivation using hbv dna during and after daa therapy in hbsag-negative and hbcab-positive patients, independently of hbsab presence, hepatic enzymes and clinical symptoms, particularly in hiv patients who are not receiving active treatment of hbv. the study was not funded. authors declare that there is no conflict of interest. recently, a notable pattern of synchrony of influenza a and b virus, and respiratory syncytial virus incidence peaks globally was reported in this journal. 1 a previous study characterized seasonal pattern of influenza a and b in china and identified three epidemiological regions featured by distinct seasonality. 2 on the basis of laboratory surveillance data from 30 chinese provinces spanning about 12 years from october 2004 through january 2017, our study further characterized seasonal patterns of circulating influenza a subtypes and influenza b lineages in the three defined epidemiological regions. our study revealed that pre-2009 a(h1n1) and a(h3n2) displayed wintertime and summertime epidemics in midlatitude and southernmost chinese provinces with subtropical climate, wavelet analysis demonstrated the two subtypes displayed twice-annual cycle in some years in mid-latitude chinese provinces ( fig. 1 (a)-(h) ). however, the two subtypes peaks in the winter with annual or longer cycle in northern chinese provinces. influenza a(h1n1)pdm09 b/victoria and b/yamagata virus all displayed epidemics in the winter or winter-spring with annual or longer cycle in all three epidemiological regions. we developed univariate and multivariate regression models to evaluate the association between climatic factors and the presence or absence of epidemics of each influenza subtype and lineage (positive proportion ≥5%) in the southernmost provinces where heating system is not generally used in the winter so temperature and relative humidity in external conditions in winter are close to those in indoor environment where people spend most of the time. we fitted the mixed-effects logistic regression model to control for the repeated measurements in each province of the region cluster. we kept one of temperature and absolute humidity (representative of vapor pressure) with smaller akaike information criterion in the model to reduce of multi-collinearity due to a high degree of correlation between the two factors. our analysis indicated temperature, humidity and rainfall were environmental predictors of influenza subtype/lineage-specific epidemics in the southernmost provinces when a 1-week lag of influenza epidemics behind climate was considered, which was similar to the findings from some ecological studies ( table 1 ). 3 our study indicated the u-shaped relationship between absolute humidity (ah) and pre-2009 a(h1n1) or a(h3n2) epidemics in the southernmost provinces, and suggest that high levels of ah in the summer, and low levels of ah in the winter increased the possibility of epidemics of the two subtypes. 3 in our analysis, lower temperature was an environmental driver of a (h1n1)pdm09 and b/yamagata epidemics in the southernmost provinces while there were bimodal associations between temperature, rainfall and b/victoria epidemics with highest probability of b/victoria epidemics at 11.7 °c of daily average temperature and 11.3 mm of daily average rainfall. although seasonal changes in human behaviors, such as school attendance or crowding indoors, and seasonal variations in immunity, such as melatonin and vitamin d levels have been proposed to account for the seasonal nature of influenza, 4 our findings suggest that the heterogeneity in influenza subtype/lineage-specific seasonality patterns could be driven by seasonal variations in virus survival, transmission and adaptive immunity by influenza subtype and lineage because of the same behavior modes and background of non-adaptive immunity in the same regions and seasons. we propose a hypothesis: under humid and hot condition the dominant transmission mode(s) for a(h1n1)pdm09, b/victoria and b/yamagata might have reduced efficiency, however, there could be effective transmission mode(s) for a(h3n2) and pre-2009 a(h1n1) virus. some experimental studies were performed to establishing a causal link between humidity, temperature and influenza virus survival/transmission. 5 transmission of influenza a (h3n2), a(h1n1)pdm09, b/victoria and b/yamagata virus by respiratory droplets or aerosols in the guinea pig model proceeds most readily under cold, dry conditions. 5 low humidity and temperature increased the stability of influenza virus in aerosols and on surfaces. furthermore, aerosol transmission of a (h3n2) virus in the guinea pig model was almost completely blocked at 30 °c, but contact transmission of a (h3n2) virus seemed to be efficient at different level of humidity and 30 °c. 5 it remains unclear if high temperature and humidity levels have effect on aerosol and contact transmission of pre-2009 a(h1n1), a(h1n1)pdm09, b/victoria and b/yamagata virus among hosts and their stability on surfaces. of note, an experimental study found the survival durations of a(h3n2) strains on swiss banknotes were significantly longer than pre-2009 a(h1n1) and b/victoria virus. 6 further studies are needed to understand how efficient are these transmission modes for different influenza subtypes or lineages, which is/are dominant mode(s) of transmission among hosts, and which of potential mechanisms is at play under humid and hot condition. one of our findings was the mutual inverse association between a(h3n2) and a(h1n1)pdm09 epidemics, which provided the evidence on interference between the two influenza a subtypes perhaps possibly due to multiple immune mechanisms. 7 however, our study showed b/yamagata epidemics were positively correlated with a(h3n2) epidemics, suggesting b/yamagata epidemics across over 12 study years that were weak could get well along with simultaneous weak h3n2 epidemics. understanding of influenza seasonality is important to define optimal timing of influenza vaccination campaigns. our study indicated that a(h3n2) virus brought about twice-annual epidemics in some years in mid-latitude chinese provinces and more frequent summertime epidemics in southernmost chinese provinces, which questions if a single annual influenza vaccination campaign starting in october can offer optimal protection against summertime epidemics of a (h3n2) virus in mid-latitude and southernmost chinese provinces. in recent years, it has been reported that mismatches of a(h3n2) virus between the influenza vaccine strains and circulating strains were identified frequently, 8 and vaccine effectiveness of a(h3n2) virus declined within 4-6 months postvaccination. 9 in conclusion, we identified the heterogeneity of seasonality pattern of pre-2009 a(h1n1) or a(h3n2) virus in three epidemiological regions of china, and different environment predictors for influenza subtypes and lineages in the southernmost provinces. further work should focus on understanding difference in virus survival, transmission by influenza subtype and lineage under humid and hot conditions. bjc has received research funding from medimmune inc. and sanofi pasteur, and consults for crucell nv. the authors report no other potential competing interests. as this study included data from the national influenza surveillance system, ethics approval was not required. not applicable. the datasets at national level analyzed during the current study are available in the world health organization flunet. the datasets with more specific information analyzed during the current study are available in chinese national influenza surveillance informatio system, but they are not open-access datasets. these influenza surveillance data can be available from chinese national influenza center on reasonable request. this study was supported by the national mega-projects for infectious diseases (grant number 2018zx102010 02-0 08-001 ), national natural science foundation of china (grant number 81302476 ) and emergency prevention and control project of ministry of science and technology (grant number 10600100000015001206 ). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. reported in zhejiang in 2017. 8 in this study, we identified ten cases of imported chikv infection in travelers returning to yunnan from southeastern asia in 2019. out of the ten patients with imported chikv infection examined in this study, nine patients had traveled back from myanmar and one patient had travelled back from thailand ( fig. 1 (a) ). all the patients displayed different degrees of symptoms, such as fever, cough, muscle pain, and rash. the details of all the symptoms are shown in table 1 . chikv infection was diagnosed using specific real-time reverse transcription-pcr. serum specimens were collected from the ten patients that tested positive for chikv by real-time pcr analysis, in yunnan between may 7, 2019 and august 1, 2019. the current study was approved by the medical ethics committee of kunming university of science and technology. written informed consent was obtained from all the participants. a new coronavirus associated with human respiratory disease in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus a pneumonia outbreak associated with a new coronavirus of probable bat origin emergence of sars-like coronavirus poses new challenge in china bat origin of a new human coronavirus: there and back again transmission of 2019-ncov infection from an asymptomatic contact in germany early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia what to do next to control the 2019-ncov epidemic? nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) national health commission of people's republic of china prevention and control of novel coronavirus pneumonia a hospital cluster combined with a family cluster of avian influenza h7n9 infection in anhui province first human infection by a novel avian influenza a(h7n4) virus contact reductions from live poultry market closures limit the epidemic of human infections with h7n9 influenza comparison between human infections caused by highly and low pathogenic h7n9 avian influenza viruses in wave five: clinical and virological findings pathogenicity and transmissibility of three avian influenza a (h5n6) viruses isolated from wild birds death of a very young child infected with influenza a (h5n6) equine influenza-a global perspective isolation and characterization of highly pathogenic avian influenza virus subtype h5n1 from donkeys molecular epidemiology of a novel re-assorted epidemic strain of equine influenza virus in pakistan in 2015-16 avian-origin h3n2 canine influenza virus circulating in farmed dogs in guangdong detection of pediatric bacterial meningitis pathogens from cerebrospinal fluid by next-generation sequencing technology next-generation sequencing combined with routine methods to detect the pathogens of encephalitis/meningitis from a chinese tertiary pediatric neurology center community-acquired bacterial meningitis rapid bacterial identification by direct pcr amplification of 16s rrna genes using the minion tm nanopore sequencer campylobacter fetus meningitis confirmed by a 16s rrna gene analysis using the minion nanopore sequencer bacterial diversity assessment in antarctic terrestrial and aquatic microbial mats: a comparison between bidirectional pyrosequencing and cultivation rapid diagnosis of bacterial meningitis by nanopore 16s amplicon sequencing: a pilot study diagnosis of haemophilus influenzae pneumonia by nanopore 16s amplicon sequencing of sputum epidemiology of invasive group b streptococcal infections among nonpregnant adults in the united states comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses impact of a poorly performing point-ofcare test during the 2017-2018 influenza season uk severe flu surveillance system icu-hdu influenza surveillance nhse) service specification 170110s. extra corporeal membrane oxygenation (ecmo) for respiratory failure in adults australia and new zealand extracorporeal membrane oxygenation (anz ecmo) influenza investigators extracorporeal membrane oxygenation for 2009 influenza a(h1n1) acute respiratory distress syndrome intensive care unit patients with 2009 pandemic influenza a (h1n1pdm09) virus infection -united states characteristics of patients with hospital-acquired influenza a (h1n1)pdm09 virus admitted to the intensive care unit cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus biomarker guided triage can reduce hospitalization rate in community acquired febrile urinary tract infection guidelines for the management of adults with community-acquired pneumonia. diagnosis, assessment of severity, antimicrobial therapy, and prevention cytokine markers as predictors of type of respiratory infection in patients during the influenza season procalcitonin as a marker of etiology in adults hospitalized with community-acquired pneumonia cd5l contributes to the pathogenesis of methicillin-resistant staphylococcus aureus-induced pneumonia orchestrating role of apoptosis inhibitor of macrophage in the resolution of acute lung injury aim inhibits apoptosis of t cells and nkt cells in corynebacterium-induced granuloma formation in mice the macrophage soluble receptor aim/api6/cd5l displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression assessment of apoptosis inhibitor of macrophage/cd5l as a biomarker to predict mortality in the critically ill with sepsis plasma long pentraxin 3 (ptx3) concentration is a novel marker of disease activity in patients with community-acquired pneumonia the updated analysis of african swine fever virus genomes: two novel genotypes are identified african swine fever virus replication and genomics phylogeographic patterns of the african swine fever virus genetic characterization of african swine fever virus isolates from soft ticks at the wildlife/domestic interface in mozambique and identification of a novel genotype the recombination hot spots and genetic diversity of the genomes of african swine fever viruses parallelization of the mafft multiple sequence alignment program rdp4: detection and analysis of recombination patterns in virus genomes structure of the african swine fever virus major capsid protein p72 detection of potential problematic cytb gene sequences of fishes in genbank assessing dna barcoding as a tool for species identification and data quality control genetic characterisation of african swine fever viruses from recent and historical outbreaks in sardinia consensus recommendations for resistance testing in the management of chronic hepatitis c virus infection: public health england hcv resistance group hepatitis c virus infection in children and adolescents challenges and perspectives of direct antivirals for the treatment of hepatitis c virus infection whole-genome characterization and resistance-associated substitutions in a new hcv genotype 1 subtype identification of 19 novel hepatitis c virus subtypes-further expanding hcv classification hepatitis c virus drug resistance associated substitutions and their clinical relevance: update identification of a new hcv subtype 6xg among injection drug users in kachin hepatitis c virus genotypes and subtypes circulating in mainland china higher relapse rate among hiv/hcv-confected patients receiving sofosbuvir/ledipasvir for 8 vs 12 weeks significance of anti-hbc alone serological status in clinical practice evaluation of hepatitis b reactivation among 62,920 veterans treated with oral hepatitis c antivirals acute fulminant hepatitis b during hepatitis c virus therapy with direct-acting antivirals in a patient co-infected with hiv spontaneous remission of hepatitis b virus reactivation during direct-acting antiviral agent-based therapy for chronic hepatitis c adverse events reporting system (faers): the public's stake in adverse event reporting hepatitis b virus reactivation during direct-acting antiviral therapy for hepatitis c: a systematic review and meta-analysis reactivation of hepatitis b in patients of chronic hepatitis c with hepatitis b virus infection treated with direct acting antivirals controversies in hepatitis c therapy: reactivation of hepatitis b virus clinical practice guidelines on the management of hepatitis b virus infection monitoring patients who are starting hcv treatment, are on treatment, or have completed therapy. hcv guidance: recommendations for testing, managing, and treating hepatitis c. electronic access significance and management of isolated hepatitis b core antibody (anti-hbc) in hiv and hcv: strategies in the daa era comparative global epidemiology of inßuenza, respiratory syncytial and parainßuenza viruses characterization of regional influenza seasonality patterns in china and implications for vaccination strategies: spatio-temporal modeling of surveillance data environmental predictors of seasonal influenza epidemics across temperate and tropical climates global influenza seasonality: reconciling patterns across temperate and tropical regions roles of humidity and temperature in shaping influenza seasonality survival of influenza virus on banknotes a systematic approach to virus-virus interactions comparing influenza vaccine efficacy against mismatched and matched strains a systematic review and meta-analysis i-move multicentre case-control study 2010/11 to 2014/15: is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination? metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers chikungunya virus infections in military deployments in tropical settings-a narrative minireview chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen east/central/south african genotype in a chikungunya outbreak caribbean and la réunion chikungunya virus isolates differ in their capacity to induce proinflammatory th1 and nk cell responses and acute joint pathology new insights into chikungunya virus emergence and spread from southeast asia mosquito-associated viruses in china chikungunya fever outbreak enhanced molecular surveillance of chikungunya virus chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes this work was supported by henan emergency project for prevention and control of 2019 novel coronavirus, the earmarked fund for modern agro-industry technology research system of china (cars-35) and the special fund for henan agriculture research system (s2012-06). the funders had no role in study de-sign, data collection and interpretation, or the decision to submit the work for publication. we thank jing li and hao-nan wang for the helpful discusses and suggestions. this work is supported by the open research fund of key laboratory of digital earth science ( 2019lde005 ), and by the fundamental research funds for the central universities under grant no. 310201911qd054 . this work was supported by the national natural science foundation of china ( 31702271 ) and the guangdong provincial natural science foundation ( 2017a030310367 ). we thank le kim thanh, le nguyen truc nhu, and lam anh nguyet for their logistic support. we are indebted to patients for their participations in this study.this study was funded by the wellcome trust of great britain ( 10 6 680/b/14/z and 204904/z/16/z ). supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.011 . we would like to thank the patients and healthy volunteers in the first affiliated hospital of chongqing medical university for their cooperation and support. this work has been financially supported by national natural science foundation of china (no. 81902134 and no. 81722001 ). we thank chinese national influenza surveillance network for contribution in influenza epidemiological and laboratory surveillance. we thank the members of the yunnan international travel healthcare center and kunming changshui airport customs for the data and sample collection. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.12.003 . supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.11.019 . recent correspondence in this journal has highlighted the current threat posed by recently-emerging imported chikungunya virus (chikv) in febrile returning travellers. 1 chikungunya fever is infection caused by the chikv and is characterized by fever, arthralgia, myalgia, headache, and rash. chikv belongs to the genus alphavirus within the togaviridae family and is transmitted to humans by the bite of infected mosquitoes-ae. aegypti and ae. albopictus . 2 since the first report of chikv infection in humans in tanzania, intermittent outbreaks have been documented in africa, south america, southern and southeastern asia, and the indian ocean islands; thus, its outbreak has become a global public health problem. 3 to date, three evolutionary distinct chikv genotypes, namely west african (wa), east/central/south african (ecsa), and asian have been identified, based on phylogenetic analyses. 4 a novel lineage of chikv-the indian ocean lineage (iol)-has also been reported, which descended from the ecsa genotype during an outbreak on the island of la reunion between 2005 and 2006. 5 recently, it has been reported that iol of chikv has spread to malaysia, singapore, thailand, and indonesia. 6 in china, the first case of imported chikv infection was found in yunnan in 1987. subsequently, several sporadic cases of nonindigenous chikv infection have been described. in 2010, the first outbreak of chikv fever with 129 cases was documented in guangdong. 7 recently, another outbreak of chikungunya fever was table 1 epidemiological information on ten febrile returning travelers infected with chikv in this study. in this study, nine complete genome sequences isolated from serum samples were successfully amplified and sequenced with 12 overlapping fragments, and then the sequence obtained was deposited in genbank under accession no. mn402883-mn402892. compared with the nucleotide sequences of the available from the ncbi database, the nine strains shared the highest 99.61-99.74% nucleotide identity with the east/central/south african lineage strain thail 2019 reported previously in thailand, 2019. further, bayesian maximum-clade-credibility tree for full-length nucleotide sequences were constructed using the beast package v.1.7.3. phylogenetic analyses revealed that the ten chikv strains clustered into the homogeneous indian ocean clade of the ecsa genotype ( fig. 1 (b) ).notably, the ten chikv strains isolated from serum samples possessed the mutation k211e in the e1 gene and v264a in the e2 gene; these mutations are associated with significant increase in viral infectivity in ae. aegypti . 9 the strains also possessed g60d and i211t substitutions in the e2 gene; these mutations contribute to increased chikv fitness in ae. albopictus. 9 however, the a226v mutation in the e1 gene that is related to significant increase in viral infectivity in ae. albopictus was not observed in any of the strains. 10 in summary, we characterized ten cases of human infection caused by imported ecsa genotype chikv in yunnan, china and successfully isolated nine infectious chikvs from the chikvpositive serum samples. the mutations associated with significant increase in viral infectivity for ae. aegypti or ae. albopictus were also observed in these strains. geographically, the yunnan province is in southeastern china and shares its border with southeast asian countries (laos, vietnam, and myanmar) that are most affected by chikv. with the increase of tourism and trade with southeast asian countries, cases of imported chikv infection are constantly increasing and may have the potential for re-emergence and autochthonous transmission to yunnan. the present study highlights the urgent need for continuous molecular screening and epidemic surveillance for chikv and its vectors to prevent future outbreaks of chikv infection among the human population of yunnan. this work was supported by the national natural science foundation of china (nsfc) ( u1702282 ), the reserve talents project for young and middle-aged academic and technical leaders of yunnan province (2019hb012), and youth talent program of yunnan "ten-thousand talents program" (ynwr-qnbj-2018-054). the authors declare no competing financial interests. key: cord-324984-ojrpsdt9 authors: ji, xingyue; li, zhuorong title: medicinal chemistry strategies toward host targeting antiviral agents date: 2020-02-14 journal: med res rev doi: 10.1002/med.21664 sha: doc_id: 324984 cord_uid: ojrpsdt9 direct‐acting antiviral agents (daas) represent a class of drugs targeting viral proteins and have been demonstrated to be very successful in combating viral infections in clinic. however, daas suffer from several inherent limitations, including narrow‐spectrum antiviral profiles and liability to drug resistance, and hence there are still unmet needs in the treatment of viral infections. in comparison, host targeting antivirals (htas) target host factors for antiviral treatment. since host proteins are probably broadly required for various viral infections, htas are not only perceived, but also demonstrated to exhibit broad‐spectrum antiviral activities. in addition, host proteins are not under the genetic control of viral genome, and hence htas possess much higher genetic barrier to drug resistance as compared with daas. in recent years, much progress has been made to the development of htas with the approval of chemokine receptor type 5 antagonist maraviroc for human immunodeficiency virus treatment and more in the pipeline for other viral infections. in this review, we summarize various host proteins as antiviral targets from a medicinal chemistry prospective. challenges and issues associated with htas are also discussed. viral infections still represent a major global public health problem. although hundreds of viruses are known to be pathogenic, only less than 10 of them can be treated in clinic with available antiviral drugs. for some highly pathogenic virus such as zika (zikv), ebola (ebov), severe acute respiratory syndrome (sars), as well as many others, there is still no effective drugs on market against them. most of the antiviral drugs approved are designed to target viral proteins to inhibit viral infections, and they are named as direct-acting antiviral agents (daas). 1 daas have been demonstrated to be very successful in clinic to combat viral infections, and are generally considered as very safe for human use because most of the targeted viral proteins have no human homologs. however, viral polymerases, one of the hottest targets for antiviral design, do share some structural similarity with their human counterparts, especially at the active sites, which is the major underlying reason for the toxicity observed with nucleoside-based antivirals. in addition, viral proteins do not normally share structural similarity among different species or even genotypes of virus, and hence one antiviral agent targeting a specific viral protein can hardly impart the same inhibitory effects against the other virus. consequently, the antiviral drugs available on market can barely be employed to treat newly emerging virus, and the magnitude of this problem is further exacerbated by the lack of broad-spectrum antiviral drugs on market. the other inherent limitation of daas is that daas, in most cases, have low genetic barrier to drug resistance because they act directly on viral proteins, and the resulted selective pressure will facilitate the mutations of virus during replication, which will make the virus refractory to the treatment of daas. 2 altogether, there still exist great unmet needs for the treatment of viral infections, and new antivirals with broad-spectrum antiviral activities as well as new mechanism of action are highly demanded. it is widely known that viruses are unable to complete their replication without the help of the host. they will hijack various host proteins or pathways throughout their replication cycle to facilitate their replication, and hence the inhibition or knockdown of such host proteins will block viral replication. therefore, host proteins as such are potential antiviral targets for drug development. the host targeting antivirals (htas) are complementary to the daas, and are superior to daas from several aspects. chiefly, a specific host protein might play crucial roles in the replication of several types of viruses, and thereby its inhibition will yield broad-spectrum antiviral activities. htas as such are likely to be effective against newly emerging virus, because they may also leverage on the same host protein for replication. for example, heat shock protein 90 (hsp90), a host chaperone responsible for the folding, assembly, and maturation of endogenous proteins, is also found to be crucial for maturation of many viral proteins, and therefore, hsp90 inhibitors are endowed with broadspectrum antiviral activities. 3 altogether, targeting host factors is a very promising strategy with possibility to address the critical challenges faced with the daas. in this review, we summarize the recent advances made in htas from a medicinal chemistry standpoint, and the host targets are generally classified into three different categories based on the development stage of their corresponding inhibitors/modulators, namely the ones which reached food and drug administration (fda) approval, that have entered clinical trials and those in preclinical studies. the focus is mainly placed on the smallmolecule based htas, and the antibody or small interfering rna (sirna)-based strategy will not be included. for several well-studied host targets with quite a lot of known inhibitors, because each one can stand alone as an independent review, and this review is not intended to be comprehensive, so for such targets, only a selection of representative inhibitors will be presented to discuss related issues. it should be noted that chemokine receptor type 5 (ccr5) antagonist but not chemokine receptor type 4 (cxcr4) antagonist has reached fda approval. however, cxcr4 is discussed in this section because it is closely related to ccr5. the chemokine receptors cxcr4 and ccr5 belong to the superfamily of g-protein-coupled receptors with seven transmembrane domains, and both are involved in the entrance of human immunodeficiency virus (hiv) virus. 4 the glycoprotein 120 (gp120) on the surface of hiv envelop will bind to the cd4 receptor on the surface of t lymphocytes, leading to the conformation change of gp120. the gp120 will then bind to coreceptor cxcr4, ccr5, or both to facilitate the entrance of hiv virus. 5 hiv-1 virus can thus be broadly classified into three types based on the coreceptor tropism, namely the x4-, r5-, and dual-tropic viruses. the feasibility of targeting cxcr4 or ccr5 for hiv therapy is supported by the fact that ccr5 mutations confer the host with resistance to hiv infection. 6 ample studies have demonstrated that cxcr4 antagonists can block hiv-1 infection through cxcr4. 7, 8 generally, cxcr4 antagonist can be divided into two types: peptide and nonpeptide based. the peptide-based antagonists were designed using the natural chemokines of cxcr4 as template, such as stromal cell-derived factor 1α (sdf-1α) and viral macrophage inflammatory protein ii (vmip-ii), both of which can bind to and then activate cxcr4 to initiate downstream signaling pathway. for example, peptide v1 (1; figure 1 ) was derived from the first 1 to 21 residues of vmip-ii, and it can block the interaction between cxcr4 and hiv-1, and thereby inhibit the entry of both x4-and dual-tropic hiv-1 virus. interestingly, replacement of all the residues in 1 with all d-amino acid lead to much more potent antiviral activities. 9 due to the inherent limitation with the peptide-based antagonist, several nonpeptide based antagonists have been developed. amd3100 (2) was discovered by random screening, and it inhibited hiv-1 infection by disrupting the interaction between cxcr4 and hiv-1. 10 encouragingly, it showed antiviral efficacy in x4 hiv-1-infected patients in a phase ii clinical trial. however, compound 2 lacks oral bioavailability due to its high positive charge, and cardiotoxicity was also reported in the clinical trial. 11 the structural optimization identified amd3465 (3) with just one macrocyclic structure retained. this compound retained all the biological profiles of 2, and it showed improved antiviral activity (ic 50 : 1-10 nm). it also inhibited the replication of drug-resistant virus strain (zidovudine). 12 amd070 (4), a noncyclam derivative, was discovered by anormed as a new cxcr4 antagonist. it showed much improved oral bioavailability (rat: 20%; dog: 80%), but hepatotoxicity was observed in preclinical studies. 13 further structural optimization led to gsk812397 (5) without the basic sidechain. it showed potent anti-hiv-1 activity with an ic 50 value of 1.5 nm, and it did not show any detectable toxicity (>1000 nm) in in vitro assay. in addition, 5 also presented acceptable pharmacokinetic profiles with the oral bioavailability in the range between 10% and 21% among different species. 14 further structural modification with constraining conformation (6) and/or opening up the tetrahydroquinoline ring (7) resulted in a series of new derivatives with retained anti-hiv activities. 15, 16 however, none of these compounds have been advanced into clinical trials. although compound 2, also known as plerixafor, was approved by the fda for autologous transplantation in patients with non-hodgkin's lymphoma or multiple myeloma, 17 no cxcr4 antagonist has been approved for the treatment of hiv infections. the underlying reasons are at least twofold: one major concern with the cxcr4 antagonist is the toxicity issue, especially that cxcr4 18, 19 or sdf-1α 20 knockout mice die prenatally with multiple neurological, cardiac/vascular, and hematopoietic defects. such potential adverse outcomes are exacerbated in the case of hiv treatment, wherein a life-long treatment is required; the other minor issue with these cxcr4 antagonists is the lack of oral bioavailability. iv injection is not practical in the case of hiv treatment, because this will definitely hurt the compliance of the patients. in comparison, the development of ccr5 antagonists for hiv treatment is much more successful. one ccr5 antagonist maraviroc (8; figure 2 ) has been approved by fda as hiv entry inhibitor in 2007. 21 with a long plasma half-life (15-23 hours), 22 8 is administrated orally once daily. the clinical data showed that 8 is well tolerated in patients and it can significantly repress the viral load in r5-infected haart-treatment experienced patients. moreover, it is active against 200 clinically derived hiv-1 envelope-recombinant pseudoviruses, with 100 of them being resistant to existing drug classes, demonstrating the merits of htas. 23 in the clinic, it is primarily recommended for the patients infected with r5-tropic hiv virus, and a tropism test (although not approved by fda) is highly required to determine the viral tropism population within the patient before the treatment to ensure treatment success. the first generation of the test (known as trofile) failed to distinguish r5 from r5/x4 mixed tropism, resulting in significant drug failure in clinic in r5/x4-infected patients. 24 therefore, 8 is just designated as a backup regimen, despite that a new reliable and cost-efficient tropism test is available in clinic now. interestingly, the combination of 8 with raltegravir/tenofovir/emtricitabine led to faster reduction of 2-long term repeat (2-ltr + ) newly infected cells and recovery of cd4 + t-cell counts after 48 weeks of treatment, 25 and 8 in combination with reverse transcriptase inhibitors is being tested in clinic for preexposure prophylaxis. 26 however, the use of 8 is accompanied with severe side effects, and in some cases even life-threatening conditions such as hepatotoxicity and heart attack were observed. to mitigate these limitations, the second generation of ccr5 antagonists have been developed. two clinical candidates vicriviroc (9) 27 and aplaviroc (10) 28 have made to phase ii trials. however, the studies were halted f i g u r e 2 the chemical structures of representative chemokine receptor type 5 antagonists due to insufficient efficacy and observed hepatotoxicity for 9 and 10, respectively. much of the subsequent medicinal chemistry effort is devoted to the structural modification toward 8 to 10, aiming to improve either efficacy and/or adme profiles. for example, pfizer discovered compound 8's structural analogue pf-232798 (11), 29 which showed not only potent anti-hiv activity (ic 50 : 2.0 nm) and moderate herg inhibition (ic 50 : 12 μm), but also superior oral bioavailability as compared to 8. in addition, compound 11 is also active against maraviroc-resistant hiv-1 isolate strain cc185. the data from phase-ii trials demonstrated its superior safety in patients with no adverse effects observed at a dosage up to 250 mg. however, no further data were disclosed after that. 30 gsk 163929 (12) with a 4,4-disubstituted piperidine scaffold exhibited potent anti-hiv activity and excellent pharmacokinetics (pk) profiles, but it did not progress to clinic trials due to toxicity concerns. 31 the other two representative new scaffolds derived from compound 8 are monocyclic piperidine amides and cyclic and acyclic urea-piperidines. some representative candidate compounds from these two series are shown in figure 2 . among these compounds, only tak-220 (13) was progressed to phase-i clinical trials. 32 however, no further update about the status of compound 13 has been reported. inspired by the scaffold of 9 and 10, several new ccr5 antagonists have been devised as depicted in figure 2 . 33 the most promising antagonist among this series is the one developed by incyte based on the structure of 10. incb9471 (16) showed potent antiviral activity against r5 hiv-1 strains at ic 50 values in sub-nanomolar range. in addition, it presented excellent pk profiles with oral bioavailability being 100% and 95% in rat and dog, respectively. 34 as such, 16 has been advanced to phase ii trials, and the data showed that it was well-tolerated in humans. however, its clinical studies were halted due to business issues. besides the chemotypes described above, ccr5 antagonists with miscellaneous scaffolds have also been discovered, yet none of them have made to clinical trials. 35 targeting cxcr4 or ccr5 alone for hiv treatment possesses several downsides. chiefly, a tropism test is required before the treatment, and tropism shifts are frequently observed in both treatment-experienced and treatment-naive hiv-infected patients. therefore, a dual antagonist against both cxcr4 and ccr5 would be perfect to mitigate these limitations. the first dual-tropic inhibitor amd3451 (17; figure 3 ) with n-pyridinylmethyl cyclam scaffold was discovered in 2004. it was moderately active against not only x4-and r5-tropic hiv strains but also dual -tropic hiv strains with ic 50 in the range of 1 to 30 μm. 36 although 17 is not potent enough for further development, it established the proof of concept of targeting cxcr4 and ccr5 simultaneously for hiv-1 treatment. ribavirin (18; figure 4 ) is an approved antiviral drug used in clinic to treat respiratory syncytial virus (rsv), hepatitis c virus (hcv) infection and viral hemorrhagic fever. although its specific antiviral mechanism of action f i g u r e 3 the chemical structure of a dual-tropic inhibitor amd3451 the chemical structures of 18 and 19 ji and li | 5 remains largely uncertain, it is widely accepted that it elicits its antiviral efficacy via modulating host pathways. for example, 18 showed moderate inhibitory potency against inosine-5′-monophosphate dehydrogenase (impdh) with a k i value of around 250 nm, which is believed to be highly involved in the replication of various viruses (see below). in addition, several other mechanism of actions have also been proposed and evidenced: (a) direct inhibition of rna polymerase by converting 18 to its triphosphate form to competitively bind to the nucleotides binding site in rna polymerase 37, 38 ; (b) 18 can act as a mutagen by inserting into the viral rna to push the virus beyond the threshold of error catastrophe 39, 40 ; (c) 18 shows immunomodulatory effect of shifting a th2 response in favor of a th1 phenotype, which helps to clear virus infections. 41 although the combination of 18 and interferon 2α (ifn-2α) used to be the soc for hcv treatment, it is notorious for several severe side effects. one major adverse effect associated with the use of 18 is hemolytic anemia. 42 to alleviate this unwanted effect, taribavirin (19) was developed as the prodrug of 18. ideally, 19 can be metabolized to 18 mainly in the liver to target hcv-infected hepatocytes, and hence the distribution of 18 within red blood cells will be significantly decreased, and thereby the development of hemolytic anemia will be subsequently eliminated. indeed, the clinical data from phase iii trials revealed that patients receiving 19 (fixed dosage 600 mg, bid) and ifn showed significantly lower rates of anemia as compared to the ones in 18 (1000-1200 mg) and ifn group (5.3% vs 24%). 43 however, the sustained virologic response (svr) rates for 19 and 18 group are 38% and 52%, respectively, failing to demonstrate the noninferiority end point for efficacy. the viser-2 trials also failed to meet the noninferiority end points. 44 with the approval of several daas such as sofosbuvir and ledipasvir, the treatment of hcv in clinic has been significantly revolutionized, and several both ribavirin (rbv)-and ifn-free regimens have been approved with better efficacy and safety profiles as compared to the old soc. initially, it was expected that the hcv treatment would not benefit much from the combination of rbv with other daas due to the safety concern associated with rbv. however, very interestingly, it has been found recently that rbv remained an indispensable component for the optimal treatment for some difficult-to-cure subgroups of hcv patients. 45 for example, in a phase 3 c-edge studies, the combination of rbv with elbasvir/grazoprevir achieved much higher svr ( [3/5] , respectively) as compared to the group without rbv in patients infected with genotype 4 hcv. 46 in addition, rbv increased the barrier to resistance, especially in patients receiving daas with low barriers to resistance. importantly, the combination of rbv with daas showed much improved safety and tolerability as compared with the combination with ifn, and the frequency and severity of anemia is significantly reduced with an adverse effect-induced discontinuation rate of less than 3%. 45 nevertheless, the last generations hcv daas (including glecaprevir/pibrentasvir, sofosbuvir/velpatasvir, sofosbuvir/velpatasvir/ voxilaprevir) are highly effective in most cases without any need to use rbv. cyclophilins are a family of cellular peptidyl-prolyl cis-trans isomerases (ppiase) and are involved in many cellular processes. the important roles cyclophilin a (cypa) plays during hcv replication were found by an unexpected clinical finding. cyclosporin a (csa; 20; figure 5 ), which shows high binding affinity to cypa, is an approved immunosuppressive drug. in a clinical trial for csa's therapeutic potential against hepatitis-associated inflammation, it was unexpectedly found that a significantly more potent antiviral response was observed in the combination group of csa and ifn-α2b as compared with the ifn-α2b alone group. 47 the function of cypa in the replication of hcv was subsequently confirmed in vitro. 48 the main cellular function of cyps is to convert the conformation (trans-to cis-form) of prolines in the protein, which is essential for trafficking of many proteins and forming protein complexes, and these functions are also indispensable for the replication of hcv. therefore, it is anticipated that the cypa inhibitors will show anti-hcv activity. although 20 is an approved drug, its main indication is suppressing immunoreaction after organ transplantation, which is an unwanted "side-effect" for antivirals. fortunately, the immnosuppressive effect of 20 is attributed to the inhibition of calcineurin (cn) but not cypa, and hence it is feasible to eliminate the immnosuppressive effect while retaining the anti-hcv activity by making new csa analogues. nim811 (21) is one of such analogues with a methyl-isoleucine at position 4 of csa. it showed anti-hcv activity in vitro with an ic 50 value of 0.12 μm, whereas its immunosuppressive effect was completely eliminated. 49 the phase i clinical trial showed that 21 is well-tolerated with no obvious adverse effects observed. however, in a subsequent double-blind, placebo-controlled study, the monotherapy with 21 failed to yield significant viral load reduction in genotype 1 hcv-infected patients. the underlying reason is attributed to a relatively low trough concentration (0.47 μm) at a dosage of 600 mg (bid), which is lower than the ic 50 value (1.5 μm) of 21 against hcv in the presence of serum, and the dosage elevation did not result in proportional exposure. 50 therefore, the clinical trial with 21 is not continued. alisporivir (22) is a more potent nonimmunosuppressive csa analogue with an anti-hcv ic 50 values of 0.045 and 0.33 μm in the absence and presence of serum, respectively. in addition, it is much more difficult to develop resistance against 22 both in vitro and in patients, as compared to other daas. 51 in phase ii trial, 22 was studied in combination with rbv as an ifn-free regimen in genotype 2 and 3 patients, and about half of the patients receiving 22 (800 mg, qd) plus rbv, and one-third of those receiving 22 only (1000 mg qd) were cured with a svr. 52 however, unfortunately, in a pivotal phase iii trial designed to study the combination of 22 with rbv and peg-ifn-α2a in treatment-naive, genotype 1 hcv patients, six cases of severe pancreatitis along with one death were reported, and the clinical trial with 22 was then put on hold. it is worth noting that all the severe side effects were observed in the triple-therapy aim including rbv and peg-ifn-α2a, both of which are notorious for their adverse effects, while the monotherapy with 22 or the ifn-free regimen did not result in the same side effects. 53 to mitigate the observed side effects with 21 and 22, the third generation of csa derivatives have been potent anti-hcv activity in vitro with an ec 50 and ec 90 value of 0.1 and 0.3 μm, respectively, and exhibited synergistic effect with ifn-α and additive effect with rbv. it also showed low potential for drug-drug interaction with no obvious induction on the major cytochrome p450 enzymes 1a2, 2b6, and 3a4. in addition, 23 also showed acceptable pk profiles with an oral bioavailability of around 20% in rat and monkey. 54 as such, 23 has been advanced into clinical trials. in patients with genotype 1 hcv infection, 900 mg/day of 23 achieved a decline in plasma viremia by 2.2log 10 after 15 days. 55 it is interesting to note that 23 showed totally different side effects from those of 21 and 22, indicating that the adverse effects may not be associated with the inhibition of cyps, but are likely resulted from the off-target effects of individual inhibitors. non-csa based cyps inhibitors have also been discovered. sanlifehins (sfa) including sanlifehin a (24), b (25), c (26) , and d (27) are a class of natural occurring polyketides isolated from the soil bacterium streptomyces sp. strain a92-308110. sfas were identified as cyps inhibitors with stronger potency as compared to csa derivatives, particular 25, of which the inhibitory potency against all cyps was 30-to 50-fold more potent than 20. it also showed much more potent antiviral activity in vitro with an ec 50 value of 70 nm against hcv genotype 1b. interestingly, albeit slightly less potent as compared to against the wild type, 24, 26, and 27 retained inhibitory effect against csa-resistant huh 9 to 13 subgenomic replicon with ec 50 values ranging from 3.3 to 6.8 μm. 56 however, the pk studies revealed that sfa suffered from poor water solubility (<25 μm) and poor oral bioavailability (<4%). moreover, sfa possessed undesirable immunosuppressive activity via an unknown mechanism. 57 structural modifications have been made to 24, and it was revealed that only the macrocyclic moiety was essential for the cyps inhibition, and modification on the sidechain had little effect on the binding affinity. 58 removal of the spirolactam moiety on the sidechain of 24 only led to the loss of immunosuppressive activity but not the cyps inhibition. such structure-activity relationships are very important for further optimization of sfa as anti-hcv agents ( figure 6 ). both csa and sfa derivatives are macrocyclic molecules with large molecular size, and as such both suffered from some limitations, including poor cell membrane permeability, high risk of drug-drug interactions and off-target toxicity, and synthetic inaccessibility for structural optimization and manufacturing. in 2016, a new family of nonpeptide based small-molecule cyps inhibitors have been designed using fragment-based strategy. 59 the crystal structure of cypa indicated that its ppiase catalytic site consisted of hydrophobic, aromatic, and polar residues, next to the catalytic site is a deep pocket called gatekeeper site, which might contribute to the substrate binding specificity ( figure 7) . a total of 34 409 fragments were docked into these two sites, and several fragments were identified to bind to these two sites separately. eventually, fragment 28 and 29 from each binding site were it has been well-established that cyps are involved in a broad range of viral infections, including hiv-1, 60 influenza virus, 61 hbv, 62 sars coronavirus, 63 human cytomegalovirus (hcmv), 64 papillomavirus, 65 and nidovirus, 66 among others. therefore, it can be envisioned that cyps inhibitors should exhibit broad-spectrum antiviral activity against these viruses. indeed, cyps inhibitors were reported to show broad-spectrum antiviral activities. 67, 68 consequently, the development of cyps inhibitors could benefit the treatment of a variety of viral infections, possibly including the newly emerging unidentified viral infections. it is widely known that virus will hijack a wide range of host factors to facilitate its replication. meanwhile, the host has also evolved an innate immune system to counteract viral infections. compounds with the capacity to mediate the host antiviral pathways are expected to confer broad-spectrum antiviral activity, and nitazoxanide (32; figure 9 ) is such a compound that regulates several host antiviral pathways to convey broad-spectrum antiviral profiles. 32 is originally an fda approved drug for the treatment of diarrhea caused by cryptosporidium parvum and gastrointestinal disorders as the most frequently observed side effects. it also showed no effect on cardiac repolarization in a clinical trial for cardiac safety. 69 in recent studies, 32 was revealed as a promising antiviral agent against a wide range of pathogenic viruses including influenza virus, 70 hbv, 71 hcv, 71 ebov, 72 denv, 73 jev, 74 hiv, 75 and zikv, 76 among others with ic 50 values ranging from 0.06 to 1.0 μg/ml 77, 78 its mechanism studies revealed that multiple host antiviral pathways were involved, and as such 32 is widely known as a polypharmacology antiviral agent. 32 activated protein kinase r (pkr), which plays vital roles in innate immune system. the activation of pkr leads to the phosphorylation of eukaryotic initiation factor 2α, an important host restriction factor against viral replications. 79 hbv viral protein hbx was found to interact with host protein damage-specific dna-binding protein 1 (ddb1) to promote transcription of covalently closed circular dna (cccdna) and degradation of a host restriction factor chromosomes 5/6 (smc5/6). 80 32 was reported to disrupt the interaction between hbx and ddb1 to block the transcription from cccdna. 81 in addition, 32 was also able to activate cellular antiviral response and induce the expression of a subset of ifn-stimulated genes, especially interferon regulatory factor 1, 82 which is known to block the replication of a wide range of viruses. 83 other host targeting mechanisms are also involved in the broad-spectrum antiviral profiles of 32. 72 since 32 has been safely used in clinic for many years, it has been advanced into clinical trials for the treatment of several viral infections. the most advanced one is the treatment of acute uncomplicated influenza with 32. in a phase 2b/3 trial, significant reductions in tci50 viral titer and alleviation in symptoms were observed in 32 (600 mg, twice daily) group as compared to the placebo. no resistance was identified in the influenza virus collected from the patients receiving 32, and no adverse effect on humoral immune response was reported. 84 a large global phase 3 trial is being conducted. several clinical trials of 32 for the treatment of other viral infections are also ongoing. in a pilot clinical trial of 32 for the treatment of chronic hbv, the serum hbv dna of eight of nine patients became undetectable after 4 to 20 weeks of treatment with 32, and more importantly, three out of nine subjects became hbeag negative, which is a rare outcome for current standard of care. this proof-of-concept study presented 32 as a very promising drug to achieve a hbv cure. 85 32 was also tested in clinical trials against hcv infections, and it showed very pronounced efficacy either as monotherapy or in combination with ifn-α and rbv. for example, administration of 500 mg 32 twice a day for 48 weeks with 180 μg of ifn-α once weekly from week 13 to 48 achieved a svr of 79% in patients infected with chronic hepatitis c (chc) genotype 4, as compared to 50% for ifn-α and rbv. however, the development of 32 as an anti-hcv drug was not further pursued due to the approval of several new daas, despite that 32 has much higher genetic barrier to resistance as compared to daas. interestingly, 32 is considered as a prodrug because it is rapidly hydrolyzed to tizoxanide (33) in the plasma with a half-life of only 6 minutes. compound 33 showed broad-spectrum antiviral activity against a panel of viruses both in vitro and in vivo. to obtain new candidate compounds with improved potency and safety profiles, structural modifications were made to 32 primarily on the phenyl ring a and the thiazole moiety. it was revealed that electron-withdrawing group such as nitro and chloro group at c-5 position was favorable for the antiviral activity, and replacement of the thiazole ring with phenyl ring retained the activity. the structural optimization led to the f i g u r e 9 the chemical structures of nitazoxanide analogues identification of a candidate compound rm-5038 (34) with a chloro group at the c-5 position, which showed comparable activity to that of 32 with ec 50 values in the submicromolar range. 34 was considered to be superior as compared to 32 due to the replacement of the potential cytotoxicity nitro group in 32. 32 also suffers from poor systemic exposure after oral administration, and it is primarily biodistributed in the gastric intestinal tract, and excreted via urine and faeces. for antiviral therapy, it is preferred to increase the systemic exposure of the active drug. for such purpose, amino acid based prodrugs for 33 were devised, 86 and they showed much improved aqueous solubility as compared to the parent drug. as such, prodrug 35 exhibited significantly improved pk profiles with oral bioavailability being around 20% in rats, as compared to 2.8% and 0 for 32 and 33, respectively. in addition, 35 also showed preferable safety profiles in laboratory animals, with a no observed adverse effect level being 25 mg/kg/day for a consecutive 28 days in beagle dogs, and it did not present any obvious toxicity in rats after a single oral dosage of 300 mg/kg, and only minor toxicity on central nervous system and respiratory system was observed at a single dosage of 1000 mg/kg. all these pk results present 35 as a very promising candidate compound for further development, and it is now undergoing phase i clinical trial. α-glucosidase is an enzyme removing glucose units from n-linked glycans attached to a nascent glycoprotein, which is essential for proper folding and functions of many glycoproteins. most viral envelope glycoproteins contain n-linked glycans, and α-glucosidase (especially endoplasmic reticulum [er] α-glucosidase) is highly involved in their proper folding and maturation. therefore, the inhibition of er α-glucosidase would yield broad-spectrum antiviral activity. indeed, er α-glucosidase inhibitors showed pronounced antiviral activity against a series of enveloped viruses both in vitro and in vivo including hiv, 87 hcv, 88 human coronavirus, 89 influenza a virus, 90 and denv. 91 although α-glucosidase is critical in the proper folding of viral envelop glycoproteins, it is less important to host cells, and the host cells can well-tolerate the complete shutdown of these er α-glucosidase. 92 moreover, several glucosidase inhibitors are being used in clinic for treating type ii diabetes and gaucher disease. consequently, targeting α-glucosidase for antiviral therapy would not raise a red flag on toxicity issues. to date, a wide range of α-glucosidase inhibitors have been discovered, 93 among which the iminosugars are the most promising inhibitors as antiviral agents. it is widely accepted that the antiviral profiles of iminosugars is attributed to the inhibition of er α-glucosidases (i and ii). the natural occurring iminosugars 1-deoxynojirimycin (dnj; 36; figure 10 ) and castanospermine (37; figure 10 ) has been used as starting points for further modifications. the modifications to 36 were primarily made at the amino position by introducing an alkyl chain. the n-butylated dnj 38 has been advanced to clinical trials for hiv treatment. in a phase ii study, although 38 showed the chemical structures of iminosugars ji and li | 11 some efficacy on viraemia, it failed to maintain a serum concentration needed to inhibit hiv replication in vitro, 87 so the clinical trials of 38 for hiv treatment have been discontinued. the other dnj analogue mon-dnj (39) also showed broad-spectrum antiviral activity, and it has recently been tested in human against denv infection. in the phase i trial, 39 has been demonstrated to be well-tolerated in human with no severe side effects observed after a single dosage of 1000 mg, and the pk data indicated a low interindividual variability and good linearity over a wide range of dosage (nct02061358). in a recent study, 39 has been tested in a proof-of-concept non-human primate trial against ebov infections. however, 39 failed to yield any survival benefit to macaques infected with ebov-makona, despite that 39 showed definitive antiviral activity against ebov in vitro. 94 celgosivir (40), a prodrug of 37, has also been tested in human against dengue fever. in a phase ib, placebo-controlled study, 40 failed to meet the primary end point in patients infected with uncomplicated dengue fever. however, in a mouse model study, it was confirmed that the dosing regime was crucial for the efficacy, 95 and therefore, a four-time daily dosing regime is planned for a phase ii trial (nct02569827). 40 was also studied for the treatment of patients infected with genotype i hcv. in a phase ii trial, 40 only showed a moderate antiviral effect as a monotherapy, but exhibited synergistic effects with ifn-based therapies. 88 however, further development of 40 as anti-hcv agents were discontinued due to inferior efficacy as compared with other approved daas. although the results from the clinical trials of 39 and 40 demonstrated the safety of iminosugars, they are all limited by poor pk profiles and insufficient efficacy. to address these limitations, further structural modifications were made to 36 by introducing various substituted alkyl sidechain, and several analogues were identified as potent antiviral activity against bovine viral diarrhea virus (bvdv), tacaribe virus, and denv with ec 50 values in the submicromolar range, which is hundreds fold more potent than 38. in addition, these compounds showed much superior pk profiles, especially compound 42, which had an oral bioavailability of 94%. most importantly, they all demonstrated significant protective effects in both ebov and bvdv infected mice models, and the in vivo glycan analysis also indicated significant er α-glucosidase suppression in the compounds treatment group. 96 it should be noted that the inhibition of other glucosidase such as intestinal glycosidase will lead to unwanted side effects. to avoid such side effects, a prodrug of compound 42 was designed by masking the free hydroxyl group by acylation. the tetrabutyrate prodrug 43 exhibited preferred stability toward simulated gastric and intestinal fluid, and yet was readily converted to the parent drug in the plasma and liver of mice. in a cell-based assay, compound 43 showed inhibitory activity against ebov with ec 50 value of 15.6 μm, which is slightly less potent as compared with the parent compound (4.1 μm). compound 43 also showed much improved overall drug exposure after either oral or intravenous administration to mice. 97 another strategy to alleviate the side effects of iminosugars is to design more specific inhibitors toward er α-glucosidase. in 2016, the crystal structure of er α-glucosidase ii in complex with 36 and 39 has been successfully resolved, which provided significant insights into the interactions between inhibitors and the active site of the enzyme, laying a firm foundation for the structure-based drug design of more specific inhibitors. 98 3.4 | inosine-5′-monophosphate dehydrogenase impdh is an enzyme catalyzing the conversion of inosine monophosphate (imp) to xanthosine monophosphate, which is a critical step in the de novo biosynthesis of guanine nucleotides. 99 inhibition of impdh will lead to the decrease in the intracellular guanosine-5'-triphosphate (gtp) level, which will disrupt the gene synthesis of both dna and rna virus, and thereby inhibit viral replication. therefore, impdh inhibitors are expected to show broadspectrum antiviral activities. indeed, impdh inhibitors exhibited broad-spectrum antiviral activities against both dna and rna virus in vitro. 100 the approved non-nucleoside-based impdh inhibitors include mycophenolic acid (44) , its prodrug mycophenolate mofetil (45) and mizoribine (46; figure 11 ), and all of them were approved for prevention of organ transplant rejection, but not for antiviral therapy, nevertheless, all these inhibitors were reported to inhibit the replication of a wide range of virus in vitro and even in vivo. for example, 44, an uncompetitive impdh inhibitor with respect to imp and nad+, is active against flaviviruses, paramyxoviruses, orthopoxviruses, avian reoviruses, and denvs with ec 50 values in low micromolar range, and its antiviral potency is closely correlated to the intracellular gtp levels, indicating the involvement of impdh inhibition in its antiviral mechanism. 101,102 however, one major limitation with the application of 44 is the presence of a phenolic hydroxyl group, which is prone to glycosylation for excretion, and thereby limits its efficacy. to overcome this limitation, a series of phenyloxazoles were developed by the vertex group and others. the representative compound from this series is vx-497 (47) , which shows high affinity to impdh with a k i value of 10 nm. it shows potent antiviral activity against a wide range of dna and rna virus including hbv, hcmv, rsv, and murine encephalomyocarditis virus, hcv, zikv, and ebov, 103 among others with ic 50 values ranging from low micromolar to submicromolar levels, and its antiviral activity can be reversed by the addition of guanosine, indicating that its antiviral mechanism is resulted from impdh inhibition. some other vx-497 derivatives also exhibited similar antiviral profiles. 104, 105 in light of the successful application of rbv in clinic for hcv treatment, as a more potent impdh inhibitor and antiviral agent as compared to rbv, 100 other mechanisms are involved in the antiviral action of rbv. in addition, rbv has to be used in combination with ifn-alpha or other anti-hcv agents in clinic, and the monotherapy with rbv failed to yield any antiviral efficacy in patients. 108 although quite a few other impdh inhibitors are under development, they are all intended for other indications. consequently, special attentions should be paid to this issue in pursuit of impdh inhibitors as antiviral agents. kinase represents a huge family of host proteins, and have been successfully targeted by a myriad of smallmolecule inhibitors for the treatment of cancers and inflammatory diseases in clinic. mounting evidence have shown that viruses hijack a variety of human kinases throughout the entire viral life cycle to facilitate their replications, and some kinases are broadly required. [109] [110] [111] therefore, it can be anticipated that the inhibition of such kinases would lead to the disruption of a broad spectrum of viral replications. since tremendous kinase inhibitors have been approved in clinic for treating cancer and inflammatory diseases, and more are under development in the pipelines, increasing efforts have been devoted to repurposing approved kinase inhibitors as broad-spectrum antiviral agents. in this section, only one type of kinases along with their respective inhibitors are picked for the discussion of related issues, and the selection of these examples is not based on the "importance" of the articles, but rather whether there are appropriate issues to discuss. the numb-associated kinases (naks) constitute a diverse family of ser/thr kinases with a broad range of cellular functions. naks have been found to play key roles in a diverse range of human diseases ranging from parkinson's and prostate cancer to viral infections. host kinases adaptor protein 2 (ap2)-associated protein kinase 1 (aak1) and cyclin g-associated kinase (gak), two kinases from this family, are found to regulate intracellular trafficking of a variety of viruses including denv and ebov. two approved kinase inhibitors sunitinib (49) and erlotinib (50) were found to potently inhibit aak1 and gak, respectively, and both are reported to exhibit potent broad-spectrum antiviral activity in vitro. in addition, the combination of 49 and 50 showed very pronounced protective effects against morbidity and mortality in denv and ebov infection mouse models. 112 a cocktail treatment containing 49 and 50 against ebov infection is being investigated in clinic trial, but no clinical data have been disclosed yet (nct02380625). gak and aak1 were also reported to regulate the binding of ap2m1 to hcv core protein, which is essential to the hcv assembly. inhibitors of gak and aak1 disrupted the interaction between ap2m1 and core, and thus inhibited hcv replication with ec 50 values ranging from 0.15 to 1.8 μm. 113 although 49 and 50 exhibited potent inhibitory activity against gak and aak1, both of which are known as pankinase inhibitors, and thus raise off-target toxicity concerns when used as antiviral agents. in 2015, herdewijn et al developed a series of highly selective inhibitors against gak. the crystal structure of gak in complex with one of these inhibitors has been resolved, showing that inhibitors as such behaved as classic type i adenosine triphosphate (atp)-competitive kinase inhibitors. these compounds showed pronounced anti-hcv activity in vitro with ec 50 in the low micromolar range. 114 further structure optimization lead to the identification of compound 51 with a k d value of 8.9 nm, which is endowed with broad-spectrum antiviral activity against a panel of viruses including denv, ebov, chikv with ec 50 values in the low micromolar range. the mechanism of action studies confirmed that the inhibition of gak is an important target underlying the broad-spectrum antiviral activity of compound 51. 115, 116 the other chemotype of gak inhibitor is quin(az)oline, 117 ,118 yet their antiviral activity was not probed. many other kinases are also involved and play essential roles in different life cycle of various viruses, and their corresponding kinase inhibitors show broad-spectrum antiviral activities against a wide range of viruses both in vitro and in vivo. [109] [110] [111] since host kinase is not under the genetic control of virus, kinase inhibitors for antiviral treatment have much higher genetic barrier to resistance. for example, denv developed resistance against the viral ns4b inhibitor sdm25n after eight passages, yet it remained sensitive to the treatment of 49 and 50 under the same conditions. 119 despite with advantages of broad-spectrum antiviral profiles and higher genetic barrier to resistance, there still remain concerns or limitations for repurposing kinase inhibitors as antiviral agents. chiefly, toxicity is the major concern associated with kinase inhibitor, because most of kinases (if not all) play essential roles to regulate the cellular functions. however, it should be noted that, in most cases, the duration of antiviral therapy can be as short as several weeks or even several days, and hence the incidence of severe side effects can be significantly decreased. in addition, the potential toxicity can be further diminished by operating in a well-defined therapeutic window. since most of the kinase inhibitors are designed to target the atp binding site, which is highly conserved among different kinase families, so nearly all kinase inhibitors possess cross inhibitory activity against other kinases, which could be problematic. first, targeting multiple kinase may result in off-target side effects; second, it is challenging to understand the mechanism underlying the antiviral action of these inhibitors because multiple kinases are involved. however, one could also argue that pan-kinase inhibition is favorable for antiviral therapy in that the off-target kinase(s) may also contribute to the viral replication, and targeting multiple kinases simultaneously may result in not only synergetic antiviral activity but also high genetic barrier to resistance as well. altogether, targeting kinases represent a promising strategy for the development of antiviral agents, especially the ones with broad-spectrum antiviral profiles and high genetic barrier to resistance. sodium taurocholate cotransporting polypeptide (ntcp) is expressed on the hepatic basolateral membranes specifically and functions as a cotransporter for bile acids and sodium ions. recently, ntcp has been identified as hbv/hdv infection receptor via interacting with hbv large surface protein, and the silence of ntcp inhibited hbv and hdv infections. 120 therefore, the inhibition of ntcp would block the entry of hbv/hdv virus, and the subsequent formation of the persistent viral reservoir: cccdna will also be halted, which cannot be achieved by current therapy. 121 were also significantly declined in all myrb groups but not the control tdf group, 122 demonstrating superiority by targeting ntcp (figure 12 ). the other discovered ntcp inhibitors include fda approved drugs (ie, csa, 123 ezetimibe, and ritonavir, 124 etc), fasiglifam, 125 oxysterol, 126 vanitaracin a, 127 nti007, 128 and among others. 129 the inhibition of ntcp by these inhibitors normally will result in the loss of transporter function of ntcp, leading to the inhibition of bile acid uptake, which might cause unwanted adverse effects. interestingly, shimura et al 130 identified several csa f i g u r e 1 2 the chemical structures of a subset of gak and aak1 inhibitors. aaak1, ap2-associated protein kinase 1; gak, cyclin g-associated kinase derivatives with anti-hbv activity in vitro via direct interaction with ntcp to inhibit viral attachment. these compounds inhibited multiple hbv genotypes including one clinically relevant nucleoside analog-resistant hbv isolate. importantly, they did not compromise the transporter function of ntcp. two analogs scy446 (56) and scy450 (57; figure 13 ) also did not show meaningful inhibition against cn, and therefore, these two compounds did not show any unwanted immunosuppressive effects. taken together, these results showed that the inhibition of viral attachment via ntcp can be functionally separated from the bile acid uptake, and future efforts should be dedicated to new ntcp inhibitors with transporter function retained to eliminate unwanted adverse effects. farnesoid x receptor (fxr) belongs to the nuclear receptor superfamily and plays regulatory roles in the metabolism of bile acid, lipid, and glucose. recently, it was identified as a proviral host factor for hbv virus. 131 silence of fxr by small hairpin rna resulted in significant decrease in the levels of cccdna, pregenomic-and precore-rnas, secreted relaxed circular dna (rcdna), and hbsag by 40%-70%, and the same effect was observed with the treatment of a fxr agonist gw4064 (58; figure 14 ). importantly, in c3h/hen adult mice infected with a recombinant aav2/8-hbv vector, gw4064 (50 mg/kg/d) significantly suppressed the rcdna and hbsag titers (mean rcdna variation: −0.52 and −0.93log10; mean hbsag variation −0.89 vs −0.73log10, at day 21 and 28, respectively). 132 interestingly, 58 together with other fxr agonists such as way362450 (59), fexaramine (60), and chenodeoxycholic acid (61) also showed inhibitory effect against hcv replications in huh7.5 cells with ic 50 values ranging from 0.75 to 7.03 μm. the mechanism of action studies showed that compound 58's anti-hcv activity is fxr-dependent. it has been reported that fxr agonist downregulated the expression of srib, which is a coreceptor for hcv entrance. as expected, the treatment of 92 also dose-dependently decreased the level of sr-bi. in addition, 58 also presented synergistic anti-hcv activity with other approved daas, and it remained sensitive against other daa-resistant hcv mutations, suggesting the advantages of targeting host factors. 133 encouragingly, one specific fxr agonist eyp001 (structure not disclosed) has successfully entered clinical trial for hbv treatment, and the data from phase i trial showed that eyp001 is very well-tolerated in humans with no treatment-related discontinuations or severe side effects. the pk of eyp001 was linear up to 500 mg with t max and t 1/2 being 2.3-2.5 and 1.4-2.4 hours, respectively. 134 the phase ii trial of eyp001 is ongoing. f i g u r e 1 3 the chemical structures of a subset of sodium taurocholate cotransporting polypeptide inhibitors. the r group in 56 and 57 was not specified in the original paper 130 diacylglycerol acyltransferases (dgats) are the key enzymes catalyzing the biosynthesis of endogenous triglycerides, which are necessary for the biogenesis of lipid droplets in the liver. it has been reported that lipid droplets are the major site for hcv particle assembly and production, and dgats, especially dgat-1, play vital roles during hcv infection. dgat-1 forms a complex with nonstructural protein 5a (ns5a) and core protein to enhance the interaction of the latter two, and the trafficking of ns5a to lipid droplets is also highly dependent on dgat-1's activity. 135 the silence of dgat-1 significantly impaired hcv entry. 136 therefore, dgat-1 could serve as a viable host target for anti-hcv agents development. one specific dgat-1 inhibitor pradigastat (62; figure 15 ) developed by novartis significantly decreased the level of hcv rna in cell supernatant at a concentration of 10 μm, indicating that compound 62 inhibited the assembly or release of virions. since compound 62 is in clinical development for the treatment of dyslipidemia, and it has demonstrated convinced efficacy and safety profiles. 137 therefore, it was directly tested in patients with genotype 1 or 3 hcv infections for safety and efficacy. however, disappointedly, 14 days of treatment with compound 62 failed to afford any significant deduction in serum hcv rna levels in either gt1 or gt3 patients, and thus the trial was terminated. 138 since the pk studies showed that the predicted concentration of compound 62 in liver is approximately 10 to 20 μm based on the plasma concentration on day 8 and 14, which is much higher than the concentration needed for dgat-1 inhibition (ic 50 : 66 nm), so the lack of efficacy is not due to pk problem. the other possibility is that hcv virus hijacked other compensate pathway to facilitate the assembly and release while dgat-1 activity is inhibited in vivo, or dgat-1 plays a nonenzymatic roles during hcv replication, so the inhibition of its enzymatic activity would not yield any inhibitory activity against hcv replication. hsp90 is a highly conserved chaperone protein that assists the maturation of its clientele proteins. there are four the viral entry and intracellular trafficking stage, hsp90 is reported to be critical for the intracellular translocation of viral proteins as well as other host factors critical for viral replications. for example, hsp90 is required for the nuclear translocation of ebv and hsv-1 dna polymerase and 139,140 rna-dependent rna polymerase of influenza virus. 141 to facilitate viral gene expression, hsp90 also activate several host signaling pathways. for instance, hsp90 is upregulated to activate akt and nuclear factor κb for viral gene expression upon hcmv infection. 142 as a chaperone protein, hsp90 is indispensable for the maturation, accurate folding, and maintenance of stability of various proteins including viral proteins. for example, hsp90 facilitates the accurate folding of ns5a of hcv virus in the replication complex to promote viral replication. 143 it can also help to maintain the stability of various viral proteins, including polymerases of vsv, 144 chikv, 145 and rsv, 146 and ribonucleoprotein complex 147 to facilitate viral genome replications. in addition, hsp90 is also involved in the formation of viral capsid. hsp90 can maintain the stability of capsid precursor p1 protein of poliovirus, 147 and it can also increase affinity between core protein dimers to facilitate hbv capsid formation. 148 market for unknown reasons. 64 was shown to inhibit rsv replication in an in vivo model of well-differentiated primary human airway epithelial cells at concentration as low as 1.9 nm. moreover, despite extensive replication in the presence of 64, no resistance against 64 was observed even after 17 passages, which is in direct contrast to previously reported rsv inhibitor. 155 similarly, 63 was reported to inhibit the replication of poliovirus both in vitro and in vivo, and no resistance against 63 was detected after 10 passages, despite that poliovirus is feature with rapid replication rate and high mutation frequency. 156 and inhibited nucleocapsid egress from the nucleus. 159 many other hsp90 inhibitors have also shown definitive antiviral activities against a panel of virus strains. 160, 161 although around 17 hsp90 inhibitors are under development in clinical trials, they are all for cancer indication and none is being tested for antiviral purpose albeit with well-established preclinical results. the major concern associated with the application of hsp90 inhibitors for antiviral treatment is the toxicity. most of the hsp90 inhibitors in clinical trials are accompanied with some severe side effects including cardiotoxicity, gastrointestinal toxicity, and/or ocular toxicity amongst other side effects. [162] [163] [164] it is now widely accepted that these unwanted toxicities are mainly attributed to paninhibition of hsp90 isoforms. for example, the observed cardiotoxicity mainly resulted from the inhibition hsp90α, which is responsible for the maturation of herg channel. 165 therefore, hsp90 isoform-selective inhibitors are expected to side-step some detrimental toxicity observed with pan-inhibitors. indeed, several hsp90 isoform-selective inhibitors against grp94 166, 167 (72) (73) and hsp90β 168 (71) have been successfully developed with much better safety profiles. these inhibitors were devised for cancer treatment, and their antiviral profiles were not investigated. very recently, two grp94 selective inhibitors 72 and 73 were found to inhibit the replication of denv and zikv with ic 50 values in the low nanomolar range, and it was further confirmed that grp94 was essential for the replication of denv and zikv. 169 therefore, it can be deduced that grp94 might be indispensable for the replications of other viruses as well. it can be envisioned that antiviral therapy with hsp90 inhibitors can benefit from hsp90 isoform inhibition, and isoform-selective show broad-spectrum antiviral activities. as compared to hsp90 inhibitors, hsp70 inhibitors are under developed, and the development of hsp70 inhibitors is faced with several challenges: hsp70 has a high affinity toward adp, and the conformation state of hsp70 makes the atp binding site less accessible. 178 therefore, it is very hard to design inhibitors targeting the atp binding site. 179 due to high sequence similarity between hsp70i and hsc70, most of the available inhibitors are unable to discriminate between those two isoforms. shown in figure 10 are a selected subset of hsp70 inhibitors, and all those inhibitors are intended for cancer indication. mkt-077 (76; figure 17 ) is the most advanced one, which has entered phase i clinical trial for cancer treatment. however, its clinical trial was halted due to severe renal dysfunction observed in patients. 180 albeit with highly structural similarity toward 80. 182 in addition, 80 suppressed the inflammation response associated with dengue fever. more importantly, no resistance to 80 was detected even after 10 passages, while significant resistance was observed with a viral ns5 inhibitor under the same conditions, highlighting the advantages of htas over daas in terms of resistance selection. 80 and its analogues have also exhibited broadspectrum antiviral profiles with inhibitory activity against hcv, 183 zikv, 184 west nile, and japanese encephalitis viruses. 182 the hsp70i is at relatively low level in unstressed cell, while its expression is significantly induced upon f i g u r e 1 7 the chemical structures of hsp70 inhibitors and downregulators, hsp, heat shock protein viral infections, indicating an integral role of hsp70i in viral infections. to achieve hsp70 isoform inhibition, several inhibitors targeting an allosteric site on hsp70 were elegantly designed with selectivity toward hsp70i. 185, 186 hs-72 (81) is one of such inhibitors, and it was shown to inhibit the entry of denv mainly by disrupting the association of hsp70i with the denv receptor complex. 187 in comparison, hsc70 selective inhibitors have not been precedented. however, in contrast to direct inhibition, hsc70 downregulators have been reported with broadspectrum antiviral activities. for example, imb-dm122 (82), an analogue derived from natural compound oxymatrine, was discovered as a hsc70 downregulator. the half-life of hsc70 messenger rna (mrna) was reduced by 78% followed by the treatment of 82 (500 μg/ml) in huh7.5 cells. as such, 82 is effective to inhibit hcv replication at a concentration of 125 μg/ml. in addition, 82 is well-tolerated in mice with no obvious toxicity at a single dosage of 1000 mg/kg (intraperitoneal [ip]). 188 intensive structural modifications were further made to the sidechain and/or substituent at the nitrogen position of 82, yielding several analogues with ic 50 s against hcv in the low micromolar range (ie, 83). [189] [190] [191] the oxymatrine analogues were also shown to inhibit both wild-type and lamivudine-resistant hbv infection via downregulating hsc70 expression with excellent safety profile in mice (ld 50 = 750 mg/kg, oral administration). 192 the activity against other virus was also reported with oxymatrine analogues. 193, 194 the other naturally occurring hsc70 downregulator is lycorine (84) . 121 it was reported to decrease the hsc70 mrna level does-dependently with definitive anti-hcv activity in vitro. the structural optimization led to several derivatives with ic 50 values ranging from low micromolar to submicromolar levels. the sar study showed that the double bond between c3 and c4 and the basic nitrogen at n-5 position are crucial to the anti-hcv activity. 195 interestingly, its naturally occurring cousin lycoricidine (85) possessed much more potent inhibitory effect against hcv with an ec 50 value of 0.55 nm, and the mechanism of action studies revealed that downregulation of hsc70 expression at least partially account for the observed antiviral activity. 196 although these compounds are confirmed to downregulate hsc70 to exert their antiviral efficacy, their respective physical binding protein(s) remain to be clarified, but it can be deduced that their physical binding partner(s) must be host factor(s). host cells have developed an innate immune system to act as the first-line defense against invading virus. in addition, a series of intracellular restriction factors are also expressed endogenously to counteract viral infections, 197 and apolipoprotein b mrna-editing enzyme catalytic polypeptide-like 3g (apobec3g, a3g) is one such factor, which possesses the capacity to restrict the replication of a panel of viruses including hiv-1, hbv, hcv, and ev71 among others via different mechanisms. a3g is known as a cytidine deaminase, catalyzing the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. its anti-hiv-1 activity is closely associated with its deaminase function, by which consistent mutation from dg to da in the positive strand of viral dna is frequently observed. 198 the high level of g to a mutation is attributed to the c to u transitions occurred on the complementary negative-strand dna by the cytidine deaminase activity of a3g. introduction of deoxyuracils in the proviral dna can ultimately lead to its cleavage and degradation by specific ap endonucleases. additionally, high percentages of g to a mutation in the hiv-1 genome will result in the loss of functions of viral proteins. 199 interestingly, the antiviral activity of a3g against other virus is independent of its deaminase activity. for example, a3g is reported to inhibit hcv replications by binding to the c-terminus of hcv ns3 protein to reduce its helicase activity, which is essential for hcv replication. 200, 201 a3g was also reported to inhibit hbv replication both in vitro and in vivo, but its anti-hbv mechanism is not related to its deaminase activity, because a3g did not yield any g to a hypermutation within the hbv genome, and the catalytically inactive a3g derivatives were able to result in the same magnitude of hbv inhibition as compared with its wild-type counterpart. 202, 203 although host cells are endowed with antiviral restriction factors such as a3g, viruses are so cunning that they developed their own mechanism to evade host innate immune system. for example, hiv-1 expresses a viral protein ji and li vif, which binds to a3g and form an ubiquitin ligase complex with cullin 5 (cul5), elongin b/c (elob/c), and cbfβ, leading to the ubiquitination and subsequent proteasomal degradation of a3g. 204 in the cases of hbv and hcv infections, endogenous a3g is also eliminated by some unknown mechanisms. therefore, agents either disrupting the formation of ubiquitin complex, stabilizing a3g or inducing the expression of a3g are expected to demonstrate antiviral activity. in 2008, chen et al identified two compounds imb-26 (86; figure 18 ) and imb-35 (87) , which directly binds to a3g and disrupted its interaction with vif, and therefore rescue a3g from vif-mediated degradation. both compounds showed a3g-dependent anti-hiv-1 activity in nonpermissive h9 cells with ec 50 values in the low nanomolar range. moreover, no cytotoxicity was observed at a concentration of around 4 μm, indicating a therapeutic index of greater than 200, and the ld 50 value for 87 is as high as greater than 1000 mg/kg (ip). 205 86 also showed strong antiviral activity against hcv in vitro via stabilizing intracellular a3g. 206 due to the presence of a bromo substituent at the α position of amide, which is highly reactive as a alkylation agent, structural modifications were made to 86, and it turned out that the bromo is not essential for the antiviral activities. most of the synthesized derivatives showed potent antiviral activity against hcv and ev71 virus with ic 50 values ranging from 0.57 to 80 μm. 207, 208 since the amide linkage is liable to hydrolysis, a methylene group was inserted between the carbonyl and amino group. however, such modification resulted in complete loss of activity. 207 to further address this issue, a ring formation strategy was employed to generate compound with 2-aryl-isoindolin-1-ones scaffold (88) . these analogues showed much-enhanced stability toward hydrolysis, and most importantly, the anti-ev71 activity was retained with ic 50 in the low micromolar range. 209 interestingly, the derivatives of 86 also showed definitive antiviral activity against both wild-type and tamiflu resistant influenza virus in vitro with ic 50 values in the low micromolar range, 210 despite the fact that a3g does not yield any inhibitory activity against influenza virus replication, 211 indicating that other a3g-independent antiviral mechanism must be involved. vif, and both inhibited viral replication via a3g pathway. the immunoprecipitation experiment confirmed that neither compound disrupted the interaction between vif and a3g, and thus inhibited the ubiquitination of a3g. in addition, neither compound impaired 20s proteasome activity, suggesting that recovery of a3g is not simply caused by inhibiting the general proteasome activities. the specific mechanism(s) underlying the recovery of a3g need further clarification. 215 both compounds also showed high cytotoxicity toward 293t cells with ic 50 values being 30 and 50 μm, respectively, necessitating further structural optimization. benzimidazole derivatives were also identified as potent anti-hiv-1 agents via stabilizing a3g. 216 the structural modifications led to the identification of compound 93 with an anti-hiv-1 ic 50 value of 3.4 nm in h9 cells, and no toxicity in mice was observed in 2 weeks after the ip injection of 1000 mg/kg of compound 93. the mechanism of action studies showed that this type of compounds stabilized a3g via disrupting the interaction between vif and eloc, which is essential for the vif-mediated a3g degradation. 217 in 2012, zuo et al 218 confirming its a3g-dependent antiviral mechanism. the coimmunoprecipitation and computational analysis suggested that 94 directly bound to eloc at the interface of eloc-vif interaction to suppress vif activity. the sar studies concluded that both the naphthoyl and benzoyl group were essential for the activity, and modifications made to the ester group were well-tolerated. 219 the abovementioned antiviral strategy is based on the recovery of a3g to inhibit viral infection. very interestingly, it was reasonably argued that hiv-1 virus might actually benefit from sub-lethal levels of a3g-induced mutation, which might contribute to the high mutation rate of hiv-1 virus and the capacity of the virus to escape innate immune system and evolve resistance against antiretroviral drugs. therefore, current antiretroviral treatment regime may benefit from the inhibition of a3g deaminase activity. 220 however, a proof of concept study needs to be established to support such hypothesis. bone marrow stromal cell antigen 2 (bst-2), also known as tetherin or cd317, is a potent ifn-induced antiviral molecule inhibiting the release of various enveloped virus particles from infected cells, including filoviruses, 221, 222 arenaviruses, 223 paramyxovirus, 223 γ-herpesviruses, 224 and among others. 225 the broad-spectrum antiviral profiles of bst-2 is attributed to its ability to target a common feature shared by these viruses: host cell-derived lipid bilayer. since the target of the bst-2 is not encoded by viral genome, so the virus cannot mutate the viral protein to evade the antiviral activity of bst-2. however, viruses have evolved other mechanisms to counteract the action of bst-2 by expressing different viral proteins to physically bind to bst-2, and thereby block the interaction between bst-2 and its target. such proteins include hiv-1 vpu, hiv-2 env, siv env, siv nef, kshv k5, and the ebov glycoprotein. for example, hiv-1 vpu, a type i integral membrane protein, can either induce the ubiquitination of bst-2 for degradation or downregulate the cell-surface bst-2 level by sequestering the de novo synthesized bst-2 away from the plasma membrane to counteract its antiviral activity. 226 therefore, it can be envisioned that disruption of the interaction between bst-2 and these viral proteins or stabilization of bst-2 could yield antiviral agents. zhang et al 227 developed a cell-based high-throughput enzyme-linked immunosorbent assay for the quantification of cell-surface bst-2 levels, 227 and a lead compound imb-la (95; figure 19 ) was identified to inhibit vpu-mediated bst-2 degradation and recover the expression of bst-2 at the cell surface. 228 human dead-box polypeptide 3, an atpase/rna helicase, was founded to be involved in a variety of cellular biogenesis process, including cellular differentiation, cell-cycle regulation, apoptosis, and cell survival. recently, it has also been identified as an essential host factors for the replication of both dna and rna virus, including hiv, 231 hcv, 232 denv, 233 and wnv, 234 among others. although the exact mechanism(s) ddx-3 employed to facilitate viral replication is yet to be elucidated, ddx-3 has been proposed as a very promising host target for broad-spectrum antiviral agent development. the crystal structure of ddx-3 in complex with amp has been resolved, 235 and a pharmacophore has been proposed for virtual screening, 236 value of 10 μm. 241 in a follow-up study, the structure-based drug design strategy was employed to devise new inhibitors with more potent activity. the structural modification was mainly made to the two phenyl rings to form more interactions with the other two unexplored binding pockets within the rna-binding site, and one inhibitor which showed much more potent antibudding against filoviruses, arenavirus, and rhabdovirus vlps. for example, both compounds inhibited egress of lfv-z vlps by more than 10-fold at a concentration of 1 μm, and as expected, showed no inhibitory activity against a ppxy mutant and budding defective virus, confirming the antibudding mechanism via inhibition of ppxy and nedd4 interaction. in addition, no cytotoxicity toward hek293t cells was observed for both compounds at concentrations tested (1 μm). 242 in a follow-up study, an intensive structural modifications were made to the lead compound 102, and several more potent analogues were identified with nanomolar activity in the antibudding assay. for example, compound 103 can achieve more than 90% inhibition of li et al identified a natural compound cajanine (105; figure 22 ) as a potent hcv inhibitor via a cell-based phenotype-screening assay with an ic 50 value of 3.12 μm. 244, 245 the structural-activity relationships study revealed that both the hydrophobic benzene ring (106) and prenyl group (107) were not essential for the anti-hcv activity, and several derivatives with more potent anti-hcv activity (ic 50 < 1 μm) were also obtained. the mechanism of action study demonstrated that 105 and its derivatives showed no inhibitory effect against hcv viral proteins, and yet led to the downregulation of a host target chondroitin sulfate n-acetylgalactosaminyltransferase 1 (csgalnact-1), which is a key enzyme responsible for the initiation of chondroitin sulfate chain. csgalnact-1 was shown to play a key role during the replication of hcv virus, and its expression was elevated upon hcv infection. knockdown of csgalnact-1 by sirna led to significant suppression of hcv replications. it is interesting to note that 105 showed no effect on the mrna level of csgalnact-1, and the downregulation of csgalnact-1 protein level was recovered by a general protease inhibitor mg132, indicating that cajanine promoted the degradation of csgalnact-1 by proteasome pathway. in consistency with such host targeting mechanism, 105 showed the same magnitude of inhibitory effect against both wild-type and drug resistant hcv virus strains, and it also showed very pronounced synergistic effects with other daas to inhibit hcv replications, indicating that 105 and its derivatives are worthy of further studies. although it is still unclear whether csgalnact-1 is broadly required for the replication of other viruses, our unpublished data showed that 105 and its derivatives also possessed inhibitory effect against other virus such as influenza virus, hiv-1, hbv, and coxsackie b virus. although it is ascertained solidly that 105 inhibits hcv replication via downregulation of csgalnact-1 protein, it remains unclear how 105 leads to the csgalnact-1 degradation, and which protein(s) 105 binds to physically. answering these types of questions would definitely provide new host targets for the development of new antiviral agents. throughout a drug repurposing campaign, estrogen receptor α (erα) inhibitor tamoxifen (108; figure 23 ) was identified as an hcv inhibitor. the known targets of 108 include erα, p-glycoprotein, calmodulin, and protein kinase c, and so forth. the inhibitors against p-glycoprotein, calmodulin and protein kinase c failed to inhibit hcv f i g u r e 2 2 the chemical structure of cajanine along with its synthesized analogues replication, excluding the possibility of these proteins as the anti-hcv targets for 108. a specific sirna against erα significantly suppressed hcv rna in replicon-containing cells, and transient transfection with erα (but not erβ) expression plasmids also augmented hcv replications. altogether, these results confirmed the important roles erα played during hcv replication. the subsequent binding assay showed that domain c of erα physically bound to ns5b but not ns3, ns4b, and ns5a, and such interaction is essential for the hcv genome replications. the selective estrogen receptor modulators (serms) toremifene (109) and clomiphene (110) were also reported to inhibited several other viruses such as hiv-1, 246 ebov, 247 and hsv. 248 however, the mechanism studies have excluded erα as potential antiviral targets, and the observed broad-spectrum antiviral property for serms resulted from the inhibition of other host factors, including protein kinase c (hiv-1) 249 and chloride channel (hsv-1). 248 these host proteins together with erα represent promising targets for the development of novel antiviral agents to combat against drug resistance and newly emerging unknown viruses. quite a few other host proteins or pathways have also been validated as feasible antiviral targets in vitro and even in laboratory animals, such as lipid biosynthesis and metabolism pathways, 250, 251 pparα, 252 and hnf4α, 253 among others. 121, [254] [255] [256] table 1 summarized the host targets included in this review along with the development stages of their respective inhibitors/modulators. it should be noted that the focus of this review is just on small-molecule based htas, and other validated antiviral host targets with only antibody or sirna-based modulators were not included. it is reasonable to anticipate that such host proteins can also be targeted by small-molecule modulators to convey antiviral activity. notably, the antiviral host targets studied only account for a very small portion of the host factors confirmed to be essential for viral replications. in 2016, ammari et al 257 released a database for hostpathogen interactions, with which one can search the known host-pathogen interactions by inputting either the genes, proteins or the viruses. it can be speculated that most of the recorded host-virus interactions must play critical roles during viral replications, and thus could serve as new targets for the development of htas. daas have shown great success in combating viral infections in clinic, and they are generally safe to use because they directly target viral proteins, which lack homologs in human. however, daas also suffer from several inherent limitations due to its viral protein targeting nature: viral proteins varied among different species and even different genotypes, and thus daa targeting one specific viral protein is unlikely to exhibit inhibitory effect against viral proteins from other viral species or variants. therefore, daas are normally narrow-spectrum antiviral agents, and this is also the underlying reason for the lack of effective antiviral drugs against newly emerging viruses; the other major downside for daas is they are prone to cause drug resistance, particular among rna viruses with very high frequency of replication errors. hta agents perfectly complement to daas in regard to narrow-spectrum activity although a large number of host factors are known to play vital roles throughout the whole life cycle of virus, only very few of them were explored as antiviral targets. this is primarily attributed to several concerns raised by htas. chiefly, targeting a host protein may potentially lead to unwanted toxicity issues, because the target protein may be indispensable for some cellular functions. however, this may not be intrinsically true for all the host proteins, and many host genes are known to be nonessential for cellular functions. therefore, the inhibition of such host proteins will be well-tolerated, and the on-target based toxicity can be minimized. even though the target host protein is essential for some cellular functions, the on-target based toxicity can be mitigated in many ways. first, it csgalnact-1 cajanine hcv, influenza, and hiv preclinical ns5b-erα tamoxifen hcv preclinical is known that the expression level or the activity of many cellular proteins are elevated to facilitate viral replications. therefore, it is possible to knockdown the target protein to a level, at which the viral replication can be blocked while the normal cellular functions can still be maintained. second, most of the time, alternative pathways or proteins exist for a cellular function, and hence the inhibition of one of these proteins or pathways may lead to the blockage of viral replication, but not the cellular function. third, the on-target based toxicity is closely associated with the duration of treatment, and the treatment of most of viral infections only lasts for several weeks or even days. therefore, the toxicity concern raised by targeting a host factor for virus treatment can be further alleviated. it is worth noting that quite a few approved drugs targeting host factors for other indications are considered to be generally safe to use in clinic for years. consequently, toxicity issue is something that one should pay attention to, but not something used to bias against host target antiviral agents. the other major challenge faced with the development of htas is that the antiviral phenotype observed with htas is merely in vitro artifact in some cases, and the correlation between in vitro and in vivo or clinical efficacy is very poor as in the case of development of impdh inhibitors as anti-hcv agents. the other example is the repurposement of statins as anti-hcv drugs. statins showed very pronounced inhibitory effects against hcv replication in vitro, but yield unsatisfactory outcomes in clinic trials. the lack of reliable predictive in vitro models indeed increased the attrition rates in the development of htas. however, despite with these challenges, development of htas can be highly rewarding because htas can potentially address the unmet needs in the treatment of viral infections. the work from the ji lab is financially supported by the national natural science foundation of china (81773608) recent advancement of direct-acting antiviral agents (daas) in hepatitis c therapy the competitive binding between inhibitors and substrates of hcv ns3/4a protease: a general mechanism of drug resistance hsp90: a promising broad-spectrum antiviral drug target the hiv coreceptors cxcr4 and ccr5 are differentially expressed and regulated on human t lymphocytes hiv: cell binding and entry. cold spring harb perspect med the geographic spread of the ccr5 delta32 hiv-resistance allele ccr5 receptor antagonists in preclinical to phase ii clinical development for treatment of hiv targeting chemokine receptor cxcr4 for treatment of hiv-1 infection, tumor progression, and metastasis exploring the stereochemistry of cxcr4-peptide recognition and inhibiting hiv-1 entry with d-peptides derived from chemokines amd3100, a small molecule inhibitor of hiv-1 entry via the cxcr4 co-receptor the bicyclam amd3100 story amd3465, a monomacrocyclic cxcr4 antagonist and potent hiv entry inhibitor multiple-dose escalation study of the safety, pharmacokinetics, and biologic activity of oral amd070, a selective cxcr4 receptor inhibitor, in human subjects blockade of x4-tropic hiv-1 cellular entry by gsk812397, a potent noncompetitive cxcr4 receptor antagonist synthesis of a novel tricyclic 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system and cxcr4 antagonists with potent activity against hiv-1 design of novel cxcr4 antagonists that are potent inhibitors of t-tropic (x4) hiv-1 replication plerixafor: in patients with non-hodgkin's lymphoma or multiple myeloma function of the chemokine receptor cxcr4 in haematopoiesis and in cerebellar development the chemokine receptor cxcr4 is essential for vascularization of the gastrointestinal tract defects of b-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the cxc chemokine pbsf/sdf-1 ccr5 inhibitors: emerging promising hiv therapeutic strategy efficacy of short-term monotherapy with maraviroc, a new ccr5 antagonist, in patients infected with hiv-1 chemokine control of west nile virus infection virological and immunological response to antiretroviral regimens containing maraviroc in hiv type 1-infected patients in clinical practice: role of different tropism testing results and of concomitant treatments intensification of a raltegravir-based regimen with maraviroc in early hiv-1 infection maraviroc and reverse transcriptase inhibitors combinations as potential preexposure prophylaxis candidates (s)-methyl-1-piperazinyl]-4-methylpiperidine (sch-417690/sch-d), a potent, highly selective, and orally bioavailable ccr5 antagonist pharmacokinetics and short-term safety of 873140, a novel ccr5 antagonist, in healthy adult subjects an imidazopiperidine series of ccr5 antagonists for the treatment of hiv: the discovery of n-{(1s)-1-(3-fluorophenyl)-3-[(3-endo)-3-(5-isobutyryl-2-methyl-4,5,6,7-tetrahydro-1h-imidazo[4, 5-c]pyridin-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]propyl}acetamide (pf-232798) is pf-232798 a possible successor to maraviroc? novel 4,4-disubstituted piperidine-based c-c chemokine receptor-5 inhibitors with high potency against human immunodeficiency virus-1 and an improved human ether-a-go-go related gene (herg) profile discovery of a piperidine-4-carboxamide ccr5 antagonist (tak-220) with highly potent anti-hiv-1 activity design, synthesis, and biological evaluation of novel 2-methylpiperazine derivatives as potent ccr5 antagonists discovery of incb9471, a potent, selective, and orally bioavailable ccr5 antagonist with potent anti-hiv-1 activity ccr5 receptor antagonists in preclinical to phase ii clinical development for treatment of hiv inhibition of human immunodeficiency virus replication by a dual ccr5/cxcr4 antagonist inhibition of influenza virus ribonucleic acid polymerase by ribavirin triphosphate molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction rna virus error catastrophe: direct molecular test by using ribavirin the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen the antiviral compound ribavirin modulates the t helper (th) 1/th2 subset balance in hepatitis b and c virus-specific immune responses ribavirin-induced anemia in hepatitis c virus patients undergoing combination therapy a phase iii study of the safety and efficacy of viramidine versus ribavirin in treatment-naive patients with chronic hepatitis c: viser1 results safety and efficacy of viramidine versus ribavirin in viser2: randomized, doubleblind study in therapy-naive hepatitis c patients ribavirin revisited in the era of direct-acting antiviral therapy for hepatitis c virus infection efficacy and safety of grazoprevir/elbasvir+/− rbv for 12 or 16 weeks in patients with hcv g1, g4 or g6 infection who previously failed peginterferon/rbv: c-edge treatment-experienced combined interferon alpha2b and cyclosporin a in the treatment of chronic hepatitis c: controlled trial specific inhibition of hepatitis c virus replication by cyclosporin a nim811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis c virus alone or in combination with alpha interferon safety, pharmacokinetics, and antiviral activity of the cyclophilin inhibitor nim811 alone or in combination with pegylated interferon in hcv-infected patients receiving 14 days of therapy alisporivir-a host-targeting antiviral, provides low viral breakthrough rate and high barrier to resistance in hcv genotype 1 treatment-naïve patients in the phase iib essential study alisporivir plus ribavirin is highly effective as interferon-free or interferon-add-on regimen in previously untreated hcv-g2 or g3 patients: svr12 results from vital-1 phase 2b study interferon (ifn)-free alisporivir (deb025) treatment in the vital-1 study has a more beneficial overall safety profile vs ifn-containing treatment scy-635, a novel nonimmunosuppressive analog of cyclosporine that exhibits potent inhibition of hepatitis c virus rna replication in vitro the cyclophilin inhibitor scy-635 suppresses viral replication and induces endogenous interferons in patients with chronic hcv genotype 1 infection preclinical characterization of naturally occurring polyketide cyclophilin inhibitors from the sanglifehrin family sanglifehrin a, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action sanglifehrin−cyclophilin interaction: degradation work, synthetic macrocyclic analogues, x-ray crystal structure, and binding data fragment-based discovery of a new family of non-peptidic smallmolecule cyclophilin inhibitors with potent antiviral activities catalysis of cis/trans isomerization in native hiv-1 capsid by human cyclophilin a cyclophilin a interacts with influenza a virus m1 protein and impairs the early stage of the viral replication activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the hbx protein involved in hepatitis b virus replication nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a cyclophilin a is required for efficient human cytomegalovirus dna replication and reactivation target cell cyclophilins facilitate human papillomavirus type 16 infection cyclophilins and cyclophilin inhibitors in nidovirus replication p0890: novel cyclophilin inhibitor cpi-431-32 shows broad spectrum antiviral activity by blocking replication of hcv, hbv and hiv-1 viruses discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance analyzing the relationship of qt interval and exposure to nitazoxanide, a prospective candidate for influenza antiviral therapy-a formal tqt study synergistic effect of nitazoxanide with neuraminidase inhibitors against influenza a viruses in vitro tizoxanide and other thiazolides are potent inhibitors of hepatitis b virus and hepatitis c virus replication the fda-approved oral drug nitazoxanide amplifies host antiviral responses and inhibits ebola virus nitazoxanide: a first-in-class broad-spectrum antiviral agent comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens systematic identification of synergistic drug pairs targeting hiv pediatric drug nitazoxanide: a potential choice for control of zika nitazoxanide inhibits paramyxovirus replication by targeting the fusion protein folding: role of glycoprotein-specific thiol oxidoreductase erp57 nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus the anti-hepatitis c agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2α via protein kinase activated by double-stranded rna activation hepatitis b virus x protein identifies the smc5/6 complex as a host restriction factor inhibition of hbv transcription from cccdna with nitazoxanide by targeting the hbx-ddb1 interaction nitazoxanide inhibits human norovirus replication and synergizes with ribavirin by activation of cellular antiviral response interferon regulatory factor-1 (irf-1) is involved in the induction of phosphatidylserine receptor (psr) in response to dsrna virus infection and contributes to apoptotic cell clearance in chse-214 cell effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial a pilot clinical trial of nitazoxanide in the treatment of chronic hepatitis b synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs the safety and efficacy of combination n-butyl-deoxynojirimycin (sc-48334) and zidovudine in patients with hiv-1 infection and 200-500 cd4 cells/mm3 an alpha-glucosidase i inhibitor for the potential treatment of hcv infection inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus nl63 spike protein-mediated entry by altering the glycan processing of angiotensin i-converting enzyme 2 in vivo therapeutic protection against influenza a (h1n1) oseltamivir-sensitive and resistant viruses by the iminosugar uv-4 dengue virus evolution under a host-targeted antiviral glucose trimming and reglucosylation determine glycoprotein association with calnexin in the endoplasmic reticulum synthetic heterocyclic candidates as promising α-glucosidase inhibitors: an overview assessment of the potential for host-targeted iminosugars uv-4 and uv-5 activity against filovirus infections in vitro and in vivo dose-and schedule-dependent protective efficacy of celgosivir in a lethal mouse model for dengue virus infection informs dosing regimen for a proof of concept clinical trial small molecule inhibitors of er α-glucosidases are active against multiple hemorrhagic fever viruses ester prodrugs of ihvr-19029 with enhanced oral exposure and prevention of gastrointestinal glucosidase interaction structures of mammalian er α-glucosidase ii capture the binding modes of broadspectrum iminosugar antivirals impdh as a biological probe for rna antiviral drug discovery: synthesis, enzymology, molecular docking, and antiviral activity of new ribonucleosides with surrogate bases. nucleosides nucleotides nucleic acids broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase antiviral activity and mode of action studies of ribavirin and mycophenolic acid against orthopoxviruses in vitro an impdh inhibitor, suppresses replication of zika virus and other emerging viral pathogens synthesis and antiviral activity of a novel class of (5-oxazolyl)phenyl amines synthesis and broad-spectrum antiviral activity of some novel benzoheterocyclic amine compounds a randomized, double-blind, placebo-controlled dose-escalation trial of merimepodib (vx-497) and interferon-alpha in previously untreated patients with chronic hepatitis c merimepodib, pegylated interferon, and ribavirin in genotype 1 chronic hepatitis c pegylated interferon and ribavirin nonresponders ribavirin as therapy for chronic hepatitis c. a randomized, doubleblind, placebo-controlled trial repurposing of kinase inhibitors as broad-spectrum antiviral drugs repurposing kinase inhibitors as antiviral agents to control influenza a virus replication new connections: kinase inhibitors as antivirals anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects identification and targeting of an interaction between a tyrosine motif within hepatitis c virus core protein and ap2m1 essential for viral assembly selective inhibitors of cyclin g associated kinase (gak) as anti-hepatitis c agents optimization of isothiazolo[4,3-b]pyridine-based inhibitors of cyclin g associated kinase (gak) with broad-spectrum antiviral activity 3-b]pyridines as inhibitors of cyclin g associated kinase: synthesis, structure-activity relationship studies and antiviral activity identification and optimization of 4-anilinoquinolines as inhibitors of cyclin g associated kinase sgc-gak-1: a chemical probe for cyclin g associated kinase (gak) anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus targeting hepatitis b virus cccdna by crispr/cas9 nuclease efficiently inhibits viral replication final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of myrcludex b in combination with tenofovir in patients with chronic hbv/hdv co-infection cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilinindependent interference with the ntcp receptor use of fda approved therapeutics with hntcp metabolic inhibitory properties to impair the hdv lifecycle inhibitory effect of fasiglifam on hepatitis b virus infections through suppression of the sodium taurocholate cotransporting polypeptide evaluation and identification of hepatitis b virus entry inhibitors using hepg2 cells overexpressing a membrane transporter ntcp isolation and structure of vanitaracin a, a novel anti-hepatitis b virus compound from talaromyces sp design and synthesis of a novel candidate compound nti-007 targeting sodium taurocholate cotransporting polypeptide [ntcp]-apoa1-hbx-beclin1-mediated autophagic pathway in hbv therapy concept of viral inhibitors via ntcp cyclosporin derivatives inhibit hepatitis b virus entry without interfering with ntcp transporter activity epigallocatechin gallate inhibits hepatitis b virus via farnesoid x receptor alpha farnesoid x receptor-alpha is a proviral host factor for hepatitis b virus that is inhibited by ligands in vitro and in vivo farnesoid x receptor agonist gw4064 indirectly inhibits hcv entry into cells via down-regulating scavenger receptor class b type i the selective fxr agonist eyp001 is well tolerated in healthy subjects and has additive anti-hbv effect with nucleoside analogues in heparg cells diacylglycerol acyltransferase-1 localizes hepatitis c virus ns5a protein to lipid droplets and enhances ns5a interaction with the viral capsid core hepatitis c virus entry is impaired by claudin-1 downregulation in diacylglycerol acyltransferase-1-deficient cells effect of the dgat1 inhibitor pradigastat on triglyceride and apob48 levels in patients with familial chylomicronemia syndrome a diacylglycerol transferase 1 inhibitor is a potent hepatitis c antiviral in vitro but not in patients in a randomized clinical trial herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus human butyrate-induced transcript 1 interacts with hepatitis c virus ns5a and regulates viral replication antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus molecular chaperone hsp90 is a therapeutic target for noroviruses heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers hsp90, an unlikely ally in the war on cancer geldanamycin, a ligand of heat shock protein 90, inhibits herpes simplex virus type 2 replication both in vitro and in vivo geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro inhibition of heat-shock protein 90 reduces ebola virus replication heat shock protein 90 controls hiv-1 reactivation from latency hsp90 inhibitors reduce influenza virus replication in cell culture hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance a novel class of geldanamycin derivatives as hcv replication inhibitors targeting on hsp90: synthesis, structure-activity relationships and anti-hcv activity in gs4.3 replicon cells synthesis and biological evaluation of heat-shock protein 90 inhibitors: geldanamycin derivatives with broad antiviral activities hsp90 inhibitor at-533 blocks hsv-1 nuclear egress and assembly inhibition of heat-shock protein 90 reduces ebola virus replication inhibition of hsp90 attenuates porcine reproductive and respiratory syndrome virus production in vitro hsp90 molecular chaperone inhibitors: are we there yet? heat shock protein 90: inhibitors in clinical trials targeting the molecular chaperone heat shock protein 90 (hsp90): lessons learned and future directions the herg channel is dependent upon the hsp90alpha isoform for maturation and trafficking paralog-selective hsp90 inhibitors define tumor-specific regulation of her2 development of a grp94 inhibitor structure-guided design of an hsp90beta n-terminal isoform-selective inhibitor small molecule grp94 inhibitors block dengue and zika virus replication involvement of endoplasmic reticulum chaperones in the folding of hepatitis c virus glycoproteins molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo posttranslational folding of vesicular stomatitis virus g protein in the er: involvement of noncovalent and covalent complexes folding, interaction with grp78-bip, assembly, and transport of the human immunodeficiency virus type 1 envelope protein heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo heat shock cognate protein 70 is involved in rotavirus cell entry in vivo and in vitro association of hsc70 with polyomavirus capsid proteins the heat shock cognate protein 70 is associated with hepatitis c virus particles and modulates virus infectivity allosteric opening of the polypeptide-binding site when an hsp70 binds atp atpases as drug targets: insights from heat shock proteins 70 and 90 a phase i and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog mkt 077 phase i trial of the selective mitochondrial toxin mkt077 in chemoresistant solid tumours defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection allosteric heat shock protein 70 inhibitors block hepatitis c virus assembly heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70 heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70 an inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating hsp70 as a host antiviral target small molecular compounds that inhibit hepatitis c virus replication through destabilizing heat shock cognate 70 messenger rna evolution of matrinic ethanol derivatives as anti-hcv agents from matrine skeleton synthesis and biological evaluation of sophocarpinic acid derivatives as anti-hcv agents synthesis, structure−activity relationship and biological evaluation of novel n-substituted matrinic acid derivatives as host heat-stress cognate 70 (hsc70) down-regulators design and synthesis of oxymatrine analogues overcoming drug resistance in hepatitis b virus through targeting host heat stress cognate 70 antiviral effect of matrine against human enterovirus 71 sar evolution and discovery of benzenesulfonyl matrinanes as a novel class of potential coxsakievirus inhibitors design, synthesis and structure-activity relationship optimization of lycorine derivatives for hcv inhibition evaluation of anti-hcv activity and sar study of (+)-lycoricidine through targeting of host heat-stress cognate 70 (hsc70) zinc finger antiviral protein inhibits coxsackievirus b3 virus replication and protects against viral myocarditis hypermutation of hiv-1 dna in the absence of the vif protein single-strand specificity of apobec3g accounts for minus-strand deamination of the hiv genome host apobec3g protein inhibits hcv replication through direct binding at ns3 host apolipoprotein b messenger rna-editing enzyme catalytic polypeptide-like 3g is an innate defensive factor and drug target against hepatitis c virus inhibition of hepatitis b virus replication by apobec3g inhibition of hepatitis b virus replication by apobec3g in vitro and in vivo ubiquitination of apobec3 proteins by the vif-cullin5-elonginb-elonginc complex small molecular compounds inhibit hiv-1 replication through specifically stabilizing apobec3g host apolipoprotein b messenger rna-editing enzyme catalytic polypeptide-like 3g is an innate defensive factor and drug target against hepatitis c virus synthesis and antiviral activity of a series of novel n-phenylbenzamide and n-phenylacetophenone compounds as anti-hcv and anti-ev71 agents synthesis and antiviral activity of n-phenylbenzamide derivatives, a novel class of enterovirus 71 inhibitors synthesis and broad antiviral activity of novel 2-aryl-isoindolin-1-ones towards diverse enterovirus a71 clinical isolates synthesis and antiviral activity of substituted bisaryl amide compounds as novel influenza virus inhibitors high level expression of the anti-retroviral protein apobec3g is induced by influenza a virus but does not confer antiviral activity small-molecule inhibition of hiv-1 vif synthesis and structure-activity relationship studies of hiv-1 virion infectivity factor (vif) inhibitors that block viral replication design, synthesis, and biological evaluation of 2-amino-n-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide derivatives as potent hiv-1 vif inhibitors small molecules that inhibit vif-induced degradation of apobec3g development of benzimidazole derivatives to inhibit hiv-1 replication through protecting apobec3g protein identification of an hiv-1 replication inhibitor which rescues host restriction factor apobec3g in vif-apobec3g complex small-molecule inhibition of human immunodeficiency virus type 1 replication by targeting the interaction between vif and elonginc indolizine derivatives as hiv-1 vif-elonginc interaction inhibitors first-in-class small molecule inhibitors of the single-strand dna cytosine deaminase apobec3g broad-spectrum inhibition of retroviral and filoviral particle release by tetherin tetherin-mediated restriction of filovirus budding is antagonized by the ebola glycoprotein infectious lassa virus, but not filoviruses, is restricted by bst-2/tetherin molecular mechanism of bst2/tetherin downregulation by k5/mir2 of kaposi's sarcoma-associated herpesvirus is tetherin a true antiviral: the influenza a virus controversy antagonism of cd317 restriction of human immunodeficiency virus type 1 (hiv-1) particle release and depletion of cd317 are separable activities of hiv-1 vpu high-throughput assay to identify inhibitors of vpu-mediated down-regulation of cell surface bst-2 a small molecule compound imb-la inhibits hiv-1 infection by preventing viral vpu from antagonizing the host restriction factor bst-2 2-thio-6-azauridine inhibits vpu mediated bst-2 degradation a novel peptide to disrupt the interaction of bst-2 and vpu requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function ddx3 dead-box rna helicase is required for hepatitis c virus rna replication strategies for development of dengue virus inhibitors p-body components lsm1, gw182, ddx3, ddx6 and xrn1 are recruited to wnv replication sites and positively regulate viral replication crystal structure of conserved domains 1 and 2 of the human dead-box helicase ddx3x in complex with the mononucleotide amp pharmacophore modeling and molecular docking led to the discovery of inhibitors of human immunodeficiency virus-1 replication targeting the human cellular aspartic acid−glutamic acid−alanine−aspartic acid box polypeptide 3 toward the discovery of novel anti-hiv drugs. second-generation inhibitors of the cellular atpase ddx3 with improved anti-hiv activity: synthesis, structure-activity relationship analysis, cytotoxicity studies, and target validation targeting ddx3 with a small molecule inhibitor for lung cancer therapy ketorolac salt is a newly discovered ddx3 inhibitor to treat oral cancer targeting the human dead-box polypeptide 3 (ddx3) rna helicase as a novel strategy to inhibit viral replication discovery of the first small molecule inhibitor of human ddx3 specifically designed to target the rna binding site: towards the next generation hiv-1 inhibitors small-molecule probes targeting the viral ppxy-host nedd4 interface block egress of a broad range of rna viruses quinoxaline-based inhibitors of ebola and marburg vp40 egress design and synthesis of cajanine analogues against hepatitis c virus through downregulating host chondroitin sulfate n-acetylgalactosaminyltransferase 1 total synthesis of cajanine and its antiproliferative activity against human hepatoma cells ligands of the antiestrogen-binding site are able to inhibit virion production of human immunodeficiency virus 1-infected lymphocytes fda-approved selective estrogen receptor modulators inhibit ebola virus infection inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and nppb effect of tamoxifen on regulation of viral replication and human immunodeficiency virus (hiv) long terminal repeat-directed transcription in cells chronically infected with hiv-1 different anti-hcv profiles of statins and their potential for combination therapy with interferon identification of the niemann-pick c1-like 1 cholesterol absorption receptor as a new hepatitis c virus entry factor naringenin inhibits the assembly and long-term production of infectious hepatitis c virus particles through a ppar-mediated mechanism effects of bezafibrate in patients with chronic hepatitis c virus infection: combination with interferon and ribavirin herpes simplex virus type 1 abrogates the antiviral activity of ch25h via its virion host shutoff protein ubiquitin c-terminal hydrolase-l3 promotes interferon antiviral activity by stabilizing type i-interferon receptor dauricine combined with clindamycin inhibits severe pneumonia co-infected by influenza virus h5n1 and streptococcus pneumoniae in vitro and in vivo through nf-kappab signaling pathway hpidb 2.0: a curated database for host-pathogen interactions he got both his bachelor's and master's degrees in chemistry from the university of science and technology of beijing in 2006 and 2008, respectively. thereafter, he moved to peking union medical college institute of medicinal biotechnology, and received his phd degree in medicinal chemistry under the supervision of professor zhuorong li in 2011. after that, he took an assistant professor position in the same institution, and he worked as a postdoctoral fellow in dr binghe wang's lab from chinese academy of medical sciences and peking union medical college. she got her bachelor's degree at peking medical school in 1984, then she moved to peking union medical college and received her master's degree in medicinal chemistry in 1988. her research interests include the development of anti-infection, antiosteoporosis, and anticancer drugs. she has published over 150 peer-reviewed scientific papers and over 30 patents medicinal chemistry strategies toward host targeting antiviral agents key: cord-344084-z4t2wkgk authors: ellwanger, joel henrique; kulmann-leal, bruna; kaminski, valéria de lima; rodrigues, andressa gonçalves; de souza bragatte, marcelo alves; chies, josé artur bogo title: beyond hiv infection: neglected and varied impacts of ccr5 and ccr5δ32 on viral diseases date: 2020-05-30 journal: virus res doi: 10.1016/j.virusres.2020.198040 sha: doc_id: 344084 cord_uid: z4t2wkgk the interactions between chemokine receptors and their ligands may affect susceptibility to infectious diseases as well as their clinical manifestations. these interactions mediate both the traffic of inflammatory cells and virus-associated immune responses. in the context of viral infections, the human c-c chemokine receptor type 5 (ccr5) receives great attention from the scientific community due to its role as an hiv-1 co-receptor. the genetic variant ccr5δ32 (32 base-pair deletion in ccr5 gene) impairs ccr5 expression on the cell surface and is associated with protection against hiv infection in homozygous individuals. also, the genetic variant ccr5δ32 modifies the ccr5-mediated inflammatory responses in various conditions, such as inflammatory and infectious diseases. ccr5 antagonists mimic, at least in part, the natural effects of the ccr5δ32 in humans, which explains the growing interest in the potential benefits of using ccr5 modulators for the treatment of different diseases. nevertheless, beyond hiv infection, understanding the effects of the ccr5δ32 variant in multiple viral infections is essential to shed light on the potential effects of the ccr5 modulators from a broader perspective. in this context, this review discusses the involvement of ccr5 and the effects of the ccr5δ32 in human infections caused by the following pathogens: west nile virus, influenza virus, human papillomavirus, hepatitis b virus, hepatitis c virus, poliovirus, dengue virus, human cytomegalovirus, crimean-congo hemorrhagic fever virus, enterovirus, japanese encephalitis virus, and hantavirus. subsequently, this review addresses the impacts of ccr5 gene editing and ccr5 modulation on health and viral diseases. also, this article connects recent findings regarding extracellular vesicles (e.g., exosomes), viruses, and ccr5. neglected and emerging topics in “ccr5 research” are briefly described, with focus on rocio virus, zika virus, epstein-barr virus, and rhinovirus. finally, the potential influence of ccr5 on the immune responses to coronaviruses is discussed. inflammatory cells play a crucial role in protecting the host from viral infections. leukocyte migration is a fundamental step of the inflammatory response to viruses, a process regulated by the interaction between chemokines and their receptors. therefore, dysregulations in the chemokine-mediated inflammatory process may contribute to viral pathogenesis (glass et al., 2003) . the c-c chemokine receptor type 5 (ccr5) interacts primarily with the chemokines ccl3 (mip-1α), ccl4 (mip-1β), and ccl5 j o u r n a l p r e -p r o o f (rantes) , which act as ccr5 agonists by stimulating cell migration and mediating inflammatory responses. on the other hand, the chemokine mcp-3/ccl7 is the main ccr5 antagonist ligand (blanpain et al., 1999; zlotnik and yoshie, 2000; glass et al., 2003; alkhatib, 2009 ). in addition to regulating the migration of non-specific leukocytes during inflammatory responses, ccr5 controls the action of specific cell types, including natural killer (nk) cells (khan et al., 2006; weiss et al., 2011) and regulatory t (treg) cells (wysocki et al., 2005; tan et al., 2009; dobaczewski et al., 2010) . ccr5 is also expressed by tissue-resident memory t cells. these ccr5 + cells support barrier immunity (davis et al., 2019) . the ccr5 is a g-protein-coupled receptor (gpcr), containing seven transmembrane α-helices, three extracellular loops, and three intracellular loops (tan et al., 2013) . figure 1 shows the structure of ccr5, highlighting its transmembrane domains and extra and intracellular loops. the specificity of interaction between ccr5 and chemokines is mediated by the second extracellular loop (samson et al., 1997) . helices 2 and 3 have a fundamental role in chemokine-induced ccr5 activation (govaerts et al., 2003) . the steps required from ligand binding culminating in cell migration encompass a series of intracellular interactions, including the g-protein heterotrimer and downstream effectors (lacalle et al., 2017) . after stimulation by chemokines or natural reactive antibodies and subsequent triggering of chemotaxis, ccr5 is phosphorylated and internalized in the cytoplasm (signoret et al., 2000; venuti et al., 2015; venuti et al., 2016; lacalle et al., 2017; venuti et al., 2017; venuti et al., 2018) . the number of available receptors on the cell surface is related to the rate of internalization and recycling of ccr5, which affects the activation of ccr5 and consequent signaling of specific pathways that culminate in chemotaxis processes (mueller et al., 2002) . of note, intracellular pools of ccr5 can be detected in the cells. these pools are probably formed by internalized or immature/precursor forms of ccr5 molecules (mirzabekov et al., 1999; kohlmeier et al., 2008; achour et al., 2009; guglielmi et al., 2011; shirvani et al., 2011) that can be rapidly expressed on the cell surface in response to viral stimuli and inflammatory responses (kohlmeier et al., 2008) . in other words, ccr5 molecules are recycled by cells. specifically, ccr5 recycling can be mediated by degradation followed by de novo synthesis (in response to stimulation by natural antibodies) or occur in the classic short-term system without de novo synthesis (in response to stimulation by ccl5, for example) (venuti et al., 2015; venuti et al., 2016) . the traffic of ccr5 between the plasma membrane and the intracellular medium is mediated by different molecules, including clathrins, β-arrestin 2, and extracellular signal-regulated kinase (erk) 1 (venuti et al., 2015; venuti et al., 2016; venuti et al., 2018) . also, intracellular cd4 regulates the expression of ccr5 on the cell surface (achour et al., 2009 ). the human ccr5 protein (352 residues) is encoded by the ccr5 gene [chromosome 3 (3p.21.31)], which is very polymorphic (blanpain et al., 2000; hoover, 2018) . among polymorphisms of the ccr5, the ccr5δ32 (rs333) has been intensively studied in different human populations. the frequency of the ccr5δ32 is quite variable. in general, the δ32 allele frequency is high in european-derived populations (for example, 16% in norway and 11% in germany) and low or absent in african and asian populations j o u r n a l p r e -p r o o f (solloch et al., 2017) . however, although the δ32 allele is more frequent in european populations, there are exceptions due to migratory events. for example, the frequency of the δ32 allele is high in south africa (13%) and chile (12%) (solloch et al., 2017) . also, the frequency of the δ32 allele can be quite variable within the same country. in brazil, the frequency of the allele in the general population is around 4-5% (silva-carvalho et al., 2016; solloch et al., 2017) . in the southern region of the country, the frequency can reach up to 9% due to the past migration of european populations to this region (boldt et al., 2009; pena et al., 2011; schauren et al., 2013) . figure 2 summarizes basic aspects of ccr5 and shows the frequency of the δ32 allele in various countries. the ccr5δ32 is the most studied genetic variant of the ccr5 gene because of its strong protective effect against hiv infection (considering susceptibility to ccr5-tropic strains). hiv entry into cd4 + t cells is mediated by the interaction of the virus with cd4 and with a co-receptor, usually ccr5. the ccr5δ32 variant is a 32 base-pair deletion in the ccr5 coding region, which causes a frameshift, resulting in a truncated protein that is not directed to the cell surface. ccr5δ32 in heterozygosis promotes a decrease in the expression of functional ccr5 on the cell surface compared to ccr5 wild-type cells. therefore, individuals with heterozygous genotype for ccr5δ32, if infected with hiv, have a small protection against disease progression due to the reduced expression of ccr5 on the surface of cd4 + t cells (reduced hiv-ccr5 interaction). in ccr5δ32 homozygous cells, no ccr5 is expressed in the plasmatic membrane. therefore, homozygous individuals for this polymorphism (δ32/δ32) show virtually total protection against hiv type 1 infection, since no ccr5 expression is verified on cell surface (no hiv-ccr5 interaction at cell surface is possible) (deng et al., 1996; dragic et al., 1996; huang et al., 1996; samson et al., 1996; wu et al., 1997; proudfoot, 2002; venkatesan et al., 2002; picton et al., 2012) . figure 3 illustrates the phenotypic effects of ccr5δ32 in human cells. the main results involving the triad "ccr5, hiv, and ccr5δ32" were published in 1996 in nature, cell and science papers by different groups (parmentier, 2015) . since then, the research involving ccr5 has explored the role of the ccr5 protein and ccr5δ32 polymorphism in different diseases, as well as the therapeutic potentials of ccr5 blockade. currently, the physical interaction of ccr5 with hiv is known in detail (shaik et al., 2019) ryst, 2015; latinovic et al., 2019) . also, a recent study has shown that molecules that inhibit ccr5 trafficking to the plasma membrane also have a therapeutic potential against hiv infection (boncompain et al., 2019) . research involving ccr5 has also brought other important advances in combating hiv infection. of note, there are already two cases of sustained remission of hiv infection following stem-cell transplantation using ccr5δ32 homozygous donor, the "berlin patient" (hütter et al., 2009) and the "london patient" (gupta et al., 2019; gupta et al., 2020) . articles that evaluated the involvement of ccr5 or ccr5δ32 in the infections caused by the mentioned viruses were analyzed. subsequently, the google scholar (https://scholar.google.com.br/) was consulted to detect relevant papers that were not indexed in pubmed, using the terms "ccr5" in association with the name of each of the viruses covered in the review. the authors of this review tried to include the largest number of original articles that addressed the topic covered in this work, intending to write a broad and complete article. however, some papers were not included because it was not possible to obtain clear conclusions from the studies. the reference lists of the consulted articles were also used to complement the selection of articles for this review. as previously mentioned in the introduction, a discussion addressing the involvement of ccr5 in tbev infection was not included in this work. articles addressing the involvement of ccr5 and ccr5δ32 in hiv infection were included only to present to the reader some basic and historical aspects related to such topics, cited mainly in the introduction section and figures, but not included as a major section. considering the importance of the coronavirus disease 19 (covid-19) pandemic, a section addressing the potential influence of ccr5 on the immune responses to coronaviruses was included in the article. review articles were also selected in pubmed and google scholar for writing the sections and paragraphs that address the basic aspects of viruses, exosomes, and diseases (e.g., epidemiological, molecular, clinical aspects). exceptionally, review articles with outstanding discussions regarding the role of ccr5 in viral infections were also cited in this work. regarding tables, it is important to note that the data available in the "population" columns are limited and often represent only general characteristics of the evaluated population. in many studies, a population can be composed of individuals from various ethnic groups. this limitation must be taken into account when evaluating the results of the studies cited in the tables. finally, also in "population" columns, "information not available" was used when this information was not clearly described in the cited article. west nile virus (wnv) is a neurotropic positive-sense single-stranded rna flavivirus endemic in various parts of the world. wnv transmission to humans occurs through the bite of infected mosquitoes, especially species of the culex genus (suthar et al., 2013) . although different animals can participate in the wnv transmission cycle, birds are the classic amplifier hosts. humans, horses and other mammals are deadend hosts (kramer et al., 2008; cdc, 2018) . among humans, blood transfusion, organ transplantation, and breast milk can also transmit the virus. vertical transmission may also occur. however, compared to transmission by mosquito bites, these routes of transmission are rare (kramer et al., 2008) . about 25% of wnv-infected individuals develop west nile fever, a clinical condition with variable symptoms and severity. in less than 1% of the infected individuals, wnv invades the central nervous system (cns), causing neurological manifestations (neuroinvasive disease), including meningitis, encephalitis, and acute flaccid paralysis. of note, west nile neuroinvasive disease shows a 10% to 20% fatality rate. severe illness is associated with older age and other factors, including genetic traces (petersen et al., 2013; sejvar, 2016) . wnv infection is considered the leading cause of arboviral encephalitis in the world (ciota, 2017) . the treatment of wnv infection is supportive (petersen et al., 2013) . it is known that the ccr5 protein interferes in the clinical course of wnv infection (glass et al., 2005; diamond, 2009; michlmayr and lim, 2014) , but the effects of ccr5δ32 on the susceptibility to this infection and disease progression are different. according to lim et al. (2010) and danial-farran et al. (2015) , ccr5δ32 has no important effect on susceptibility to wnv infection. in accordance, loeb et al. (2011) found no association between ccr5δ32 and wnv infection. conversely, there is robust populationbased data showing a strong association between the ccr5δ32 homozygous genotype and increased risk of developing symptomatic wnv infection lim et al., 2008; lim et al., 2010) . also, bigham et al. (2011) showed that the ccr5δ32 variant was associated with symptomatic wnv infection when the dominant model of inheritance was considered in the analysis. a recent meta-analysis confirmed the role of the ccr5 gene in wnv infection, specifically the association between the ccr5δ32 with severe disease (cahill et al., 2018) . table i summarizes the results of the studies that evaluated the ccr5δ32 genetic variant in the context of human wnv infection. in agreement with studies showing that ccr5δ32 homozygous genotype is a risk factor for symptomatic wnv infection in humans, ccr5-/-wnv-infected mice showed a reduced capacity of viral control, increased disease severity, impaired leukocyte trafficking towards the brain, and high mortality rates than ccr5 wild-type mice. these findings reinforce that ccr5 is a key molecule in the immune response against wnv (glass et al., 2005; durrant et al., 2015) . taking together, these pieces of evidence robustly support the role of ccr5 as a protective molecule on wnv pathogenesis. specifically, ccr5+ leukocytes play a fundamental role in combating wnv in the brain (glass et al., 2005; lim et al., 2006; michlmayr and lim, 2014; durrant et al., 2015) . in this sense, ccr5 plays a specific and non-redundant role in controlling wnv infection (lim and murphy, 2011; durrant et al., 2015; ellwanger et al., 2020a) . based on the data mentioned above, the lack of ccr5 expression linked to ccr5δ32 homozygosis is an important risk factor for increased severity of wnv-associated disease. the opposite effects of the ccr5δ32 genetic variant on both hiv and wnv infections are summarized in figure 5 . hereupon, individuals homozygous for ccr5δ32 and living in endemic areas of the wnv should take additional care to prevent wnv infection (e.g., use of repellents, mosquito nets). also, the use of ccr5 blockers to treat hiv infection may have a negative impact on populations living in wnv-endemic areas. to avoid this negative impact, hiv-infected individuals who live in such areas and who use ccr5 blockers must apply robust measures against mosquito bites lim et al., 2006; lim and murphy, 2011). j o u r n a l p r e -p r o o f influenza infection affects humans seasonally, causing recurrent epidemics and even pandemics in some years. in humans, the infection is caused basically by influenza a and influenza b, both enveloped negative-sense single-stranded rna viruses belonging to orthomyxoviridae family (krammer et al., 2018; petrova and russell, 2018) . influenza a is a zoonotic disease, and influenza b circulates primarily in humans. influenza infection affects mainly the respiratory tract, which can cause mild to severe disease depending on viral and host characteristics. secondary bacterial infection may also occur (krammer et al., 2018) . human co-infection with multiple influenza types is an important neglected problem (gregianini et al., 2019) . influenza is a prevalent infection worldwide, and new vaccines are produced annually, based on strains circulating each year in the northern and southern hemispheres (krammer et al., 2018; petrova and russell, 2018) . antivirals can be used in the treatment of influenza infection (krammer et al., 2018) . investments in new vaccines, antiviral drugs, and surveillance systems are needed to reduce the global burden associated with influenza infection (petrova and russell, 2018) . the severity of influenza infection is related to the intensity of proinflammatory responses and the predominant profile of cytokine production by the host . a body of evidence has shown that both ccl5 and ccr5 participate in the modulation of the immune response to influenza virus infection (matsukura et al., 1998; tyner et al., 2005; sládková and kostolanský, 2006; kohlmeier et al., 2008; oslund and baumgarth, 2011) . of note, ccr5 mediates the recruitment of nk cells to the lungs in influenza a infection (carlin et al., 2018) and participates in neutrophil action in the lungs during influenza pneumonia (rudd et al., 2019) . although flow cytometry data did not indicate significant changes regarding ccr5 expression on the surface of human monocytes after experimental influenza a infection (salentin et al., 2003) , various studies have shown that, in mice, the lack of ccr5 expression is associated with a higher risk of death by influenza infection (dawson et al., 2000; tyner et al., 2005; fadel et al., 2008; tavares et al., 2020) . based on these findings, it was postulated that pharmacological ccr5 blockade may have some undesirable effect on the immune response against the influenza virus in humans (fadel et al., 2008) . importantly, more research on this aspect is needed since this data suggests that, in humans, the ccr5 absence due to the ccr5δ32 polymorphism could affect pathogenesis and the lethality rate of influenza infection. falcon et al. (2015) evaluated the frequency of the ccr5δ32 in pandemic h1n1-infected spanish individuals and revealed an association between the polymorphism with fatal outcome. the ccr5δ32 was also associated with increased disease severity in other studies (keynan et al., 2010; rodriguez et al., 2013) . however, these results should be interpreted with caution since both studies were based on very small sample sizes (keynan et al., 2010; rodriguez et al., 2013) . importantly, other studies addressing humans reported no association between ccr5δ32 and severity of influenza infection (sironi et al., 2014; maestri et al., 2015; matos et al., 2019) . taking together, the body of evidence suggests that ccr5δ32 has little j o u r n a l p r e -p r o o f influence on severity of influenza infection (table 2) . results described by falcon et al. (2015) seem to be specific to that studied population, composed of individuals from 13 regions of spain. human papillomavirus (hpv) is a double-stranded dna virus belonging to the papillomavirus family. hpv is transmitted by direct contact (for example, through sexual intercourse). the hpv infection is quite common worldwide, and is usually controlled by the immune system. viral clearance occurs in most cases within 1-2 years after infection. however, if the infection is not controlled, hpv can cause noncancerous mucosal lesions or different types of malignant lesions such as anal cancer, penile cancer, vulvar cancer, head cancer, neck cancer, and especially cervical cancer. hpv is an oncogenic pathogen because it inhibits the activity of p53 and prb (retinoblastoma protein) tumor suppressor molecules through the action of e6 and e7 viral proteins, respectively. these proteins also exhibit other oncogenic mechanisms. worldwide, between 5-10% of all cancers in women are due to hpv infection (schiffman et al., 2016; sanjosé et al., 2018) . there are several hpv genotypes (>200), and those most associated with cancer are: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and probably 68 (schiffman et al., 2016) . of note, the genotype 16 (hpv16) has a prominent role in cancer development. vaccination is one of the most effective ways to prevent hpv infection, having important positive impacts on multiple aspects of population health. hpv vaccine protects the vaccinated individual from infection per se and from hpv-related cancer (schiffman et al., 2016; sanjosé et al., 2018) . the expression of ccr5 is increased in cervical cancer tissues (sales et al., 2014; che et al., 2016) . also, in vitro growth, proliferation, and invasive capacity of cervical cancer cells can be inhibited through ccr5 downregulation (che et al., 2016) . these findings suggest the involvement of ccr5 in the development of cervical lesions. in a study performed with indian individuals (singh et al., 2008) , the genotype and allele frequencies of ccr5δ32 were not different between cervical cancer patients and controls. however, when the patients were stratified by cancer stage (stages 1b to 4), the ccr5 heterozygous genotype was associated with stage 1b of cervical cancer. the hpv positivity rate among the evaluated patients was not described (singh et al., 2008) . the relationship between ccr5δ32, hpv infection, and cervical lesions is addressed in other studies (table 3 ). ccr5δ32 homozygous genotype was associated with increased susceptibility to hpv infection in a study performed with swedish individuals (zheng et al., 2006) . mangieri et al. (2019) investigated the potential influence of ccr5δ32 on susceptibility to hpv infection and cervical lesions in brazilian women. however, the genetic variant was not significantly associated with susceptibility to hpv infection (considering allele frequency, codominant model and dominant model) or hpv-associated lesions (mangieri et al., 2019) . in accordance, previous studies performed with brazilian women did not find an association between ccr5δ32 and susceptibility to hpv infection (suzuki et al., 2008) or between the polymorphism and hpv-related cervical lesions (suzuki et al., 2008; santos et al., 2016) . lastly, ccr5δ32 j o u r n a l p r e -p r o o f did not affect susceptibility to hpv infection in lithuanian individuals with laryngeal cancer (stumbrytė-kaminskienė et al., 2020) . in conclusion, although tissue analysis and evidence obtained in vitro suggest that the ccr5 is potentially involved in the pathogenesis of hpv, most studies point to a lack of involvement of ccr5δ32 in susceptibility to hpv infection or hpv-associated diseases. the the ccr5 and its ligands regulate the action of t cells and other leukocytes in the liver. thus, ccr5 regulates liver inflammation and participates in the local immune response against viruses (ajuebor et al., 2006; sanchooli et al., 2014) . in mice models of hepatitis, ccr5 deficiency was associated with increased liver inflammation, tissue injury, and liver failure (ajuebor et al., 2005; moreno et al., 2005; stevens et al., 2019) . deficiency of ccr5 expression is generally associated with reduced migration of inflammatory cells, which would translate into less inflammation. this reasoning is correct and applies to different situations and tissues (braunersreuther et al., 2007; muntinghe et al., 2009; kaminski et al., 2019a) . however, ccr5 is an immunoregulatory molecule (doodes et al., 2009; dobaczewski et al., 2010; christmann et al., 2011; hütter et al., 2011) and therefore its deficiency can also cause deregulation in the action of various immune cell types (e.g., nk and treg cells), increasing the inflammatory status in some tissues. in humans, multiple evidence has shown the involvement of ccr5 (protein and gene) in distinct aspects of hbv infection (ahn et al., 2006; trehanpati et al., 2009; ahmadabadi et al., 2013; yang et al., 2018) . interestingly, ccr5δ32 and other host genetic factors can affect the immunogenicity of the hbv vaccine (ganczak et al., 2017; ellwanger and chies, 2019b) . considering these aspects, the influence of the ccr5δ32 on susceptibility/resistance to hbv and disease severity is quite plausible. several studies investigated the role of the ccr5δ32 polymorphism in hbv infection. some of them found no association between those variables (arababadi et al., 2010; khorramdelazad et al., 2013; safari et al., 2017; zhang et al., 2018; moudi et al., 2019) . in other studies, the absence of ccr5δ32 allele in the sample, due to ethnic features of the evaluated population, precluded the analysis concerning the potential impact of this genetic variant on hbv infection (ahn et al., 2006; li et al., 2011) . some authors have reported significant influences of ccr5δ32 on hbv infection. suneetha et al. (2006) reported an association between the ccr5δ32 heterozygous genotype and chronic hbv infection. in contrast, thio et al. (2007) found an association between the ccr5δ32 allele and infection recovery in a study that analyzed individuals with persistent hbv infection and individuals who recovered from the infection. subsequently, thio et al. (2008) associated the hbv infection recovery with an epistatic effect between the ccr5δ32 and the rantes-403a promoter polymorphisms. finally, abdolmohammadi et al. (2016) found a protective effect of the ccr5δ32 genetic variant against hbv infection, once the δ32 allele was more frequent in controls compared to hbv-infected individuals. recently, we investigated the frequency of ccr5δ32 in hbv mono-infected and hbv/hiv coinfected brazilian individuals (ellwanger et al., 2020b) . a control group and hiv mono-infected individuals were also evaluated in our study. a total of 1113 individuals were studied, which represents the largest study involving ccr5δ32 and hbv infection to date (see table 4 for comparisons with other studies). we found a significant protective effect of ccr5δ32 on hbv/hiv co-infection, a result probably due to the partial protective effect of ccr5δ32 against hiv infection since no important impact of ccr5δ32 on susceptibility to hbv mono-infection was observed (ellwanger et al., 2020b) . the hepatitis c virus (hcv) was formally described in 1989 (choo et al., 1989) and, currently, hcv infection is one of the most important infectious diseases in terms of global public health burden. hcv is an enveloped single-stranded positive-sense rna virus, has seven genotypes, and belongs to the flaviviridae family, hepacivirus genus (pietschmann and brown, 2019) . it is estimated that 71 million individuals are chronically infected by hcv worldwide (viganò et al., 2019) . similar to the hbv infection, hcv-infected individuals can eliminate the virus naturally or develop chronic infection, which occurs in 55-85% of the cases. chronic infection can cause liver inflammation, cirrhosis, and hepatocarcinoma (lingala and ghany, 2015) . in addition to liver damage, hcv causes a series of immune-mediated extrahepatic manifestations, including rheumatologic, dermatologic, ophthalmologic, renal, pulmonary, neuropsychiatric, cardiovascular, and hematologic manifestations, especially mixed cryoglobulinemia (romano et al., 2018) . hcv therapy using direct-acting antivirals (daas) shows cure rates over 95% (pietschmann and brown, 2019) . early treatment of infected patients decreases death rates from hcv-associated liver disease, reduces disease transmission, and alleviates extrahepatic health problems. focusing the efforts on hcv treatment is extremely important because there is no effective hcv vaccine (viganò et al., 2019) . hcv seropositivity is an important risk factor for hiv infection (zwolińska et al., 2013) . like hiv, hcv is primarily transmitted through blood transfusion and sexual intercourse, and hcv/hiv co-infection is a major problem worldwide. depression of the immune system due to uncontrolled hiv infection may contribute to hcv progression (schlabe and rockstroh, 2018) . both susceptibility to hcv infection and j o u r n a l p r e -p r o o f disease progression are affected by viral and environmental factors and physio-metabolic, immune, and genetic components of the host (ellwanger et al., 2018a) . chemokines and chemokine receptors participate in the recruitment and activity of inflammatory cells in the liver, acting on anti-hcv immune responses and ultimately modifying the rate of inflammation and other histological manifestations observed during infection. based on this rationale, the ccr5 molecule was postulated as having an impact on hcv-induced liver injury, susceptibility to hcv infection, and modulation of the possibilities of viral clearance. the downregulation of ccr5 due to ccr5δ32 may interfere in these processes (ahlenstiel et al., 2004; coenen and nattermann, 2010) . in agreement, several polymorphisms in other immune system genes [especially human leukocyte antigen (hla), mannosebinding lectin (mbl), toll-like receptor (tlr), interleukins (il), and interferon (ifn) gene families] indeed modify both susceptibility to hcv infection and disease progression (ellwanger et al., 2018a) . especially the focus of this review, clinical response to hcv therapy is influenced by ccr5 gene polymorphisms (konishi et al., 2004; omran et al., 2013) . also, there is evidence showing that ccr5 haplotypes can affect susceptibility to hcv infection (huik et al., 2013) . a recent in vitro study suggested that ccr5 blockage could have a beneficial effect on the treatment of hcv infection since ccr5 antagonists (maraviroc and cenicriviroc) inhibit hcv replication (blackard et al., 2019) . the use of ccr5 antagonists in humans is safe (fätkenheuer et al., 2010; gulick et al., 2014; giaquinto et al., 2018) and these drugs have the potential to treat a number of diseases in which ccr5 is involved, including hcv-associated liver disease. although the use of ccr5 antagonists on hcv monoinfection is not yet approved, it can be useful specifically for co-infected hiv/hcv patients, where ccr5 blocking (maraviroc) is already recommended (haïm-boukobza et al., 2013; blackard et al., 2019) . however, the detailed patterns of ccr5 expression in different tissues and at various points in the clinical course of hcv infection are still poorly understood. according to a recent study, the expression of ccr5 in cd8+ t cells is increased in the liver of chronic hcv-infected patients (pirozyan et al., 2019) , but other studies have found mixed results regarding ccr5 expression on t cells in the context of hcv infection (lichterfeld et al., 2002; vincent et al., 2005; larrubia et al., 2007; zahran et al., 2020) . therefore, considering that the role of ccr5 in hcv infection is still uncertain, the potential use of ccr5 blockers to treat hcv mono-infection should be cautious. the influence of the ccr5δ32 variant on hcv infection susceptibility was investigated by several studies. woitas et al. (2002) found a significantly higher frequency of the ccr5δ32 homozygous genotype in hcv-infected individuals compared to controls, hiv-infected and hcv/hiv co-infected individuals, suggesting the ccr5δ32 as a risk factor for hcv infection. in agreement, the δ32 allele was a significant risk factor for infection when the authors compared the hcv-infected group to both controls and hivinfected individuals. moreover, the ccr5δ32 homozygous genotype was associated with increased hcv loads. in their study, it was observed an important deviation from the hardy-weinberg equilibrium in data from hcv-infected individuals; and a high portion of the individuals included in the study was hemophiliac j o u r n a l p r e -p r o o f (woitas et al., 2002) . hemophiliac individuals were at high risk of exposure to hcv and hiv until the mid-1980s (promrat et al., 2003; zhang et al., 2003) . considering that the ccr5δ32 homozygous genotype provides protection against hiv infection, a high frequency of this genotype in an hcv-infected group may be due to hiv resistance, but not to hcv, among individuals highly exposed to both viruses. due to those and other reasons, the results of woitas et al. (2002) were criticized by different authors (klein, 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003) . in this sense, no influence of ccr5δ32 on susceptibility to hcv infection were reported in studies performed with various populations (glas et al., 2003; mangia et al., 2003; poljak et al., 2003; promrat et al., 2003; zhang et al., 2003; ruiz-ferrer et al., 2004; wald et al., 2004; wasmuth et al., 2004; thoelen et al., 2005; goyal et al., 2006) . reinforcing the observations of those different studies, our group found no association between the ccr5δ32 and susceptibility to hcv infection or hcv/hiv co-infection in a study that evaluated a large number of brazilian individuals (ellwanger et al., 2018b) . bineshian et al. (2018) did not detect the ccr5δ32 allele in any iranian hcv-infected individual and controls included in their study, preventing any conclusion in terms of susceptibility in that population. finally, it is important to mention that in addition to the study by woitas et al. (2002) , a study performed in 2013 also found an association between the ccr5δ32 homozygous genotype with chronic hcv infection in europeans, but the authors of the study mentioned that specific factors regarding selection bias (e.g., co-exposure to hiv) may have influenced their results (suppiah et al., 2013) . taken together, the above-mentioned results called attention for the importance of performing genetic variant studies in different populations, exposed to different social and environmental factors and presenting distinct ethnic backgrounds. considering multiple clinical and histological parameters, two main different results were obtained when the ccr5δ32 genetic variant was evaluated in the context of hcv-related diseases: reduced liver inflammation in δ32 allele carriers (hellier et al., 2003; wald et al., 2004; goulding et al., 2005) ; and no association between the ccr5δ32 and clinical variables (glas et al., 2003; mangia et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; ruiz-ferrer et al., 2004; morard et al., 2014; ellwanger et al., 2018b) . in the context of persistence/resolution of hcv infection and viral control, in the one hand, studies described association of the ccr5δ32 allele with reduced rates of spontaneous viral clearance (nattermann et al., 2011; morard et al, 2014) , higher viral load (yilmaz et al., 2014) , and reduced anti-hcv immune response (ahlenstiel et al., 2009 ). on the other hand, studies have reported association between the ccr5δ32 variant and increased rates of spontaneous viral clearance (goulding et al., 2005; el-moamly et al., 2013) . no significant effect of the ccr5δ32 on viral clearance was reported by other authors (mascheretti et al., 2004) . the potential impact of the ccr5δ32 polymorphism on response to hcv therapy was also evaluated by some authors. ahlenstiel et al. (2003) found an association between the ccr5δ32 allele and reduced response rates to interferon-α monotherapy, but the polymorphism did not affect the response to the combined interferon/ribavirin therapy. this finding shows that the use of more robust therapeutic regimens j o u r n a l p r e -p r o o f compensates the undesirable effects of ccr5δ32 on hcv therapy with interferon-α monotherapy. of note, the effect of ccr5δ32 may be negligible in the context of modern hcv therapies (ahlenstiel et al., 2003) . other studies did not report significant effects of the polymorphism on response to hcv therapy (glas et al., 2003; promrat et al., 2003; goyal et al., 2006; mascheretti et al., 2004; suppiah et al., 2013; morard et al., 2014) . again, it is important to mention that the ethnic distribution of the ccr5δ32 allele could interfere with study results. konishi et al. (2004) , for example, did not detect the ccr5δ32 allele in any japanese individual included in their study focused on host genetic factors involved in the response to interferon therapy. lastly, the polymorphism also does not appear to be associated with any specific hcv genotype (glas et al., 2003; wasmuth et al., 2004; goyal et al., 2006) . ahlenstiel et al. (2004) highlighted that the impact of ccr5 on hcv infection was controversial. in 2020, many controversies remain, although some points are better defined. table 5 summarizes the main findings of the studies that evaluated ccr5δ32 on hcv infection. although some studies indicate an influence of the polymorphism on susceptibility to infection, most studies indicate that the δ32 allele has little (or no) influence on hcv susceptibility. the impact of the ccr5δ32 on hcv-related liver disease is quite variable and context-dependent. finally, available data suggest some benefit of ccr5 antagonists for the treatment of hcv mono-infection. however, these data are still limited and further studies evaluating this topic are needed. poliovirus (pv) is a single-stranded rna enterovirus of the family picornaviridae. there are three types of pv: wild pv type 1 (wpv1), type 2 (wpv2), and type 3 (wpv3). the pv replicates in the tonsils and intestinal tract. in few infection cases (~1%), the virus invades the cns and can cause poliomyelitis resulting in paralysis. poliomyelitis is a condition characterized by inflammation of the gray matter of the spinal cord and muscle paralysis unleashed by pv replication in motor neurons (racaniello, 2006; kew and pallansch, 2018; keohane et al., 2020) . polioencephalitis can also occur and is characterized by the pv outbreak occurred in that country (table 6 ). the authors found no statistically significant effect of the ccr5δ32 on pv infection; only a trend of association between the δ32 allele and increased risk of pv infection was observed (rosenberg et al., 2013) . however, this study had a very small sample size (only seven cases of severe pv infection were evaluated) and therefore the results were not conclusive. in addition, due to the declining number of pv infection cases in the world, the effect of ccr5δ32 will be increasingly difficult to be assessed in population-based studies. dengue virus (denv) is a single-stranded rna virus that belongs to the flaviviridae family, flavivirus genus. there are four denv serotypes, being all transmitted to humans by aedes mosquitoes (aedes aegypti and aedes albopictus) (guzman et al., 2016) . denv infection is a global health problem with huge impacts on public health systems, especially in tropical countries (bhatt et al., 2013) , being considered the most common arbovirosis in the world (stanaway et al., 2016) . globally, the incidence of symptomatic denv infection is within the range of 50 to 100 million cases per year, resulting in ~10,000 deaths each year (stanaway et al., 2016) . clinically, dengue illness is divided into three basic phases: acute febrile phase, critical phase, and recovery (convalescent) phase. dengue disease occurs with/without warning signs or severe dengue. warning signs (suggestive signs or symptoms of important fluid loss, capillary leakage, and shock, such as severe abdominal pain and mucosal bleeding; observed at the end of the febrile phase) allow the rapid identification of patients who need more clinical attention and supportive therapy, in an attempt to avoid severe dengue. when severe disease occurs, this condition can lead to serious organ involvement, shock, and hemorrhage, among other signals and symptoms (guzman et al., 2016) . infection with a denv serotype triggers long-term immunity to that specific serotype (homotypic denv). immunity to heterotypic denv also occurs, but it is transitory. therefore, an individual can have dengue disease more than once. severe dengue occurs more frequently in recurrent infection with a different viral serotype (murphy and whitehead, 2011; st john and rathore, 2019 ). an immune response mediates denv clearance and the resolution of dengue diseases, but it is also involved in the disease pathogenesis (murphy and whitehead, 2011) . some evidence suggests the participation of ccl5/ccr5 axis in the protection against denv (sierra et al., 2014) , as well as in the pathogenesis of dengue disease (islam et al., 2019) . indeed, denv infection is associated with increased frequency of human ccr5+ t cells (de-oliveira-pinto et al., 2012; badolato-corrêa et al., 2018) . in a study performed by marques et al. (2015) , lower viral replication was found in macrophages treated with ccr5 blockers. in the same study, ccr5-/-mice were protected from denv infection. these findings suggest that ccr5δ32 could be a protective factor against denv infection. however, xavier-carvalho et al. (2013) found no statistical difference in the frequency of ccr5δ32 polymorphism between brazilian children with j o u r n a l p r e -p r o o f severe denv infection and healthy controls. in the same direction, no effect of ccr5δ32 on susceptibility to denv infection was found in a small sample-size study performed with individuals from western australia (brestovac et al., 2014) and recent data suggested no important involvement of ccr5 gene or ccr5 polymorphisms in denv infection (cahill et al., 2018; ornelas et al., 2019) . finally, the ccr5δ32 allele was not identified in a small group of indian denv-infected individuals (islam et al., 2019) . table 6 shows some details of the studies involving ccr5δ32 and denv infection. taking together, the data mentioned above indicate that the effects of ccr5 on denv infection are very different between humans and rodents. however, it should be noted that the approach of each mentioned study is quite particular, and we cannot exclude some potential effects of ccr5 and ccr5δ32 on denv infection in humans. cmv is also linked to cancer development, once several cmv proteins (e.g., pul122, pul123, pus28, pul83, pul111a) activate pro-oncogenic pathways, including angiogenesis, escape of immune control and tumor suppressors, tumoral inflammation, invasion and metastasis, genome instability, and increased cell survival and proliferation (herbein, 2018) . poor socioeconomic condition favors cmv infection. antibodies indicating past cmv infection are found in ~60% of adults from high-income countries. in low-income countries, the rate of past infections can reach 100% (griffiths et al., 2015) . cmv can manipulate the immune system producing virokines (virus-encoded cytokine/chemokine homologs) and viroceptors (virus-encoded cytokine/chemokine receptor homologs), molecules that enable the virus to evade host immune defenses. such molecules can also facilitate viral replication (lucas et al., 2001; froberg, 2004; vomaske et al., 2012) . importantly, cmv-encoded proteins can interact with ccr5, (johnson et al., 2015) . however, mixed results were reported regarding the effect of cmv on ccr5 expression since there is evidence indicating that cmv infection may reduce ccr5 expression in various cell types (lecointe at al., 2002; varani et al., 2005) . interestingly, these mixed results may not be contradictory. cmv-infected cells may indeed exhibit decreased ccr5 expression, limiting hiv infection in these cells. however, cmv-infected cells release cmv-associated soluble factors that increase ccr5 expression in non-infected bystander cells, then facilitating hiv replication in such cells and, consequently, contributing to hiv pathogenesis (king et al., 2006) . there is evidence showing that variants in the ccr5 gene can influence multiple aspects of cmv infection (loeffler et al., 2006; sezgin et al., 2011) . for example, the ccr5 promoter polymorphism rs1800023 affects cmv replication (bravo et al., 2014; corrales et al., 2015) . in a study evaluating children, kasztelewicz et al. (2017) found no influence of ccr5δ32 on susceptibility to congenital cmv infection, severity of congenital cmv disease, or cmv-related sensorineural hearing loss at birth. as an individual genetic factor, ccr5δ32 was not statistically associated with the progression of cmv retinitis, a condition that cmv can cause in immunocompromised individuals (sezgin et al., 2011) (table 6 ). bunyaviridae. cchfv circulates in africa, europe, middle east, and asia countries, and can infect a variety of domestic animals and wild species, but without causing symptomatic illness. humans are accidental hosts of cchfv, for which the virus is transmitted mainly by tick-bites (especially ticks of the genus hyalomma), although other routes of transmission also exist, such as exposure to blood of infected animals. most cchfv-infected individuals have no symptoms or have mild nonspecific febrile syndrome. however, in some individuals, the infection can cause the crimean-congo hemorrhagic fever, a severe disease characterized by fever, myalgia, hemorrhage, among other manifestations. neurological complications can also occur, being the spectrum and intensity of the disease quite variable (ergönül, 2006; bente et al., 2013; garrison et al., 2019) . hemorrhagic fever (ergönül, 2006; saksida et al., 2010; bente et al., 2013; garrison et al., 2019) . in a study performed by arasli et al. (2015) , expression of the ccr5 ligands ccl2, ccl3, and ccl4 was increased in cchfv-infected adults compared to controls. considering these same chemokines in cchfv-infected children, only ccl4 was significantly increased compared to pediatric controls (arasli et al., 2015) . engin et al. (2009) evaluated the ccr5δ32 in 15 turkish cchfv-infected individuals and observed the wild-type homozygous genotype in all cases. in a subsequent study evaluating the turkish population, rustemoglu et al. (2017) found a protective effect of ccr5δ32 heterozygous genotype and δ32 allele on cchfv infection, since the genotype and allele frequencies were higher in controls than in cchfv-infected individuals. conversely, the wild-type genotype (normal ccr5 expression) was prevalent among infected j o u r n a l p r e -p r o o f individuals. these findings suggest that ccr5 contributes to susceptibility to cchfv infection and that ccr5 down-regulation due to ccr5δ32 results in some protection against the infection. however, further studies are needed to explain the mechanisms by which ccr5 participates in cchfv infection. in the same study, the ccr5δ32 was not significantly associated with disease severity, clinical parameters, or mortality rate (rustemoglu et al., 2017) . together, these findings indicate that the effect of ccr5δ32 is given specifically on resistance against cchfv infection, without affecting the pathogenesis/outcome of crimean-congo hemorrhagic fever. the genus enterovirus is composed of non-enveloped, positive-stranded rna viruses, belonging to the picornaviridae family. enteroviruses (ev) can infect the gastrointestinal tract, cns, and other organs, including heart (tapparel et al., 2013) . cardiomyopathy is a common consequence of ev infection in the heart. ev infection is associated with myocardial inflammation (myocarditis) and other damages to heart tissues (badorff et al., 2000; cooper, 2009; sagar et al., 2012; tapparel et al., 2013; weintraub et al., 2017) . damage heart tissues. viral persistence in individuals with enteroviral cardiomyopathy is associated with an increased mortality rates (kühl et al., 2005; lassner et al., 2018) . some studies suggest that ccr5 influences different aspects of the pathogenesis of viruses belonging to the picornaviridae family, including encephalomyocarditis virus (christmann et al., 2011; shaheen et al., 2015) , coxsackievirus b3 (valaperti et al., 2013) , and rhinovirus (muehling et al., 2017) , once ccr5 participates in the regulation of the host immune response during infection by these viruses. considering the effects of the genetic variant ccr5δ32 on ccr5 expression and ccr5-related immune responses, it is possible that ccr5δ32 also shows some impact on ev-related diseases. in a german study that evaluated patients with enteroviral (chronic/inflammatory) cardiomyopathy (lassner et al., 2018) , the ccr5δ32 was strongly associated with spontaneous viral clearance and better clinical outcome (reduced mortality rate) ( table 6 ). these findings indicate a critical involvement of the ccr5 molecule in the pathogenesis of ev cardiomyopathy. it was suggested that the ccr5δ32 genotyping could be used to assist in the prediction of the clinical progression of enteroviral cardiomyopathy: the δ32 allele as a predictor of a better prognosis, without the need of antiviral interferon-β (ifn-β) therapy; and the wild-type genotype as a predictor of a worse prognosis and immediate need of antiviral ifn-β therapy (lassner et al., 2018) . the clinical use of ifn-β is effective to eliminate the virus, avoid irreversible cardiac injury, and reduce mortality rates, but it is also associated to numerous adverse effects (kühl et al., 2012; lassner et al., 2018) . considering the prognostic value of the ccr5δ32 on the clinical course of enteroviral cardiomyopathy, it is necessary to evaluate the relationship between the ccr5δ32 and the disease in different human populations, mainly through genetic association studies. if this association is confirmed in other populations, the ccr5δ32 genotyping will be a broad-spectrum clinical tool, enhancing and driving the treatment of enteroviral cardiomyopathy. jev is the etiological agent of most cases of viral encephalitis in many countries, reaching ~30% mortality rate (van den hurk et al., 2009; tiwari et al., 2012; le flohic et al., 2013) . some evidence obtained in laboratory conditions (in vitro analysis and murine models) showed that jev infection induces the up-regulation of ccr5 gene (gupta and roa, 2011; pereek et al., 2014; zhang et al., 2019) . also, increased infiltration of ccr5 + cd8 + t cells was observed in the brains of jev-infected mice (zhang et al., 2019) . in this context, using a mouse model of japanese encephalitis, larena et al. (2012) showed that ccr5 protects the host against jev infection in the cns, being essential for disease recovery. ccr5-deficient mice showed higher viral loads in the brain and spinal cord as well as increased mortality rate, as compared to control mice (larena et al., 2012) . also using a mouse model of japanese encephalitis, kim et al. (2016) demonstrated that ccr5 controls the infiltration of cd4 + foxp3 + t regulatory cells (treg) in the cns, contributing to protection against japanese encephalitis. the infiltration and action of inflammatory cells in the brain are important to limit neuroinvasive infections, processes that are regulated by multiple factors, especially cytokines, chemokines and their receptors, including ccr5. however, the uncontrolled action of inflammatory cells can cause damage to the cns. of note, cd4 + foxp3 + treg cells regulate the immune responses, avoiding undesirable or excessive action of inflammatory cells (bardina and lim, 2012; veiga-parga et al., 2013; simonetta and bourgeois, 2013; campbell, 2015; kim et al., 2016) . according to these pieces of evidence, ccr5-mediated action of treg cells is critical for the control of japanese encephalitis. however, the role of ccr5 in jev infection may be more complex than it appears at first. liu et al. (2018) demonstrated that the use of the ccr5 antagonist maraviroc reduces the jev-induced inflammation in mice brain, increasing survival rate. this result suggests that potential deleterious effects of ccr5-mediated inflammation in the brain may override the effects of ccr5-mediated action of treg cells and, therefore, the use of ccr5 antagonists to treat japanese encephalitis may be beneficial. based on these complex and contradictory results, it can be concluded that ccr5 indeed has an important influence on japanese encephalitis, but it is not yet possible to state its specific roles, once they are varied and appear to be context-dependent. also, the results obtained in animal models may not fully represent the disease course in humans. indeed, some evidences suggest the involvement of ccr5 in the pathogenesis of japanese encephalitis in humans. in indian individuals with mild cases of japanese encephalitis, a significant dowregulation of the ccr5 gene was observed as compared to healthy controls (chowdhury and khan, 2019) . however, the ccr5δ32 does not have a relevant influence on the disease. deval et al. (2019) investigated the effect of various genetic variants, including ccr5δ32, on the development of japanese encephalitis in individuals from north india (table 6 ). no statistically significant difference was found when the ccr5δ32 (as an individual factor) was compared between cases and controls (deval et al., 2019) . considering that both studies mentioned above were performed in the indian population, studies evaluating the potential effects of ccr5 and ccr5δ32 on japanese encephalitis in other populations are necessary. puumala virus (puv) infection is a zoonosis endemic in europe. puv is an enveloped singlestranded rna virus, belonging to the bunyaviridae family, genus hantaviridae. most puv infections are mild or subclinical. in some infected individuals, puv is responsible for the development of nephropathia epidemica, a milder form of hemorrhagic fever with renal syndrome, a condition typically caused by other hantaviruses (settergren, 2000; mustonen et al., 2013; lebecque and dupont, 2019 ). an in vitro study found increased ccr5 gene expression in primary monocytes infected by hantaviruses (hantaan virus, sin nombre virus and andes virus) (markotić et al., 2007) . in this sense, host genetic factors and the immune responses affect different aspects of puv infection and progression of nephropathia epidemica (mustonen et al., 2013) , although the role of ccr5 in this disease is little known. (table 6 ). of note, in their study, the authors stated hantavirus infection as the cause of nephropathia epidemica, not specifying the puv detection. no statistical difference was observed in ccr5δ32 genotype frequencies between cases and controls, indicating that ccr5δ32 does not influenced the susceptibility to hantavirus infection in the studied population. considering only nephropathia epidemica cases, the study revealed an association between the wild-type homozygous genotype (functional ccr5) and increased severity of the disease. conversely, the heterozygous genotype was considered a protective factor against increased disease severity ( in 2019, the multiple roles of ccr5 in physiological and pathological conditions gained the attention of the scientific community and lay public due to ccr5 gene editing in human embryos, the "crispr babies", performed by a chinese biophysicist. the researcher claimed to have used crispr gene editing technology to edit the ccr5 of germline cells to mimic the effects of ccr5δ32, aiming to generate babies resistant to hiv infection. the ethical, safety, and legal aspects related to this procedure have caused an intense and broad discussion in the media and scientific literature (cohen, 2019; daley et al., 2019; greely, 2019; rosenbaum, 2019) , and this case culminated in a three years prison sentence for the biophysicist responsible for the procedure (cyranoski, 2020). also, the consequences of the ccr5 absence have brought many concerns to light, once the ccr5 protein participates in tissue development processes, control of immune responses, and several other physiological processes. considering these concerns, our group and others described some undesirable effects associated to natural ccr5 absence (due to the ccr5δ32 homozygous genotype) and ccr5 editing (ellwanger et al., 2019; wang and yang, 2019; xie et al., 2019) . besides gene editing techniques, the expression of ccr5 can be artificially modulated through the use of pharmacological antagonists (e.g. maraviroc), chemokine ligands, and monoclonal antibodies (fantuzzi et al., 2019) . the use of ccr5 antagonists is well established for the treatment of hiv infection and is currently being tested for the treatment of many other diseases, such as cancer (pervaiz et al., 2019; huang et al., 2020a) , stroke (joy et al., 2019), and cocaine addiction-related disorders (hall et al., 2020; nayak et al., 2020) . taking together, it is increasingly clear that the specific inhibition of ccr5 expression through different techniques is gaining pace in different clinical contexts. the pharmacological ccr5 blockade has many benefits in different clinical situations, particularly in the treatment of hiv infection. also, the potential of gene editing (especially in somatic cells) for the treatment of genetic diseases is very promising. however, considering viral infections, two aspects must be considered, as follows: i) the non-redundant characteristics of the ccr5 should be taken into consideration when studying the ccr5 protein and the effects of ccr5δ32 on viral infections: the traditional concepts of redundancy and robustness of the chemokine system consider that the absence of a specific chemokine or chemokine receptor is adequately compensated by other molecules. although these concepts are generally correct for some chemokines/receptors and for some physiological conditions, the lack of ccr5 expression may affect the protection against some specific infectious diseases once the ccr5 absence is not perfectly compensated for by other receptors (ellwanger et al., 2020a) . the nonredundancy of ccr5 is relevant especially for infections by neuroinvasive flaviviruses (bradina and lim, 2012) . for example, the absence of ccr5 due to ccr5δ32 is considered deleterious in wnv infection lim et al., 2008) and in some aspects of tbev infection (ellwanger and chies, 2019a) . the non-redundant role of ccr5 is also likely in jev infection (larena et al., 2012) . it is possible that those individuals using ccr5 antagonists (e.g., for the treatment of hiv infection) and living in areas endemic for neuroinvasive viruses, especially wnv and tbev, may be at increased risk of j o u r n a l p r e -p r o o f developing viral encephalitis-related problems. although, it is still necessary that studies consistently evaluate this assumption, since the available evidence does not support risks for the use of ccr5 antagonists in endemic areas of flaviviruses (martin-blondel et al., 2016) . as mentioned in the topic addressing wnv in this review, it is prudent to recommend to individuals using ccr5 antagonists to adopt measures to minimize the risk of neuroinvasive infections, such as the use of mosquito repellents and mosquito nets (considering the risk of wnv infection), and to limit their exposure to tick-infested areas (considering the risk of tbev infection). concerns regarding the use of ccr5 antagonists in areas of jev circulation have also been raised by other authors (kim et al., 2016; larena et al., 2012) . if the connection between the use of ccr5 antagonists and increased risk of neuroinvasive infections is confirmed in methodologically wellcontrolled studies, these recommendations should be considered of essential importance for users of ccr5 antagonists. "extracellular vesicles" (evs) is a general term used to designate different membranous vesicles released by various cell types. evs include microparticles, microvesicles, nanovesicles, nanoparticles, ectosomes, exosomes, exovesicles, exosome-like vesicles, oncosomes, among other vesicle types. evs act in the transport of different molecules (e.g. micrornas, mrnas, proteins) between cells and tissues in a regulated and precise manner. evs promote the maintenance of physiological processes, also participating in the pathogenesis of various diseases, such as cancer and inflammatory diseases (colombo et al., 2014; théry et al., 2018) . besides, the regulation of multiple aspects of the immune system is strongly influenced by evs (o'neill and quah, 2008; colombo et al., 2014; ellwanger et al., 2016) . the multiple roles of evs in viral infections began to be studied more intensively in recent years, representing an emerging topic in the field of infectious diseases. currently, the relationship between evs and viral infections has already been explored (to a greater or lesser extent) in the context of the following . evs participate in immune evasion processes, ultimately allowing viruses to bypass host defenses (kaminski et al., 2019b) . for example, hiv is likely to usurp the exosome biogenesis pathway in such a way that enhances its infectivity, while increasing its evading strategies from the host immune defenses (ellwanger et al., 2017a) . regarding tbev, exosomes were indicated as important participants in both viral infection and pathogenesis; in this case, tick-derived exosomes facilitate tbev transmission to the host. also, exosomes derived from tbev-infected host neurons can facilitate the spread of the virus in the cns . exosomes have shown multi-dimensional roles in denv life cycle. they can modulate negatively or positively denv pathogenesis, where they are suggested as instrumental for dengue j o u r n a l p r e -p r o o f hemorrhagic fever development in reason of the transportation of specific micrornas (mishra et al., 2019) . since hcv can be found inside exosomes (liu et al., 2014) , it is not suprising that blood-derived exosomes from hcv-infected patients can transmit the virus to other cells (bukong et al., 2014) . these findings suggest that exosomes and other evs assist in the transport of hcv particles/components between cells, ultimately facilitating viral infection. of note, evs can transport multiple viral components or molecules from virus-infected cells that end up facilitating the spread of the virus in the host. on the other hand, evs can act in favor of the host, transporting molecules that assist host defenses in the elimination or control of infections (kaminski et al., 2019b) . evs can transport a range of cytokines and chemokines, such as il-1, il-2, ifn-α, ifn-β, ccl2, ccl3, ccl4, and cxcl10 (chen et al., 2011; konadu et al., 2015; hosseinkhani et al., 2018; kodidela et al., 2018; gao et al., 2019a; gao et al., 2019b; aiello et al., 2020; chiantore et al., 2020) . besides, evs and microparticles also transport chemokine receptors (shen et al., 2016; liang et al., 2018) , including ccr5 tokarz et al., 2019) . interestingly, evs from ccr5 wild type cells can deliver ccr5 molecules to ccr5 δ32/δ32 cells, making them susceptible to hiv infection . a similar phenomenon has been reported involving microparticles, hiv, and cxcr4 (rozmyslowicz et al., 2003) . ccr5 expression is also influenced by the release of evs, specifically microparticles (renovato-martins et al., 2017) . therefore, evs have the potential to attribute greater complexity to ccr5 roles in viral infections. it is possible that the presence of ccr5 in cells is not only dependent on mechanisms of membrane expression and internalization of the receptor. it can also be dependent on ccr5 delivery mediated by evs. however, the actual impact of evs on ccr5-mediated immune response in infections remains to be determined and deserves further investigation. finally, a truncated ccr5 soluble form (tsimanis et al., 2005) , as well as soluble cxcr4, have also been reported in the plasma of humans (malvoisin et al., 2011) . of note, the truncated ccr5 soluble form has ~22 kda (half the size of the ccr5 found on cell membranes) and is truncated at the end of the second extracellular loop (the third extracellular loop and the subsequent three transmembrane regions are absent) (tsimanis et al., 2005) . however, the existence of cell-free soluble ccr5 is still controversial and does not represent a consensus in the scientific community. such doubts occur mainly in consideration of the structure of the receptor, which is highly characteristic of a cell membrane-associated molecule. we believe that evscontaining ccr5 can explain potential (misleading) detections of soluble ccr5. the importance of circulating evs-containing ccr5 on viral infections represents an additional and interesting open question to be investigated. chávez et al. (2013) (ellwanger et al., 2017b) . specifically, using a mouse model of rocv-associated encephalitis, ccr5-deficient mice showed increased survival rate and reduced levels of brain inflammation compared to mice expressing ccr5, indicating the participation of ccr5 in rocv-associated encephalitis (chávez et al., 2013) . although other studies discussed in this review have shown an important involvement of ccr5 in infection by flaviviruses [especially as an important molecule for the resolution of neuroinfection, in opposition to results of chávez et al., (2013) ], the role of the ccr5 in rocv infection represents a neglected topic. this knowledge gap should be addressed in further studies since the reemergence of rocv in brazil is a concern in terms of public health (figueiredo, 2007; ellwanger et al., 2017b) . although no other rocv outbreaks have been reported in brazil after the 1980s, there is evidence of rocv circulation in animals (casseb et al., 2014; pauvolid-correa et al., 2014; silva et al., 2014) . ccr5 expression on t cells. of note, zikv is another mosquito-borne flavivirus that recently reemerged in many countries, affecting brazil in particular. zikv infection is associated with microcephaly and other human development problems (baud et al., 2017) . zachova et al. (2019) showed that epstein-barr virus (ebv)-infected b cells have increased ccr5 expression compared to ebv-non-infected cells. also, recent evidence points to a pivotal role of the ccr5 in the attenuation of rhinovirus-associated inflammation, by controlling the activity of cd4 + foxp3 + treg cells (hossain et al., 2020) . altered levels of cytokines and chemokines are associated with several aspects of zikv (barros et al., 2018; naveca et al., 2018; khaiboullina et al., 2019; lima et al., 2019) , ebv (piovan et al., 2005; ehlin-henriksson et al., 2009; cohen et al., 2017) , and rhinovirus infections (rajan et al., 2014; shelfoon et al., 2016; hansel et al., 2017) . however, the potential role of the chemokine receptor ccr5 in the pathogenesis of zikv, ebv, and rhinovirus represents open questions in the field of ccr5 research. coronaviruses are positive-sense rna viruses that belong to the coronaviridae family (li et al., 2020) . coronaviruses infect a wide range of species, including humans. until 2019, humans have faced two major outbreaks of high pathogenic coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov) outbreak and the middle east respiratory syndrome coronavirus (mers-cov) outbreak (fung and liu, 2019) . in 2019, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan (hubei province, china) and rapidly spread to more than 180 countries/regions (interactive dashboard of global covid-19 cases -johns hopkins university: https://arcg.is/0fhmtx ; dong et al., 2020) . of note, sars-cov-2 infection causes the "coronavirus disease 2019" and therefore is also called "covid-19". considering the ongoing sars-cov-2 pandemic and the clinical variability observed in the affected individuals, ranging from mild to severe respiratory disease j o u r n a l p r e -p r o o f 2020b), the following question arose: does ccr5 have any clinically relevant influence on sars-cov-2 or other coronaviruses infections? recent data indicated that sars-cov-2 binds to the ace2 receptor (andersen et al., 2020) , using this receptor for entry into human cells. therefore, it is unlikely that ccr5 or ccr5δ32 have some significant effect on the susceptibility or resistance to sars-cov-2 infection. however, ccr5 may have some impact on the clinical course of sars-cov-2 infection. the pattern and intensity of the human immune responses regulate the clinical progression and even the outcome of infections by coronaviruses (li et al., 2020) , including sars-cov-2 (qin et al., 2020; shi et al., 2020) . sars-cov-infected mice showed increased expression of ccr5 mrna in lungs along with the production of ccl2, ccl3 and ccl5 chemokines, the major ccr5 natural agonists (chen et al., 2010) . intracranial infection of mice with mouse hepatitis virus (mhv, a murine coronavirus) induces an increased ccr5 expression, which contributed to mhv-induced demyelination through ccr5-mediated macrophage recruitment to the cns (glass et al., 2001) . subsequent studies using mhv-infected mice showed that ccr5 participates in the regulation of cd4 + and cd8 + t cell activities against the virus in the cns (glass and lane, 2003a; glass and lane, 2003b) . also, sars-cov-infected human monocyte-derived dendritic cells showed increased ccr5 expression in vitro (law et al., 2009) . taking together, the findings mentioned above suggest that ccr5 could have some influence on the clinical course of sars-cov-2 infection. however, future studies addressing humans are needed to clarify this suggestion. in this sense, we emphasize that so far, there is no evidence showing a clear involvement of ccr5 in sars-cov-2 human infection. the ccr5δ32 is a highly penetrating polymorphism, and exerts a robust phenotypic effect on ccr5. however, the expression of ccr5 is regulated by other polymorphisms in addition to ccr5δ32 (mehlotra, 2019) . also, the ccr5 expression is influenced by non-coding genetic elements. the effect of the genetic backround of the host can also be exacerbated or diminished depending on the environmental conditions to which the individual is exposed (ellwanger et al., 2018c; kulkarni et al., 2019) . taking together, these factors help to explain why many of the effects exerted by ccr5 on a given disease are specific to a certain population, ethnic group, or individual. research involving ccr5 and hiv began in the mid-1990s. since then, many achievements in the field of ccr5/hiv research have been made, such as the identification of ccr5δ32 as a strong protective factor against hiv infection and the development of ccr5 antagonists for the treatment of hiv infection. currently, these drugs are being tested for the treatment of other inflammatory and infectious diseases. in this context, the use of ccr5 antagonists has raised some concerns, since the blockade of ccr5 can affect j o u r n a l p r e -p r o o f or even weaken the host defenses against certain infections, especially neuroinvasive infections by flaviviruses. however, to date, there is no strong evidence indicating that the use of ccr5 antagonists has increased the susceptibility of individuals to problematic flaviviruses infections. the frequency of ccr5δ32 is quite varied among human populations, and therefore the effects of the allele in a particular population may not apply to another population. moreover, the effects of ccr5 and ccr5δ32 are disease-specific. for instance, the ccr5δ32 does not significantly affect susceptibility to wnv infection. however, the effect of the ccr5 and ccr5δ32 on wnv disease progression is very robust. some animal-derived findings suggest the involvement of the ccr5 in the pathogenesis of denv infection. however, the effects of ccr5 on denv infection are very different between humans and rodents. of note, ccr5δ32 does not significantly affect susceptibility to denv infection or disease progression. moreover, the effect of ccr5δ32 on hbv-related disease is population-specific. interestingly, cmv release virokines, which are molecules that can manipulate the host immune response and affect the ccr5 function. the examples cited above highlight the varied impacts of ccr5 and ccr5δ32 on viral infections. the few studies involving cchfv, ev, and hantavirus infection have indicated important effects of ccr5δ32 on these conditions. based on these findings, further studies should investigate the role of ccr5δ32 and ccr5 protein in such infections, considering populations with distinct genetic backgrounds. some evidence suggested the participation of ccr5 in infections by zikv, ebv, and rhinovirus. also, a mouse model of rocv-associated encephalitis suggested an important role for ccr5 in host immune responses against the virus. however, the roles of ccr5 and ccr5δ32 in infections by zikv, ebv, rhinovirus, and rocv are still poorly understood and need to be investigated in future studies. the role of evs in the transport of ccr5 between cells indicates that the expression of ccr5 on the cell surface may also depend on the release of evs containing ccr5. also, the transport of host molecules and viruses through evs adds complexity to the topics covered in this review and should be taken into account in future studies that investigate the role of ccr5 in viral infections. gene editing technologies have the potential to be used to treat different diseases, mainly when applied to somatic cells. however, ccr5 gene editing in human embryos presents a number of ethical problems. besides, the absence of ccr5 can have deleterious effects in certain conditions, such as increased susceptibility to symptomatic wnv infection. finally, this article showed that the participation of ccr5 in different viral infections is complex and varied and, therefore, cannot be generalized. this article also pointed out neglected gaps in knowledge involving ccr5 that should be addressed in future studies. the authors declare no conflicts of interest. ccr5 polymorphism as a protective factor for hepatocellular carcinoma in hepatitis b virus-infected iranian patients cd4-ccr5 interaction in intracellular compartments contributes to receptor expression at the cell surface effects of the ccr5-δ32 mutation on antiviral treatment in chronic hepatitis c effects of the ccr5-δ32 mutation on hepatitis c virus-specific immune responses in patients with haemophilia cc-chemokine receptor 5 (ccr5) in hepatitis c--at the crossroads of the antiviral immune response? downregulation of ccr5 expression on the peripheral blood + t cells of southeastern iranian patients with chronic hepatitis b infection association of genetic variations in ccr5 and its ligand, rantes with clearance of hepatitis b virus in korea an emerging interplay between extracellular vesicles and cytokines lack of chemokine receptor ccr5 promotes murine fulminant liver failure by preventing the apoptosis of activated cd1d-restricted nkt cells ccr5 in t cell-mediated liver diseases: what's going on? the biology of ccr5 and cxcr4 gene editing of hiv-1 co-receptors to prevent and/or cure virus infection the proximal origin of sars-cov-2 host genetics, innate immune responses, and cellular death pathways in poliomyelitis patients peripheral blood cd8 + t cells ccr5 expression and its δ32 mutation in iranian patients with occult hepatitis b infections elevated chemokine levels during adult but not pediatric crimean-congo hemorrhagic fever human t cell responses to dengue and zika virus infection compared to dengue/zika coinfection enteroviral cardiomyopathy: bad news for the dystrophinglycoprotein complex ccr5 proinflammatory allele in prostate cancer risk: a pilot study in patients and centenarians from sicily the role of chemokines in the pathogenesis of neurotropic flaviviruses acute zika virus infection in an endemic area shows modest proinflammatory systemic immunoactivation and cytokine-symptom associations an update on zika virus infection crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity the global distribution and burden of dengue host genetic risk factors for west nile virus infection and disease progression a study on the association between ccrδ32 mutation and hcv infection in iranian patients ccr5 receptor antagonism inhibits hepatitis c virus (hcv) replication in vitro multiple nonfunctional alleles of ccr5 are frequent in various human populations ccr5 binds multiple cc-chemokines: mcp-3 acts as a natural antagonist analysis of the ccr5 gene coding region diversity in five south american populations reveals two new non-synonymous alleles in amerindians and high ccr5*d32 frequency in euro-brazilians targeting ccr5 trafficking to inhibit hiv-1 infection ccr5 but not ccr1 deficiency reduces development of dietinduced atherosclerosis in mice looking for biological factors to predict the risk of active cytomegalovirus infection in non-immunosuppressed critically ill patients ccr5 revisited: how mechanisms of hiv entry govern aids pathogenesis primary acute dengue and the deletion in chemokine receptor 5 (ccr5δ32) exosomes from hepatitis c infected patients transmit hcv infection and contain replication competent viral rna in complex with ago2-mir122-hsp90 identification of genetic variants associated with dengue or west nile virus disease: a systematic review and meta-analysis control of regulatory t cell migration, function, and homeostasis natural killer cell recruitment to the lung during influenza a virus infection is dependent on cxcr3, ccr5, and virus exposure dose seroprevalence of flaviviruses antibodies in water buffaloes (bubalus bubalis) in brazilian amazon division of vector-borne diseases (dvbd) mip-1 α axis in the pathogenesis of rocio virus encephalitis in a mouse model downregulation of ccr5 inhibits the proliferation and invasion of cervical cancer cells and is regulated by microrna-107 cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4 + t cells are important in control of sars-cov infection epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type i t cells infiltration into central nervous system chemokine-containing exosomes are released from heatstressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine human papillomavirus and carcinogenesis: novel mechanisms of cell communication involving extracellular vesicles sickle cell disease: a chronic inflammatory condition isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome differential expression levels of inflammatory chemokines and tlrs in patients suffering from mild and severe japanese encephalitis ccr5 regulates inflammatory gene expression in response to encephalomyocarditis virus infection west nile virus and its vectors regulation of inflammatory chemokine receptors on blood t cells associated to the circulating versus liver chemokines in dengue fever identification of a major co-receptor for primary isolates of hiv-1 association of single nucleotide polymorphisms in tnfa and ccr5 genes with japanese encephalitis: a study from an endemic region of north india virus and host determinants of west nile virus pathogenesis ccr5 signaling suppresses inflammation and reduces adverse remodeling of the infarcted heart, mediating recruitment of regulatory t cells an interactive web-based dashboard to track covid-19 in real time ccr5 is involved in resolution of inflammation in proteoglycan-induced arthritis hiv-1 entry into cd4 + cells is mediated by the chemokine receptor cc-ckr-5 ccr5 limits cortical viral loads during west nile virus infection of the central nervous system changes in chemokines and chemokine receptor expression on tonsillar b cells upon epstein-barr virus infection host immunogenetics in tick-borne encephalitis virus infection-the ccr5 crossroad host genetic factors can impact vaccine immunogenicity and effectiveness exosomes are possibly used as a tool of immune regulation during the dendritic cell-based immune therapy against hiv-i rocio virus: an overview ccr5 gene editing -revisiting pros and cons of ccr5 absence what we say and what we mean when we say redundancy and robustness of the chemokine system -how ccr5 challenges these concepts immunogenetic studies of the hepatitis c virus infection in an era of pan-genotype antiviral therapies -effective treatment is coming role of the genetic variant ccr5δ32 in hbv infection and hbv/hiv co-infection ccr5δ32 in hcv infection, hcv/hiv co-infection, and hcv-related diseases exosomes in hiv infection: a review and critical look microrna-related polymorphisms in infectious diseases-tiny changes with a huge impact on viral infections and potential clinical applications role of ccr5δ32 mutation in protecting patients with schistosoma mansoni infection against hepatitis c viral infection or progression cytochrome p450 2d6 and mdr1 gene mutation in relation to mortality in patients with crimean-congo hemorrhagic fever: a preliminary study crimean-congo haemorrhagic fever cxcr3-deficiency protects influenza-infected ccr5-deficient mice from mortality ccr5 deficiency predisposes to fatal outcome in influenza virus infection dual ccr5/ccr2 targeting: opportunities for the cure of complex disorders short-term administration of the ccr5 antagonist vicriviroc to patients with hiv and hcv coinfection is safe and tolerable emergent arboviruses in brazil human coronavirus: host-pathogen interaction possible impact of 190g > a ccr2 and δ32 ccr5 mutations on decrease of the hbv vaccine immunogenicity-a preliminary report exosomes derived from septic mouse serum modulate immune responses via exosome-associated cytokines association between cytokines and exosomes in synovial fluid of individuals with knee osteoarthritis animal models for crimean-congo hemorrhagic fever human disease pharmacokinetics, safety and efficacy of maraviroc in treatment-experienced pediatric patients infected with ccr5-tropic hiv-1 the δ32 mutation of the chemokine-receptor 5 gene neither is correlated with chronic hepatitis c nor does it predict response to therapy with interferon-α and ribavirin functional expression of chemokine receptor ccr5 on cd4 + t cells during virus-induced central nervous system disease functional analysis of the cc chemokine receptor 5 (ccr5) on virus-specific + t cells following coronavirus infection of the central nervous system chemokine receptor ccr5 promotes leukocyte trafficking to the brain and survival in west nile virus infection reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor ccr5 following infection with a neurotropic coronavirus ccr5 deficiency increases risk of symptomatic west nile virus infection chemokine regulation of inflammation during acute viral infection the ccr5-δ32 mutation: impact on disease outcome in individuals with hepatitis c infection from a single source activation of ccr5 by chemokines involves an aromatic cluster between transmembrane helices 2 and 3 ccr5δ32 mutation does not influence the susceptibility to hcv infection, severity of liver disease and response to therapy in patients with chronic hepatitis c controls for lung dendritic cell maturation and migration during respiratory viral infection crispr'd babies: human germline genome editing in the 'he jiankui affair dual and triple infections with influenza a and b viruses: a case-control study in southern brazil the pathogenesis of human cytomegalovirus circulating human cd4 + t cells have intracellular pools of ccr5 molecules five-year safety evaluation of maraviroc in hiv-1-infected treatment-experienced patients transcriptomic profile of host response in japanese encephalitis virus infection hiv-1 remission following ccr5δ32/δ32 haematopoietic stem-cell transplantation evidence for hiv-1 cure after ccr5δ32/δ32 allogeneic haemopoietic stem-cell transplantation 30 months post analytical treatment interruption: a case report dengue infection blockade of ccr5 to protect the liver graft in hiv/hcv co-infected patients ccr5 and responses to cocaine: addiction is not just about the brain a comprehensive evaluation of nasal and bronchial cytokines and chemokines following experimental rhinovirus infection in allergic asthma: increased interferons (ifn-γ and ifn-λ) and type 2 inflammation (il-5 and il-13) association of genetic variants of the chemokine receptor ccr5 and its ligands, rantes and mcp-2, with outcome of hcv infection the human cytomegalovirus, from oncomodulation to oncogenesis intragenus (homo) variation in a chemokine receptor gene (ccr5) ccr5 attenuates neutrophilic airway inflammation exacerbated by infection with rhinovirus extracellular vesicles work as a functional inflammatory mediator between vascular endothelial cells and immune cells clinical features of patients infected with 2019 novel coronavirus in wuhan the ccr5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction the role of a mutant ccr5 allele in hiv-1 transmission and disease progression ccr5 haplotypes influence hcv serostatus in caucasian intravenous drug users the effect of the ccr5-delta32 deletion on global gene expression considering immune response and inflammation long-term control of hiv by ccr5 delta32/delta32 stemcell transplantation significance of rantes-ccr5 axis and linked downstream immunomodulation in dengue pathogenesis: a study from guwahati, india human cytomegalovirus enhances placental susceptibility and replication of human immunodeficiency virus type 1 (hiv-1), which may facilitate in utero hiv-1 transmission cytomegalovirus upregulates expression of ccr5 in central memory cord blood mononuclear cells, which may facilitate in utero hiv type 1 transmission immunological cure of hbv infection the chemokine receptor ccr5, a therapeutic target for hiv/aids antagonists, is critical for recovery in a mouse model of japanese encephalitis the role of ccr5/cxcr3 expressing cd8+ cells in liver damage and viral control during persistent hepatitis c virus infection ccr5del32 genotype in human enteroviral cardiomyopathy leads to spontaneous virus clearance and improved outcome compared to wildtype ccr5 ccr5 inhibitors and hiv-1 infection toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells puumala hantavirus: an imaging review human cytomegalovirus infection reduces surface ccr5 expression in human microglial cells, astrocytes and monocyte-derived macrophages updates on chronic hbv: current challenges and future goals review of climate, landscape, and viral genetics as drivers of the japanese encephalitis virus ecology coronavirus infections and immune responses polymorphisms of ccl3l1/ccr5 genes and recurrence of hepatitis b in liver transplant recipients horizontal transfer of exosomal cxcr4 promotes murine hepatocarcinoma cell migration, invasion and lymphangiogenesis the transcriptional and protein profile from human infected neuroprogenitor cells is strongly correlated to zika virus microcephaly cytokines phenotype evidencing a persistent inflammation in the cns the chemokine receptor ccr1 is identified in mast cell-derived exosomes reduced cc chemokine receptor (ccr) 1 and ccr5 surface expression on peripheral blood t lymphocytes from patients with chronic hepatitis c infection ccr5: no longer a 'good for nothing' gene--chemokine control of west nile virus infection genetic deficiency of chemokine receptor ccr5 is a strong risk factor for symptomatic west nile virus infection: a meta-analysis of 4 cohorts in the us epidemic ccr5 deficiency is a risk factor for early clinical manifestations of west nile virus infection but not for viral transmission chemokine control of west nile virus infection natural history of hepatitis c chemokine receptor antagonist block inflammation and therapy japanese encephalitis virus infection in mouse model the cytokine storm of severe influenza and development of immunomodulatory therapy exosome-associated hepatitis c virus in cell cultures and patient plasma genetic variants and susceptibility to neurological complications following west nile virus infection polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation viral escape mechanisms -escapology taught by viruses transfer of the chemokine receptor ccr5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection the ccr5δ32 (rs333) polymorphism is not a predisposing factor for severe pandemic influenza in the brazilian admixed population soluble chemokine receptor cxcr4 is present in human sera hcv chronic infection and ccr5-δ32/δ32 ccr5 genetic variants and epidemiological determinants for hpv infection and cervical premalignant lesions pathogenic hantaviruses elicit different immunoreactions in thp-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells dengue virus requires the ccchemokine receptor ccr5 for replication and infection development ccr5 blockade for neuroinflammatory diseases-beyond control of hiv genetic variants in the ccr gene cluster and spontaneous viral elimination in hepatitis c-infected patients expression of rantes by normal airway epithelial cells after influenza virus a infection ccr5 promoter polymorphism -2459g > a: forgotten or ignored? cells chemokine receptors as important regulators of pathogenesis during arboviral encephalitis enhanced expression, native purification, and characterization of ccr5, a principal hiv-1 coreceptor dengue haemorrhagic fever: a job done via exosomes? clinical significance of the ccr5delta32 allele in hepatitis c ccr5 deficiency exacerbates t-cell-mediated hepatitis in mice ccr5, mcp-1 and vdr gene polymorphisms are associated with the susceptibility to hbv infection pathways for internalization and recycling of the chemokine receptor ccr5 single-cell tracking reveals a role for pre-existing ccr5 + memory th1 cells in the control of rhinovirus-a39 after experimental challenge in humans ccr5δ32 genotype leads to a th2 type directed immune response in esrd patients ccr5 deletion protects against inflammation-associated mortality in dialysis patients immune response to dengue virus and prospects for a vaccine the pathogenesis of nephropathia epidemica: new knowledge and unanswered questions the predictive value of il28b gene polymorphism for spontaneous clearance in a single source outbreak cohort is limited in patients carrying the ccr5δ32 mutation analysis of the immunological biomarker profile during acute zika virus infection reveals the overexpression of cxcl10, a chemokine linked to neuronal damage chemokine ccr5 and cocaine interactions in the brain: cocaine enhances mesolimbic ccr5 mrna levels and produces place preference and locomotor activation that are reduced by a ccr5 antagonist why and where an hiv cure is needed and how it might be achieved a study of cc-chemokine receptor 5 (ccr5) polymorphism on the outcome of hcv therapy in egyptian patients exosomes secreted by bacterially infected macrophages are proinflammatory association between mbl2 haplotypes and dengue severity in children from rio de janeiro, brazil influenza-induced innate immunity: regulators of viral replication, respiratory tract pathology & adaptive immunity mir-155 induction in microglial cells suppresses japanese encephalitis virus replication and negatively modulates innate immune responses ccr5 and hiv infection serological evidence of widespread circulation of west nile virus and other flaviviruses in equines of the pantanal, brazil the genomic ancestry of individuals from different geographical regions of brazil is more uniform than expected ccr5 blockage by maraviroc: a potential therapeutic option for metastatic breast cancer west nile virus: review of the literature the evolution of seasonal influenza viruses marked differences in ccr5 expression and activation levels in two south african populations hepatitis c virus chemokine-regulated recruitment of antigen-specific t-cell subpopulations to the liver in acute and chronic hepatitis c infection chemokine receptor expression in ebv-associated lymphoproliferation in hu/scid mice: implications for cxcl12/cxcr4 axis in lymphoma generation frequency of the 32-base pair deletion in the chemokine receptor ccr5 gene is not increased in hepatitis c patients associations of chemokine system polymorphisms with clinical outcomes and treatment responses of chronic hepatitis c chemokine receptors: multifaceted therapeutic targets dysregulation of immune response in patients with covid-19 in wuhan, china one hundred years of poliovirus pathogenesis human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells metabolomics as an approach to characterise the contrasting roles of ccr5 in the presence and absence of disease microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of cd16 + , ccr5 + and tlr8 + monocytes characterization in vitro and in vivo of a pandemic h1n1 influenza virus from a fatal case uncommon immune-mediated extrahepatic manifestations of hcv infection the future of gene editing -toward scientific and social consensus ccr5 deficiency and severe polio infection in the 1984 outbreak in finland platelet-and megakaryocyte-derived microparticles transfer cxcr4 receptor to cxcr4-null cells and make them susceptible to infection by x4-hiv neutrophils induce a novel chemokine receptors repertoire during influenza pneumonia analysis of ccr5-δ32 and ccr2-v64i polymorphisms in a cohort of spanish hcv patients using real-time polymerase chain reaction and fluorescence resonance energy transfer technologies the possible role of ccr5δ32 mutation in crimean-congo hemorrhagic fever infection the ccr5-δ32 mutation: impact on disease outcome in individuals with hepatitis b infection in the southern khorasan population (east of iran) interacting roles of immune mechanisms and viral load in the pathogenesis of crimean-congo hemorrhagic fever chemokine receptor expression and chemotactic responsiveness of human monocytes after influenza a virus infection ccr5 expression is elevated in cervical cancer cells and is up-regulated by seminal plasma host genetic risk factors for community-acquired pneumonia the second extracellular loop of ccr5 is the major determinant of ligand specificity resistance to hiv-1 infection in caucasian individuals bearing mutant alleles of the ccr-5 chemokine receptor gene ccr5 plays important roles in hepatitis b infection the natural history of human papillomavirus infection ccr2 and ccr5 genes polymorphisms in women with cervical lesions from pernambuco, northeast region of brazil: a case-control study ccr5delta32 in systemic lupus erythematosus: implications for disease susceptibility and outcome in a brazilian population carcinogenic human papillomavirus infection west nile virus infection structural basis of coreceptor recognition by hiv-1 envelope spike clinical aspects of nephropathia epidemica (puumala virus infection) in europe: a review association of host genetic risk factors with the course of cytomegalovirus retinitis in patients infected with human immunodeficiency virus ccr5-dependent activation of mtorc1 regulates translation of inducible no synthase and cox-2 during encephalomyocarditis virus infection chemokine release from human rhinovirus-infected airway epithelial cells promotes fibroblast migration ccr2 positive exosome released by mesenchymal stem cells suppresses macrophage functions and alleviates ischemia/reperfusion-induced renal injury covid-19 infection: the perspectives on immune responses evidence for internal stores of ccr5 in blood cells role of cc chemokine receptor 1 and two of its ligands in human dengue infection. three approaches under the cuban situation endocytosis and recycling of the hiv coreceptor ccr5 a saint louis encephalitis and rocio virus serosurvey in brazilian horses frequency of the ccr5-delta32 allele in brazilian populations: a systematic literature review and meta-analysis cd4+foxp3+ regulatory t-cell subsets in human immunodeficiency virus infection ccr5-δ32 polymorphism and susceptibility to cervical cancer: association with early stage of cervical cancer the ccr5δ32 allele is not a major predisposing factor for severe h1n1pdm09 infection the role of cytokines in the immune response to influenza a virus infection frequencies of gene variant ccr5-δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national dkms donor centers adaptive immune responses to primary and secondary dengue virus infections the global burden of dengue: an analysis from the global burden of disease study advances in the treatment of hiv/hcv coinfection in adults ccr5 deficiency enhances hepatic innate immune cell recruitment and inflammation in a murine model of acute hepatitis b infection combined effect of hpv and several gene snps in laryngeal cancer association between vitamin d receptor, ccr5, tnf-α and tnf-β gene polymorphisms and hbv infection and severity of liver disease interaction between rantes promoter variant and ccr5δ32 favors recovery from hepatitis b frequency of the ccr5-δ32 mutant allele is not increased in belgian hepatitis c virus-infected patients japanese encephalitis: a review of the indian perspective retinopathy severity correlates with rantes concentrations and ccr 5-positive microvesicles in diabetes soluble chemokine ccr5 receptor is present in human plasma ccl5-ccr5 interaction provides antiapoptotic signals for macrophage survival during viral infection ccr5δ32 mutation, mycoplasma pneumoniae infection, and asthma innate immune interleukin-1 receptor-associated kinase 4 exacerbates viral myocarditis by reducing ccr5 + cd11b + monocyte migration and impairing interferon production ecology and geographical expansion of japanese encephalitis virus maraviroc -a ccr5 antagonist for the treatment of hiv-1 infection human cytomegalovirus inhibits the migration of immature dendritic cells by down-regulating cell-surface ccr1 and ccr5 role of regulatory t cells during virus infection reduced cell surface expression of ccr5 in ccr5δ32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration the role of natural antibodies to cc chemokine receptor 5 in hiv infection class b β-arrestin2-dependent ccr5 signalosome retention with natural antibodies to ccr5 the abrogation of phosphorylation plays a relevant role in the ccr5 signalosome formation with natural antibodies to ccr5 erk1-based pathway as a new selective mechanism to modulate ccr5 with natural antibodies real life experiences in hcv management t-cell surface ccr5 density is not correlated with hepatitis severity in hepatitis c virus/hivcoinfected individuals: implications for the therapeutic use of ccr5 antagonists cytomegalovirus cc chemokine promotes immune cell migration the ccr5δ32 allele is associated with reduced liver inflammation in hepatitis c virus infection gene-edited babies: what went wrong and what could go wrong cc chemokine receptor 5 δ32 polymorphism in two independent cohorts of hepatitis c virus infected patients without hemophilia dilated cardiomyopathy ccr5 deficiency regulates the proliferation and trafficking of natural killer cells under physiological conditions frequency of the hiv-protective cc chemokine receptor 5-δ32/δ32 genotype is increased in hepatitis c a new coronavirus associated with human respiratory disease in china ccr5 levels and expression pattern correlate with infectability by macrophagetropic hiv-1, in vitro critical role for ccr5 in the function of donor cd4 + cd25 + regulatory t cells during acute graftversus-host disease single nucleotide polymorphisms in candidate genes and dengue severity in children: a case-control, functional and meta-analysis study the potential risks of c-c chemokine receptor 5-edited babies in bone development bioinformatic identification of key genes and pathways that may be involved in the pathogenesis of hbv-associated acute liver failure effects of the chemokine receptor 5 (ccr5)-delta32 mutation on hepatitis c virus-specific immune responses and liver tissue pathology in hcv infected patients hepatitis b virus infection multiparametric flow cytometry analysis of peripheral blood b cell trafficking differences among epstein-barr virus infected and uninfected subpopulations differential expression of tim-3, pd-1, and ccr5 on peripheral t and b lymphocytes in hepatitis c virus-related hepatocellular carcinoma and their impact on treatment outcomes correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1a and c-c motif chemokine receptor 5 genes and infection with the hepatitis b virus in three ethnic groups in china pd1 + ccr2 + cd8 + t cells infiltrate the central nervous system during acute japanese encephalitis virus infection high frequency of ccr5-δ32 homozygosity in hcv-infected, hiv-1-uninfected hemophiliacs results from resistance to hiv-1 genetic polymorphism of chemokine receptors ccr2 and ccr5 in swedish cervical cancer patients host micrornas and exosomes that modulate influenza virus infection structure of cc chemokine receptor 5 with a potent chemokine antagonist reveals mechanisms of chemokine recognition and molecular mimicry by hiv exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral rna and proteins to the vertebrate neuronal cells chemokines: a new classification system and their role in immunity protective effect of ccr5-δ32 against hiv infection by the heterosexual mode of transmission in a polish population american 395 symptomatic wnv+ individuals; 145 symptomatic but noninfected individuals; 1318 controls (blood donors)the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection glass et al. (2006) american 224 symptomatic wnv+ individuals; 1318 controls (blood donors)corroborating data from glass et al. (2006) , the ccr5δ32 homozygous genotype was strongly associated with increased risk of symptomatic wnv infection lim et al. (2008) american 634 wnv+ individuals; 422 non-infected individuals ccr5δ32 homozygous genotype was associated with clinical symptoms of wnv infection; no association between ccr5δ32 and susceptibility to wnv infection lim et al. (2010) american, canadian 385 symptomatic wnv+ individuals; 328 asymptomatic wnv+ individuals; 1318 controls [blood donors from glass et al. (2006)] no association of ccr5δ32 and wnv infection considering symptomatic and asymptomatic wnv+ individuals; considering a dominant model of inheritance of ccr5δ32 and using controls from glass et al. (2006) , the ccr5δ32 was associated with symptomatic wnv infection and infection risk bigham et al. (2011) key: cord-017012-yl0vanuh authors: herberg, jethro; pahari, amitava; walters, sam; levin, michael title: infectious diseases and the kidney date: 2009 journal: pediatric nephrology doi: 10.1007/978-3-540-76341-3_52 sha: doc_id: 17012 cord_uid: yl0vanuh the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. the kidney is involved in a wide range of bacterial, viral, fungal, and parasitic diseases. in most systemic infections, renal involvement is a minor component of the illness, but in some, renal failure may be the presenting feature and the major problem in management. although individual infectious processes may have a predilection to involve the renal vasculature, glomeruli, interstitium, or collecting systems, a purely anatomic approach to the classification of infectious diseases affecting the kidney is rarely helpful because most infections may involve several different aspects of renal function. in this chapter, a microbiologic classification of the organisms affecting the kidney is adopted. although they are important causes of renal dysfunction in infectious diseases, urinary tract infections and hemolytic uremic syndrome (hus) are not discussed in detail because they are considered separately in chapters 54 and 48 respectively. elucidation of the cause of renal involvement in a child with evidence of infection must be based on a careful consideration of the geographic distribution of infectious diseases in different countries. a history of foreign travel; exposure to animals, insects, or unusual foods or drinks; outdoor activities such as swimming or hiking; and contact with infectious diseases must be sought in every case. the clinical examination should include a careful assessment of skin and mucous membranes and a search for insect bites, lymphadenopathy, and involvement of other organs. a close collaboration with a pediatric infectious disease specialist and hospital microbiologist will aid the diagnosis and management of the underlying infection. a tantalizing clue to the pathogenesis of glomerular disease is the marked difference in the incidence of nephrosis and nephritis in developed and underdeveloped areas of the world. in several tropical countries, glomerulonephritis (gn) accounts for up to 4% of pediatric hospital admissions; the incidence in temperate climates is 10-to 100-fold less. this difference might be explained by a complex interaction of several different factors, including nutrition, racial and genetically determined differences in immune responses, and exposure to infectious diseases. a growing body of evidence, however, suggests that longterm exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. renal involvement in infectious diseases may occur by a variety of mechanisms: direct microbial invasion of the renal tissues or collecting system may take place in conditions such as staphylococcal abscess of the kidney as a result of septicemic spread of the organism or as a consequence of ascending infection; damage to the kidney may be caused by the systemic release of endotoxin or other toxins and activation of the inflammatory cascade during septicemia or by a focus of infection distant from the kidney; ischemic damage may result from inadequate perfusion induced by septic shock; the kidney may be damaged by activation of the immunologic pathways or by immune complexes resulting from the infectious process. in many conditions, a combination of these mechanisms may be operative. in the assessment of renal complications occurring in infectious diseases, the possibility of druginduced nephrotoxicity caused by antimicrobial therapy should always be considered. the nephrotoxic effects of antibiotics and other antimicrobial agents are not addressed in this chapter but are covered in chapter 53. bacterial infections associated with renal disease and the likely mechanisms causing renal dysfunction are shown in > impaired renal function is a common occurrence in systemic sepsis (1) . depending on the severity of the infection and the organism responsible, the renal involvement may vary from insignificant proteinuria to acute renal failure requiring dialysis. the organisms causing acute renal failure as part of systemic sepsis vary with age and geographic location and also differ in normal and immunocompromised children. in the neonatal period, group b streptococci, coliforms, staphylococcus aureus, and listeria monocytogenes are the organisms usually . responsible. in older children, neisseria meningitidis, streptococcus pneumoniae, and s. aureus account for most of the infections. in people who are immunocompromised, a wide range of bacteria are seen, and, similarly, in tropical countries other pathogens, including haemophilus influenzae, salmonella species, and pseudomonas pseudomallei, must be considered. where h. influenzae type b vaccine has been introduced, however, the incidence of severe systemic infections due to this organism has shown a sharp fall. systemic sepsis usually presents with nonspecific features: fever, tachypnea, tachycardia, and evidence of skin and organ underperfusion. the pathophysiology of renal involvement in systemic sepsis is multifactorial (1, 2) . hypovolemia with diminished renal perfusion is the earliest event and is a consequence of the increased vascular permeability and loss of plasma from the intravascular space. hypovolemia commonly coexists with depressed myocardial function because of the myocardial depressant effects of endotoxin or other toxins. the renal vasoconstrictor response to diminished circulating volume and reduced cardiac output further reduces glomerular filtration, and oliguria is thus a consistent and early event in severe sepsis (1, 3) . a number of vasodilator pathways are activated in sepsis, including nitric oxide and the kinin pathways. this may lead to inappropriate dilatation of vascular beds. vasodilation of capillary beds leading to warm shock is common in adults with sepsis due to gram-negative organisms but is less commonly seen in children, in whom intense vasoconstriction is the usual response to sepsis. if renal underperfusion and vasoconstriction are persistent and severe, the reversible prerenal failure is followed by established renal failure with the characteristic features of vasomotor nephropathy or acute tubular necrosis. other mechanisms of renal damage in systemic sepsis include direct effects of endotoxin and other toxins on the kidney, and release of inflammatory mediators such as tumor necrosis factor (tnf) and other cytokines, arachidonic acid metabolites, and proteolytic enzymes (3) . nitric oxide (no) is postulated to play a key role in the pathophysiology of renal failure in sepsis. whether the renal effects of increased no are beneficial or harmful remains unclear. trials of selective no synthetase inhibition did not offer any advantages over saline resuscitation (4) . no in endotoxemia is possibly beneficial because it maintains renal blood flow and glomerular filtration. activation of coagulation is an important component of the pathophysiology of septic shock and may contribute to renal impairment. activation of multiple prothrombotic and antifibrinolytic pathways occurs, together with downregulation of antithrombotic mechanisms such as the protein c pathway. treatment with activated protein c has been shown to improve outcome of adult septic shock, but has not been confirmed to have benefit in pediatric sepsis, and may carry a risk of bleeding particularly in infants (5) . the renal findings early in septic shock are oliguria, with high urine/plasma urea and creatinine ratios, low urine sodium concentration, and a high urine/plasma osmolarity ratio. once established, renal failure supervenes, and the urine is of poor quality with low urine/ plasma urea and creatinine ratios, elevated urine sodium concentration, and low urine osmolarity. proteinuria is usually present, and the urine sediment may contain red cells and small numbers of white cells (1) . the management of acute renal failure in systemic sepsis depends on early diagnosis and administration of appropriate antibiotics to cover the expected pathogens. . in addition, management is directed at improving renal perfusion and oxygenation. volume replacement with crystalloid or colloid should be undertaken to optimize preload. central venous pressure or pulmonary wedge pressure monitoring is essential to guide volume replacement in children in severe shock (1, 2) . the use of low-dose (2-5 pg/kg/min) dopamine to reduce renal vasoconstriction together with administration of inotropic agents such as dobutamine or epinephrine to improve cardiac output may reverse prerenal failure. early elective ventilation should be undertaken in patients with severe shock. if oliguria persists despite volume replacement and inotropic therapy, dialysis should be instituted early, because septic and catabolic patients may rapidly develop hyperkalemia and severe electrolyte imbalance. in most children who develop acute renal failure as part of systemic sepsis or septic shock, the renal failure is of short duration, and recovery can be expected within a few days of achieving cardiovascular stability and eradication of the underlying infection. occasionally, renal cortical necrosis or infarction of the kidney may result in prolonged or permanent loss of renal function. meningococcus n. meningitidis continues to be a major cause of systemic sepsis and meningitis in both developed and underdeveloped parts of the world (6) . in developed countries, most cases are caused by group b and y strains, particularly after introduction of meningococcal c vaccination, whereas epidemics of meningococcus groups a, c and w135 continue to occur in many underdeveloped regions of the world (6, 7) . infants and young adults are most commonly affected, but cases in adolescents and young children are also common. there are two major presentations of meningococcal disease (6) : meningococcal meningitis presents with features indistinguishable from those of other forms of meningitis, including headache, stiff neck, and photophobia. lumbar puncture is required to identify the causative agent and distinguish this from other forms of meningitis. despite the acute nature of the illness, the prognosis is good, and most patients with the purely meningitic form of the illness recover without sequelae. meningococcemia with purpuric rash and shock is the second and more devastating form of the illness. affected patients present with nonspecific symptoms of fever, vomiting, abdominal pain, and muscle ache. the diagnosis is only obvious once the characteristic petechial or purpuric rash appears. patients with a rapidly progressive purpuric rash, hypotension, and evidence of skin and organ underperfusion have a poor prognosis, with a mortality of 10-30%. adverse prognostic features include hypotension, a low white cell count, absence of meningeal inflammation, thrombocytopenia, and disturbed coagulation indices (8) . renal failure was seldom reported in early series of patients with meningococcemia, perhaps because most patients died rapidly of uncontrolled septic shock. with advances in intensive care, however, more children are surviving the initial period of profound hemodynamic derangement, and renal failure is more often seen as a major management problem. approximately 10% of children with fulminant meningococcemia develop renal failure, which usually occurs 24-48 h after the onset of illness (9) . the pathophysiology of meningococcal septicemia involves the activation of cytokines and inflammatory cells by endotoxin (6, 7) . mortality is directly related to both the plasma endotoxin concentration and the intensity of the inflammatory response, as indicated by levels of tnf and other inflammatory markers (10) . patients with meningococcemia have a profound capillary leak leading to severe hypovolemia. loss of plasma proteins from the intravascular space is probably the major cause of shock (11) , but myocardial suppression secondary to il-6 production is also important (12) . intense vasoconstriction further impairs tissue and organ perfusion, and vasculitis with intravascular thrombosis and consumption of platelets and coagulation factors is also present (6) . oliguria is invariably present in children with meningococcemia during the initial phase of the disorder. this is prerenal in origin and may respond to volume replacement and inotropic support. if cardiac output cannot be improved and renal underperfusion persists, established renal failure supervenes. occasionally, cortical necrosis or infarction of the kidneys occurs. children with meningococcemia should be aggressively managed in a pediatric intensive care unit, with early administration of antibiotics (penicillin or a third-generation cephalosporin), volume replacement, hemodynamic monitoring, and the use of inotropic agents and vasodilators. if oliguria persists despite measures to improve cardiac output, elective ventilation and dialysis should be instituted early (6, 7) . because activation of coagulation pathways occurs, severe acquired protein-c deficiency may result and is usually associated with substantial mortality (13) . protein c is a natural anticoagulant which also has important antiinflammatory activity. despite evidence for impaired function of the activated protein c pathway in meningococcal diseases (14) , and adult trials suggesting benefit of activated protein c administration in septic shock (prowess trial) (15) , pediatric trials of activated protein c showed no clear benefit, and were associated with increased risk of intracranial bleeding in very young infants (5) . the role of apc therapy in pediatric sepsis remains unclear. most patients who survive the initial 24-48 h of the illness and regain hemodynamic stability will ultimately recover renal function even if dialysis is required for several weeks. the least common presentation of meningococcal sepsis is chronic meningococcemia. patients with this form of the illness present insidiously with a vasculitic rash, arthritis, and evidence of multiorgan involvement. the features may overlap those of henoch-schonlein purpura or subacute bacterial endocarditis (sbe), and the diagnosis must be considered in patients presenting with fever, arthritis, and vasculitic rash, often accompanied by proteinuria or hematuria. response to antibiotic treatment is good, but some patients may have persistent symptoms for many days resulting from an immunecomplex vasculitis. staphylococcal infections may affect the kidneys by direct focal invasion during staphylococcal septicemia, forming a renal abscess; by causing staphylococcal bacteremia; or by toxin-mediated mechanisms, as in the staphylococcal toxic shock syndrome. staphylococcal abscess. staphylococcal renal abscess presents with fever, loin pain and tenderness, and abnormal urine sediment, as do abscesses caused by other organisms (16) . the illness often follows either septicemia or pyelonephritis. the diagnosis is usually considered only when a patient with clinical pyelonephritis shows an inadequate response to antibiotic treatment. the diagnosis is confirmed by ultrasonography or computed tomographic scan, which shows swelling of the kidney and intrarenal collections of fluid. antibiotic therapy alone may result in cure, but if the patient remains unwell with evidence of persistent inflammation despite use of appropriate antibiotics, surgical intervention may be required. percutaneous drainage under ultrasonographic or computed tomographic scan guidance is often effective and may avoid the need for a more direct surgical approach (16, 17) . staphylococcal toxic shock syndrome. the staphylococcal toxic shock syndrome is a systemic illness characterized by fever, shock, erythematous rash, diarrhea, confusion, and renal failure. the disorder was first described by todd et al. in 1978 in a series of seven children (18) . during the 1980s, thousands of cases were reported in the united states. most cases were in menstruating women, in associated with tampon use. although most cases worldwide are seen in women and are associated with menstruation, children of both sexes and of all ages are affected (19) . the illness usually begins suddenly with high fever, diarrhea, and hypotension, together with a diffuse erythroderma (20) . mucous membrane involvement with hyperemia and ulceration of the lips and oral mucosa or vaginal mucosa, strawberry tongue, and conjunctival injection are usually seen. desquamation of the rash occurs in the convalescent phase of the illness. confusion is often present in the early stages of the illness and may progress to coma in severe cases. multiple organ failure with evidence of impaired renal function, elevated levels of hepatic transaminases, thrombocytopenia, elevated cpk and disseminated intravascular coagulation (dic) is often seen. according to cdc criteria, the diagnosis is made on the basis of the clinical features of fever, rash, hypotension, and subsequent desquamation along with deranged function of three or more of the following organ systems: gastrointestinal (gi), mucous membranes, renal, hepatic, hematologic, central nervous system, and muscle. other disorders causing a similar picture, such as rocky mountain spotted fever, leptospirosis, measles, and streptococcal infection, must be excluded. the staphylococcal toxic shock syndrome is now known to be due to infection or colonization with strains of s. aureus that produce one or more protein exotoxins (21) . most cases in adults are associated with toxic shock toxin i; in children, many of the isolates associated with the syndrome produce other enterotoxins (a to f). the staphylococcal enterotoxins appear to induce disease by acting as superantigens (22) , which activate t cells bearing specific v beta regions of the t-cell receptor; this causes proliferation and cytokine release (23) . the systemic illness and toxicity are believed to result largely from an intense inflammatory response induced by the toxin. the site of toxin production is often a trivial focus of infection or simple colonization, and bacteremia is rarely observed. renal failure in toxic shock syndrome is usually caused by shock and renal hypoperfusion. in the early stages of the illness, oliguria and renal impairment are usually prerenal and respond to treatment of shock and measures to improve perfusion. in severe cases and in patients in whom treatment is delayed, acute renal failure develops as a consequence of prolonged renal underperfusion, and dialysis may be required. in addition to underperfusion, direct effects of the toxin or inflammatory infectious diseases and the kidney mediators may also contribute to the renal damage. recovery of renal function usually occurs, but in severe cases with cortical necrosis or intense renal vasculitis, prolonged dialysis may be required. the management of staphylococcal toxic shock syndrome depends on early diagnosis and aggressive cardiovascular support with volume replacement, inotropic support, and, in severe cases, elective ventilation. if oliguria persists despite optimization of intravascular volume and administration of inotropic agents, dialysis should be commenced early (20) . anti-staphylococcal antibiotics should be started as soon as the diagnosis is suspected and the site of infection identified. initial empiric antimicrobial therapy should include an anti-staphylococcal antibiotic effective against betalactamase-resistant organisms and a protein synthesisinhibiting antibiotic such as clindamycin to stop further toxin production (24) . if there is a focus of infection such as a vaginal tampon, surgical wound, or infected sinuses, the site should be drained early to prevent continued toxin release into the circulation. the intravenous administration of immune globulins may be considered when infection is refractory to several hours of aggressive therapy, an undrainable focus is present, or persistent oliguria with pulmonary edema occurs (24) . with aggressive intensive care, most affected patients survive, and renal recovery is usual, even in patients who have had severe shock and multiorgan failure. relapses and recurrences of staphylococcal toxic shock syndrome occur in a proportion of affected patients because immune responses to the toxin are ineffective in some individuals. panton valentine leucocidin (pvl) producing staphylococcal infection: in recent years there have been increasing reports of severe staphylococcal disease, associated with shock and multiorgan failure, caused by strains of staphylococci producing the pvl toxin. panton-valentine leukocidin (pvl) is a phage-encoded toxin, which profoundly impairs the host response due to its toxic effect on leucocytes (see review (25) ). pvl producing strains are associated with tissue necrosis and increased propensity to cause abscesses in lung, bone, joint, and soft tissue infections. perinephric abscesses have been reported (26) . there are increasing numbers of children and adults admitted with fulminant sepsis, and shock due to pvl producing strains, and renal failure is a significant component of the multiorgan failure. in addition to intensive care support, antibiotic treatment of pvl strains should include antibiotics which reduce toxin production, such as clindamycin, linezolid or rifampicin, as well as vancomycin if the strain is resistant to methicillin. beta-lactam antibiotics should be avoided, as there is some data to suggest that pvl toxin production can increased by these antibiotics under some conditions (27) . immunoglobulin infusion may also be of benefit. aggressive surgical drainage of all collections requires close consultation with orthopedic and surgical teams. the group a streptococci (gas) are a major worldwide cause of renal disease, usually as poststreptococcal nephritis. however, in addition to this post-infection immunologically mediated disorder, in recent years there have been increasing reports of gas causing acute renal failure as part of an invasive infection with many features of the staphylococcal toxic shock syndrome (28) . acute poststreptococcal glomerulonephritis. acute poststreptococcal gn (apsgn) is a delayed complication of pharyngeal infection or impetigo with certain nephritogenic strains of gas. different strains can be serotyped according to the antigenic properties of the m protein found in the outer portion of the bacterial wall. apsgn after pharyngeal infection is most commonly associated with serotype m12. in contrast, in apsgn after impetigo, serotype m49 is most commonly identified (29) . on occasions, other serotypes and non-typeable strains have been described as causing gn. the pathology and pathogenesis of the disorder is discussed in detail in chapter 30. apsgn has a worldwide distribution. epidemiologic differences are observed between pharyngitis-associated and impetigo-associated streptococcal infections. pharyngitis-associated apsgn is most common during school age and has an unexplained male/female ratio of 2:1. it occurs more often in the cooler months, and familial occurrences are commonly described. the latent period is 1-2 weeks, in notable contrast to impetigo-associated cases, which have a latent period of 2-6 weeks. in many developing countries, children have chronic skin infections, and it may be difficult to establish the latent period with accuracy. impetigoassociated cases are more common in the warmer months, sex distribution is equal, and children tend to be younger. introduction of a nephritogenic strain into a family often results in the occurrence of several cases within that family, and in some cases, attack rates of up to 20% have been described (30) . the incidence is linked to poor socioeconomic conditions. renal involvement in apsgn can be mild, and in many patients, the disease may not be manifested clinically. studies of epidemics with nephritogenic strains of streptococci have shown that up to 50% of those infected had subclinical evidence of renal disease (30, 31) . in a typical case a sudden onset of facial or generalized edema occurs. hypertension is usually modest but is severe in 5% of cases, and occasionally may lead to encephalopathy or left ventricular failure. the urine is smoky or tea colored in 30-50% of cases. pallor, headache, backache, lethargy, malaise, anorexia, and weakness are all common nonspecific features. the urine volume is decreased. proteinuria is present (up to 100 mg/dl), and microscopy shows white cells, red cells, and granular and hyaline casts. urea, electrolyte, and creatinine levels are normal in subclinical cases but show features of acute renal failure in severe cases. it may be possible to culture gas from the skin or the throat in some patients. other evidence of infection with a gas can be obtained through the antistreptolysin-o titer (asot), which is increased in 60-80% of cases. early antibiotic treatment can reduce the proportion of cases with elevated asot to 30%. anti-deoxyribonuclease b and anti-hyaluronidase testing has been shown to be of more value than asot in confirming group a streptococcal infection in impetigo-associated cases. measurement of anti-m protein antibodies is of more value for epidemiologic purposes than for the diagnosis of individual cases (31) . decreased c3 and total hemolytic complement levels are found in 90% of cases during the first 2 weeks of illness and return to normal after 4-6 weeks. penicillin should be given to eradicate the gas organisms. erythromycin, clindamycin, or a first-generation cephalosporin can be given to patients allergic to penicillin. antibiotic treatment probably has no influence on the course of renal disease but will prevent the spread of a nephritogenic strain (32) . close contacts and family members who are culture-positive for gas should also be given penicillin, although antibiotic treatment is not always effective in eliminating secondary cases. recurrent episodes are rare, and immunity to the particular nephritogenic strain that caused the disease is probably lifelong. antibiotic prophylaxis is therefore unnecessary. most studies suggest that the prognosis for children with apsgn is good, with more than 90% making a complete recovery. however, 10% of cases may have a prolonged and more serious course with long-term chronic renal failure (33) . other streptococci. apsgn has also been described after outbreaks of group c streptococcus infection (34) . this has occurred after consumption of unpasteurized milk from cattle with mastitis. patients developed pharyngitis followed by apsgn. endostreptosin was found in the cytoplasm of these group c strains, and during the course of the illness, patients developed anti-endostreptosin antibodies. this antigen has been postulated to be the nephritogenic component of gas. in addition, strains of group g streptococci have been implicated in occasional cases of apsgn (35) . isolates possessed the type m12 protein antigen identical to the nephritogenic type m12 antigen of some group a streptococcal strains. streptococcal toxic shock syndrome and invasive group a streptococcal infection. since 1988, there have been several reports of an illness with many similarities to the staphylococcal toxic shock syndrome, occurring in both children (36) and adults, associated with invasive group a streptococcal disease (32, 37, 38) . patients with this syndrome present acutely with high fever, erythematous rash, mucous membrane involvement, hypotension, and multiorgan failure. unlike staphylococcal toxic shock syndrome, in which the focus of infection is usually trivial and bacteremia is seldom seen, the streptococcal toxic shock syndrome is usually associated with bacteremia or a serious focus of infection such as septic arthritis, myositis, or osteomyelitis (36, 38) . laboratory findings of anemia, neutrophil leukocytosis, thrombocytopenia, and dic are often present, together with impaired renal function, hepatic derangement, and acidosis. acute renal failure requiring dialysis occurs in a significant proportion of cases. it is not clear why there are increasing numbers of cases with invasive disease caused by gas, nor why there has been an emergence of streptococcal toxic shock syndrome, and indeed a similar syndrome caused by some pseudomonas and klebsiella strains. the most common antecedent of invasive gas disease is varicella infection, with the streptococcal infection developing after the initial vesicular phase of the disease is subsiding. strains causing toxic shock syndrome and invasive disease appear to differ from common isolates of gas in producing large amounts of pyrogenic toxins that may have superantigenlike activity. another important mechanism is the production by invasive gas of an il8 protease. il8 serves as a molecular bridge between receptors on neutrophils and the vascular endothelium. cleavage of this protein prevents neutrophil attachment to the endothelium, and results in uncontrolled spread of the bacteria through the tissues (39) . in severe cases necrotizing fasciitis occurs with extensive destruction of the subcutaneous tissues, and is often associated with multiorgan failure. the pathophysiology of streptococcal toxic shock syndrome and that of invasive disease is similar in that superantigen toxins that induce release of cytokines and other inflammatory mediators play a role in both conditions. however gas toxic shock is usually more severe, carries a higher mortality, and is more often associated with focal collections or necrotizing fasciitis. treatment of streptococcal toxic shock syndrome depends on the administration of appropriate antibiotics, aggressive circulatory support, and treatment of any multiorgan failure. surgical intervention to drain the infective focus in muscle, bone, joint, or body cavities is often required. antibiotic therapy with beta-lactams should be supplemented by treatment with a protein synthesisinhibiting antibiotic, such as clindamycin, and it is suggested that this limits new toxin production (40, 41) . a number of new therapies are in development. firstly, pooled intravenous immunoglobulins are now in widespread use in the treatment of toxic shock, particularly when caused by streptococcus (42, 43) . the role of steroids remains unclear, with their hemodynamic benefit set against the detrimental effects of hyperglycemia secondary to gluconeogenesis. (44) . the benefit of insulin therapy to control hyperglycemia is unclear. a recent study in adults found that intensive insulin therapy increased the risk of serious adverse events (45) . in contrast to adult patients, in children with severe sepsis, the use of activated protein c (drotrecogin) cannot be recommended, as in a multicenter trial, fatality was increased in the treatment group (5) . recovery of renal function occurs in patients who respond to treatment of shock and the eradication of the infection. leptospirosis is an acute generalized infectious disease caused by spirochetes of the genus leptospira (46) . it is primarily a disease of wild and domestic animals, and humans are infected only occasionally through contact with animals. most human cases occur in summer or autumn and are associated with exposure to leptospiracontaminated water or soil during recreational activities such as swimming or camping. in adolescents and adults, occupational exposure through farming or other contact with animals is the route of infection. the spirochete penetrates intact mucous membranes or abraded skin and disseminates to all parts of the body, including the cerebrospinal fluid (csf). although leptospires do not contain classic endotoxins, the pathophysiology of the disorder has many similarities to that of endotoxemia. in severe cases, jaundice occurs because of hepatocellular dysfunction and cholestasis. renal functional abnormalities may be profound and out of proportion to the histologic changes in the kidney (47) . renal involvement is predominantly a result of tubular damage, and spirochetes are commonly seen in the tubular lesions. the inflammatory changes in the kidney may result from either a direct toxic effect of the organism or immunecomplex nephritis. however, hypovolemia, hypotension, and reduced cardiac output caused by myocarditis may contribute to the development of renal failure. in severe cases, a hemorrhagic disorder caused by widespread vasculitis and capillary injury also occurs (47, 48) . the clinical manifestations of leptospirosis are variable. of affected patients, 90% have the milder anicteric form of the disorder, and only 5-10% have severe leptospirosis with jaundice. the illness may follow a biphasic course. after an incubation period of 7-12 days, a nonspecific flu-like illness lasting 4-7 days occurs, associated with septicemic spread of the spirochete. the fever then subsides, only to recur for the second, ''immune,'' phase of the illness. during this phase, the fever is low grade and there may be headache and delirium caused by meningeal involvement, as well as intense muscular aching. nausea and vomiting are common. examination usually reveals conjunctival suffusion, erythematous rash, lymphadenopathy, and meningism. the severe form of the disease (weil's disease) presents with fever, impaired renal and hepatic function, hemorrhage, vascular collapse, and altered consciousness. in one series the most common organs involved were the liver (71%) and kidney (63%). cardiovascular (31%), pulmonary (26%), neurologic (5%), and hematologic (21%) involvements were less common (49) . vasculitis, thrombocytopenia, and uremia are considered important factors in the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis (50) . urinalysis results are abnormal during the leptospiremic phase with proteinuria, hematuria, and casts. uremia usually appears in the second week, and acute renal failure may develop once cardiovascular collapse and dic are present (48) . the clinical features of leptospirosis overlap with those of several other acute infectious diseases, including rocky mountain spotted fever, toxic shock syndrome, and streptococcal sepsis. the diagnosis of leptospirosis should be considered in febrile patients with evidence of renal, hepatic, and mucous membrane changes and rash, particularly if a history of exposure to fresh water is found. diagnosis can be confirmed by isolation of the spirochetes from blood or csf in the first 10 days of the illness or from urine in the second week (48) . the organism may be seen in biopsy specimens of the kidney or skin or in the csf by dark-field microscopy or silver staining. serologic tests to detect leptospirosis are now sensitive and considerably aid the diagnosis. immunoglobulin m (igm) antibody may be detected as early as 6-10 days into the illness, and antibody titers rise progressively over the next 2-4 weeks. some patients remain seronegative, and negative serologic test results do not completely exclude the diagnosis. in one series levels of igm and igg anticardiolipin concentrations were significantly increased in leptospirosis patients with acute renal failure (50) . leptospirosis is treated with intravenous penicillin or other beta-lactam antibiotics. the severity of leptospirosis is reduced by antibiotic treatment, even if started late in the course of the illness (51) . supportive treatment with volume replacement to correct hypovolemia, administration of inotropes, and correction of coagulopathy is essential in severe cases. dialysis may be required in severe cases and may be needed for prolonged periods until recovery occurs. infection with s. pneumoniae is one of the most common infections in humans and causes a wide spectrum of disease, including pneumonia, otitis media, sinusitis, septicemia, and meningitis. despite the prevalence of the organism, significant renal involvement is relatively rare but is seen in two situations: pneumococcal septicemia in asplenic individuals or in those with other immune deficiencies presents with fulminant septic shock in which renal failure may occur as part of a multisystem derangement. the mortality from pneumococcal sepsis in asplenic patients is high, even with early antibiotic treatment and intensive support. the second nephrologi syndrome associated with s. pneumoniae is a rare form of hus. in 1955, gasser and colleagues described hus as a clinical entity in children, and they included two infants with pneumonia among the five patients they described (52) . hus associated with pneumococcal infection is induced by the enzyme neuraminidase released from s. pneumoniae (53, 54) . thomsen-friedenrich antigen (t antigen) is present on the surface of red blood cells, platelets, and glomerular capillary endothelia against which antibodies are present in normal serum. neuraminidase causes desialation of red blood cells, and possibly other blood cells and endothelium, by the removal of terminal neuraminic acid, which leads to unmasking of the t antigen. the resultant widespread agglutination of blood cells causes intravascular obstruction, hemolysis, thrombocytopenia, and renal failure. results of the direct coombs test are frequently positive, either from bound anti-t igm or from anti-t antibodies. the diagnosis of thomsen-friedenrich antibody-induced hus should be suspected in patients with acute renal failure, thrombocytopenia and hemolysis after an episode of pneumonia or bacteremia caused by s. pneumoniae. fragmented red blood cells will usually be present on blood film. association with s. pneumoniae is defined by culture of pneumococci from a normally sterile site within a week before or after onset of signs of hus. clues to a pneumococcal cause, in addition to culture results, include severe clinical disease, especially pneumonia, empyema, pleural effusion, or meningitis; hemolytic anemia without a reticulocyte response; positive results on a direct coombs test; and difficulties in abo crossmatching or a positive minor crossmatch incompatibility (55) . however, when renal disease is seen in the context of severe pneumococcal infection, it is important to maintain a broad diagnostic perspective, because the occurrence of acute tubular necrosis due to septic shock and dic is well described (56, 57) . therapy for this syndrome should be with supportive treatment and antibiotics (usually a third generation cephalosporin); dialysis may be required if renal failure occurs. because normal serum contains antibodies against the thomsen-friedenrich antigen, blood transfusion should be undertaken with washed red blood cells resuspended in albumin rather than plasma (53, 54) . exchange transfusion and plasmapheresis have been used in some patients, with the rationale that these procedures may improve outcome by eliminating circulating neuraminidase (53, 57, 58) . intravenous igg has been used in a patient and was shown to neutralize neuraminidase present in the patient's serum (59) . in comparison to patients with the more common diarrhea-associated hus, s. pneumoniae-induced hus patients have a more severe renal disease. they are more likely to require dialysis. their long-term outcome maybe affected by the severity of the invasive streptococcal disease itself, and a significant proportion of surviving patients (30-70%) develop end-stage renal failure (60, 61) . a recent review of uk cases found an eightfold increase in early mortality as compared to diarrhoea-induced hus (62) . gastrointestinal infections (escherichia coli, salmonella, campylobacter, yersinia, shigella, vibrio cholerae) the diarrheal diseases caused by escherichia coli, salmonella, shigella, campylobacter, vibrios, and yersinia remain important and common bacterial infections of humans. although improvements in hygiene and living conditions have reduced the incidence of bacterial gastroenteritis in developed countries, these infections remain common in underdeveloped areas of the world, and outbreaks and epidemics continue to occur in both developed and underdeveloped countries. renal involvement in the enteric infections may result from any of four possible mechanisms. regardless of the causative organism, diarrhea results in hypovolemia, abnormalities of plasma electrolyte composition, and renal underperfusion. if severe dehydration occurs and is persistent, oliguria from prerenal failure is followed by vasomotor nephropathy and established renal failure. e. coli, shigella, and salmonella (particularly salmonella typhi) may invade the bloodstream and induce septicemia or septic shock. acute renal failure is commonly seen in infants with e. coli sepsis but is also reported with klebsiella, salmonella, and shigella infections. its pathophysiology and treatment were discussed previously. enteric infections with e. coli, yersinia, campylobacter, and salmonella have been associated with several different forms of gn, including membranoproliferative gn (mpgn), interstitial nephritis, diffuse proliferative gn, and iga nephropathy (63) (64) (65) . in typhoid fever, gn ranging from mild asymptomatic proteinuria and hematuria to acute renal failure may occur (64, (66) (67) (68) . renal biopsy findings show focal proliferation of mesangial cells, hypertrophy of endothelial cells, and congested capillary lumina. immunofluorescent studies show igm, igg, and c3 deposition in the glomeruli, with salmonella antigens detected within the granular deposits in the mesangial areas. in the iga nephropathy after typhoid fever, salmonella vi antigens have been demonstrated within the glomeruli. yersinia infection has been reported as a precipitant of gn in several studies (65, 69) . transient proteinuria and hematuria are found in 24% of patients with acute yersinia infection, and elevated creatinine levels in 10%. renal biopsy reveals mild mesangial gn or iga nephropathy. yersinia antigens, immunoglobulin, and complement have been detected in the glomeruli. yersinia pseudotuberculosis is well recognized as one of the causes of acute tubulointerstitial nephritis causing acute renal failure, especially in children; patients have histories of drinking untreated water in endemic areas (70) (71) (72) . the illness begins with the sudden onset of high fever, skin rash, and gi symptoms. later in the course, periungual desquamation develops, mimicking kawasaki disease. elevated erythrocyte sedimentation rate, c-reactive protein level, and thrombocytosis are noticeable, and mild degrees of proteinuria, glycosuria, and sterile pyuria are common. acute renal failure, which typically develops 1-3 weeks after the onset of fever, follows a benign course with complete recovery. renal biopsy mainly reveals findings of acute tubulointerstitial nephritis. antibiotic therapy, although recommended, does not alter the clinical course, but reduces the fecal excretion of the organism (73, 74) . hus is characterized by three distinct clinical signs: acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. it was first described in 1955 and was associated with infection by shiga toxin-producing shigella dysenteriae. a major breakthrough in the search for the cause of hus occurred in the 1980s when karmali et al. reported that 11 of 15 children with diarrheaassociated hus had evidence of infection with a strain of e. coli that produced a toxin active on vero cells (75) . in diarrhea-associated hus in the united states and most of europe, e. coli 0157:h7 is the most important of these strains. e. coli 0157:h7 occurs naturally in the gi tract of cattle and other animals, and humans become infected through contaminated food products. most outbreaks have been associated with consumption of undercooked meat, but unpasteurized milk and cider, drinking water, and poorly chlorinated water for recreational use have also been implicated as vehicles for bacterial spread. hus is discussed in detail in chapter 67. the global epidemic of mycobacterium tuberculosis is growing. several factors have contributed to this increase, including the emergence of the human immunodeficiency virus (hiv) infection epidemic, large influxes of immigrants from countries in which tuberculosis (tb) is common, the emergence of multiple-drug-resistant m. tuberculosis, and breakdown of the health services for effective control of tb in various countries. it is generally estimated that, overall, one-third of the world's population is currently infected with the tb bacillus. there are more than 8 million cases of tb, which result in the death of approximately 2 million people each year. furthermore, 5-10% of people who are infected with the tb bacillus develop tb disease or become infectious at some time during their lives (76, 77) . after respiratory illness in children, mycobacteria are widely distributed to many organs of the body during the lymphohematogenous phase of childhood tb (78) . tubercle bacilli can be recovered from the urine in many cases of miliary tb. hematogenously-spread tuberculomata develop in the glomeruli, which results in caseating, sloughing lesions that discharge bacilli into the tubules. in most cases, the renal lesions are asymptomatic and manifest as mycobacteria in the urine or as sterile pyuria. tuberculomata in the cortex may calcify and cavitate or may rupture into the pelvis, discharging infective organisms into the tubules, urethra, and bladder. dysuria, loin pain, hematuria, and pyuria are the presenting features of this complication, but in many cases, the renal involvement is asymptomatic, even when radiologic and pathologic abnormalities are very extensive. continuing tuberculous bacilluria may cause cystitis with urinary frequency and, in late cases, a contracted bladder (79) . the intravenous urogram is abnormal in most cases. early findings are pyelonephritis with calyceal blunting and calyceal-interstitial reflux. later, papillary cavities may be seen, indicating papillary necrosis. ureteric strictures, focal calcification, hydronephrosis, and cavitation may also be seen. renal function is usually well preserved, and hypertension is uncommon. in some cases, either the infection itself or reactions to the chemotherapeutic agents may result in renal failure with evidence of an interstitial nephritis (79) (80) (81) . classic symptomatic renal tb is a late and uncommon complication in children, rarely occurring less than 4 or 5 years after the primary infection, and therefore is most commonly diagnosed after adolescence (78, 80) . adult studies have shown that 26-75% of renal tb coexists with active pulmonary tb and 6-10% of screened sputumpositive pulmonary tb patients have renal involvement. the diagnosis is established by isolation of mycobacteria from the urine or by the presence of the characteristic clinical and radiographic features in a child with current or previous tb. renal tb is treated with drug regimens similar to those used for other forms of tb, with isoniazid, rifampicin, pyrazinamide and ethambutol administered initially for 2 months, and isoniazid and rifampicin then continued for a further 7-10 months. late scarring and urinary obstruction may occur in cases with extensive renal involvement, and such patients should be followed by ultrasonography or intravenous urogram. mycobacteria, both m. tuberculosis and atypical mycobacteria, have also emerged as important causes of opportunistic infection in immunocompromised patients undergoing dialysis and in patients undergoing renal transplantation. the possibility of mycobacterial disease must be considered in patients with fever of unknown origin or unexplained disease in the lungs or other organs. results of the mantoux test are often negative, and diagnosis depends on maintaining a high index of suspicion and isolating the organism from the infected site. renal involvement has been well documented in both congenital and acquired syphilis, with an estimated occurrence of 0.3% in patients with secondary syphilis and up to 5% in those with congenital syphilis (82, 83) . the most common manifestation of renal disease in congenital syphilis is the nephrotic syndrome, with proteinuria, hypoalbuminemia, and edema. in some patients, hematuria, uremia, and hypertension may be seen. the renal disease is usually associated with other manifestations of congenital syphilis, including hepatosplenomegaly, rash, and mucous membrane findings. nephritis in congenital syphilis is usually associated with evidence of complement activation, with depressed levels of clq, c4, c3, and c5. histologic findings are a diffuse proliferative gn or a membranous nephropathy. the interstitium shows a cellular infiltrate of polymorphonuclear and mononuclear cells (84) . immunofluorescent microscopy reveals diffuse granular deposits of igg and c3 along the glomerular basement membrane (gbm). mesangial deposits may also contain igm. on electron microscopy, scattered subepithelial electron-dense deposits are seen, with fusion of epithelial cell foot processes (84) . good evidence exists that renal disease is due to an immunologically mediated reaction to treponemal antigens. antibodies reactive against treponemal antigens can be eluted from the glomerular deposits, and treponemal antigens are present in the immune deposits. treatment of both congenital and acquired syphilis with antibiotics results in rapid improvement in the renal manifestations (82, 84) . renal involvement is surprisingly rare in mycoplasma pneumoniae infection considering the prevalence of this organism and its propensity to trigger immunologically mediated diseases such as erythema multiforme, arthritis, and hemolysis. acute nephritis associated with mycoplasma infection may occur 10-40 days after the respiratory tract infection (85, 86) . renal histopathologic findings include type 1 mpgn, proliferative endocapillary gn, and minimal change disease (87) . antibiotic treatment of the infection does not appear to affect the renal disease, which is self-limited in most cases (85, 86) . since its recognition in 1976, legionnaires' disease, caused by legionella pneumophila, has emerged as an important infectious diseases and the kidney cause of pneumonia. the disease most commonly affects the elderly but has been reported in both normal and immunocompromised children (97, 98) . renal dysfunction occurs in a minority of patients (98) . patients who develop renal impairment present with oliguria and rising urea and creatinine levels. they are usually severely ill, with bilateral pulmonary infiltrates, fever, and leukocytosis. shock may be present, and the renal impairment has been associated with acute rhabdomyolysis with high levels of creatine phosphokinase and myoglobinuria. renal histologic examination usually shows a tubulointerstitial nephritis or acute tubular necrosis (98, 99) . the pathogenesis of the renal impairment is uncertain, but the organism has been detected within the kidney on electron microscopy and immunofluorescent studies, which suggests a direct toxic effect. myoglobinuria and decreased perfusion may also be contributing factors, however. mortality has been high in reported cases of legionnaires' disease complicated by renal failure. treatment is based on dialysis, intensive care, and antimicrobial therapy with erythromycin (98) . steroid therapy may be effective for tubulointerstitial nephritis (99) . the rickettsial diseases are caused by a family of microorganisms that have characteristics common to both bacteria and viruses and that cause acute febrile illnesses associated with widespread vasculitis. with the exception of q fever, all are associated with erythematous rashes. there are four groups of rickettsial diseases: 1. the typhus group includes louse-borne and murine typhus, spread by lice and fleas, respectively. 2. the spotted fever group includes rocky mountain spotted fever, tick typhus and related mediterranean spotted fever and rickettsial pox, which are spread by ticks and mites, with rodents as the natural reservoir. 3. scrub typhus, which is spread by mites. 4. q fever, which is spread by inhalation of infected particles from infected animals. rickettsial diseases have a worldwide distribution and vary widely in severity, from self-limited infections to fulminant and often fatal illnesses (88) . in view of the widespread vasculitis associated with these infections, subclinical renal involvement probably occurs in many of the rickettsial diseases. however, in rocky mountain spotted fever, tick typhus, and q fever, the renal involvement may be an important component of the illness. rocky mountain spotted fever is the most severe of the rickettsial diseases (89, 90) . the onset occurs 2-8 days after the bite of an infected tick. high fever develops initially, followed by the pathognomonic rash, which occurs between the second and sixth days of the illness. the rash initially consists of small erythematous macules, but later these become maculopapular and petechial, and in untreated patients, confluent hemorrhagic areas may be seen. the rash first appears at the periphery and spreads up the trunk. involvement of the palms and soles is a characteristic feature (88) . headache, restlessness, meningism, and confusion may occur together with other neurologic signs. cardiac involvement with congestive heart failure and arrhythmia are common. pulmonary involvement occurs in 10-40% of cases. infection is associated with an initial leucopenia, followed by neutrophil leukocytosis. thrombocytopenia occurs in most cases. histopathologically, the predominant lesions are in the vascular system (91) . rickettsiae multiply in the endothelial cells, which results in focal areas of endothelial cell proliferation, perivascular mononuclear cell infiltration, thrombosis, and leakage of red cells into the tissues. the renal lesions involve both blood vessels and interstitium, and acute tubular necrosis may occur. acute gn with immune-complex deposition has been reported (92) , but in most cases the pathology appears to be a direct consequence of the invading organism on the renal vasculature (90, 93) . renal dysfunction is an important complication of rocky mountain spotted fever. elevation of urea and creatinine levels occurs in a significant proportion of cases, and acidosis is common. prerenal renal failure caused by hypovolemia and impaired cardiac function may respond to volume replacement and inotropic support, but acute renal failure may subsequently occur, necessitating dialysis. rocky mountain spotted fever is diagnosed by the characteristic clinical picture, the exclusion of disorders with similar manifestations (e.g., measles, meningococcal disease, and leptospirosis), and detection of specific antibodies in convalescence. culture of rickettsia rickettsii, immunofluorescent staining, and polymerase chain reaction (pcr) testing of blood and skin biopsy specimens are available only in reference laboratories. antibiotics should be administered in suspected cases without awaiting confirmation of the diagnosis (93) . doxycycline is the drug of choice for children of any age. chloramphenicol is also effective (94) . intensive support of shock and multiorgan failure may be required in severe cases, and peritoneal dialysis or hemodialysis may be required until renal function returns. before the advent of specific therapy, mortality was 25%. today the overall mortality in the united states is still 5-7%. death predominantly occurs in cases in which the diagnosis is delayed. q fever is caused by coxiella burnetii and has a worldwide distribution, with the animal reservoir being cattle, sheep, and goats. human infection follows inhalation of infected particles from the environment. the clinical manifestations range from an acute self-limited febrile illness with atypical pneumonia to involvement of specific organs that causes endocarditis, hepatitis, osteomyelitis, and central nervous system disease (95) . proliferative gn may be associated with either q fever endocarditis, rhabdomyolysis or a chronic infection elsewhere in the body (96) . renal manifestations range from asymptomatic proteinuria and hematuria to acute renal failure, hypertension, and nephrotic syndrome. renal histologic findings are those of a diffuse proliferative gn, focal segmental gn, or mesangial gn. immunofluorescent studies reveal diffuse glomerular deposits of igm in the mesangium, together with c3 and fibrin. c. burnetii antigen has not been identified within the renal lesions. treatment of the underlying infection may result in remission of the renal disease, but prolonged treatment may be required for endocarditis. tetracycline has been used in conjunction with rifampicin, co-trimoxazole, or a fluoroquinolone. nephritis has been reported in association with the presence of a wide range of microorganisms that cause chronic or persistent infection (> table 52 -2) (63, 100). it is likely that any infectious agent that releases foreign antigens into the circulation, including those of very low virulence, can cause renal injury either by deposition of foreign antigens in the kidney or by the formation of immune complexes in the circulation, which are then deposited within the kidney. nephritis is most commonly seen in association with intravascular infections such as sbe or infected ventriculoatrial shunts, but it is also seen after focal extravascular infections; ear, nose, and throat infections; and abscesses. renal involvement is one of the diagnostic features of bacterial endocarditis. virtually all organisms that cause endocarditis also produce renal involvement ( > table 52 -2). although endocarditis caused by bacteria is the most common and is readily diagnosed by blood culture (100), unusual but important causes of culture-negative endocarditis include q fever (101) and legionella infection (102) . in the immunocompromised individual, opportunistic pathogens such as fungi and mycobacteria are important causes. the usual renal manifestations of sbe are asymptomatic proteinuria, hematuria, and pyuria. loin pain, hypertension, nephrotic syndrome, and renal failure may occur in more severe cases. the renal lesions occurring in endocarditis are variable, and focal embolic and immune-complex-mediated features may coexist (100, 103, 104) . embolic foci may be evident as areas of infarction, intracapillary thrombosis, or hemorrhage. more commonly, there is a focal necrotizing or diffuse proliferative gn. immunofluorescent studies show glomerular deposits of igg, igm, iga, and c3 along the gbm and within the mesangium. electron microscopy reveals typical electron-dense deposits along the gbm and within the mesangium (100, 103, 104) . early reports suggested that the renal lesions were caused by microemboli from infected vegetations depositing in the kidney, a hypothesis supported by the occasional presence of bacteria within the renal lesions. most subsequent evidence, however, indicates that immunologic mechanisms rather than emboli are involved in the pathogenesis in most cases: bacteria are rarely found within the kidney, and renal involvement occurs with lesions of the right side of the heart, which would not be likely to embolize to the kidney. immune complexes containing bacterial antigens are present in the circulation, and both bacterial antigens and bacteria-specific antibodies can be demonstrated within the immune deposits in the kidney. serum c3 level is usually low, and complement can be found within both the circulating and the deposited immune complexes. these features all support an immune-complex-mediated pathogenesis of the renal injury (100, 103, 104) . treatment of the endocarditis with antibiotics usually results in resolution of the gn and is associated with the disappearance of immune complexes from the circulation and return of c3 levels to normal. the prognosis of the renal lesions in sbe generally depends on the response of the underlying endocarditis to antibiotics or, in cases of antibiotic failure, to surgical removal of the infective vegetations (105) . in patients previously treated by shunting for hydrocephalus, there is a well-documented association of gn with infected ventriculoatrial shunts. this condition is another example of an immune-complex nephritis similar to that seen in endocarditis (106) . coagulase-negative staphylococci are the causative organisms in 75% of cases. the clinical and pathologic findings are similar to those in sbe. presenting features are proteinuria, hematuria, and pyuria, and they may progress to renal failure. immune complexes containing the bacterial antigens and complement are present in the serum, and c3 is depressed. histologic findings are those of a diffuse mesangiocapillary gn. immunofluorescent microscopy demonstrates deposits of immunoglobulin and c3 along the gbm, and bacterial antigen can be demonstrated in the renal lesions (107) . the prognosis for the renal lesion is good if the infection is treated early. this usually involves removal of the infected shunt and administration of appropriate antibiotics (106, 108) . the possible progression to end-stage renal disease requires frequent nephrologic monitoring of patients with ventriculoatrial shunts (106) . there are a few reports in the literature of a similar renal complication occurring in chronic infection of ventriculoperitoneal shunts. gn has been reported after chronic abscesses (63), osteomyelitis, otitis media, pneumonia, and other focal infections ( > table 52 -2). acute renal failure has been the presenting feature of focal infections in various sites, including the lung, pleura, abdominal cavity, sinuses, and pelvis. many different organisms have been responsible, including s. aureus, pseudomonas, e. coli, and proteus species. this is probably another example of immunecomplex gn. c3 level is decreased in approximately onethird of reported cases, and immunofluorescent studies reveal diffuse granular deposits of c3 in the glomeruli of all reported instances, with a variable presence of immunoglobulin. the renal lesion is that of mpgn and crescentic nephritis. the renal outcome is reported to be good with successful early treatment of the underlying infection. the role of viral infections in the causation of renal disease has been less well defined than that of bacterial infections. clearly defined associations of renal disease have been made with hepatitis b virus (hbv), hepatitis c virus (hcv), hiv, and hantaviruses, but the role of most other viruses in the pathogenesis of renal disease is not clearly defined. most viruses causing systemic infection may trigger immunologically mediated renal injury. with increasing application of molecular techniques, it may be that a significant proportion of gns currently considered to be idiopathic will ultimately be shown to be virus induced. in children with immunodeficiency states and those undergoing renal transplantation, viruses such as cytomegalovirus (cmv) and polyoma virus have been recognized to be associated with nephropathy. since the discovery of hepatitis b surface antigen (hbsag) in 1964, hepatitis virus has been shown to infect more than 5% of the world's population and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma worldwide. some 350 million people have hbsag in the circulation (who figures). the infection is most common in africa and the orient, where it is acquired in childhood by vertical transmission from infected mothers or by horizontal transmission from other children or adults. in developed countries, transmission in adults occurs more often by blood product exposure, sexual contact, or intravenous drug use. the epidemiology of hbv infection in children is changing following the widespread use of effective vaccination at birth, in both developed and developing countries. hbv is a complex dna virus with an outer surface envelope (hbsag) and an inner nucleocapsid core containing the hepatitis b core antigen (hbcag), dna polymerase, protein kinase activity, and viral dna. incomplete spherical and filamentous viral particles consisting solely of hbsag are the major viral products in the circulation and may be present in concentrations of up to 1014 particles per ml of serum. hepatitis b e antigen (hbeag) can be released from hbcag by proteolytic treatment and may be found in the circulation either free or complexed to albumin or igg antibodies. the presence of hbeag correlates with the presence of complete viral particles and the infectivity of the individual (109) . infection with hbv may result in either a self-limited infectious hepatitis followed by clearance of the virus and complete recovery, or a chronic or persistent infection in which the immune response is ineffective in eliminating the virus. chronic hbv infection with continued presence of viral antigens in the circulation caused by an ineffective host immune response provides the best-documented example of immunologically mediated renal injury caused by persistent infection (110) . development of chronic hbv infection is positively associated with infection at a younger age, particularly in infancy, where the rate of chronic infection is up to 90%. in contrast, the likelihood of an acute, symptomatic illness increases with age, to a level where approximately 40% of infections are symptomatic in children above 15 years. in the early prodromal phase of hbv hepatitis, before the onset of jaundice, some patients develop fever, maculopapular or urticarial rash, and transient arthralgias or arthritis. occasionally, proteinuria, hematuria, or sterile pyuria are observed. the syndrome usually lasts 3-10 days and often resolves before the onset of jaundice (110, 111) . there have been no histologic studies of the renal changes during this prodromal period. since 1970, numerous reports have linked hbv infection with polyarteritis nodosa (pan). most of these cases have been in adults, but the disorder has also been reported in children (112, 113) , where it is estimated that approximately one third of pan cases are caused by hbv (114) . hbv pan appears to be uncommon in africa and the orient, where infection is usually acquired in childhood, and has declined in incidence following the introduction of hbv vaccination (115) . hbv pan presents weeks to months after a clinically mild hepatitis but may occasionally predate the hepatitis. after a prodromal illness, frank vasculitis affecting virtually any organ appears. abdominal pain, fever, mononeuritis multiplex, and pulmonary and renal involvement may occur. the renal involvement may appear as hypertension, hematuria, proteinuria, or renal failure (see chapter 61) . laboratory investigations reveal a florid acute-phase response, leukocytosis, and anemia. transaminase levels are usually elevated, and hbsag is present in the circulation. the pathology consists of focal inflammation of small and medium-sized arteries, with fibrinoid necrosis, leukocyte infiltration, and fibrin deposition. renal pathology may be limited to the medium-sized arteries or may coexist with gn (110, 116) . circulating immune complexes containing hbsag and anti-hbs antibodies are usually present in the circulation (110, 116) . c3, c4, and total hemolytic complement levels are depressed. hbsag, igg, and igm antibodies to hbv and c3 have been identified by immunofluorescence in the blood vessels (116) . a positive anca excludes hbv-pan (115) . although most evidence suggests that the pathogenesis involves an immunecomplex-mediated vasculitis, autoantibody or cell-mediated vascular injury may coexist. if the condition is untreated, the mortality is high (112) . most studies suggest that steroids or immunosuppressants help to suppress the vasculitis but potentially predispose to chronic infection . (110, 112) . successful treatment of hepatitis b-associated pan with nucleoside analogues such as lamivudine or newer anti-viral drugs, either alone or in combination with interferon-alpha and conventional immunosuppressive therapy, has been reported (117) (118) (119) . hbv is now the major cause of membranous gn (mgn) in children worldwide. the proportion of patients with mgn caused by hbv is directly related to the incidence of hbsag in the population, with 80-100% of all cases of mgn in some african and oriental countries being associated with hbv (110, 120) (see chapter 26) . hbv mgn usually presents in children aged 2-12. there is a striking male predominance; in the united states, 80% of patients are males (121) . the virus is usually acquired by vertical transmission from infected mothers or horizontally from infected family members. unlike adults with hbv mgn, children do not usually have a history of hepatitis or of active liver disease, but liver function test results are generally mildly abnormal. liver biopsy specimens may show minimal abnormalities, chronic persistent hepatitis, or (occasionally) more severe changes (121) . the renal manifestations are usually of proteinuria, nephrotic syndrome, microscopic hematuria, or (rarely) macroscopic hematuria. hypertension occurs in less than 25% of cases, and renal insufficiency is rare. hbsag and hbcag are usually present in the circulation, and hbe antigenemia is seen in a high proportion of cases. occasionally, hbsag may be found in the glomeruli but is absent from the circulation. c3 and c4 levels are often low, and circulating immune complexes are found in most cases. immunohistologic study reveals deposits of igg and c3 and (less commonly) igm and iga in subepithelial, subendothelial, or mesangial tissue. hbv particles may be seen on electron microscopy, and all the major hepatitis b antigens, including hbsag, hbcag, and hbeag, have been localized in the glomerular capillary wall on immunofluorescence. immunologic deposition of hbv and antibody in the glomerular capillary wall is clearly involved in the glomerular injury, but the underlying immunologic events are incompletely understood (110, 122) . passive trapping of circulating immune complexes may be involved, but the circulating immune complexes containing hbsag are usually larger than would be expected to penetrate the basement membrane. hbsag and hbcag are anionic and are therefore unlikely to penetrate the glomerular capillary wall. in contrast, hbeag forms smaller complexes with anti-hbe antibodies and may readily penetrate the gbm. this may explain the observation that hbeag in the circulation frequently correlates with the severity of the disease (110) . an alternative mechanism for immunemediated glomerular injury is the trapping of hbv antigens by antibody previously deposited in the kidney. anti-hbe antibodies are cationic and may readily localize in the glomerulus and subsequently bind circulating antigen and complement. the third possibility is that the depositions of hbv and antibodies are consequences of glomerular injury by cellular mechanisms or autoantibodies. little evidence supports this view at present (110) . a transgenic mouse model of hbv-associated nephropathy has been developed, in which hbsag and hbcag is expressed in liver and kidney, particularly tubular epithelial cells, without viral replication. in these mice, gene expression analysis revealed upregulation of acute-phase proteins, particularly c3, although measurable serum c3 levels were reduced. this supports the notion that local persistent expression of hbv viral proteins contributes to hbv-associated nephropathy (123) . hbv infection has been associated with a variety of other forms of gn in both adults and children. in one small series in children, mpgn was found to be equal in incidence to mgn in the spectrum of hbv-associated gns (124) . both mpgn and mesangial proliferative gn may be triggered by hbv. in several countries where hbv is common, the proportion of patients with these forms of nephritides who test positive for hbv greatly exceeds the incidence of positivity in the general population (122) . as with mgn and hbv-associated pan, circulating immune complexes and localization of hbv antigens in the glomeruli have been reported in both mpgn and mesangial proliferative gn, and it is likely that similar mechanisms are occurring (110, 125) . several other forms of gn have been associated with hbv, including iga nephropathy, focal glomerulosclerosis, crescentic nephritis, and systemic lupus erythematosus, but the evidence for these associations is less consistent than for the entities discussed earlier (125) . hbv is normally cleared as a result of cell-mediated responses in which cytotoxic t cells and natural killer cells eliminate infected hepatocytes. it is not surprising, therefore, that the administration of steroids and immunosuppressive agents either may have no effect on hbv disease or may increase the risk of progressive disease (126) . children with hbv mgn have a good prognosis, and two-thirds undergo spontaneous remission within 3 years of diagnosis. steroid therapy does not appear to provide any additional benefit (110, 120, 110) . antiviral therapy with pegylated interferon-alpha and lamivudine shows promise in facilitating clearance of hbv, and in some cases, elimination of the infection with antiviral therapy in both children and adults is associated with improvement or resolution of the coexisting renal disease. there is considerable effort being put into the development of newer anti-viral agents which avoid the common problems of resistance associated with lamivudine (127-129). hcv is an enveloped, single-stranded rna virus of approximately 9.4 kb in the flaviviridae family. there are six major hcv genotypes. hepatitis c is a common disease affecting approximately 400 million people worldwide. in the united states, 4.1 million persons are estimated to be anti-hcv positive, and 3.2 million may be chronically infected (130) . an estimated 240,000 children in the united states have antibody to hcv and 68,000-100,000 are chronically infected (131) . children become infected through receipt of contaminated blood products or through vertical transmission. the risk of vertical transmission increases with higher maternal viremia and maternal co-infection with hiv. acute hcv infection is rarely recognized in children outside of special circumstances such as a known exposure from an hcv-infected mother or after blood transfusion. most chronically infected children are asymptomatic and have normal or only mildly abnormal alanine aminotransferase levels. although the natural history of hcv infection during childhood seems benign in the majority of instances, the infection can take an aggressive course in a proportion of children, leading to cirrhosis and end-stage liver disease during childhood. the factors responsible for this more aggressive course are unidentified (131) . even in adults, the natural history of hcv infection has a variable course, but a significant proportion of patients will develop some degree of liver dysfunction, and 20-30% will eventually have end-stage liver disease as a result of cirrhosis. the risk of hepatocellular carcinoma is significant for those who have established cirrhosis. hepatitis c is currently the most common condition leading to liver transplantation in adults in the ''western world.'' gn has been described as an important complication of chronic infection with hcv in adults. the clinical presentation is usually of nephrotic syndrome or proteinuria, hypertension, or hematuria, with or without azotemia (132) . mpgn, with or without cryoglobulinemia, and mgn are most commonly described. isolated case reports of other, more unusual patterns of glomerular injury, including iga nephropathy, focal segmental glomerulosclerosis, crescentic gn, fibrillary gn, and thrombotic microangiopathy, have also been associated with hcv infection (132, 133) . glomerular deposition of hepatitis antigens and antibodies has been described and is believed to play a role in pathogenesis. cryoglobulinemia is a common accompaniment of gn that is associated with the depression of serum complement levels (132) . renal failure may develop in 40-100% of patients who have mpgn (132, 134) . the presence of virus-like particles as well as viral rna within the kidney sections of patients with hcv-associated glomerulopathies has been reported (135) . the diagnosis should be suspected if glomerular disease is associated with chronic hepatitis, particularly with the presence of cryoglobulins, but renal biopsy is necessary to establish a definitive diagnosis. hcv infection is relatively common in children with end-stage renal disease and is an important cause of liver disease in this population. acquisition of hcv infection continues to occur in dialysis patients because of nosocomial spread (136) . elevation of transaminase level is not a sensitive marker of infection in children and hcv enzymelinked immunosorbent assay or pcr testing should be used to increase sensitivity (137) . hcv-infected renal transplant recipients had higher mortality and hospitalization rates than other transplant recipients (138) , and hcv infection has been reported to be associated with de novo immune-mediated gn, especially type 1 mpgn, in renal allografts, resulting in accelerated loss of graft function (139, 140) . no large randomized, controlled trials of treatment of children with chronic hepatitis c have been performed, although one study (peds-c) is currently recruiting patients into a trial of pegylated interferon +/ã� ribavirin (141) . small heterogeneous studies of interferon monotherapy have reported sustained virologic response rates of 35-40% (131) . in adults, improvement of proteinuria and renal function often follows interferon-alpha treatment (132, 134) , but relapses are common after cessation of treatment. combination of interferon with ribavirin in infectious diseases and the kidney patients with chronic liver disease has been shown to increase the rate of sustained response in these patients (142) . as yet, however, there are few data regarding the use of combination therapy with interferon and ribavirin in children. moreover, interferon-alpha therapy is associated with acute or subacute renal failure in more than one-third of the patients with renal transplants (143) . hepatitis c may be complicated by systemic mixed cryoglobulinemic (mc) vasculitis, and in some cases by a polyarteritis nodosa (pan)-type non-cryoglobulinemic vasculitis (144) . treatment with interferon-a (ifn-a) and ribavirin mostly is associated with an improvement of vasculitic symptoms. in some cases, exacerbation and rarely new onset of vasculitis of the peripheral nervous system have been described after this treatment. in fulminant cases immunosuppressive therapy with steroids, and cyclophosphamide, or rituximab may be needed to control life threatening vasculitis prior to antiviral treatment (144) . cytomegalovirus cmv is one of the eight human herpes viruses. transmission of the virus requires exposure to infected body fluids such as breast milk, saliva, urine, or blood. individuals initially infected with cmv may be asymptomatic or display nonspecific flu-like symptoms. after the initial infection cmv, like all herpes viruses, establishes latency for life but will be periodically excreted by an asymptomatic host. cmv replicates within renal cells, and on biopsy samples from immunocompromised hosts, viral inclusions can be visualized by light microscopy in cells of the convoluted tubules and collecting ducts (145) . glomerular cells and shed renal tubular cells may have characteristic inclusions, but clinically evident renal disease is rare and is seen virtually only in immunocompromised or congenitally infected children (145, 146) . the clinical manifestations of cmv-induced renal disease in congenitally infected infants are variable and range from asymptomatic proteinuria to nephrotic syndrome and renal impairment. in congenital cmv infection, histologic changes of viral inclusions commonly occur in the tubules. in addition, proliferative gn has been reported, with evidence on electron microscopy of viral immune deposits in glomerular cells (146, 147) . in cmv-infected immunocompromised patients, immunecomplex gn has been documented with mesangial deposits of igg, iga, c3, and cmv antigens within glomeruli. eluted glomerular immunoglobulins have been shown to contain cmv antigens (148) . cmv is the most common viral infection after kidney transplantation. experience with pediatric kidney transplant recipients suggests a 67% incidence of cmv infection (149) . the direct and indirect effects of cmv infection result in significant morbidity and mortality among kidney transplant recipients. cmv-negative patients who receive a cmv-positive allograft are at risk for primary infection and graft dysfunction. patients who are cmv seropositive at the time of transplantation are also at risk of reactivation and superinfection. tubulointerstitial nephritis is a well-characterized pathologic feature of renal allograft cmv disease, which can be difficult to distinguish from injury caused by rejection. histologic evidence of endothelial cell injury and mononuclear cell infiltration in the glomeruli has been reported (148) . cmv glomerular vasculopathy in the absence of tubulointerstitial disease, causing renal allograft dysfunction, has also been reported (150) . beyond the acute allograft nephropathy associated with cmv viremia, cmv is known to cause chronic vascular injury. this may adversely affect the long-term outcome of the allograft and may be the explanation for the observed association with chronic allograft nephropathy (151) . newer techniques for rapidly diagnosing cmv infection are becoming widely available and include shell vial culture, pp65 antigenemia assay, pcr, and the hybridcapture rna-dna hybridization assay for qualitative detection of cmv pcr. quantitative plasma pcr testing (pcr viral load) is increasingly used for diagnosis and monitoring of cmv viremia in renal transplant recipients. antiviral agents that have been shown to be effective against cmv include ganciclovir, valganciclovir, foscarnet, and cidofovir. ganciclovir remains the drug of choice for treating established disease. intravenous ganciclovir therapy is preferred in children because of the erratic absorption of oral ganciclovir. major limitations of ganciclovir therapy are the induction of renal tubular dysfunction and bone marrow toxicity, principally neutropenia and thrombocytopenia. dosage adjustments are necessary for recipients with renal dysfunction. oral valganciclovir is now used for cmv prophylaxis post-transplant (152) . use of other antiviral agents such as foscarnet and cidofovir is limited because of nephrotoxicity and difficulty of administration. a number of reports have demonstrated the effectiveness of high-titer cmv immune globulin therapy in reducing severe cmv-associated disease when used in combination with ganciclovir (149, 153) . the association of varicella with nephritis has been known for more than 100 years since henoch reported on four children with nephritis that occurred after the appearance of varicella vesicles. varicella, however, is rarely associated with renal complications (154) . in fatal cases with disseminated varicella and in the immunocompromised individual, renal involvement is more common. cases in which varicella infection caused gn in renal transplant recipients have been reported (155) . histologic findings in fatal cases include congested hemorrhagic glomeruli, endothelial cell hyperplasia, and tubular necrosis. in mild and nonfatal cases and in non-immunocompromised individuals, varicella is occasionally associated with a variety of renal manifestations, ranging from mild nephritis to nephrotic syndrome and acute renal failure (156) . histologic findings include endocapillary cell proliferation, epithelial and endothelial cell hyperplasia, and inflammatory cell infiltration (154) . rapidly progressive nephritis has also been reported. immunohistochemical studies reveal glomerular deposition of igg, igm, iga, and c3. on electron microscopy, granular electron-dense deposits have been found in the paramesangial region, and varicella antigens may be deposited in the glomeruli. the features suggest an immune-complex nephritis. elevated circulating levels of igg and iga immune complexes and depressed c3 and c4 levels support this possibility (154) . fulminant disseminated varicella and varicella in immunocompromised patients should be treated with intravenous acyclovir. renal involvement is common during acute infectious mononucleosis, usually manifesting as an abnormal urine sediment, with hematuria in up to 60% of cases. hematuria, either microscopic or macroscopic, usually appears within the first week of the illness and lasts for a few weeks to a few months. proteinuria is usually absent or low grade. more severe renal involvement with proteinuria, nephrotic syndrome, or acute nephritis with renal failure is much less common. acute renal failure may be seen during the course of fulminant infectious mononucleosis with associated hepatic failure, thrombocytopenia, and encephalitis. it is usually caused by interstitial nephritis that is likely the result of immunopathologic injury precipitated by epstein-barr virus (ebv) infection. however, the identification of ebv dna in the kidney raises the possibility that direct infection might play a role (157) . the renal involvement must be distinguished from myoglobinuria caused by rhabdomyolysis, which may occur in infectious mononucleosis, and from bleeding into the renal tract as a result of thrombocytopenia. renal histologic findings in ebv nephritis are an interstitial nephritis with mononuclear cell infiltration and foci of tubular necrosis. glomeruli may show varying degrees of mesangial proliferation. on immunohistochemical study, ebv antigens are seen in glomerular and tubular deposits. the prognosis for complete recovery of renal function is good. treatment with corticosteroids may have a role in the management of ebv-induced acute renal failure and may shorten the duration of renal failure (158) . ebv-associated post-transplantation lymphoproliferative disease is a recognized complication in renal transplant recipients. latent infection of ebv in renal proximal tubular epithelial cells has recently been described as causing idiopathic chronic tubulointerstitial nephritis (159) . the herpes simplex virus (hsv) causes persistent infection characterized by asymptomatic latent periods interspersed with acute relapses. as with other chronic and persistent infections, immunologically mediated disorders triggered by hsv are well recognized, and it is perhaps surprising that hsv has rarely been linked to nephritis. acute nephritis and nephrotic syndrome have been associated with herpes simplex encephalitis. renal histology shows focal segmental gn with mesangial and segmental deposits of igm, c3, and hsv antigens. as with other herpes viruses, hsv has been suggested as a trigger for iga nephritis, mpgn, and membranous nephropathy. elevated levels of hsv antibodies have been reported in patients with a variety of forms of gn, but no conclusive evidence exists of an etiologic role for hsv (160) . adenovirus and enterovirus, are unrelated ubiquitous pathogens that infect large proportions of the population annually and yet are rarely associated with renal disease. the literature contains scattered reports of acute nephritis after infection with each of these viruses. adenovirus is a major cause of hemorrhagic cystitis and was implicated as the cause of hemorrhagic cystitis in 23-51% of children with this disorder (161) . boys are affected more often than girls, and hematuria persists for 3-5 days. microscopic hematuria, dysuria, and frequency may occur for longer periods. adenovirus types 11 and 21 are the usual strains isolated. picornaviruses, including enteroviruses, echovirus and coxsackieviruses, have been linked with acute nephritis and acute renal failure associated with rhabdomyolysis. coxsackie b virus can be isolated in urine. direct infection of kidney cells is supported by in vitro work demonstrating lytic infection of human podocyte and proximal tubular epithelial cell cultures, although different strains exhibit variable degrees of nephrotropism. renal damage in vivo may have both a direct lytic mechanism and an immune-complex basis (162) . in the newborn, enteroviruses cause fulminant disease with dic, shock, and liver failure, and acute renal failure may occur. renal involvement from measles virus is uncommon, although measles virus can be cultured from the kidney in fatal cases. an acute gn has been reported to follow measles with evidence of immune deposits containing measles virus antigen within the glomeruli. the nephritis is generally self-limiting (163) . mild renal involvement is common during the acute phase of mumps infection. one-third of children with mumps have abnormal urinalysis results, with microscopic hematuria or proteinuria. mumps virus may be isolated from urine during the first 5 days of the illness, at a time when urinalysis findings are abnormal. plasma creatinine concentrations usually remain normal, despite the abnormal urine sediment, but more severe cases in adults have been associated with evidence of acute nephritis with impaired renal function. renal biopsy specimens demonstrate an mpgn with deposition of iga, igm, c3, and mumps virus antigen in the glomeruli, which suggests an immune-complex-mediated process (164) . despite the increasing availability of interventions to limit vertical hiv transmission, an estimated 1,500 children renal involvement in hiv infection was first described in 1984 in adults (165) (166) (167) and in children (168) , and renal involvement occurs in 2-15% of hiv-infected children in the united states (169) (170) (171) . since the development of highly active antiretroviral therapy (haart), however, the incidence of end-stage renal disease in hiv infection in both adults and children in industrialized countries has declined, but it is predicted that the dramatic decline in aids-related deaths will lead to an ageing population of hiv-infected individuals who will be at risk of non-hiv related renal problems, such that the numbers of hiv-positive esrd patients will increase in the united states (172) . hiv infection is associated with a number of renal pathologies. hiv-associated nephropathy (hivan) is a syndrome of glomerular and tubular dysfunction, which can progress to end-stage renal failure. it is discussed more fully below. glomerular syndromes other than hivan include mgn that resembles lupus nephritis and immune-complex gn, with iga nephropathy and hcv-associated mpgn being the most common forms. there have also been several case reports of amyloid kidney (171, 173, 174) . the kidneys may be affected by various other mechanisms. opportunistic infections with organisms such as bk virus (bkv) that give rise to nephropathy and hemorrhagic cystitis have been reported in association with hiv infection (175) . systemic infections accompanied by hypotension can cause prerenal failure leading to acute tubular necrosis. acute tubular necrosis has also been reported in hiv patients after the use of nephrotoxic drugs such as pentamidine, foscarnet, cidofovir, amphotericin b, and aminoglycosides. intratubular obstruction with crystal precipitation can occur with the use of sulfonamides and intravenous acyclovir. indinavir is well recognized to cause nephropathy and renal calculi (176) . mpgn associated with mixed cryoglobulinemia and thrombotic microangiopathy/atypical-cal hus in association with hiv infections have been reported (177, 178) . hivan is characterized by both glomerular and tubular dysfunction, the pathogenesis of which is not entirely known. hivan is a clinico-pathologic entity that includes proteinuria, azotemia, focal segmental glomerulosclerosis or mesangial hyperplasia, and tubulointerstitial disease (171) . in adults in the united states, there is a markedly increased risk of nephropathy among african american persons with hiv infection. this appears to be true in children as well, but the data are sparse. the spectrum of hivan seems to be coincident with the degree of aids symptomatology. it is thought that hivan can present at any point in hiv infection, but most patients with hivan have cd4 counts of less than 200 ã� 10 6 /200 cells/ml, which suggests that it may be primarily a manifestation of late-stage disease (179) . although a spectrum of clinicopathologic entities including mesangial hyperplasia, focal segmental glomerulosclerosis, minimal change disease, and systemic lupus erythematosus nephritis has been described, the classic pathologic feature of hivan is the collapsing form of focal and segmental glomerulosclerosis (180) . in the affected glomeruli, visceral epithelial cells are hypertrophied and hyperplastic, and contain large cytoplasmic vacuoles and numerous protein resorption droplets. there is microcystic distortion of tubule segments, which contributes to increasing kidney size. podocyte hyperplasia can become so marked that it causes obliteration of much of the urinary space, forming ''pseudocrescents'' (173) . capillary walls are wrinkled and collapsed with obliteration of the capillary lumina. the interstitium is edematous with a variable degree of t-cell infiltration (181) . the bowman capsule can also be dilated and filled with a precipitate of plasma protein that represents the glomerular ultrafiltrate. one of the most distinctive features of hivan, however, is the presence of numerous tubuloreticular inclusions within the cytoplasm of glomerular and peritubular capillary endothelial cells (173) . immunofluorescence testing is positive for igm and c3 in capillary walls in a coarsely granular to amorphous pattern in a segmental distribution (180, 181) . the presence of the hiv genome in glomerular and tubular epithelium has been demonstrated using complementary dna probes and in situ hybridization. proviral dna has been detected by pcr in the glomeruli, tubules, and interstitium of micro dissected kidneys from patients who had pathologic evidence of hivan, but it has also been detected in the kidneys of hiv-positive patients with other glomerulopathies (182) . a combination of both proliferation and apoptosis of renal cells may cause the loss of nephron architecture. apoptosis has been demonstrated in cells in the glomerulus, tubules, and interstitium of biopsy specimens from hiv-positive patients with focal segmental glomerulosclerosis. in addition, the role of various cytokines and growth factors, specifically transforming growth factor beta (tgf-beta), in the development of sclerosis has been studied (183, 184) . transgenic murine models provide some of the strongest evidence for a direct role of hiv-1 in the induction of hivan. these mice do not produce infectious virus but express the hiv envelope and regulatory genes at levels sufficient to re-create the hivan that is seen in humans (183) . serial deletion experiments have concluded that the nef and vpr genes are necessary though not sufficient for hivan pathogenesis. additional factors such as genetic predisposition are thought to explain the fact that african americans have a far greater likelihood of developing hivan than other racial groups, and that hivan is more likely in patients with a family history of esrd. hivan can manifest as mild proteinuria, nephrotic syndrome, renal tubular acidosis, hematuria, and/or acute renal failure (168) (169) (170) (171) . nephrotic syndrome and chronic renal insufficiency are late manifestations of hivan. children with hivan are likely to develop transient electrolytic disorders, heavy proteinuria, and acute renal failure due to systemic infectious episodes or nephrotoxic drugs. early stages of hivan can be identified by the presence of proteinuria and ''urine microcysts'' along with renal sonograms showing enlarged echogenic kidneys. urinary renal tubular epithelial cells are frequently grouped together to form these microcysts, which were found in the urine of children with hivan who had renal tubular injury (171) . advanced stages of hivan typically present with nephrotic syndrome with edema, heavy proteinuria, hypoalbuminemia, and few red or white blood cells in urinary sediments. hypertension may be present, but usually blood pressure is within or below the normal range. hivan in adults follows a rapidly progressive course, with end-stage renal disease developing within 1-4 months, but in children this rapid progression does not necessarily occur. definitive diagnosis of hivan should be based on biopsy results, and biopsy should be performed if significant proteinuria is present, because in approximately 50% of hiv-infected patients with azotemia and/or proteinuria (>1 g/24 h) who undergo renal biopsy, the specimen will have histologic features consistent with other renal diseases (179) . when available, haart should be given to children with symptomatic hiv disease. specific treatment of hivan remains controversial. several studies have looked at the role of haart, angiotensin i-converting enzyme (ace) inhibitors, steroids, and even cyclosporin with somewhat encouraging results. however, as yet no randomized case-controlled trials have been undertaken. most of the studies have been small and retrospective, and many have included patients both with and without renal biopsy-proven hivan. cyclosporin has been used to treat hivan in children with remission of nephrotic infectious diseases and the kidney syndrome (169) . similar responses have been reported to treatment with corticosteroids in various studies (185) (186) (187) (188) . ace inhibitors have been used with encouraging results (189) . the general regimen used to treat patients with hiv, including haart, should be applied to children with hivan. the dosages of some medications must be adjusted to the patients glomerular filtration. there are reports of spontaneous regression of hivan with supportive management and treatment with haart, particularly with regimes containing protease inhibitors (190) (191) (192) (193) . it should be emphasized that the improvement reported with other modalities of treatment such as corticosteroids, cyclosporin, and ace inhibitors always occurs when these agents are given in conjunction with antiretroviral therapy. the kidneys of transgenic mice have been found to have elevated levels of tgf-beta messenger rna and protein (184) . furthermore, gene expression analysis on tubular epithelial cells from a patient with hivan found upregulation of several inflammatory mediator genes downstream of interleukin 6 and of the transcription factor nfkb (194) . several other therapeutic options have been suggested, aimed specifically at the presumed role of tgfbeta in the pathogenesis of hivan. treatment directed at its synthesis using gene therapy to block tgf-beta gene expression is being explored. therapy directed at decreasing the activity of tgf-beta using anti-tgf-beta antibodies or other inhibitory substances is also an area of investigation. in addition, blocking renal receptors for chemokines such as rantes (regulated upon derivation, normal t cell expressed and secreted), interleukin-8, and monocyte-chemoattractant protein-1 has been proposed as another possible treatment alternative (195) . in the haart era, the outlook for hiv patients with esrd has improved, but these patients fare worse than esrd patients without hiv (196) . most reports of hivinfected patients on hemodialysis have shown poor prognosis, with mean patient survival times ranging from 14-47 months. mortality is therefore still close to 50% within the first year of dialysis. in general, improved survival is associated with younger age at initiation of hemodialysis and with higher cd4 counts. access complications such as infection and thrombosis tend to occur at a higher rate in hiv-infected hemodialysis patients. cross infection with hiv in dialysis patients is very rare. no patient-topatient hiv transmission has yet been reported in a hemodialysis unit in the united states, although several such cases have occurred in south america (195, 197) . peritoneal dialysis is an alternative for hiv-infected patients. the incidence of peritonitis varies across studies, but some studies did report a higher incidence of pseudomonas and fungal peritonitis in the hiv-positive population (195) . infections with unusual organisms such as pasteurella multocida, trichosporon beigelii, and mycobacterium avium intracellulare complex have also been reported. several studies, however, have suggested that there is no significant difference between the hiv-infected and non-hiv-infected populations. of note is that virus capable of replication in vitro has been recovered from the peritoneal dialysis effluent, and it can be recoverable for up to 7 days in dialysis bags at room temperature and for up to 48 h in dry exchange tubing (195) . previously, long-term dialysis had been thought to be preferable to renal transplantation, primarily because of the concern that the immunosuppressive therapy required after transplantation could promote progression of hiv/ aids. a multicenter prospective study has been addressing these questions (198) . data so far indicate that the outcome for liver and kidney transplantation is not considerably different from patients without hiv, with good graft persistence, and a low rate of development of opportunistic infections in those with well-controlled hiv and relatively high cd4 counts (199) . the human polyoma viruses are members of the papovavirus family and have received increasing attention as pathogens in immunocompromised patients. they are nonenveloped viruses ranging in size from 45-55 nm, with a circular, double-stranded dna genome that replicates in the host nucleus. the best-known species in this genus are the bkv, the jc virus (jcv), and the simian virus sv40. bkv was first isolated from the urine of a 39-year-old man who developed ureteral stenosis 4 months after renal transplantation (200) . the name of the virus refers to the first patients initials, which is also true of jcv. bkv establishes infection in the kidney and the urinary tract, and its activation causes a number of disorders, including nephropathy and hemorrhagic cystitis. bkv-associated nephropathy has become an increasingly recognized cause of renal dysfunction in renal transplantation patients (201) (202) (203) (204) (205) . jcv establishes latency mainly in the kidney, and its reactivation can result in the development of progressive multifocal leukoencephalopathy. there are a few reports of nephropathy in association with jcv infection (see references in (206) ), but bkv poses a much bigger problem in this regard. recent studies have reported sv40 in the allografts of children who received renal transplants and in the urine, blood, and kidneys of adults with focal segmental glomerulosclerosis, which is a cause of end-stage renal disease and an indication for kidney transplantation (207) . seroprevalence rates as high as 60-80% have been reported among adults in the united states and europe. the peak incidence of primary infection (as measured by acquisition of antibody) occurs in children 2-5 years of age. bkv antibody may be detected in as many as 50% of children by 3 years of age, and in 60-100% of children by 9 or 10 years of age; antibodies wane thereafter. bkv infection may be particularly important in the pediatric transplantation population, in whom primary infection has a high probability of occurring while the children are immunosuppressed (208) . primary infection with bkv in healthy children is rarely associated with clinical manifestations. mild pyrexia, malaise, vomiting, respiratory illness, pericarditis, and transient hepatic dysfunction have been reported with primary infection. investigators hypothesize that after an initial round of viral replication at the site of entry, viremia follows with dissemination of the virus to distant sites at which latent infection is established. the most frequently recognized secondary sites of latent infection are renal and uroepithelial cells. secondary infection has been reported to cause tubulointerstitial nephritis and ureteral stenosis in renal transplantation patients. it may be that renal impairment in immunocompromised patients and in non renal solid organ transplant recipients is found to be frequently associated with bkv infection. the reported prevalence of bkv nephropathy in renal allografts is between 1 and 8% (201, 202, 205, 209, 210) . asymptomatic infection is characterized by viral shedding without any apparent clinical features. viruria, resulting from either primary or secondary infection, can persist from several weeks to years. tubulointerstitial nephritis associated with bkv in renal transplant recipients is accompanied by histopathologic changes, with or without functional impairment. ''infection'' and ''disease'' must be differentiated carefully. bkv infection (either primary or reactivated) can progress to bkv disease, but will not always do so (208) . furthermore, not all cases of bkv disease lead to renal impairment. however, infection can progress to transplant dysfunction and graft loss, although the diagnosis may be complicated by the coexistence of active allograft rejection. bkv nephritis is reported to have a bimodal distribution, with 50% of bkv-related interstitial nephritis cases occurring 4-8 weeks after transplantation and the remainder of patients developing disease months to years after transplantation (211) . allograft failure is due mainly to extensive viral replication in tubular epithelial cells leading to frank tubular necrosis (203) . although damage is potentially fully reversible early in the disease, persisting viral damage leads to irreversible interstitial fibrosis. tubular atrophy and allograft loss has been observed in 45% of affected patients (203, 212) . in most cases, bkv nephropathy in adult renal transplant recipients represents a secondary infection associated with rejection and its treatment. in children, however, primary bkv infection giving rise to allograft dysfunction may occur (208) . the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathologic features include severe tubular injury with cellular enlargement, marked nuclear atypia, epithelial necrosis, denudation of tubular basement membranes, focal intratubular neutrophilic infiltration, and mononuclear interstitial infiltration, with or without concurrent tubulins. this constellation of histologic features, particularly severe tubulitis, is often misinterpreted as rejection, even by the experienced pathologist. the presence of well-demarcated basophilic or amphophilic intranuclear viral inclusions, primarily within the tubular and parietal epithelium of the bowman capsule, can help distinguish bkv disease from rejection (202, 203, 205) . additional tests such as immunohistochemistry, pcr analysis, or electron microscopy of biopsied tissue aimed at the identification of bkv may be required. a practical diagnostic approach for identifying bkv in renal transplant patients is summarized in > table 52-3. bkv infection may cause ureteral obstruction due to ureteral ulceration and stenosis at the ureteric anastomosis. bkv-associated ureteral stenosis has been reported in 3% of renal transplant patients and usually occurs between 50 and 300 days after transplantation. ulceration due to inflammation, proliferation of the transitional epithelial cells, and smooth muscle proliferation may lead to partial or total obstruction. high-level bkv replication is implicated in acute, late-onset, long-duration hemorrhagic cystitis after bone marrow transplantation (213) . there are two case reports in children of renal carcinomas arising in the transplanted kidney in association with bk virus nephropathy. it remains unclear whether infectious diseases and the kidney bk virus itself has oncogenic potential in the transplant setting, but this is possible given that the big t antigen (t-ag) expressed by polyomavirus family viruses has been shown to have the ability to disrupt chromosomal integrity (214, 215) . whether patients with asymptomatic viremia or viruria need specific therapeutic intervention is not certain. review of the literature suggests that careful reduction of immune suppression, combined with active surveillance for rejection, will result in clinical improvement. reduction in immunosuppression may precipitate episodes of acute cellular rejection, which need to be judiciously treated with corticosteroids. the outcome of bkv nephropathy is unpredictable, and stabilization of renal function may occur regardless of whether maintenance immunotherapy is altered or not (216) . some reports favor the use of cidofovir. cidofovir has important nephrotoxic side effects in the usual therapeutic dosage recommended for the treatment of cmv infection, and for bkv nephropathy a reduced dosage regime is generally used. the efficacy of cidofovir in reducing viremia has been demonstrated (see review in (210)). however, spontaneous clearance of viral infection after reduction of immunosuppression (without cidofovir) has also been reported. there are also case studies of the use of leflunamide. presence of bkv by pcr or decoy cells in urine signifies bkv replication. decoy cells are caused by infection of the urinary epithelial cells with human polyoma viruses. the nuclei are enlarged and nuclear chromatin is completely homogenized by viral cytopathic effect. positive pcr results for bkv viruria and presence of decoy cells have poor predictive value. specificity is increased if >10 cells/ cytospin along with presence of inflammatory cells. presence of antibody is usually indicative of previous infection; however, positive results for bkv dna pcr on serum signifies bk viremia. bkv pcr testing of plasma has proven to be a sensitive (100%) and specific (88%) means to identify bkv-associated nephropathy in adults. viral load has also been used to monitor infection and clearance. however, because primary infection occurs in childhood, it might not be applicable to the pediatric population. the definitive diagnosis of bkv nephropathy requires renal biopsy. histopathology might mimic rejection or drug toxicity. however, characteristic findings have been described. electron microscopy and immune staining are helpful in confirming the diagnosis. pcr assays of viral load in tubular cells have been reported to be a sensitive marker for diagnosis and monitoring. viral hemorrhagic fever involves at least 12 distinct rna viruses that share the propensity to cause severe disease with prominent hemorrhagic manifestations ( > table 52 -4) . the viral hemorrhagic fevers, widely distributed throughout both temperate and tropical regions of the world, are important causes of mortality and morbidity in many countries. most viral hemorrhagic fevers are zoonoses (with the possible exception of dengue virus), in which the virus is endemic in animals and human infection is acquired through the bite of an insect vector. aerosol and nosocomial transmissions from infected patients are important for lassa, junin, machupo, and congo-crimean hemorrhagic fevers, and marburg and ebola viruses (217) . viral hemorrhagic fevers have many clinical similarities but also important differences in their severity, major organs affected, prognosis, and response to treatment. in all viral hemorrhagic fevers, severe cases occur in only a minority of those affected; subclinical infection or nonspecific febrile illness occurs in the majority. fever, myalgia, headache, conjunctival suffusion, and erythematous rash occur in all the viral hemorrhagic fevers (218) . hemorrhagic manifestations range from petechiae and bleeding from venepuncture sites to severe hemorrhage into the gi tract, kidney, and other organs. a capillary leak syndrome, with evidence of hemoconcentration, pulmonary edema, oliguria, and ultimately shock, occurs in the most severely affected patients (218) . renal involvement occurs in all the viral hemorrhagic fevers, proteinuria is common, and prerenal failure is seen in all severe cases complicated by shock. however, in congo-crimean hemorrhagic fever and hemorrhagic fever with renal syndrome (hfrs), an interstitial nephritis, which may be hemorrhagic, is characteristic, and renal impairment is a major component of the illness. dengue is caused by a flavivirus that is endemic and epidemic in tropical america, africa, and asia, where the mosquito vector aedes aegypti is present (219). classic dengue is a self-limited nonfatal disease; dengue hemorrhagic fever and dengue shock syndrome, which occur in a minority of patients, have a high mortality if not aggressively treated with fluids. after an incubation period of 5-8 days, the illness begins with fever, headache, arthralgia, weakness, vomiting, and hyperesthesia. in uncomplicated dengue the fever usually lasts 5-7 days. shortly after onset a maculopapular rash appears, sparing the palms and the soles, and is occasionally followed by desquamation. fever may reappear at the onset of the rash. in dengue hemorrhagic fever and dengue shock syndrome, the typical febrile illness is complicated by hemorrhagic manifestations, ranging from a positive tourniquet test result or petechiae to purpura, epistaxis, and gi bleeding with thrombocytopenia and evidence of a consumptive coagulopathy. increased capillary permeability is suggested by hemoconcentration, edema, and pleural effusions (219) . in severe cases, hypotension and shock supervene, largely as a result of hypovolemia. renal manifestations include oliguria, proteinuria, hematuria, and rising urea and creatinine. acute renal failure occurs in patients with severe shock, primarily as a result of renal underperfusion. however, glomerular inflammatory changes may also occur. children with dengue hemorrhagic fever show hypertrophy of endothelial and mesangial cells, mononuclear cell infiltrate, thinning of basement membranes, and deposition of igg, igm, and c3. electron microscopy shows viral particles within glomerular mononuclear cells (220) . the diagnosis of dengue is made by isolation of the virus from blood or by serologic testing. there is no specific antiviral treatment, and management of patients with dengue shock syndrome or dengue hemorrhagic fever depends on aggressive circulatory support and volume replacement with colloid and crystalloid (221, 222) . with correction of hypovolemia, renal impairment is usually reversible, but dialysis may be required in patients with established acute renal failure. yellow fever is caused by a flavivirus, and is transmitted by mosquito bites, typically aedes species. it remains an important public health problem in africa and south america. renal manifestations are common and include albuminuria and oliguria. over the next few days after first manifestation of infection, shock, delirium, coma, and renal failure develop, and death occurs 7-10 days after onset of symptoms. laboratory findings include thrombocytopenia and evidence of hemoconcentration, rising urea and creatinine levels, hyponatremia, and deranged liver function test results. pathologic findings include necrosis of liver lobules, cloudy swelling and fatty degeneration of the proximal renal tubules, and, often, petechiae in other organs. the oliguria appears to be prerenal and is due to hypovolemia; later, acute tubular necrosis supervenes. at present, there is no effective antiviral agent for yellow fever. . congo-crimean hemorrhagic fever, first recognized in the soviet union, is now an important human disease in eastern europe, asia, and africa (223) . severely affected patients become stuporous or comatose 5-7 days into the illness, with evidence of hepatic and renal failure and shock. proteinuria and hematuria are often present. the disease is fatal in 15-50% of cases. the virus is sensitive to ribavirin, but in one small trial of i.v. ribavirin versus supportive treatment only, there was no significant improvement in outcome in the treatment group (224) . rift valley fever is found in many areas of sub-saharan africa. in humans, most infections follow mosquito bites or animal exposure. the infection may present as an uncomplicated febrile illness, with muscle aches and . (226) . clinical entities include korean hemorrhagic fever, nephropathia epidemica in scandinavia, and epidemic hemorrhagic fever in japan and china. in general, hfrs due to hantaan, porogia, and belgrade viruses is more severe and has higher mortality than that due to puumala virus (nephropathia epidemica) or seoul virus. hantaan is predominant in the far east, porogia and belgrade in the balkans, and puumala in western europe; seoul has a worldwide distribution (225) . the clinical features of the disease vary. the incubation period is 4-42 days. although hfrs occurs with the same clinical picture in children as in adults, both incidence rates and antibody prevalence rates are very low in children under 10 years of age. men of working age make up the bulk of clinical cases (226) . mild cases are indistinguishable from other febrile illnesses. in more severe cases, fever, headache, myalgia, abdominal pain, and dizziness are associated with the development of periorbital edema, proteinuria, and hematuria. there is often conjunctival injection, pharyngeal injection, petechiae, and epistaxis or gi bleeding. the most severely affected patients develop shock and renal failure. the disease usually passes through five phases: febrile, hypotensive, oliguric, diuretic, and convalescent. laboratory findings include anemia, lymphocytosis, thrombocytopenia, prolonged prothrombin and bleeding times, and elevated levels of fibrin degradation products. liver enzyme levels are elevated, and urea and creatinine levels are elevated during the oliguric phase. proteinuria and hematuria are consistent findings. the renal histopathologic findings are those of an interstitial nephritis with prominent hemorrhages in the renal medullary interstitium and renal cortex. acute tubular necrosis may also be seen. immunohistochemical analysis reveals deposition of igg and c3, and the gbm, mesangial, and subendothelial deposits may be seen on electron microscopy (227) . recovery from hantavirus-associated disease is generally complete, although chronic renal insufficiency is a rare sequela of hfrs. in mildly affected patients, the disease is self-limiting and spontaneous recovery occurs. however, in severe cases, with shock, bleeding, and renal failure, dialysis and intensive circulatory support may be required (228) . mortality rates vary depending on the strain of virus; rates are 5-15% for hemorrhagic fever and renal syndrome in china and significantly lower for the milder finnish form associated with the puumala virus strain. ribavirin is active against hantaan viruses in vitro, and clinical trials indicate that both mortality and morbidity can be reduced by treatment with this antiviral agent if it is administered early in the course of illness. dosages of 33 mg/kg followed by 16 mg/kg every 6 h for 4 days and then 8 mg/kg every 8 h for 3 days have been used (229) . lassa fever is a common infection in west africa, caused by an arenavirus, and usually manifests as a nonspecific febrile illness. in 10% of cases, a fulminant hemorrhagic disease occurs. in severe cases, proteinuria and hematuria are usually present, and renal failure may occur. ribavirin is effective in decreasing mortality. as in other hemorrhagic fevers, intensive hemodynamic support and correction of the hemostatic derangements are important components of therapy (230) . junin and machupo viruses, the agents of argentine and bolivian hemorrhagic fever, respectively, cause hemorrhagic fevers with prominent neurologic features and systemic and hemorrhagic features similar to those of lassa fever. oliguria, shock, and renal failure occur in the most severe cases. marburg and ebola viruses have been associated with outbreaks of nosocomially transmitted hemorrhagic fever. both viruses cause fulminant hemorrhagic fever. onset is with high fever, headache, sore throat, myalgia, and profound prostration. an erythematous rash on the trunk is followed by hemorrhagic conjunctivitis, bleeding, impaired renal function, shock, and respiratory failure. the mortality rate is high. renal histopathologic findings in fatal cases are of tubular necrosis, with fibrin deposition in the glomeruli. there is no specific treatment for these disorders. the important role played by a number of other recently characterized viruses is only now being recognized, as improved molecular diagnostic techniques allow identification of hitherto unrecognized viruses. two examples of recently described viruses are metapneumovirus (237) and bocavirus (238) . while both have significant prevalence, and may make an important contribution to the burden of childhood viral infection, as yet there are no reports indicative of significant renal pathology in association with these infections. influenza virus has been linked with nephritis and acute renal failure. an emerging infectious disease is avian flu, caused by highly pathogenic h5n1 strains which have hitherto been confined to an avian reservoir, and there have been several outbreaks of infection in humans, particularly in the first part of this decade. commonly, these patients develop a flu-like illness with prominent respiratory and gastrointestinal symptoms. renal failure may develop alongside multi-organ failure in the context of acute respiratory distress syndrome (231) . as yet, there is no clear correlation of degree of initial renal insufficiency, and outcome (232) . there is little data available on treatment, but based on the known resistance patterns of h5n1 strains, oseltamivir and zanamivir are the preferred agents to be used for treatment of infection with h5n1. severe acute respiratory syndrome (sars) is a newlyemerged infectious disease which was first seen in south china in 2002. it is caused by a sars coronavirus (sars cov). predominantly, it causes a viral pneumonia, with diffuse alveolar damage; it has considerable mortality (233) . renal effects are not generally significant in the pathophysiology of sars. however, sars cov has been found in kidney tissue at post-mortem (234) (235) . sars cov enters cells via angiotensin converting enzyme 2 (ace2) (236) , and it is thought that the invasion of kidney tissue reflects the virus' tropism for ace2, which is expressed on kidney cells. chronic exposure to infectious agents is a major factor in the increased prevalence of glomerular diseases in developing countries. malaria is the best-documented parasitic infection associated with glomerular disease, but other parasitic infections including schistosomiasis, filariasis, leishmaniasis, and possibly helminth infections may also induce nephritis or nephrosis. malaria is estimated to cause up to 500 million clinical cases of illness and more than 1 million deaths each year (239) . the association of quartan malaria and nephritis has been well known in both temperate and tropical zones since the end of the nineteenth century. epidemiologic studies provide the most conclusive evidence for a role of plasmodium malariae in glomerular disease (240, 241) . chronic renal disease was a major cause of morbidity and mortality in british guiana in the 1920s. the frequent occurrence of p. malariae in the blood of these patients led to detailed epidemiologic studies that implicated malaria as a cause of the nephrosis. after the eradication of malaria from british guiana, chronic renal disease ceased to be a major cause of death in that country (240) . the link between malaria and nephrotic syndrome was strengthened by studies in west africa in the 1950s and 1960s that demonstrated a high prevalence of nephrotic syndrome in the nigerian population (242) . the pattern of nephrotic syndrome differed from that in temperate climates, with an older peak age, extremely poor prognosis, and unusual histologic features. the incidence of p. malariae parasitemia in patients with the nephrotic syndrome in nigeria was vastly in excess of that occurring in the general population, whereas the incidence of plasmodium falciparum parasitemia was similar to that in the general population. the age distribution of nephrotic syndrome also closely paralleled that of p. malariae infection (242) . in some affected patients, circulating immune complexes and immunoglobulin, complement, and antigens were present in the glomeruli that were recognized by p. malariae-species antisera. there is now a view that the patterns of childhood renal disease described in the last century may no longer be representative of the current situation. the variable patterns of renal disease throughout africa may no longer reflect a dominant role for ''malarial glomerulopathy,'' and the relative causative role of tropical infections in nephropathy remains an unanswered question (243) . most patients have poorly selective proteinuria and are unresponsive to treatment with steroids or immunosuppressive agents. the characteristic lesions of p. malariae nephropathy are capillary wall thickening and segmental glomerular sclerosis, which lead to progressive glomerular changes and secondary tubular atrophy (242) . cellular proliferation is conspicuously absent. electron microscopy shows foot-process fusion, thickening of the basement membrane, and increase in subendothelial basement membrane-like material. immunofluorescent studies show granular deposits of immunoglobulin, complement, and p. malariae antigen in approximately one-third of patients. in addition to the histologic pattern, termed quartan malaria nephropathy, p. malariae infection is associated with a variety of other forms of histologic appearance, including proliferative gn and mgn (244) . although quartan malaria nephropathy has been clearly linked to p. malariae infection in nigeria, a number of studies from other regions in africa have not revealed the typical histopathologic findings described in the nigerian studies (245) . furthermore, quartan malaria nephropathy may be seen in children with no evidence of p. malariae infection or deposition of malaria antigens in the kidney. this, together with the fact that antimalarial treatment does not affect the progression of the disorder, raises the possibility that factors other than malaria might be involved in the initiation and perpetuation of the disorder. although there is undoubtedly a strong association between p. malariae infection and nephrotic syndrome on epidemiologic grounds, the direct causal link is not proven. most likely, a number of different infectious processes, including malaria, hepatitis b, schistosomiasis, and perhaps other parasitic infections that cause chronic or persistent infections and often occur concurrently in malaria areas, may all result in glomerular injury and a range of overlapping histopathologic features. the prognosis for the nephrotic syndrome in most african studies has been poor, regardless of whether the histologic findings were typical of quartan malaria nephropathy or whether p. malariae parasitemia was implicated. treatment with steroids and azathioprine is generally ineffective, and a significant proportion of patients progress to renal failure. p. falciparum appears to be much less likely to cause significant glomerular pathology. epidemiologic studies have failed to show a clear association between p. falciparum parasitemia and the nephrotic syndrome. whereas renal failure appears to be a common complication of severe malaria in adults, it seldom occurs in children. renal biopsy specimens from adult patients with acute p. falciparum infections who have proteinuria or hematuria show evidence of glomerular changes, including hypercellularity, thickening of basement membranes, and hyperplasia and hypertrophy of endothelial cells (246) . electron microscopy reveals electron-dense deposits in the subendothelial and paramesangial areas. deposits of igm, with or without igg, are localized mainly in the mesangial areas. p. falciparum antigens can be demonstrated in the mesangial areas and along the capillary wall, which suggests an immune-complex gn. the changes, generally mild and transient, are probably unrelated to the acute renal failure that may complicate severe p. falciparum infection (246) . heavily parasitized erythrocytes play a central role in the various pathologic factors (247) . renal failure occurring in severe p. falciparum malaria is usually associated with acidosis, volume depletion, acute intravascular hemolysis or heavy parasitic infection that leads to acute tubular necrosis. recent studies have confirmed an important role for volume depletion in children with severe falciparum malaria, who characteristically have evidence of tachycardia, tachypnoea, poor perfusion and in severe cases hypotension (248) . volume expansion with either colloid or crystalloid results in improvement in hemodynamic indices and reduction in acidosis (249) . there is growing evidence that volume expansion with albumin is associated with a better outcome than saline or synthetic colloids (250, 251) . treatment with antimalarials, correction of hypoglycemia and infectious diseases and the kidney electrolyte imbalance, and volume expansion reduces mortality to less than 5%. although renal failure is usually associated with infection by p. falciparum, acute renal failure has been described with plasmodium vivax infection and mixed infections (252) . the term blackwater fever refers to the combination of severe hemolysis, hemoglobinuria, and renal failure. it was more common at the start of the twentieth century in nonimmune individuals receiving intermittent quinine therapy for p. falciparum malaria. blackwater fever has become rare since 1950, when quinine was replaced by chloroquine. however, the disease reappeared in the 1990s, after the reuse of quinine because of the development of chloroquine-resistant organisms. since then, several cases have been described after therapy with halofantrine and mefloquine, two new molecules similar to quinine (amino-alcohol family) (253) . renal failure generally occurred in the context of severe hemolytic anemia, hemoglobinuria, and jaundice. the pathophysiology of the disorder is unclear; however, it appears that a double sensitization of the red blood cells to the p. falciparum and to the amino-alcohols is necessary to provoke the hemolysis. histopathologic findings include swelling and vacuolization of proximal tubules, necrosis and degeneration of more distal tubules, and hemoglobin deposition in the renal tubules. recent studies indicates a better outcome with earlier initiation of intensive care and dialysis combined with necessary changes in antimalarial medications. schistosomiasis affects 200 million people living in endemic areas of asia, africa, and south america (254) . the infection is usually acquired in childhood, but repeated infections occur throughout life. schistosoma japonicum is found only in the orient, whereas schistosoma haematobium occurs throughout africa, the middle east, and areas of southwest asia. schistosoma mansoni is widespread in africa, south america, and southwest asia. human infection begins when the cercarial forms invade through the skin, develop into schistosomula, and move to the lungs via the lymphatics or blood. they then migrate to the liver and mature in the intrahepatic portal venules, where male:female pairing takes place. the adult worm pairs then migrate to their final resting site -the venules of the mesenteric venous system of the large intestine (s. mansoni) or in the venules of the urinary tract (s. haematobium). the females release large numbers of eggs, which may remain embedded in the tissues, embolize to the liver or lungs, or pass into the feces or urine. clinical manifestations may occur at any stage of the infection. cercarial invasion may cause an intense itchy papular rash. katayama fever is an acute serum sicknesslike illness that occurs several weeks after infection, as eggs are being deposited in the tissues. deposition of the eggs in tissues results in inflammation of the intestines, fibrosis of the liver, and portal hypertension. with s. haematobium, chronic inflammation and fibrosis of the ureters and bladder may lead to obstructive uropathy (255) . renal manifestations of schistosomiasis occur most commonly in s. mansoni infection. schistosomal nephropathy usually presents with symptoms including granulomatous inflammation in the ureters and bladder, but glomerular disease (probably on an immune-complex basis) may also occur. renal disease usually occurs in older children or young adults with long-term infection, but serious disease may also occur in young children (255) . the early renal tract manifestations of schistosomiasis are suprapubic discomfort, frequency, dysuria, and terminal hematuria. in more severe cases, evidence of urinary obstruction appears. poor urinary stream, straining on micturition, a feeling of incomplete bladder emptying, and a constant urge to urinate may be severely disabling symptoms. the fibrosis and inflammation of ureters, urethra, and bladder may be followed by calcification and may result in hydroureter, hydronephrosis, and bladder neck obstruction. renal failure may ultimately develop, and there is a suspicion that squamous cell carcinoma of the bladder may be linked to the chronic infective and inflammatory process. secondary bacterial infection is common within the obstructed and inflamed urinary tract (254) . the hepatosplenic form of s. mansoni infection may be accompanied by a glomerulopathy in 12-15% of cases, manifested in the majority as nephrotic syndrome (256) . histopathologic findings include mesangioproliferative gn, focal segmental glomerulosclerosis, mesangiocapillary gn, mgn, and focal segmental hyalinosis (257) . immune complexes may be detected in the circulation of these patients, and glomerular granular deposition of igm, c3, and schistosomal antigens are seen on immunofluorescence. usually schistosoma-specific nephropathy is a progressive disease and is not influenced by antiparasitic or immunosuppressive therapy (258) , but isolated case reports of remission after treatment with praziquantel have been reported (259) . the diagnosis is confirmed by the detection of schistosoma eggs in feces, urine, or biopsy specimens. eggs are shed into the urine with a diurnal rhythm, and urine collected between 11 am and 1 pm is the most useful. urinary sediment obtained by centrifugation or filtration through a nuclepore membrane should be examined. in cases in which studies of urine and feces yield negative results in patients in whom the diagnosis is suspected, rectal biopsy specimens taken approximately 9 cm from the anus have a high diagnostic yield for both s. mansoni and s. haematobium infection. biopsy of liver or bladder may be required to establish the diagnosis. antibodies indicating previous infection can be detected using enzyme-linked immunosorbent assay or radioimmunoassay. the tests are sensitive but lack specificity and may not differentiate between past exposure and current infection. praziquantel is the drug of choice for treatment of schistosomiasis. a single oral dose of 40 mg/kg is effective in s.haematobium and s. mansoni infection and is usually well tolerated. the alternative drug for s. mansoni infection is oxamniquine. complete remission of urinary symptoms may occur in renal disease of short duration, but in late disease with extensive fibrosis, scarring, and calcification, obstructive uropathy and renal failure may persist after the infection has been eradicated. there are reports of a drastic decrease in the number of severe hepatosplenic forms of s. mansoni infection after mass treatment of the population in endemic areas with oxamniquine. this also reduced schistosomal nephropathy (256) . visceral leishmaniasis is a chronic protozoon infection characterized by fever, hepatosplenomegaly, anemia, leukopenia, and hyperglobulinemia. proteinuria and/or microscopic hematuria or pyuria have been reported in 50% of patients with visceral leishmaniasis (260) . acute renal failure in association with interstitial nephritis has also been reported (261) . renal histologic analysis in patients with visceral leishmaniasis reveals glomerular changes, with features of a mesangial proliferative gn or a focal proliferative gn, or a generalized interstitial nephritis with interstitial edema, mononuclear cell infiltration, and focal tubular degeneration. immunofluorescence reveals deposition of igg, igm, and c3 within the glomeruli, as well as electron-dense deposits in the basement membrane and mesangium on electron microscopy (260) . circulating immune complexes together with immunoglobulin and complement deposition in the glomeruli suggests an immune-complex cause. renal disease in leishmaniasis is usually mild and may resolve after treatment of the infection. renal dysfunction may be associated with treatment for visceral leishmaniasis with antimony compounds. proteinuria is more common in filarial hyperendemic regions of west africa than in nonfilarial areas. renal histologic analysis has shown a variety of different histopathologic appearances; the most common is diffuse mesangial proliferative gn with c3 deposition in the glomeruli (262) . renal biopsy specimens also demonstrate large numbers of eosinophils in the glomeruli, and microfilariae may be seen in the lumen of glomerular capillaries. filarial antigens have been detected within immune deposits within the glomeruli. echinococcus granulosus causes chronic cysts within a variety of organs. in addition, nephrotic syndrome in association with hydatid disease has been reported. membranous nephropathy, minimal change lesions, and mesangiocapillary gn have been described in association with hydatid disease (263, 264) . immunofluorescence reveals deposits of immunoglobulin, complement, and hydatid antigens within the glomeruli. remission of nephrotic syndrome has been reported with treatment by antiparasitic agents such as albendazole (263, 264) . few reports have been published of renal disease occurring in patients with trypanosomiasis. the trypanosomal antigens can induce gn in a variety of experimental animals (265) . nephrotic syndrome has occasionally been reported as a manifestation of congenital toxoplasmosis. dissemination of previously latent toxoplasma infection in patients undergoing treatment with immunosuppressive drugs has been increasingly recognized in recent years. reactivation of toxoplasmosis or progression of recently acquired primary infection should be considered in patients undergoing renal transplantation or immunotherapy for renal disease who develop unexplained inflammation of any organ. fungal infections of the kidneys and urinary tract occur most commonly as part of systemic fungal infections in patients with underlying immunodeficiency, as focal urinary tract infections in patients with obstructive lesions, or as a result of indwelling catheters. although candida infection is the most common fungal infection in both immunocompromised and non immunocompromised hosts, virtually all other fungal pathogens may invade the renal tract during severe immunocompromise. urinary infection with candida albicans is most commonly a component of systemic candidiasis in patients who are severely immunocompromised. systemic candidiasis is also seen in premature and term infants with perinatally acquired invasive candidiasis. presentation is usually with systemic sepsis, fever or hypothermia, hepatosplenomegaly, erythematous rash, and thrombocytopenia. systemic candidiasis may be seen on ophthalmologic investigation as microemboli in the retina. the first clue to the underlying diagnosis may be the presence of yeasts in the urine (266) . candida involvement of the urinary tract may affect all structures including the glomeruli, tubules, collecting system, ureters, and bladder. microabscesses may form within the renal parenchyma, and large balls of fungi may completely obstruct the urinary tract at any level. acute renal failure caused by systemic candidiasis or obstruction of the renal tracts with fungal hyphae is a wellrecognized complication of systemic candidal infection (266, 267) . indwelling catheters (which form a nidus for persistent infection) should be removed. successful treatment of non-obstructing bilateral renal fungal balls by fluconazole either alone or in combination with liposomal amphotericin b has been reported (268, 269) . in the presence of obstruction, however, percutaneous nephrostomy to relieve the obstruction with antegrade amphotericin b irrigation, coupled with systemic antifungal therapy, is the mainstay of treatment (267) . amphotericin b is the most effective antifungal agent, but it is not excreted in the urine. local irrigation via nephrostomy provides good results, however. for treatment of urinary tract candidiasis, it is usually combined with fluconazole or 5-flucytosine, both of which are excreted in high concentrations in the urine. treatment is required for weeks to months to ensure complete elimination of the fungus, and the ultimate outcome is largely dependent on whether there is a permanent defect in immunity. in 1983, levin et al. first described hemorrhagic shock and encephalopathy, which appeared to be distinct from previously recognized pediatric disorders (270) . other cases have subsequently been reported from several centers in the united kingdom, europe, israel, the united states, and australia, and the syndrome is now recognized as a new and relatively common severe childhood disorder (271) . hemorrhagic shock and encephalopathy usually affects infants in the first year of life, with a peak onset at 3-4 months of age. a prodromal illness with fever, irritability, diarrhea, or upper respiratory infection occurs 2-5 days before the onset in two-thirds of cases. affected infants develop profound shock, coma, convulsions, bleeding and evidence of dic, diarrhea, and oliguria. laboratory findings include acidosis, falling hemoglobin and platelet levels, elevated urea and creatinine levels, and elevated levels of hepatic transaminases. despite vigorous intensive care, the prognosis is poor, and most affected infants die or are left severely neurologically damaged (271, 272) . a small number of patients have been reported to survive without residual sequelae. the renal impairment appears to be largely prerenal in origin, and when aggressive volume replacement and treatment of the shock results in improved renal perfusion, rapid improvement in renal function is usually observed. in patients with profound shock unresponsive to initial resuscitation, vasomotor nephropathy supervenes and dialysis may be required. myoglobinuria in association with hemorrhagic shock and encephalopathy has been reported. following the description of the mucocutaneous lymph node syndrome by kawasaki in 1968, kawasaki disease has been recognized as a common and serious childhood illness with a worldwide distribution. although the etiology remains unknown, epidemiologic features clearly suggest an infective cause. the disease occurs in epidemics, and wavelike spread has been demonstrated during outbreaks in japan. deposition of amyloid within the kidney is an important complication of chronic and persistent infection. amyloidosis is most common in patients with chronic osteomyelitis and chronic pulmonary infections such as bronchiectasis and is seen occasionally in those with persistent infections such as leprosy or malaria (273) (274) (275) . paediatric emergencies, 2nd edn. black ja shock in the paediatric patient vasoactive hormones in the renal response to systemic sepsis renal effects of nitric oxide in endotoxemia drotrecogin alfa (activated) in children with severe sepsis: a multicentre phase iii randomised controlled trial treatment of meningococcal disease in childhood pathophysiology and management of meningococcal septicaemia severity of meningococcal disease: assessment by factors and scores and implications for patient management case records of the massachusetts general hospital. case 4 plasma endotoxin as a predictor of multiple organ failure and death in systemic meningococcal disease hemodynamic patterns of meningococcal shock in children role of interleukin 6 in myocardial dysfunction of meningococcal septic shock dysfunction of endothelial protein c activation in severe meningococcal sepsis dysfunction of endothelial protein c activation in severe meningococcal sepsis efficacy and safety of recombinant human activated protein c for severe sepsis renal abscess in childhood: diagnostic and therapeutic progress percutaneous drainage of renal and perirenal abscesses: results in 30 patients toxic shock syndrome associated with phage group 1 staphylococci toxic shock syndrome not associated with menstruation: a review of 54 cases toxic shock syndrome phenotypic distinctiveness of staphylococcus aureus strains associated with toxic shock syndrome the staphylococcal enterotoxins and their relatives toxic shock syndrome: associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumour necrosis factor toxic shock syndrome bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes perinephric abscess caused by communityacquired methicillin resistant staphylococcus aureus impact of antibiotics on expression of virulence? associated exotoxin genes in methicillin, sensitive and methicillin, resistant staphylococcus aureus clinical and bacteriologic observations of a toxic shock-like syndrome due to streptococcus pyogenes acute poststreptococcal glomerulonephritis. a review of recent developments occurrence of acute glomerulonephritis in sibling contacts of children with sporadic acute glomerulonephritis epidemic glomerulonephritis in israel does antimicrobial therapy of streptococcal pharyngitis or pyoderma alter the risk of glomerulonephritis? medium and long-term prognosis of patients with acute post-streptococcal glomerulonephritis infectious diseases and the kidney nephritis caused by streptococcus zooepidemicus (lancefield group c) acute glomerulonephritis following group g streptococcus infection streptococcus associated toxic shock changing group a streptococcus: the reappearance of streptococcal ''toxic shock severe group a streptococcus infections associated with a toxic shock-like syndrome and scarlet fever toxin a specific c-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of streptococcus pyogenes penicillin and clindamycin differentially inhibit the production of pyrogenic exotoxins a and b by group a streptococci improved outcome of clindamycin compared with beta-lactam antibiotic treatment for invasive streptococcus pyogenes infection intravenous immunoglobulin g therapy in streptococcal toxic shock syndrome: a european randomized, double-blind, placebo-controlled trial intravenous immunoglobulin therapy for streptococcal toxic shock syndrome -a comparative observational study. the canadian streptococcal study group a history of adjunctive glucocorticoid treatment for pediatric sepsis: moving beyond steroid pulp fiction toward evidence-based medicine intensive insulin therapy and pentastarch resuscitation in severe sepsis philadelphia renal lesions in leptospirosis leptospirosis and acute renal failure: clinical experiences and a review of the literature clinical profile of leptospirosis in south gujarat evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis placebo controlled trial of intravenous penicillin for severe and late leptospirosis hã¤molytich-urã¤miche syndrome: bilaterale nierenrindennekrosen bei akuten erworbenen hã¤molytischen anã¤mien hemolytic-uremic syndrome associated with neuraminidase-producing micro-organism: treatment by exchange transfusion hemolytic uremic syndrome associated with pneumococcal sepsis recent advances in understanding the pathogenesis of the hemolytic uremic syndromes glomerular and arteriolar thrombosis in pneumococcal septicemia hemolytic uremic syndrome associated with invasive streptococcus pneumoniae infection hemolytic-uremic syndrome associated with neuraminidase-producing microorganisms: treatment by exchange transfusion hã¤molytisch-urã¤misches syndrome bei pneumokok-ken-menigitis und -sepsis. bedeutung der t-transformation prognosis of streptococcus pneumoniaeinduced hemolytic uremic syndrome invasive pneumococcal disease and hemolytic uremic syndrome hemolytic uremic syndrome associated with invasive pneumococcal disease: the united kingdom experience acute renal failure of glomerular origin during visceral abscess glomerulitis in typhoid fever yersinia and chronic glomerulopathy in the savannah region of nigeria typhoid glomerulonephritis acute glomerulonephritis in multi-drug resistant salmonella typhi infection acute glomerulonephritis in salmonella typhi infection urinary tract and renal findings in acute yersinia infection acute tubulointerstitial nephritis in association with yersinia pseudotuberculosis infection acute renal failure associated with yersinia pseudotuberculosis infection in children acute renal failure associated with yersinia pseudotuberculosis infection ampicillin vs. placebo for yersinia pseudotuberculosis infection in children yersinia enterocolitica and yersinia pseudotuberculosis infections sporadic cases of haemolytic uraemic syndrome associated with faecal cytotoxin and cytotoxin producing escherichia coli in stools fact sheet no. 104. revised. geneva: world health organization global burden of tuberculosis: estimated incidence, prevalence, and mortality by country mycobacterium tuberculosis. in principles and practice of infectious diseases genitourinary tuberculosis revisited urogenital tuberculosis in children genitourinary tuberculosis: review of 102 cases the glomerulopathy of congenital syphilis: an immune deposit disease nephropathy of secondary syphilis: a clinical and padiological spectrum immunopathogenesis of syphilitic glomerulonephritis membranoproliferative glomerulonephritis and mycoplasma pneumoniae infection mycoplasma pneumoniae associated with acute glomerulonephritis mycoplasma pneumoniaeassociated nephritis in children textbook of pediatric infectious diseases rocky mountain spotted fever: clinical, laboratory and epidemiological features in 262 cases acute renal failure in rocky mountain spotted fever kidney lesion in rocky mountain spotted fever acute glomerulonephritis in a patient with rocky mountain spotted fever rocky mountain spotted fever rocky mountain spotted fever q fever: current concepts glomerulonephritis associated with coxiella burnetii endocarditis disseminated legionella pneumophila infection in an infant with severe combined immunodeficiency legionnaire's disease and acute renal failure: base report and review tubulointerstitial nephritis associated with legionnaires' disease the immune complex glomerulonephritis of bacterial endocarditis glomerular nephropathy associated with chronic q fever legionella prostheticvalve endocarditis glomerulonephritis in bacterial endocarditis the immune nature of subacute bacterial endocarditis (sbe) nephritis infective endocarditis in children the clinical spectrum of shunt nephritis the role of complement, immunoglobulin and bacterial antigen in coagulase-negative staphylococcal shunt nephritis membranoproliferative glomerulonephritis associated with infected ventriculoatrial shunt: report of two cases recovered after removal of the shunt the hepatitis b virus hepatitis b infection and renal disease: clinical, immunopathogenetic and therapeutic considerations extrahepatic manifestations of viral hepatitis christian cl vasculitis with hepatitis b antigenemia. long term observation in nine patients hepatitis b-associated vasculitis in an infant polyarteritis nodosa related to hepatitis b virus. a prospective study with long-term observation of 41 patients hepatitis b virus-associated polyarteritis nodosa: clinical characteristics, outcome, and impact of treatment in 115 patients immune complexes of heparins b surface antigen in the pathogenesis of periarteritis nodosa. a study of seven necropsy cases successful treatment of polyarteritis nodosa related to hepatitis b virus with interferon alpha as first-line therapy lamivudine in the treatment of polyarteritis nodosa associated with acute hepatitis b successful treatment of hepatitis b virus associated polyarteritis nodosa with a combination of prednisolone, afpha-interferon and iamivudine infectious diseases and the kidney membranous glomerulonephropathy in childhood hepatitis b surface antigenemia in north american children with membranous glomerlonephropathy the clinico-pathologic features of hepatitis b virus-associated glomerulonephritis gene expression profile of transgenic mouse kidney reveals pathogenesis of hepatitis b virus associated nephropathy hepatitis-b virus associated nephropathies: a clinicopathological study in 14 children strong association between iga nephropathy and hepatitis b surface antigenemia in endemic areas approaches to the treatment of hepatitis b virus and delta-related liver disease hbv associated nephrotic syndrome: resolution with oral lamivudine management of chronic hepatitis b virus infection: a new era of disease control lamivudine and hbv-associated nephropathy the prevalence of hepatitis c virus infection in the united states children with hepatitis c hepatitis c virusassociated glomerulonephritis focal segmental glomerular sclerosis among patients infected with hepatitis c virus hepatitis c virus-associated glomerulonephritis. effect of alpha-interferon therapy a comprehensive study of the association between hepatitis c virus and glomerulopathy hepatitis c infection and the patient with end-stage renal disease hepatitis c infection in children and adolescents with end-stage renal disease hepatitis c virus seropositivity at the time of renal transplantation in the united states: associated factors and patient survival de novo membranoproliferative glomerulonephritis in hepatitis c virus-infected renal allograft recipients hepatitis c virus infection and de novo glomerular lesions in renal allografts design of the peds-c trial: pegylated interferon +/ã�ribavirin for children with chronic hepatitis c viral infection effect of combination therapy (ribavirin and interferon) in hcv-related glomerulopathy acute renal failure in kidney transplant patients treated with interferon alpha 2b for chronic hepatitis c vasculitic complications of interferon-alpha treatment for chronic hepatitis c virus infection: case report and review of the literature widespread presence of histologically occult cytomegalovirus glomerulonephritis in congenital cytomegalic inclusion disease immune complexes in congenital and natal cytomegalovirus infection of man glomerulopathy associated with cytomegalovirus virenmia in renal allografts epstein-barr virus, cytomegalovirus and other viral infections in children after transplant cytomegalovirus-induced glomerular vasculopathy in renal allografts: a report of two cases the association of viral infection and chronic allograft: nephropathy with graft dysfunction after renal transplantation antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients infection in organ transplant recipients acute glomerulonephritis associated with varicella infection varicella infection in a renal transplant recipient associated with abdominal pain, hepatitis, and glomerulonephritis nephrotic syndrome associated with varicella infection epstein-barr virus-associated acute interstitial nephritis: infection or immunologic phenomenon? acute interstitial nephritis secondary to infectious mononucleosis an atypical pattern of epstein-barr virus infection in a case with idiopathic tubulointerstitial nephritis an in situ hybridization study of herpes simplex and epstein barr viruses in iga nephropathy and non-immune glomerulonephritis a review of adenoviruses in the aetiology of haemorrhagic cystitis coxsackie b viruses and the kidney -a neglected topic measles and acute glomerulonephritis mumps associated with nephritis associated focal and segmental glomerulosclerosis in the acquired immunodeficiency syndrome renal disease in patients with aids: a clinicopathologic study glomerular lesions in the acquired immunodeficiency syndrome urinary and renal histological changes in children with the acquired immunodeficiency syndrome (aids) nephrotic syndrome associated with acquired immunodeficiency syndrome in children human immunodeficiency virus nephropathy human immunodeficiency virus (hiv)-associated nephropathy in children from the hiv-1 and hiv-associated nephropathy 25 years later renal pathology of human immunodeficiency virus infection protease inhibitor therapy for hiv infection: the effect on hiv-associated nephrotic syndrome bk virus renal infection in a patient with the acquired immunodeficiency syndrome indinavir-associated interstitial nephritis and urothelial inflammation: clinical and cytologic findings mixed cryoglobulinemia in hiv-1 infection: the role of hiv-1 a typical hemolytic uremic syndrome in human immunodeficiency virus-1-infected children hiv-associated nephropathy is a late, not early, manifestation of hiv-1 infection hiv-associated nephropathy: current concepts hiv-associated nephropathy glomerulosclerosis and viral gene expression in hiv-transgenic mice: role of nef pathogenesis of human immunodeficiency virus (hiv)-associated nephropathy increased levels of transforming growth factor-[beta] in hiv-associated nephropathy a steroid responsive nephrotic syndrome in a patient with human immunodeficiency virus infection clinicopathologic correlates of prednisone treatment of human immunodeficiency virus-associated nephropathy cohort study of the treatment of severe hfv-associated nephropathy with corticosteroids prednisone improves renal function and proteinuria in human immunodeficiency virus-associated nephropathy effect of angiotensin-converting enzyme inhibition in hiv-associated nephropathy spontaneous improvement of the renal function in a patient with hiv-associated focal glomerulosclerosis hiv-1-associared nephropathy and response to highly-active antiretroviral therapy protease inhibitor therapy for hiv infection: the effect on hiv-associated nephrotic syndrome zidovudine is beneficial in human immunodeficiency virus associated nephropathy hiv-1 infection initiates an inflammatory cascade in human renal tubular epithelial cells treatment of human immunodeficiency virus (hiv)-associated nephropathy hivan and medication use in chronic dialysis patients in the united states: analysis of the usrds dmms wave 2 study maintenance hemodialysis in patients with hiv-associated nephropathy organ transplant multi-site study. clinical, immunologic and pharmacologic consequences of kidney transplantation in people with hiv infection hivinfected liver and kidney transplant recipients: 1-and 3-year outcomes new human papovavirus (bk) isolated from urine after renal transplantation polyomavirus disease under new immunosuppressive drugs: a cause of renal graft dysfunction and graft loss diagnosis and management of bk polyomavirus interstitial nephritis in renal transplant recipients polyomavirus infection of renal allograft recipients: from latent infection to manifest disease infectious diseases and the kidney testing for polyomavirus type bk dna in plasma to identify renal-allograft recipients with viral nephropathy nephropathy due to polyomavirus type bk polyomavirus-associated nephropathy: update in diagnosis pathogenesis and management of polyomavirus infection in transplant recipients bk virus infection in renal transplant recipients prospective study of polyomavirus type bk replication and nephropathy in renal-transplant recipients bk virus infection, replication, and diseases in pediatric kidney transplantation occurrence and significance of papovaviruses bk and jc in the urine polyoma-virus induced interstitial nephritis in two renal transplant recipients: case reports and review of the literature late-onset hemorrhagic cystitis associated with urinary excretion of polyoma-viruses after bone marrow transplantation collecting duct carcinoma arising in association with bk nephropathy posttransplantation in a pediatric patient. a case report with immunohistochemical and in situ hybridization study association of renal adenocarcinoma and bk virus nephropathy post-transplantation clinical course of polyoma virus nephropathy in 67 renal transplant patients epidemiology of haemorrhagic fever viruses virus and hemostasis dengue and dengue haemorrhagic fever glomerular changes in dengue haemorrhagic fever intravenous fluids -getting the balance right comparison of three fluid solutions for resuscitation in dengue shock syndrome epidemiology and clinical features of crimean-congo haemorrhagic fever in southern africa viral hemorrhagic fever-induced acute kidney injury hantavirus: an increasing problem? haemorrhagic fever with renal syndrome, virological and epidemiological aspects different pathohistological presentations of acute renal involvement in hantaan virus infection: report of two cases clinical characteristics of haemorrhagic fever with renal syndrome in children prospects for treatment of viral haemorrhagic fever with ribavirin, a broad spectrum antiviral drug lassa fever: effective therapy with ribavirin human infection with highly pathogenic h5n1 influenza virus renal insufficiency on presentation of bird flu infection: is it correlated to outcome? pathogenesis of severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease cloning of a human parvovirus by molecular screening of respiratory tract samples malaria in 2002 malaria and renal disease with special reference to british guiana. ii. the effect of malaria eradication on the incidence of renal disease in british guiana immunopathology of malaria the nephrotic syndrome and other diseases in children in western nigeria malaria-induced renal damage: facts and myths clinicopathological features of childhood nephrotic syndrome in northern nigeria quartan malarial nephrotic syndrome renal disease in acute plasmodium falciparum infection in man current knowledge in falciparum malaria-induced acute renal failure severe p. falciparum malaria in kenyan children: evidence for hypovolaemia response to volume resuscitation in children with severe malaria volume expansion with albumin compared to gelofusine in children with severe malaria: results of a controlled trial randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit severe acute renal failure in malaria recurrence of blackwater fever: triggering of relapses by different antimalarials philadelphia schistosomal specific nephropathy leading to end-stage renal failure is glomerulopathy due to schistosomiasis mansoni disappearing? characterisation of kidney lesions in early schistosomal-specific nephropathy schistosoma mansoni-induced mesangiocapillary glomerulonephritis: influence of therapy minimal change glomerulonephritis associated with schistosoma haematobium infectionresolution with praziquantel treatment renal involvement in visceral leishmaniasis acute renal failure in visceral leishmaniasis the nephrotic syndrome associated with filariasis minimal change glomerulonephritis associated with hydatid disease reversible nephrotic syndrome due to mesangiocapillary glomerulonephritis secondary to hepatic hydatid disease immune complexmediated glomerulopathy in experimental chagas' disease anuria in a newborn secondary to bilateral ureteropelvic fungus balls the role of percutaneous nephrostomy in the management of obstructing candidiasis of the urinary tract in infants successful treatment of bilateral renal fungal balls with liposomal amphotericin b and fluconazole in an extremely low birth weight infant renal fungus ball in a premature infant successfully treated with fluconazole haemorrhagic shock and encephalopathy: a new syndrome with high mortality in young children haemorrhagic shock and encephalopathy: reflections about a new devastating disorder that affects normal children haemorrhagic shock and encephalopathy: clinical, pathologic, and biochemical fea-mks use of protein-c concentrare, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans quantitative viral load monitoring and cidofovir therapy for the management of bk virus associated nephropathy in children and adults antiviral therapy and prophylaxis for influenza in children infectious diseases and the kidney key: cord-286719-1xjmlwqr authors: draz, mohamed shehata; shafiee, hadi title: applications of gold nanoparticles in virus detection date: 2018-02-15 journal: theranostics doi: 10.7150/thno.23856 sha: doc_id: 286719 cord_uid: 1xjmlwqr viruses are the smallest known microbes, yet they cause the most significant losses in human health. most of the time, the best-known cure for viruses is the innate immunological defense system of the host; otherwise, the initial prevention of viral infection is the only alternative. therefore, diagnosis is the primary strategy toward the overarching goal of virus control and elimination. the introduction of a new class of nanoscale materials with multiple unique properties and functions has sparked a series of breakthrough applications. gold nanoparticles (aunps) are widely reported to guide an impressive resurgence in biomedical and diagnostic applications. here, we review the applications of aunps in virus testing and detection. the developed aunp-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-aunp hybrid structures, virus detection targets, and assay modalities and formats. we pay particular attention to highlighting the functional role and activity of each core au nanostructure and the resultant detection improvements in terms of sensitivity, detection range, and time. in addition, we provide a general summary of the contributions of aunps to the mainstream methods of virus detection, technical measures, and recommendations required in guidance toward commercial in-field applications. viruses are remarkable pathogens that are causing prominently increasing morbidity and mortality worldwide. their highly contagious nature and the absence of immediate and efficient control systems are the main reasons behind their potential health impacts. currently, viral infections and associated diseases are major causes of death in mankind, and under the present context of industrialization and immigration, they continue to emerge at a rapid pace, causing significant human, social, and financial costs [1] . additionally, gaps in currently applied detection systems potentially contribute to increasing the likelihood of international incidences and outbreak of viral infections [2] [3] [4] [5] . the implementation of highly sensitive and specific diagnostic tools has the potential to rapidly identify viral infections, initiate and guide judicious controls, and subsequently curtail their dissemination. toward this endeavor and beyond the pitfalls of current immunological and molecular techniques commonly applied to virus detection, several new approaches based on nanoparticles (nps) have recently been developed. aunps are widely described to be suitable for numerous biosensing functions and applications. their unique photonic, electric, and catalytic properties, coupled with the molecular interaction specificity of various biomolecules (e.g., antibodies, single-stranded (ss) dna, and rna aptamers, among others), represent the design principles of a wide range of virus detection systems [6] [7] [8] . the advantages of being simple, rapid, and sensitive and facilitating quantitative detection with excellent multiplexing capabilities have greatly promoted these systems to be envisioned as state-of-the-art technologies for virus ivyspring international publisher detection [9, 10] . however, there are no available reviews of the applications of bio-aunp hybrid structures for sensing and detecting viruses. in this article, we provide a current review of aunp-based virus detection at the level of virus type, including the types and structures of the applied aunps. we highlight their role in enhancing sensory and detection performance in comparison with current techniques in terms of analytical sensitivity, detection range, and time. furthermore, this review specifically summarizes detection designs, formats, functions, and contributions, which are of special importance to both scientific and applied research. viruses are particulate in nature and usually exist in different morphological forms that generally range in size from 20 to 900 nm [11] [12] [13] . their intact, mature infectious particles are typically composed of definite units of proteins and nucleic acid that self-assemble to form nanoparticulate structures called virions. the viral proteins are usually arranged in a surface layer called the capsid and sometimes in an outer envelope surrounding an inner core of nucleic acids. this core of viral nucleic acids can be single-or double-stranded (ds), dna or rna, one or several molecules, linear or circular in shape, and a few thousands of nucleotides to one million base pairs in size. the nanoscale size and the relatively simple structure of viruses tend to impose technical difficulties in establishing wide-use and long-term systems for virus detection. the small size of viruses increases the difficulty of their isolation and visualization compared with other microbes, such as bacteria and fungi that can be readily examined using ordinary light microscopes. only electron microscopy (em) with a high-magnification power of ~100,000× can allow the direct visualization of viruses and the study of their structures [14, 15] . therefore, em remains crucial for many purposes in virus research. however, em is certainly inappropriate for routine clinical diagnosis because of the required time and cost, as well as many safety concerns. furthermore, although the simple structure of virus particles allows the features of diagnostic relevance to be easily defined and tested, it presents limitations on the use of their characteristics for practical applications, especially with the increasing number of discovered viruses and recorded viral infections [14, 16, 17] . in addition, this structural simplicity confers viruses with rapid rates of spontaneous adaptation and evolution that may occur through direct genetic mutation, genetic substitution, or recombination. in this way, viruses not only outpace our attempts to develop sustainable control strategies but also raise more questions about the appropriateness and validity of current diagnostic techniques for long-term use [18, 19] . along with these limitations in virus detection and control, the possibility of viral infection emergence or reemergence has become increasingly prevalent, and severe pandemics and epidemics have occurred around the world. in the past, many outbreaks of viruses, such as human immunodeficiency virus (hiv), severe acute respiratory syndrome (sars), influenza virus (h5n1 and h1n1) and zika virus, started as local events and then expanded to have global consequences. hiv was discovered in central africa as a new virus causing acquired immune deficiency syndrome (aids) three decades ago, and it now represents one of the most significant public health threats worldwide [20, 21] . the highly contagious respiratory virus sars has fueled global fears of pandemics since its first appearance in china [22, 23] . in late 2012, the world health organization (who) raised a global alert for a new sars-like respiratory coronavirus, which is now called middle east respiratory syndrome (mers) coronavirus. mers is now classified as one of the most prominent viral threats to the middle east and has caused hundreds of deaths and thousands of infections in a short time. moreover, in the past few years, the world witnessed the worst ebola outbreak ever that started to hit west africa in early 2014 and the pandemic reemergence of zika virus in 2016 [11, 12, 24] . with these evident possibilities of massive outbreaks and implications, the situation is rapidly growing more serious, and the demand for the development of rapid diagnostics and effective control strategies is becoming more urgent. the first studies on virus isolation and detection were started early in the 1950s when the first cell culture system and electron microscope were developed [25, 26] . for decades, these techniques represented the main tools for studying and investigating the biochemical and morphological properties of viruses that remain the main foundation of all known classification and detection systems. however, their practical use in virus detection has remained greatly debated due to several considerations, including laboratory equipment expenses and time and safety concerns [27, 28] . in the early 1980s, the field of diagnostic virology was boosted with two other major developments: 1) the birth of various immunoassays; and 2) the invention of polymerase chain reaction (pcr). this was followed by the development of a very wide range of serological and molecular detection techniques, which rapidly evolved to constitute the mainstream approaches of both laboratory research and the clinical diagnosis of viruses ( fig. 1) [24, 29] . serology remains the standard method for virus detection. it primarily relies on testing for the presence of specific viral antigens or the corresponding antibody responses of the immune system. the most common types of serological tests include the neutralization assay, complement-fixation test, immunoprecipitation assay (ipa), hemagglutination-inhibition (hai) assay, enzyme immunoassay (eia), radioimmunoassay (ria), chemiluminescent immunoassay (cia), particle agglutination, immunostaining, immunofluorescence assay, single radial hemolysis, immunoblotting assay (iba), and immunochromatographic test (ict). the main working principle of these techniques is the use of specific antibodies in conjugation with different signal reporting systems, such as red blood cells, enzymes, and radioactive or fluorescent materials [30, 31] . most of these techniques are relatively easy to perform and flexible in their timing of specimen collection, and most of the needed reagents are usually commercially available. furthermore, they are generally unrestricted by the practical limitations of virus isolation and propagation usually involved with other direct detection methods, such as cell culture and em [9, 32] . therefore, serological methods are widely accepted and recognized techniques for simple, safe, and cheap virus detection [25, 30] . in addition, they are usually described as the first choice for large-scale testing in epidemiological studies and for evaluating antiviral therapies and vaccinations. however, the accuracy and reliability of serological methods are usually challenged by the cross-reactivity of the used antibodies, and the risk of false-positive results is usually very high. in addition, most immune responses can only be detected for a period of time after the initial virus infection; thus, serological detection does not normally benefit patients. molecular techniques are attracting more interest and have found an increasing number of applications in virus detection. the discovery of the genetic enzyme systems involved in the cellular machinery of nucleic acid replication and the stunning invention of an in vitro nucleic acid amplification system, commonly called pcr, by mullis in the early 1980s, opened new frontiers in nucleic acid-based detection [33] . in addition, the known high specificity of nucleic acid hybridization and the absolute availability of its synthesis and modification guided the development of many figure 1 . the onset of nanotechnology in virus detection applications compared with the development of the most common virus detection techniques. cell culture and electron microscopy techniques that are now commonly applied in the direct testing for and detection of viruses were discovered in the mid-20th century [25] . then, different serological and molecular techniques were developed. the serological detection of viruses with immunoassays was first reported in 1970: a radioimmunoassay was applied for the detection of the australia antigen, later called hepatitis b virus surface antigen [218] . pcr was discovered in the 1980s and first reported in virus detection in 1988 for acquired immune deficiency syndrome detection [219] . later, many molecular techniques, including amplification-and nonamplification-based techniques, were reported in virus detection. the concept of nanotechnology was envisioned as early as 1959 by the renowned physicist richard feynman [36] . however, nanotechnology was only applied to virus detection in 1997, when gold nanoparticles were employed for the detection of single-copy human papillomavirus [39] . nanotechnology has recently come to represent one of the most outstanding trends in virus detection and diagnosis via the wide variety of assays described in this review. detection and genotyping techniques known for the rapid and specific detection of viruses. these techniques can be classified as amplification-or nonamplification-based molecular detection techniques. amplification-based molecular techniques usually employ one or more forms of nucleic acid amplification to allow the indirect detection of the target virus. these techniques represent the majority of molecular techniques applied in virus detection and include various types of target amplification techniques (e.g., pcr, loop-mediated isothermal amplification (lamp), transcription-mediated amplification, and nucleic acid sequence-based amplification), signal amplification techniques (e.g., branched dna and hybrid capture), and probe amplification techniques (e.g., ligase chain reaction and strand-displacement amplification). nonamplification-based techniques are mainly applied to the direct testing for the presence of a specific virus in clinical samples (e.g., in situ hybridization, southern blot hybridization, and dot blot hybridization). generally, molecular methods are relatively rapid and more sensitive than immunoassays and can be applied either in a simple form for the manual detection of viruses or as an embedded component of more advanced systems. numerous fully automated and high-throughput detection systems are now available and widely used in clinical settings. in fact, the development of advanced molecular detection systems has revolutionized the way in which diagnostic tests are delivered and greatly enhanced the control of many viral infections, whether in hospitals or communities. in addition, these systems have eliminated the biosafety and time concerns usually associated with the clinical and academic study of viruses. however, despite these wide and promising applications, most molecular techniques still have several potential limitations in repeatability, accuracy, sensitivity, and specificity that are mainly caused by the high genetic variability of some viruses. furthermore, these assays are expensive and time consuming and typically call for specialized laboratory instruments and skilled personnel [34, 35] . the concept of nanotechnology was introduced in early 1959 [36] . subsequently, nanotechnology was realized via various types of new materials that rapidly emerged as promising tools for biological and chemical analyses. nanomaterials are known to possess multiple unique optical, electronic, magnetic, and mechanical properties enabling very attractive applications, especially in the fields of biomedical imaging and clinical diagnosis [37, 38] . the first reported application of nanomaterials in the detection of viruses was attempted in the late 1990s: aunps were coupled with silver staining and applied for the detection of human papillomavirus in cervical carcinoma cells (fig. l) [39] . currently, there is a very wide range of nanomaterials, including metal nps, carbon nanotubes, silica nps, quantum dots (qds), upconversion nps, and polymeric nps, that are being heavily investigated for virus detection [37, 38, 40] . one of the most common approaches for exploiting these nanostructures in virus detection is the development of nanobio hybrid systems that contain one or more biomolecules derived from viruses (e.g., dna, rna, antibody, pentabody, antigen, or peptide) conjugated to the surface of different np forms. these systems leverage the significant labeling properties and signal transduction functions of nps and the specific activity of the conjugated biomolecules to act as multivalent-np probes [37, 38, 41] . such virus-specific np probes have surprisingly been used to build up various optical, fluorometric, electrochemical, and electrical assays that have been extensively reported for single and multiple detection modes ( table 1 ). the results of most of these studies clearly demonstrate the inherent potential of these probes, along with numerous advantages over traditional approaches, in terms of size, performance, specificity, signal sensitivity, and stability. additionally, these studies have extensively described their application to allow simple, rapid, highly sensitive and label-free detection. aunps are a leading class of metal nanostructures that is widely known for its chemical stability, water solubility, and broad size and shape controllability. aunps can range from 1 to 800 nm in size and have different morphological shapes, including spheres, rods, prisms, tetrapods, dog bones, cubes, shells and several hollow structures [42, 43] . the synthesis of aunps can be performed using different methods such as chemical reduction of salts, ultraviolet irradiation, lithography, aerosol technologies, laser ablation, ultrasonic fields, photochemical reduction of au, and biological synthesis [9, 32] . aunps possess a high surface density of free electrons that results in inherent optical, electrical, and catalytic properties. the excitation of aunps with light can cause these free surface electrons, i.e., "plasmons," to oscillate to one side away from the atomic core, which remains as a positive charge on the other side, thereby creating a dipole or plasmon polariton [44, 45] . this dipole plasmon can change its direction in accordance with the frequency of incident light, and the resonance condition is reached when their frequency is approximately the same. this condition has been referred to as surface plasmon resonance (spr). the most widely used aunps exhibit intense spr bands that usually exist between 510-1100 nm and are known to be weakly dependent on the size of the aunps and the refractive index of the surrounding media but very sensitive to both the shape of the nps and the interparticle distance [46] . the spr of aunps has been observed to cause intense enhancing or quenching effects upon interactions with nearby photon emitters. these distance-dependent coupling effects are dipole-dipole interactions that usually include surface plasmon-mediated energy nanotransfer processes similar to fluorescence resonance energy transfer (fret) and may be either destructive (resulting in quenched emission) or constructive (resulting in enhanced emission). compared to other types of nanomaterials, metal nanoparticles, particularly aunps, constitute ideal tools in virus detection for numerous reasons, including the ease of synthesis, characterization, and surface modification, outstanding stability, biocompatibility, and exceptionally high absorption coefficients [47, 48] . furthermore, as labeling agents, aunps are easily visualized due to their intense colors and are known to form stable and highly active bioconjugates with common targeting biomolecules, such as dna and proteins, thereby enabling highly sensitive and specific sensing and detection applications [48] . aunps have been preferentially employed to perform numerous optical signal transduction functions in virus detection, such as resonance light scattering [49] [50] [51] [52] [53] , color amplification [54] [55] [56] [57] [58] [59] , and fluorescence quenching or enhancing [60] [61] [62] [63] [64] [65] (fig. 2) . resonance light-scattering-based detections usually involve measuring the amount of light scattered by different light spectroscopy techniques, including localized surface plasmon resonance (lspr) [52] , raman spectroscopy [49, 50] and dynamic light scattering (dls) [51, 53] . the colorimetric detection of viruses using aunps usually relies on two main techniques: 1) a color amplification technique in which aunps are applied to act as direct coloring labels with their characteristic, intense red color [55, 58, 59, 66] ; and 2) color change technique in which a color change from red to purple occurs in response to particle aggregation. aunp aggregation can be noncrosslinked aggregation caused by the target-triggered removal of stabilizing ligands from the surfaces of aunps [54, 56] or interparticle-crosslinked aggregation caused by the binding of ligands on modified aunps with target analytes [57, 67] . analogous to fret, aunp-based fluorescence quenching is a distance-dependent process in which aunps act to reduce the radiative rate of fluorophores in their proximity. thus, as efficient acceptors, aunps have been paired with dyeand qd-based fluorophores in different analyte-induced donor-acceptor crosslinking and coupling protocols [60-62, 64, 65] . aunps are widely described as electroactive and catalytic tags in various electrochemical assays applied to virus detection (fig. 2 ). based on their redox and electrical properties, aunps can directly act as electrochemical tags detected either by their acid dissolution followed by electrochemical stripping measurements of gold ions [68] or by their direct deposition on the surface of electro transducers, allowing an enhanced electrical conduction and resistance change [69] [70] [71] [72] [73] . aunps can indirectly perform electrochemical transduction functions based on their catalytic activity toward some chemical reduction reactions of metal ions (e.g., silver and copper) [72, 74, 75] or other species, such as h2o2 [76] [77] [78] . this potential to catalyze metal deposition is preferentially called np-metal enhancement amplification, and it represents the basic function of multiple scanometric [50, 70, [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] , photoelectric [89] , light-scattering [90] , and colorimetric schemes [91] for virus detection. additionally, aunp-based catalysis for the oxidation-reduction reaction of hydroquinone has been reported in another simple, colorimetricbased virus detection method [92] . other studies have exploited aunps to enhance the detection sensitivity of some bioanalytical techniques, such as quartz crystal microbalance (qcm) [93] , inductively coupled plasma mass spectrometry (icp-ms) [94] , and atomic force microscopy (afm) [95] in virus detection. through these schemes, aunps have been applied as effective nanoparticulate amplification tags to enhance mass, elemental, and topographic signal transduction, respectively (fig. 2) . interestingly, the enhanced surface area-to-volume ratio of aunps has allowed them to further act as exquisite scaffolds for biorecognition and detection reactions (fig. 2 ); in addition, it has granted these structures unprecedented capabilities for bioimmobilization, which is a basic principle of most aunp-based diagnostic designs. by coupling these bioimmobilization and signal transduction functions, aunps can achieve virus detection with highly improved analytical sensitivity and specificity. furthermore, the direct increase in loading efficiency allows new possibilities of controlled multifunctionalization with different biomolecules. based on this principle, developed aunp tags are characterized by enhanced reactivity and stability. increased loading efficiency also allows the design of new probes modified with multiple structures for biotargeting (e.g., antibodies and dna), together with other signal amplification structures (e.g., enzymes and dna oligonucleotides) [69, 76, 77, [96] [97] [98] , both types of which are stridently included in the development of new diagnostic schemes and designs. for example, the aunpfigure 2 . a generalized scheme for the basic characteristics and functions of aunps applied in virus detection. the size and metal nature of aunps are the key characteristics that enable them to directly transduce multiple types of signals, including optical, catalytic and electrical signals that can be detected by dynamic light scattering (dls), localized surface plasmon resonance (lspr), surface-enhanced raman scattering (sers), ultraviolet-visible (uv-vis) spectroscopy, fluorometry and other electrochemical analysis techniques. in addition, aunps act as nanoparticulate tags to enhance the signal detection of several analytical techniques, such as quartz crystal microbalance (qcm), atomic force microscopy (afm), and inductively coupled plasma mass spectrometry (icp-ms). their large surface area-to-volume ratio is known to permit them to function as an excellent scaffold for bioimmobilization and target-probe interactions with highly enhanced specificity and sensitivity. barcoding assay and its descendent technique of immuno-pcr universally rely on aunp tags modified with antibodies and dna oligonucleotides [81, 96, 97] , and aunp-based enzymatic electrochemical assays are based on using aunps dually modified with dna and antibodies together with an enzyme to induce a chemical reduction reaction allowing electrochemical signal transduction [76, 77] . several recent studies have attempted to harness the unique properties of aunps to develop various advanced schemes for virus detection. the developed assays are greatly variable in design and underlying principle. however, the utilization of aunps conjugated with specific virus-targeting biomolecules is a key component in most of these assays. various aunp bioconjugates have been widely employed in many colorimetric, scanometric, electrochemical, and fluorometric systems for the detection of many groups of well-known human viruses (table 1 and fig. 3 ). each of these groups will be discussed in more detail in the following sections. the bunyaviridae family comprises more than 300 members that are primarily organized into four main genera: hantavirus, orthobunyavirus, phlebovirus and nairovirus [99] . bunyaviruses are spherical, enveloped rna viruses 80-100 nm in size. their genome is composed of three segments of negative-sense, ssrna: a large segment (l, 6.3-12 kb) that encodes rna polymerase, a medium segment (m, 3.5-6 kb) that encodes viral glycoprotein, and a small segment (s, 1-2.2 kb) that encodes nucleocapsid protein [100] . these viruses are diverse in their host range and are frequently involved in a wide range of diseases in plants, animals, and humans. human pathogenic bunyaviruses, such as crimean-congo hemorrhagic fever virus, hantaan virus, la crosse virus, oropouche virus, rift valley fever virus, and toscana virus, continue to present increasingly important health concerns worldwide [101, 102] . the genus hantavirus was recently expanded to include more than 24 antigenically and genetically distinct htnvs [99] . among them, rodent-borne htnvs can cause serious diseases in humans, including hemorrhagic fever with renal syndrome, and hantavirus cardiopulmonary syndrome, and hospitalizes more than 150,000 persons each year with a mortality rate reaching 10%. recently, the health impacts of htnvs are expected to dramatically increase in the near future due to the increasing number of reports on newly discovered htnvs [102] [103] [104] . aunps were utilized to develop a novel immuno-pcr assay for the immunological detection of htnv nucleocapsid protein [96] . this assay mainly relies on the enhanced surface area of aunps to prepare dually functionalized antibody-oligonucleotide conjugates that exceptionally carry specific monoclonal antibodies to label the target htnv antigen; the assay also involves barcoding dna for signal amplification (fig. 4a ). this assay could optimally detect concentrations as low as 200 am of purified or spiked antigen samples, which is ~7 orders of magnitude more sensitive than conventional elisa. the high detection sensitivity together with the targeting of htnv nucleocapsid protein [105] , which is the most abundant viral component synthesized post virus infection, makes this assay a promising candidate method for the early diagnosis and control of htnv. rvfv is an arthropod-borne pathogen primarily known to affect animals and later discovered to infect humans. rvfv infection in humans can progress to serious hemorrhagic fevers that often lead to death, with prominent mortality rates of up to 10-12%. infected persons can also develop other clinical manifestations, including retinitis, encephalitis, and paralysis [106, 107] . rvfv is historically endemic to africa and has had a long history of outbreaks ranging from its reemergence in egypt in 1977 to the most recent outbreak in south africa in 2011 [108, 109] . thus far, rvfv outbreaks are unpredictable and usually associated with very high socio-economic and public health consequences [110, 111] . therefore, diagnostic methods that can rapidly identify the virus have become crucial not only for avoiding and preventing such outbreaks but also for monitoring cross-border dissemination. a novel immunoassay based on aunps coupled with a surface-enhanced raman scattering (sers) technique has been developed for the detection of rvfv capsid antigen [49] . in this assay, aunps are applied as both immobilization scaffolds for the target-capturing antibodies and metal promoters to enhance the raman reporter dyes. the target antigen is first captured by antibody-magnetic particle conjugates and then labeled with dye/antibody-aunp conjugates, forming a three-component immunocomplex that is magnetically concentrated and finally detected by sers (fig. 4b) . this approach has shown an outstanding sensitivity level down to 5 fg/ml of the target antigen. this high sensitivity with the possibility for the direct detection of rvfv in complex media or samples allowed by magnetic particles support the application of this assay for the rapid and sensitive detection of rvfv and the control of any future outbreaks. immuno-pcr assay for htnv detection using aunp probes dually functionalized with antibody (ab) and double-stranded (ds)dna. au-nanoprobes are directly applied to label virus antigens precaptured on a microplate. the aunps are carrying dsdna that includes barcode single-stranded (ss) dna for signal amplification. the barcode dna is separated, amplified and detected by gel electrophoresis [96] . additionally, this assay can be modified in a more complex detection scheme that has been reported for human immunodeficiency virus detection [81] . (b) surface-enhanced raman spectroscopy (sers)-based assay for detection of rvfv using raman reporter dye-coated aunps and magnetic nps (mnps). aunps and mnps are conjugated with a polyclonal ab specific for the target virus antigen forming aunp/virus antigen/mnp complexes; then, a 785 nm laser excites the magnetically concentrated aunp/virus antigen/mnp complexes. the presence of the target antigen yields a reduction in the intensification of raman dye signature spectrum peaks, thereby providing an estimation of its concentration [49] . this assay has been applied for the detection of wnv infection [49] . the coronaviridae family comprises two subfamilies, coronavirinae and torovirinae, that were recently expanded to include many viruses. coronavirinae are classified into four genetically distinct genera (alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus) [112] . coronaviruses are enveloped rna viruses that are pleomorphic in shape (spherical and 120-160 nm or bacilliform and 170-200 nm by 75-88 nm). they have a relatively large, positive-sense, ssrna genome of 27-32 kb that encodes four to five structural proteins (s, the spike glycoprotein; m, the membrane glycoprotein; n, the nucleocapsid interrupt phosphoprotein; e, the envelope protein; and he, the hemagglutinin-esterase glycoprotein) and 2 nonstructural polyprotein precursors (pp1a, polyprotein 1a; and pp1ab, polyprotein 1ab), which are later processed into several other nonstructural proteins and viral polymerase [113, 114] . coronaviruses are clinically significant to humans and usually associated with different respiratory, intestinal, hepatic, or neurological diseases [115] . hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 are among the most disseminating coronaviruses that usually infect the upper respiratory tract in humans, causing mild common cold-like diseases [116] [117] [118] [119] . other coronaviruses, such as sars-cov identified in china in 2002 and mers-cov discovered in the middle east in 2012, are notorious pathogens able to cause widespread outbreaks of pneumonia and pneumonialike conditions with very high morbidity and mortality rates of up to 35% [120, 121] . due to their high clinical significance and contagious nature, there has recently been great interest in testing and developing advanced sars detection methods. sars detection using aunps is primarily focused on developing rapid and specific molecular detection through two main assays: 1) a colorimetric assay for pp1ab gene detection; and 2) an electrochemical assay for nucleocapsid protein gene detection ( table 1 ). the colorimetric assay involves the ability of aunps to preferentially adsorb ssdna over dsdna and specifically sense the presence of the target dna [54] . specific, short ssdna probes adsorb to the surface of aunps, resulting in an increased particle colloidal stability and an increased ability to withstand slightly elevated salt concentrations without significant aggregation or color changes. upon addition, the target dna specifically forms dsdna with the adsorbed ssdna probes, which then easily detach, leaving the aunps to aggregate due to the salt. the particle aggregation causes the red color of the solution to change to blue, indicating the presence of target sars nucleic acids; this change can be directly assessed by the naked eye or precisely quantified by ultraviolet-visible (uv-vis) spectroscopy in correlation to the target dna concentration (fig. 5a ). in the electrochemical assay, aunps are applied to enhance the electrode conductivity and increase the surface area available for detection probe immobilization [72] . sars-specific dna-capturing probes are first immobilized on the surface of an electrode structured with aunps; then, they are allowed to hybridize with the biotinylated targets. then, streptavidin-labeled alkaline phosphatase is applied to catalyze the indirect reduction and deposition of silver ions, which are eventually measured by anodic stripping voltammetry in correlation with the target dna concentration (fig. 5b) . the aunp-based assays developed for the molecular detection of sars are relatively rapid and simple; especially the colorimetric assay, which completely eliminates the need for instrumentation or trained personnel and yields results exclusively in the liquid phase that can be rapidly identified as positive/negative by the naked eye within 5 min. this technique can allow the detection of target sars nucleic acids with a sensitivity limit down to 100 fm; thus, it can be very beneficial for the early diagnosis of the sars virus, which is critical for such a contagious pathogen. in addition, the simplicity and very high sensitivity of aunp-based colorimetric assays for nucleic acids have promoted the application of aunps for the detection of other viruses either using slightly modified procedures or in combination with other sensing characteristics of aunps [56] . the family filoviridae can be classified into two main genera, ebolavirus [119, 122] . filoviruses are filamentous, enveloped rna viruses 80 nm in diameter and approximately 800-1000 nm in length. their genome is nonsegmented, negative-sense, ssrna 19 kb in size that encodes for at least 7 proteins (nucleoprotein, viral proteins vp24, vp30, vp35, and vp40, glycoprotein, and polymerase protein) [123] . aunps stabilized by single-stranded (ss)dna probes. in the presence of the target dna sequence, double-stranded (ds)dna is formed and desorbs from the surface of citrate-reduced aunps, leaving them to aggregate. this aggregation results in a color change from red to blue, indicating the presence of target nucleic acids [54, 56] . (b) enzymatic electrochemical detection of sars by anodic stripping voltammetry using a aunp-screen-printed carbon electrode. the aunp-modified electrode is modified with capture dna probes that are allowed to hybridize with biotinylated targets. streptavidin-alkaline phosphatase (s-ap) is then applied for the indirect reduction of silver ions in the solution into a metallic deposit. the formed silver metals are then electrochemically measured to determine the target virus dna concentration [72] . most ebovs primarily originate from africa, where they are frequently associated with large outbreaks of ebola hemorrhagic fever (ehf), which is now known to be one of the most deadly and virulent diseases affecting humans, with a case fatality approaching 90% [124] . the early outbreaks of ehf were confined to sudan and its neighbor, democratic republic of the congo, from 1976 to 1978, followed by several independent outbreaks in different countries: [124, 125] . most of these outbreaks were caused by zebov and sebov, while some were caused by new species later named ciebov and bebov [125] . these heavy series of ehf outbreaks are continuing, and most recently, multiple countries in west africa have struggled against a massive outbreak of ebov beginning in march 2014. ebola is expected to remain a major global public health threat, especially with the current absence of effective treatments; therefore, it is necessary to have flexible rapid-detection schemes in place to diagnose and control ebov outbreaks [126] . a very interesting aunp-based molecular assay has been developed for the bimodal scanometric and sers-based detection of ebov by using aunps to promote silver staining on their large surface area [50] . the target ebov dna sequence is first captured on chip surfaces by specific, short dna probes and labeled with aunp-dna-cy3 probes. then, the formed 3-component hybridization complexes are subjected to a silver enhancement step. the deposited silver metal eventually enables scanometric detection (based on a silver-caused dark color) and sers detection (based on using cy3 as a raman-active tag) (fig. 6) . this assay has additional benefits beyond its high specificity and sensitivity that reach down to 20 fm of the target rna. because a very large number of probes can be designed based on using different raman tags, this assay can be readily adapted for multiplex detection, allowing the simultaneous detection of different diagnostic targets of the same virus or even completely different viruses. furthermore, it does not require dna amplification and eliminates the need for specific thermal cyclic equipment. in addition, the capability for bimodal detection simplifies evaluation of the results, as the output can be positive/negative either by traditional scanner systems or by sers when the probes are labeled with raman dyes. such simplicity based on bimodal detection without the need for complicated thermal amplification equipment, multiplexing, and high sensitivity supports the application of this technique for ebov, which is very contagious and usually necessitates the detection of several targets. it is a chip-based detection scheme in which the target dna is captured by immobilized, specific dna oligonucleotides and then directly labeled by aunp-dna probes, followed by silver enhancement. the aunps enhance the surface area available for silver deposition and dna immobilization to achieve highly sensitive scanometric detection signals. modification of the aunp probes with cy3 allows the additional sers-based detection of target dna. this scheme has been reported for the detection of a wide range of viral nucleic acids, including those of hcv, hbv, and hav, which belong to different virus groups [50] . furthermore, the scanometric detection reported in this scheme has been considered a universal step in other detection schemes described for other viruses, such as hev [82] and hiv [81, 84] . this assay has been reported by the same authors for multiplex detection of hepatitis a virus (hav), hepatitis b virus (hbv), hiv, and variola virus (smallpox, vv), which confirms the broad capability of this assay for virus detection [50] . flaviviridae is a large virus family comprising three main genera: flavivirus (53 species, type species is yellow fever virus), hepacivirus (one species, hepatitis c virus; hcv), and pestivirus (four species, type species is bovine virus diarrhea) [127] . flaviviruses are spherical, enveloped rna viruses 40-65 nm in size. their genome is nonsegmented, positive-sense ssrna approximately 10-11 kb in size and includes one open reading frame (orf) encoding three structural proteins (c capsid; prm, premembrane; and e, envelope), and seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5). several well-known human pathogens are included in this family, such as dengue virus (denv), hcv, yellow fever virus, and west nile virus (wnv), japanese encephalitis virus, and tick-borne encephalitis virus. these viruses have become increasingly serious and have been frequently involved in causing many endemics and epidemics around the world [128, 129] . denv is the most rapidly spreading vector-borne virus in the world. denv is now known to be epidemic in more than 100 subtropical and tropical countries, threatening up to 2.5 billion people. denv causes more than 350 million new annual infections, including 90 million with symptoms varying from flu-like, mild, undifferentiated fever to classic dengue fever (df) or df with hemorrhagic manifestations [130, 131] . although many of these infections are self-limiting and resolve without hospitalization, some progress to severe disease that needs to be quickly identified and treated [132, 133] . currently, there are no specific treatments or protective vaccines available against denv, and as a result of its rapid expansion, accurate and sensitive diagnostic techniques have become crucial for effective control and treatment applications [134] . aunps have been combined with the well-known analytical techniques qcm and icp-ms to develop two novel molecular assays for denv detection. for the first time, these assays implemented aunps to increase the mass of target dna to allow its detection by qcm in a highly quantitative and sensitive manner (fig. 7a) or to amplify the detection signal by releasing many gold ions during icp-ms analysis of the target dna (fig. 7b ). in the aunp-based qcm assay, the presence of target dna initiates a layer-by-layer hybridization of the target dna and several specific multivalent aunp-dna probes, resulting in significant mass changes detected by qcm [93] (fig. 8a) . whereas, in the icp-ms-based assay, two types of probes prepared from specific aunp-dna and mnp-dna conjugates are applied to specifically recognize and sandwich the target virus dna sequences. the mnp probes then help to magnetically separate the formed sandwich complexes, and the target dna concentration is indirectly estimated by detection of the au concentration existing in the sample [94] (fig. 8b) . it is worth noting that both techniques are among the most novel applications of aunps in virus detection. the implementation of aunps extends the ability of qcm and icp-ms to detect dna. moreover, aunp-based icp-ms is among the most sensitive molecular dna assays applied for virus detection, with a detection limit down to 80 zmol, which can greatly help to control denv and other viruses [94] . the bio-barcode ssdna is released and employed to establish multilayered aunps over the electrodes to enhance the produced electric current [72] . (b) dry-reagent strip-based dna assay using aunp-oligo (dt) reporters. aunps with poly (dt) are applied to label biotinylated target dna modified with a poly (da)-tail pre-captured by immobilized streptavidin in the test zone of the strip and generate characteristic red bands [55] . (c) single-particle fluorometric detection of hcv using cy3-tagged ssrna that is electrostatically adsorbed or covalently coupled through thiol (sh) chemistry to the surface of aunps. the coupling of aunps and ssrna probes brings cy3 in the vicinity of the aunp surface and eventually results in emission quenching; upon hybridization with the target rna, the quenched emission is released [55] . hcv is one of the leading causes of chronic liver diseases in humans. approximately 3% of the global population has experienced hcv infection. currently, there are 170 million chronic carriers, and more than three million new infections occur globally each year [135, 136] . chronic hcv infection is frequently associated with marked hepatic injuries, including cirrhosis, liver failure or hepatocellular carcinoma. these types of injuries can be very severe and often end in death or liver transplantation [135, 137] . hcv infection is generally resilient and usually requires treatment that combines numerous medications. the most popular treatment regimen for hcv is a combination of pegylated interferon alfa (peg-ifnα) and ribavirin (rbv) [138] . other three-drug regimens based on the addition of the serine protease inhibitor telaprevir (tvr) or boceprevir (bec) to the standard peg-ifnα/rbv treatment have also been described [139] . in addition, several recently developed oral drugs, such as simeprevir and sofosbuvir, have been approved by the fda for more effective care and shorter treatment duration [140] [141] [142] [143] . despite these numerous drugs and due to the lack of efficient vaccines against hcv, reliable and sensitive detection methods are essential for viral infection prevention and therapeutic responses. numerous aunp-based scanometric, fluorometric, electrochemical, and colorimetric assays have been reported for the molecular detection of hcv (table 1 ). in these assays, aunps are utilized for different sensing functions: 1) aunps have been modified with poly(dt)-tailed dna and directly applied to label target dna sequences terminally modified with a poly(da)-tail in a lfa assay to allow the colorimetric detection of hcv (fig. 7b) [55] . 2) aunps electrostatically or covalently modified with cy3-tagged ssrna sequences have been applied for the fluorometric detection of target hcv sequences, in which the fluorophores are quenched in a process analogous to fret. the presence of cy3 in the vicinity of the surface of aunps eventually results in cy3-emission quenching. upon hybridization with target viral rna, the electrostatically adsorbed probes labeled with cy3 are detached from the surface of the particles, releasing the quenched fluorescence. the covalently bound probes form rigid dsrna with a linear configuration, causing cy3 to move away from the particle surface and release the quenched emission [60] (fig. 7c) . 3) aunps have also been applied to enhance electrical conduction and catalyze a reduction reaction on the surface of detection electrodes. a specifically designed hairpin dna probe terminally modified with aunps and bound to the surface of a glassy carbon electrode is utilized to detect the amplified viral rna. the presence of target dna results in the formation of rigid dsdna, and the hairpin conformation consequently changes, moving the aunps away from the electrode surface, which eventually appears as a detectable decline in the current value in proportion to the target dna concentration [78] . 4) aunps decorated with a specific nucleic acid probe were used for colorimetric detection of hcv. the assay is based on using cationic aunps to induce the aggregation of aunps probes. in the absence of hcv, the cationic aunps electrostatically bind to negatively charged aunps-dna probes causing their aggregation and change of the solution color to blue. the presence of hcv nucleic acid protects the probes and prevents their aggregation by cationic aunps and the solution color remains red. this assay was specific and showed a sensitivity of 93.3% and a detection limit of 4.57 iu/μl [13] . in the immunological detection of hcv, aunps dually modified with capturing antibodies and barcoding dna, similar to those described for the immuno-pcr technique, in combination with magnetic np (mnp)-antibody conjugates, have been applied to capture and separate the target antigen. subsequently, the barcode dna is further allowed to act as a bridge, guiding the formation of a multilayer of aunp-dna probes over the surface of a nanogap electrode (fig. 7a) . the formation of such barcode dna-guided aunp multilayers significantly increases the detected electrical current and is utilized to quantitatively measure the target hcv antigen concentration in applied solutions. the results of this immunoassay indicate a detection limit of 1 pg/µl [69] . in a similar protocol, the barcoding dna was further applied to hcv core antigen detection based on an enzyme to release the barcode dnas to be quantified with rt-pcr. this assay showed a detection limit of 1 fg/ml, which is one magnitude greater than the standard elisa (2 ng/ml) [14] . it is worth mentioning that the primary focus of the developed aunp-based assays was the detection of hcv rna rather than hcv antigens or antibodies. this is due to the broader potential of molecular assays for the detection and management of active hcv infections. hepadnaviridae comprises two main genera, orthohepadnavirus and avihepadnavirus. the genus orthohepadnavirus includes 4 species (hbv, woodchuck hepatitis virus, ground squirrel hepatitis virus, and woolly monkey hbv) that infect mammals, while the genus avihepadnavirusincludes2 species (duck hbv and heron hbv) infect birds [144] . hepadnaviruses are spherical, enveloped dna viruses 40-48 nm in size. they possess a unique gapped genome of 3.2-kb circular dsdna, which contains 4 partly overlapping orfs encoding the different viral proteins: the core protein (c), e antigen, polymerase protein (pol), envelope proteins, and transcriptional transactivator protein [144, 145] . the prototype member of the family hepadnaviridae, hbv, is a health threat to ~88% of the global population [145, 146] . hbv is the etiological agent of hepatitis type b in humans. it is estimated that nearly 2 billion people around the world have been exposed to hbv infection, and there are more than 360 million chronic carriers suffering from the serious risk of developing liver cirrhosis and cancer [146, 147] . the availability of an effective protective vaccine against hbv has greatly reduced the number of new infections. in addition, there have been significant advances in the available treatments for chronic hbv infection. viral dna polymerase inhibitors and peg-ifnα are now well known to significantly control hbv infection and prevent its progression to chronic hepatitis b-associated liver failure and hepatocellular carcinoma [148, 149] . however, chronic hbv infections remain highly contagious, and the virus can easily be transmitted to other persons through any close contact allowing the transfer of infected bodily fluids. the highly contagious nature of hbv and its ability to spread among people are the main reasons why this pathogen continues to threaten the public. therefore, diagnostic methods that are highly sensitive to low concentrations of virus are needed to curtail this virus and prevent its dissemination. hbv is by far the most common virus reported in the published literature regarding the applications of aunps in virus detection (fig. 3) . interestingly, the developed assays cover most of the known diagnostic markers of hbv through many different electrochemical, scanometric, fluorometric, lightscattering, colorimetric, and sers detection schemes (table 1 and fig. 6, 14-18) . several aunp-based electrochemical assays have been developed for the detection of hbv antigens and dna. two enzyme-amplified electrochemical assays have been reported for the detection hbv surface antigen (hbsag) using conjugates of aunps and horseradish peroxidase (hrp)-labeled antibodies against hbv surface antigen (hbsabs) [76, 77] . the target hbsag is captured and labeled with aunp-hbsab/hrp as secondary antibody conjugates. then the reduction of h2o2 catalyzed by the bound hrp is measured either by using an aunp/thionin/dna-modified au electrode coupled with a cyclic voltammeter [76] or through using a nanoporous au electrode coupled with differential pulse voltammeter [77] (fig. 9a) . alizadeh et al. reported aunps modified with horseradish peroxidase mimicking dnazyme to label hbsag magnetically captured and concentrated on the surface of a au sheet-like electrode. due to the efficient catalytic activity of hrp-mimicking dnazyme, the proposed immunoassay allowed quantitative detection of hbsag with a linear concentration range of 0.3-1000 pg/ml and detection limit of 0.19 pg/ml [15] . other studies described the electrochemical detection of hbv based on aunp-metal enhancement amplification, including copper and silver metal enhancement [74, 75] . using copper metal enhancement, hbsag is sandwiched by mnp-hbsab conjugates and aunp-hbsab probes to form a 3-component immunocomplex, which is magnetically separated and stained through a copper enhancement step (fig. 9b ). after copper acidic dissolution, the resulting ions are measured by anodic stripping voltammetry (asv) [75] . using silver enhancement, hbv dna is detected using probes prepared of streptavidin-modified aunps. biotinylated hbsag gene sequences are magnetically pre-separated and concentrated using magnetic bead-dna conjugates. the deposited silver ions are then analyzed by an electrochemical stripping technique to detect the target hbv dna quantitatively [74] . hbv detection through aunp-based scanometric assays mainly relies on the well-described aunp-promoted silver-staining protocol, in which aunp probes are applied to facilitate the reduction of silver ions into metallic silver that is deposited as visible black spots, indicating the presence of hbv. following this protocol, aunps modified with staphylococcal protein a (spa) [88] or specific dna probes [50, 80] have been utilized in multiple chip-based assays to permit the detection of different hbv antibodies and dna (fig. 6 ). in addition, wang et al. combined this aunp-promoted silver-staining protocol integrated with a bio-barcode-amplification (bca) technique to allow an enhanced scanometric detection of hbv dna [85] . this bca-based scanometric assay employs two different sets of dna conjugates to capture and detect a specific hbv sequence. the first set is a group of aunps modified with the short dsdna of a signal amplification dna strand (barcode dna) partially complemented with a detection probe strand, while the second set is a group of magnetic microparticles (mmps) modified with ssdna specific to the target hbv dna. the presence of the complementary target sequences of dna guides the assembly of aunp/dna/mmp sandwich hybrids. subsequently, the sandwiched hybrids are magnetically isolated and washed, and the barcode dna is eventually released from the aunps. the barcode dna is then added to the surface of a chip modified with specific dna-capturing probes and directly labeled with aunp-dna conjugates, followed by silver staining to further amplify the detection signal (fig. 10) . the aunps-fluorometric assays for hbv are performed through different schemes that basically depict the quenching efficiency of aunps in a manner remarkably similar to the traditional fret protocol. zheng et al. developed a multiplex detection system based on applying gold nanorods (aunrs) as acceptors and qds of different colors as donors to simultaneously detect hbsag and hbv e antigen [62] . this assay follows the direct sandwich immunoassay format, and the presence of target antigens is manifested by the fret-induced quenching of qd fluorescence in the formed sandwich nanostructure of aunr-ab1/ag/qd-ab2 (fig. 9c ). draz et al. further coupled multiple aunp-peptide conjugateacceptors with single-core qd-antibody fab conjugate-donors, forming a preassembled hybrid nanocluster plasmonic resonator complex for hbsag and hbv particle detection (fig. 9c) . this scheme follows a competitive assay format, and the addition of hbv target surface antigen or particles to the preassembled nanocluster releases the quenched fluorescence signal of the qds in proportion to the in this assay, the target hbsag is captured and labeled with aunp-ab/hrp, and the detection signal is measured by using an ab-modified au electrode to assess the reduction of h2o2 catalyzed by the bound hrp using differential pulse voltammetry (dpv) technique [76] . (b) copper (cu)-enhanced electrochemical immunoassay. aunps serve as a scaffold for a horseradish peroxidase (hrp) enzyme that catalyzes a redox reaction in the first scheme or enhances metal deposition in the second scheme [75] . (c) fluorometric detection by the fret-induced quenching of fluorophores on the surface of aunps. aunps and gold nanorods (aunrs) are applied to quench the fluorescence of fluorophores, such as (i and ii) quantum dots (qds) [62, 65] and (iii) fluorescein amidite (fam) [61] , through a fret-based interaction in the presence of the target virus. a similar aunp fret-based scheme has been reported for the detection of h1n1, which belongs to the family orthomyxoviridae [63] . (d) light-scattering assays using aunp-antibody probes. (i) light-scattering immunoassay of virus antigen based on using aunps with different sizes (10 nm and 50 nm) conjugated with antibodies (abs) to sandwich target antigens and enhance the dynamic light scattering [51] . additionally, this scheme has been reported with the use of aunps of the same size for h1n1 detection [53] . (ii) plasmonic immunoassay based on allowing aunr-ab conjugates to interact with the target antigen, thus changing the aunr localized surface plasmon resonance (lspr) behavior [52] . target concentration [65] . furthermore, lu et al. developed a fluorometric assay for hbv dna detection in which positively charged aunrs are employed to chelate fluorescein amidite (fam)tagged ssdna electrostatically onto their surface, forming fam-ssdna-aunr ternary complexes [61] . the formation of these complex results in the fret-based quenching of fam fluorescence, and when complementary target dna is present in the system, a fam-ssdna/cdna duplex is formed. this dna duplex is comparatively more negative than the fam-ssdna and can mediate stronger electrostatic interactions with aunrs, leading to increased fret efficiency, which manifests as a further decline in the fluorescence intensity of fam. this change in fluorescence intensity is utilized to sense and estimate the exact concentration of the added target dna (fig. 9c) . aunp-based light-scattering assays have been described for the detection of hbsag through two main schemes: the first scheme applies spherical aunps of different diameters (10 nm and 50 nm) modified with hbsab to sandwich hbsag, and the presence of hbsag results in specific immuno-based aggregations that can be estimated by measuring the increases in hydrodynamic diameter using dls (fig. 9d) [47, 51] ; the second scheme relies on the lspr characteristic behavior of aunrs to directly sense the binding of hbsag to the antibodies conjugated to their surface by measuring lspr peak shifts via uv-vis spectroscopy (fig. 9d) [52] . a aunps-based colorimetric lateral flow assay (lfa) assay was developed for the detection of hbsag. this assay relies on the characteristic red color of different sized aunps to label hbsag on an immunochromatographic test strip. a strip composed of a sample pad, conjugation pad, control line, test line and absorbent pad was used to capture hbsag labeled with aunps and the test line becomes red in color. the results showed that the signal visibility of lfa could be improved by increasing the diameter of aunps, and ~43 nm aunp enabled lfa assay with a detection limit of 100 ng/ml [16] . a aunps-based sers assay for hbv dna detection was developed following a sandwich assay format performed on a heat-responsive hybrid silicon substrate modified with aunps. the target hbv dna is captured on the surface of the silicon substrate and forms a sandwich complex with specifically designed aunps modified with dnaindocyanine green proximally to the surface, leading to high sers signals. this heat-responsive hybrid silicon substrate-based sers platform can detect remarkably low hbv dna concentrations of ~0.44 fm at 25 °c and ~0.14 fm at 37 °c [17] . aunp-based fluorometric detection is the most sensitive among the reported methods for hbv detection, with a detection limit of 1 particle/μl. due to the highly infectious nature of hbv, this limit of detection can be very useful for exploiting these assays for efficient control and management of hbv infection. in addition, the simplicity of scanometric and colorimetric detections is very interesting, allowing flexibility in the detection design and target, and was reported for single and multiple target detection for more efficient and accurate screening of hbv. on the other hand, the developed dls assay is very novel to hbv research, simple and sensitive; however, the need for bulky equipment for dls measurement remains the major challenge for its wide use and real application in hbv detection. the family hepeviridae is monogeneric with one exclusive member called hev, which is a spherical, small, nonenveloped rna virus with a ~7.2 kb ssrna genome [150] . the viral rna is a positive-sense molecule organized into 3 partially overlapping orfs flanked by short untranslated regions at both ends. the first orf encodes 4 mature nonstructural proteins (methyltransferase, protease, helicase and replicase), the second orf encodes 2 structural proteins (c and vp1), and the third orf encodes a small protein of unknown function [150] . hev is known for causing human liver inflammation figure 10 . aunp-based nucleic acid assay for hepatitis b virus (hbv) detection. hbv dna was detected using a scanometric detection that integrates magnetic separation and aunps-based bio-barcode-amplification method. aunps provide a large surface area for silver deposition and dna immobilization, thereby achieving highly sensitive scanometric detection signals through a chip-based silver deposition scheme [85] . called hepatitis e, which is widely disseminated in developing countries with poor sanitation conditions and has recently become apparently endemic in some industrialized countries, including the usa [151] . one aunp-based colorimetric assay has been reported for the molecular detection of hev [152] . this assay targets a conservative fragment of hev in orf1 that is pre-amplified by one-step rt-pcr/pcr amplification, and the generated cdna is labeled by aunp-dna conjugates, forming three-component sandwich hybrids in a manner very similar to that previously depicted in fig. 6 . this hybridization is followed by a silver-staining step to allow the scanometric detection of target dna either by the naked eye or using simple scanners, with a detection limit of 100 fm of hev amplicon. one of the major advantages of this hev detection assay is its sensitivity, which can help control hev. however, this assay suffers from the same disadvantages of conventional pcr because of the requirements of the amplification step. herpesviridae is a taxonomically large family of viruses, including 3 major subfamilies (alphaherpesvirinae, betaherpesvirinae and gammaherpesvirinae), which together comprise nearly 9 genera with over 100 viruses [153] . repeats. the rearrangements of these repeated sequences result in different genome isomers. in epstein-barr virus (ebv) and kaposi sarcoma-associated herpes virus (kshv), from the subfamily gammaherpesvirinae, there are repeated sequences at the termini of their genomes that differ from those of other human pathogenic herpesviruses, including herpes simplex virus type 1 (hsv-1) and type 2 (hsv-2), human cytomegalovirus (hcmv), and varicella zoster virus (vzv); these viruses are characterized by the definite presence of us and ul regions in their genomes [153] [154] [155] . herpesviruses can infect humans and many animals. they account for significant and recurrent human cutaneous diseases, including oral herpes, genital herpes, and many cancers. eight herpesviridae family members are commonly involved in human infection: hsv-1 (human herpesvirus1; hhv-1), hsv-2 (hhv-2), vzv (hhv-3), ebv (hhv-4), hcmv (hhv-5), hhv-6, hhv-7, and kshv (hhv-8). hsvs are among the most prevalent viruses in humans. there are two serotypically and genetically different hsvs, types 1 (hsv-1) and 2 (hsv-2) [156] . hsv-1 is estimated to infect up to one-third of the world population, while hsv-2 infects nearly 500 million people around the globe, with more than 20 million new cases occurring every year [157] [158] [159] . both hsv types are renowned for infecting epithelial cells of the skin or mucosal surfaces and then infecting the central nervous system, causing lifelong, incurable infections in humans. these infections are primarily asymptomatic but can progressively result in serious health complications. for example, hsv-1 infection is the main cause of infectious blindness in the world and has emerged as an important cause of genital disease in the developed world, and hsv-2 infection is the leading cause of genital ulcers worldwide [159, 160] . nevertheless, these viruses are involved in many other clinical conditions, such as encephalitis, conjunctivitis, zosteriform skin lesions, pneumonia, and systemic infections that compromise vital organs in the body [161] . therefore, desperate efforts have been directed toward developing an effective vaccine against hsvs. however, there are no protective vaccines against hsvs, and only a few anti-herpes drugs, such as acyclovir, valacyclovir, and famciclovir, are commercially available [162] [163] [164] . these drugs are only helpful to control symptoms and signs of infection and do not eradicate the virus or even decrease the frequency or severity of infection recurrence and due to the drug resistance rapidly developed by hsvs during treatment, the efficacy of anti-herpes drugs is increasingly hampered over time. thus, the definitive diagnosis of hsv virus infection is essential for effectively controlling its severity and applying treatment regimens. with their characteristic red color, aunps have been exploited as colorimetric labels in a lateral-flow immunochromatographic assay (lfia) to allow the immunological detection of hsv antigen. in this assay, the lfia strip is preloaded with the labeling aunp-anti-human igg conjugates onto the conjugate pad, and the capturing igg-1 and igg-2 antigens, along with the anti-igg antibody, are immobilized onto separate test and control lines directly next to the sampling area (fig. 11a) . the sample is loaded and allowed to migrate across the strip with the chase buffer to react with the capturing antigens (igg-1 and igg-2) loaded on the test lines, as well as the anti-igg antibody loaded on the control line. as a result, hsv antibodies are captured on the test lines and labeled with the loaded aunp-dna conjugates, causing characteristic red lines to appear. according to the color of the test lines, the samples can be qualitatively determined to be positive or negative within 20 min [59] . lateral-flow immunochromatographic assay of hsv-2 antibodies using aunp anti-igg probes. sample addition followed by chase buffer addition causes gold nanoparticle (aunp)-anti-igg conjugates and the sample to migrate, contacting the test lines sequentially and resulting in the capture of type-specific antibodies to hsv-2. the concentration of hsv-2 antibody-aunp complexes on the test line causes a corresponding pink color to form [59] . this scheme is similar to that reported for the detection of h5n1 [58] and hiv-1 [91] . (b) electrochemical detection of hcmv by the acidic dissolution of aunps. hcmv dna is directly labeled with specific aunp-dna probes. after the acidic dissolution of aunps, au iii ions are measured by anodic stripping voltammetry (asa) [68] . (c) colorimetric one-pot np-based assay of kshv. this assay has been reported for the differential diagnosis of kshv dna and dna from a frequently confounding bartonella species. upon the addition of sample dna to a mixture of silver np (agnp)-dna and aunp-dna conjugates specific for kshv and bartonella dna, respectively, complementary dna hybridization triggers the aggregation of bare np conjugates and the subsequent decrease or disappearance of its corresponding color [60] . hcmv/hhv-5 is the largest known endemic virus in humans. it persistently infects over 70% of the population worldwide [165] . hcmv can infect a broad range of cells, including endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, neuronal cells, hepatocytes, and many types of immune cells [165, 166] . nearly all hcmv infections are asymptomatic, except in immunocompromised individuals and neonates, and can lead to severe symptoms. hcmv infection is the leading cause of congenital malformations in live births worldwide, while in immunocompromised individuals hcmv is usually opportunistic, causing very serious clinical manifestations, such as pneumonia, hepatitis, encephalitis, colitis, and retinitis [167, 168] . moreover, hcmv has been described to be associated with several human malignancies, especially in the brain, breast, prostate, skin and colon [169] . numerous hcmv vaccine candidates have been developed and tested, but a vaccine has yet to be licensed. antiviral therapy with several anti-hcmv drugs, such as ganciclovir, valganciclovir, foscarnet, and cidofovir, which target the viral dna polymerase, is currently the main strategy for controlling hcmv infection [166, 170, 171] . unfortunately, these drugs result in substantial hematotoxicity and nephrotoxicity in patients, and hcmv remains a considerable public health threat without an efficient treatment or control strategy. in such conditions, the need for new diagnostic methods that can help to control the dissemination of this pathogen is very important. a novel aunp-based electrochemical assay for the molecular detection of hcmv dna has been reported [68] . in this assay, aunps are applied to amplify the detected electrochemical signal by releasing many au ions upon exposure to acid dissolution. typically, the immobilized target hcmv ssdna is initially labeled with oligonucleotidemodified aunp probes followed by a metal dissolution step, releasing many gold ions. the concentration of solubilized gold ions is then indirectly assessed by asv (fig. 11b ). this assay allows the highly sensitive detection of target dna concentrations down to 5 pm [68] . kshv/hhv-8 is a leading cancer-causing virus. it is commonly responsible for inducing a considerable range of neoplastic diseases in immunocompromised patients, such as kaposi sarcoma, primary effusion lymphoma, and some kinds of multicentric castleman disease [172, 173] . owing to the dramatically increasing rates of immunocompromised patients over the past two decades, the clinical significance of kshv is clearly increasing; additionally, kshv has a prevalence of 3-40% in this class of patients worldwide [173, 174] . while kshv is a relatively newly discovered virus, there are no standard diagnostic approaches, and the detection of this virus through traditional serological and molecular methods remains challenging. this difficulty is mainly due to the inconsistency observed among serological assays targeting different antigens, and the low levels of viral dna that are usually present in the peripheral blood of infected individuals [175, 176] . hence, there is a continued interest in developing new reliable and sensitive methods for kshv detection. through using a mixture of aunps and silver nps (agnps), a colorimetric assay has been established for the multiplex, one-pot molecular detection of kshv and its frequently confounding disease bacillary angiomatosis [57] . in this assay, two types of probes are prepared from aunps and agnps separately functionalized with kshv and bartonellaspecific dna-capturing dna probes, respectively, and mixed with the target dna. then, the color of the reaction mixture changes in a manner dependent on the type of target dna added to the reaction solution: the addition of kshv dna causes aunp conjugates to aggregate and the solution to appear murky yellow-orange, i.e., the specific color of the nonaggregated agnps, while the addition of bartonella dna causes agnp conjugates to aggregate and the solution to appear pink, i.e., the color of nonaggregated aunps (fig. 11c ). in addition to being simple, this multicolor-change system successfully allows the multiplex detection of targets present in nanomolar concentrations. the family orthomyxoviridae includes five genera: influenzavirusa, influenzavirus b, influenzavirus c, isavirus, and thogotovirus [177] . the first three genera are widely known to infect birds, humans, and other mammals, causing influenza. the influenza-virusa genus contains many strains and can be further divided into several antigenic subtypes according to the antigenic properties of their envelope glycoproteins, i.e., hemagglutinin (ha) and neuraminidase (na). in contrast, the influenzavirusb genus has no distinct antigenic subtypes of hemagglutinin or neuraminidase. influenzavirusc is similar to types a and b but less common and lacks the na protein. the genus isavirus is known to infect salmon, while thogotoviruses usually cause infection in some vertebrates and invertebrates [177, 178] . orthomyxoviruses can be spherical (80-120 nm) or filamentous (20 nm in diameter and 200-300 nm long) in shape, and their genome contains 7 or 8 segments of linear negative-sense ssrna with a total length of 12-15 kb encoding different structural and nonstructural proteins, including nucleoprotein, three large proteins (pb1, pb2, and pa), matrix proteins (m1 and m2), the glycoproteins ha and na, and two nonstructural proteins (ns1 and ns2) [177, 179, 180] . several strains of the genus influenzavirusa cause seasonal and occasional acute respiratory diseases called influenza, which is profoundly and rapidly spread in humans. influenza is reported to affect up to 5% of adults and 20% of children of the global population each year [181, 182] . over the past decade, the world witnessed two major influenza outbreaks caused by newly emerging influenzavirusa strains: h5n1 in 2003 (avian influenza), and h1n1 in 2009 (swine influenza). both were severe enough to incite the who to issue a pandemic warning alert, which reached the highest level, i.e., "phase 6," for h1n1 in 2009 [21, 183] . the severity of influenza infection depends on both the virus strain and the age of the infected person. in elderly persons, influenza can result in hospitalization or death, while in younger adults, influenza-associated morbidity usually results in restricted activity, impaired work performance, and increased health care use [184, 185] . most people with the flu recover within one to two weeks without treatment. however, serious complications usually require medications to reduce the severity and duration of infection. some antiviral drugs, such as oseltamivir and zanamivir, can effectively treat or prevent influenza [186] . on the other hand, immunization with vaccine preparations of the most-circulating virus strains (trivalent or quadrivalent vaccines) remains the key strategy for preventing both influenza and its serious complications [187, 188] . in fact, both the treatment and management of influenza infection, either by antivirals or vaccination, depend on the influenza virus strains and the timing and severity of the infection. therefore, the rapid and accurate testing and typing of influenza is critical for treating infection and plays an important role in public health measures. the application of aunps in iav detection has primarily been focused on developing assays that enable the direct detection of whole iav particles [53, 58, 92] or viral rna [64, 66, 67, 70, 71, 83, 86] through numerous aunp-based fluorometric, sers, scanometric, light-scattering, colorimetric, and electrochemical detection schemes ( table 1) . the aunp-based fluorometric assays developed for h5n1 detection have simply exploited the intense plasmonic-based quenching properties of aunps through different detection formats. one of these assays targets the detection of the whole virus particle through a lateral-flow immunoassay format that is similar to that previously described for the detection of hsv-2, as shown in fig. 11a . in this assay, the virus is first captured on a lateral-flow test strip by pre-immobilized antibodies; then, aunp-antibody conjugates are applied to label the captured virus particles. after washing, the aunps are collected and dissolved to release gold ions that are eventually utilized to quench the fluorescence of qds in an additional, separate step [58] . in addition, ganbold et al. have developed a bimodal assay for the fluorometric and sers detection of viral rna using aunps modified with ssdna-dye probes [64] . the surface ssdna-dye probes are weakly adsorbed to the surface of aunps by electrostatic interactions, and with the addition of the complementary target dna, perfectly matched dsdna forms and desorbs from the surface of aunps, resulting in measurable changes in the conjugated dye fluorescence and raman fingerprint that are more substantial than those resulting from either mismatched dsdna or ssdna (fig. 12a) . aunp-based colorimetric assays have been developed for iav detection through four main detection schemes: 1) the first scheme involves the application of aunps conjugated with anti-digoxigenin as colorimetric labels to detect the target viral dna premodified with digoxigenin and electrophoresed through a membrane-based lateral-flow technique [66] . 2) the second scheme is simply based on the electrostatic interactions between the negatively charged phosphate groups of the target dna amplified with lamp and the positively charged ctab bilayer on aunrs, resulting in the aggregation of aunrs and a color change from red to purple [67] . 3) the third scheme relies on a magnetic-based hydroquinone oxidation reaction involving two metal np bioconjugates: iav-specific pentabody-mnps as capture probes and anti-aiv monoclonal antibody-aunps as detection probes. in the presence of virus particles, an immunocomplex forms and is separated by a magnetic field; after washing, aunps in the separated complex act to catalyze the oxidation of hydroquinone, which is detected optically in correlation with the aiv sample concentration [92] . 4) the fourth scheme relies on the peroxidase-like catalytic activity of au np films and colloidal aunps toward tmb-h2o2 mixtures. the virus is captured on a 96-well plate modified with aunp film biofunctionalized with ha antibody. then it is labeled with aunps modified with na antibody for signal generation. tmb-h 2 o 2 is added, and rapid color changes are observed because of the oxidation of peroxidase substrate tmb. using h1n1, the response of the developed assay was linear in the range from 10 pg/ml to 10 μg/ml and the limit of detection was 50.5 pg/ml, while with h3n2, the optical density of the developed color was dependent on the virus concentration in the range of 10-50,000 pfu/ml and the limit of detection was 24.3 pfu/ml [22] . aunp-based electrochemical assays rely on using aunps to directly modify the target dna or electron transducer surfaces, allowing enhanced electrical conduction and increased surface area for probe immobilization and target interaction. following this protocol, liu et al. [71] successfully detected aiv ha gene sequences using a dna aptamer immobilized onto an electrode; its surface was modified with a composite of carbon nanotubes, polypyrrole nanowires and aunps (fig. 12b) . furthermore, bonanni et al. [70] developed an impedimetric electrochemical assay for the detection of aiv m gene sequences based on measuring changes in the impedimetric behavior of the electrode when the target dna hybridizes with the capture dna probes immobilized onto its surface and is subsequently labeled by aunps via streptavidin/ biotin interaction (fig. 12c) . aunp-based scanometric assays for detecting aiv are mainly performed using the well-described aunp-promoted metal staining protocol shown in fig. 12d . in this protocol, the target nucleic acid is recaptured on the surface of glass chips modified with specific capture dna probes [86] . then, the captured probes are labeled with aunps either modified with specific dna probes or with the renowned dsdna intercalator daunorubicin [83] to mediate the metal staining. following this labeling step with aunp probes, the presence of the target nucleic acids is visually determined with an additional silver-or gold-staining step. specific dna aptamers immobilized on a gold electrode (ge) modified with a layer of multiwall carbon nanotubes (mwcnts), polypyrrole nanowires (ppnws) and aunps are collectively applied as an electrochemical platform for the detection of dna hybridization using dpv [71] . (c) impedimetric electrochemical assay using streptavidin-aunps probes. the direct/sandwich hybridization of biotinylated target dna with capture probes immobilized on the surface of a screen-printed electrode (spe) and additional conjugation with streptavidin-aunps results in impedimetric signal enhancement [70] . (d) label-free scanometric detection using positively charged aunps. the electrostatic interaction between positively charged aunps and negatively charged dna is utilized to visualize the double-stranded (ds)dna formed by the hybridization of single-stranded probe dna and complementary target dna on the array of chips through aunp-mediated metal staining [83, 86, 87] . the light-scattering detection of h1n1 virus using aunps relies on dls to measure the aggregation of aunp-antibody conjugates induced by the addition of target virus particles (similar to fig. 9d ). the magnitude of aunp aggregation and the corresponding increase in their hydrodynamic size is correlated with the concentration of virus particles [53] . finally, the immunological detection of h1n1 hemagglutinin (ha) antigen has only been reported through a novel lspr-based immunoassay. in this assay, the presence of target antigen induces a sandwich immunoreaction between the fluorophorelabeled detection antibodies and the aunp-capturing antibody conjugates, directly resulting in enhanced fluorescence caused by the coupling of fluorophore emission with the localized surface plasmons of aunps in a manner similar to that shown in fig. 9c . the sensitivity of this method has been described to be 10 3 -fold, improved over that of the conventional elisa technique, with a reported limit of detection of 13.9 pg/ml [63] . papillomaviridae is a widely diverse virus family that includes hundreds of recognized viruses classified into 16 taxonomically distinct genera designated with specific greek alphabet letters, from alpha to pi. the first five genera (alphapapillomavirus, betapapillomavirus, gammapapillomavirus, mupapillomavirus and nupapillomavirus) include the human papillomaviruses, while the remaining genera specifically include other mammalian and avian papillomaviruses [189, 190] . papillomaviruses are spherical, nonenveloped dna viruses 40-55 nm in size. their genome consists of a single molecule of circular, supercoiled, dsdna 5.3-8 kbp in size and encodes 6 early expressed (e) and 2 late (l) expressed proteins, in addition to a noncoding long control region [189, 191] . hpv infections are the most epidemic sexually transmitted viral infections worldwide [192, 193] . hpvs are highly epitheliotropic pathogens that usually infect the deepest layer of the skin or the genital surfaces. most hpv infections cause no symptoms and usually self-clear without causing clinical problems, but persistent infections lead to serious clinical complications [194] . this persistence typically depends on the type of hpv causing the infection. among more than 100 types of hpvs, approximately 40 types infect the mucous membranes and can be either high-or low-risk hpvs. high-risk types, such as hpvs 16, 18, 31, 51 and 52, are usually associated with many cancers of the cervix, vagina, vulva, penis, anus, neck, and head, as well as oropharyngeal cancer, while low-risk types, such as hpvs 6 and 11, are mainly related to anogenital warts and recurrent respiratory papillomatosis [195, 196] . currently, hpv is considered the most important cancer-causing virus, as it is associated with ~4.8% of all human cancers [197] . despite this high clinical significance of hpv, there are no available treatments, and vaccination using the bivalent vaccine against hpvs 16 and 18 or the quadrivalent vaccine against hpvs 6, 11, 16, and 18 remains the first and only choice for controlling the global hpv disease burden [198] [199] [200] . in fact, these vaccines are relatively expensive, and their efficacy is very restricted to specific hpv types. therefore, the development of accurate diagnostic techniques for hpv detection and typing is essential for acquiring surveillance data and initiating appropriate vaccination programs. aunp-based assays have been developed for the molecular detection of hpv through different photoelectric, fluorometric, electric and colorimetric schemes [89, 98] . the photoelectric assay essentially leverages aunps to enhance the deposition of silver metal on their surfaces, as previously described for several other assays. however, the enhanced precipitation of silver metal in this assay is used to block the light generated from a photodiode array (pda), and the target dna concentration is measured in proportion to the decrease in light intensity as electrically detected using a bipolar integrated circuit pda chip [89] . specifically, pcr-amplified biotinylated target dna is captured on the surface of the pda chip, and then labeled with aunp-biotin antibody conjugates; subsequently, a silver enhancement step decreases the light intensity, which is quantitatively translated into an electrical signal depending on the amount of target dna (fig. 13a) . on the other hand, a fluorometric assay integrating aunps and a microfluidic bead-based array has been developed as a rapid and controlled hpv dna detection system [98] . in this assay, aunps dually functionalized with hrp enzyme and capture dna strands are applied to label the target dna captured on the surface of microbeads (fig. 4a) , causing hrp to be in proximity to biotin-tyramine loaded on the surface of the microbeads and catalyze their oxidation by hydrogen peroxide. this process eventually increases the deposition of biotin moieties on the surface of the beads, subsequently increasing the labeling efficiency with qd-streptavidin conjugates and yielding an enhanced fluorescence signal that varies significantly based on the target dna concentration (fig. 13b) . in the electrical detection of hpv, aunps were used as an immobilization scaffold for dna immobilization on a silicon substrate carrying nanogapped interdigitated electrodes. this aunps deposition technique was shown to reduce the size of the nanogaps, allowing enhanced electric detection of the target dna captured on the silicon substrate by specific hpv dna probes. the results demonstrated that this assay was able to specifically detect hpv dna within 30 min and at concentrations down to 1 pm [201] . based on the color change of aunps upon aggregation, aunps carrying hpv dna probe were coupled with the lamp technique for colorimetric detection of hpv. lamp reaction was performed at a temperature of 65°c for 20 min and the generated products were hybridized with aunp-dna probes for 5 min. then the presence of the target dna was detected by the addition of magnesium salt. the addition of the salt causes aunps aggregation and the color changes from red to blue. the presence of lamp amplicons can protect aunps from aggregation and prevents aunps aggregation. the sensitivity of the developed lamp-aunp assay was greater than the lamp turbidity assay by up to 10-fold with detection limits of 10 2 and 10 0 copies for hpv16 and hpv18, respectively [27] . the family picornaviridae is taxonomically very diverse and has recently been expanded to involve 12 genera: eight are originally identified genera (aphthovirus, cardiovirus, enterovirus, erbovirus, hepatovirus, kobuvirus, parechovirus, and teschovirus), and four newly proposed genera (avihepatovirus, sapelovirus, senecavirus, and tremovirus) [202] . picornaviruses are spherical, relatively small (22-30 nm) , nonenveloped rna viruses. their genome is positive-sense ssrna that is 7.0-9.5 kb in size and terminally bonded to a noncapsid viral protein (vpg) at its 5' end and to a polyadenylated tail at its 3' end. the expression of this genome produces a single large polyprotein that undergoes a cascade of cleavage and processing reactions to form 10-12 final structural (vp1-4) and nonstructural (2a-c and 3a-d) proteins [203] . picornaviruses can infect a broad spectrum of hosts, including birds, humans, and other mammals [202, 204] . the genera enterovirus, hepatovirus, parechovirus, kobuvirus, and cardiovirus include several important species that affect humans. particularly, the genus hepatovirus encompasses hav, which is the most likely causal agent of acute viral hepatitis in humans. hav infection is usually asymptomatic and self-limiting, and the related symptoms can be mild or sporadically progress to fulminant hepatic failure [205, 206] . the availability of an effective vaccine against hav has substantially reduced the number of people who become infected with hav [207, 208] . however, the ability of this virus to survive harsh environmental conditions and remain in seawater, fresh water, wastewater, and soil for extended periods increases the possibility of outbreaks, especially in developing countries where sanitation is not typically available [205] . the transmission of the virus usually occurs through the consumption of contaminated materials. as such, the virus does not replicate in the environment, water, or foods, and traces of viral contamination are difficult to identify, which remains a challenge for controlling hav. the need for sensitive assays for hav remains an urgent public health demand. hav detection using aunps was accomplished through scanometric and sers-based assays. both assays rely on the extensively described virtue of aunps to enhance silver staining, thereby allowing the molecular detection of hav dna in a microarray chip format (table 1 and fig. 6 ). in the scanometric aunps modified with anti-biotin igg are applied to label biotinylated target dna captured on the pda chip. after a silver enhancement step, the precipitated silver metal at the surfaces of the aunps blocks the light irradiated from above by light-emitting diodes (leds) and decreases the resulting photocurrent [89] . (b) microfluidic bead-based enzyme-aunp amplification method for the fluorometric detection of hpv. streptavidin-coated beads are dually functionalized with biotin-electron-rich protein and biotin-dna capture probes. the captured target dna first reacts with horseradish peroxidase (hrp)-functionalized aunp-dna labels, bringing hrp close to the surface of the microbeads. the oxidation of tyramine-biotin by hydrogen peroxide results in the deposition of multiple biotin moieties onto the surface of the beads. this deposition is markedly increased in the presence of immobilized electron-rich proteins. streptavidin-labeled quantum dots (qds) are then allowed to bind to the deposited biotin moieties and display amplified fluorescence signals in relation to the target dna concentration [98] . detection scheme, aunp-dna conjugates are applied as specific detection probes to label target hav rna captured by specific vp1 probes on the surface of a microarray chip. a silver metal enhancement step is subsequently used for signal amplification, followed by the visual detection of nucleic acids [79] . in the sers detection, the captured hav dna is labeled by aunp-dna probes loaded with the raman reporter dye cy3; this approach has been applied as previously described for ebov and depicted in fig. 6 of this review [50] . these assays are very simple and can detect target concentrations ranging from 3.37×10 -7 -3.37×10 -8 m, with 100 fm as a limit of detection, which can be superior to the relatively expensive pcr-based approaches currently used for hav detection and management. the family retroviridae is broadly classified into 7 genera (alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus, lentivirus, and spumavirus), which include nearly 51 viruses [209] . retroviruses are spherical, small (80-100 nm), enveloped rna viruses. their viral genome is typically bipartite and composed of two positive-sense ssrna molecules (10 kb in total length). they comprise 4 major genes that encode the different structural and nonstructural proteins of the virus: gag encodes the large protein form matrix, capsid, and nucleocapsid; pro encodes the protease enzyme; pol encodes the reverse transcriptase and integrase enzymes; and env encodes the envelope glycoproteins and transmembrane proteins [209] [210] [211] . retroviruses infect a wide variety of vertebrates, including humans. their infection in humans is usually very serious and can potentially cause oncogenesis and variable hematopoietic and neurological conditions. among retroviruses, hiv is a well-known, formidable human pathogen responsible for the most devastating viral pandemic that has ever occurred in human history [212] . hiv infection is usually correlated with the progression of what is clinically called aids, in which the immune system is harshly destroyed, and the entire body becomes vulnerable to terrible, life-threatening opportunistic infections and cancers. people with aids also suffer from other systemic symptoms, such as fevers, sweats, swollen glands, chills, weakness, and weight loss [213] . currently, there is no effective vaccine against hiv, and most of the treatment strategies rely on using antiretroviral drugs, which in most cases maintain the viral load at low levels to prevent the formation of acute hiv infection rather than cure the virus [214] . therefore, the accurate detection of hiv is a key determinant for monitoring hiv treatment and reducing its transmission and dissemination. currently, aunps have been extensively tested for the immunological and molecular detection of hiv through various scanometric, colorimetric, electrochemical, ipcr, and afm assays ( table 1) . the scanometric assays target the detection of the hiv p24 antigen and hiv nucleic acid using the silver metal enhancement protocol. for hiv p24, the target antigen is precaptured by magnetic particles [81] or isolated in a microplate [84] , followed by a silver enhancement step for the quantitative visual immunological detection of hiv (fig. 4a, b and fig. 6 ). for hiv nucleic acid detection, aunp-dna probes specific to the hiv pol gene are applied to detect hiv nucleic acids precaptured and isolated by magnetic particles, followed by a silver metal enhancement step (fig. 14a) . the deposition of silver on the aunp surface increases the np size and scattering efficiency and additionally allows the target detection via dls [90] . in aunp-based colorimetric detection of hiv, the large surface area of aunps was exploited to amplify the detection signal by enhancing the gold metal staining on their surfaces, allowing naked-eye detection of hiv nucleic acids (hiv gag gene) in a lateral-flow format, similar to the scheme presented in fig. 11a [91] . on the other hand, the ability of aunps to enhance electron transfer has been applied to develop an electrochemical assay for hiv detection (fig. 14b) . in this electrochemical assay, the target hiv genome is detected in correlation with the change in electrical impedance of graphene sheets decorated with spherical aunp-dna probes specific to the hiv pol gene [73] . the developed aunp-based rna detection of hiv using the aunp-lfa technique has a sensitivity limit of ~11 log10 copies/ml, while the best dna detection can be achieved through the light-scattering scheme with a sensitivity limit of 10 fm. furthermore, afm coupled with aunps as nanoparticulate labels has been applied for the detection of hiv p24 as follows: the target antigen is precaptured on the surface of a nanoarray specifically coated with anti-p24 and labeled with aunp-antibody conjugates that significantly changes the surface topography of the array, allowing the detection of p24 (fig. 14c) [95] . so far the developed aunps-based assays for hiv are among the most innovative in virus detection and offer a significantly improved detection limit down to attomolar range, which is hundreds of times lower than that of conventional techniques used in hiv detection, including pcr and elisa. therefore, aunps assays can be very beneficial in detecting hiv early, preventing its dissemination, and treatment monitoring. furthermore, simple detection methods such as lateral flow and scanometric assays can be easily incorporated into the point-of-care detection of hiv, which is currently of great interest and more suitable for developing countries. aunps have made a tremendous impact in the detection of viruses, as demonstrated by the numerous assays for most of the clinically relevant groups of human viruses (fig. 3) . coupled with different biomolecules, aunps have enabled the development of a completely new class of probes that are specifically composed of a aunp core (spherical or rod-like in shape) for signal readout and a surface layer of one or multiple types of biomolecules for target recognition and interaction. these probes have mostly been incorporated as a new set of functional and highly interactive components in different traditional techniques (e.g., eias and pcr) and have derived substantial improvements in design, detection performance, sensitivity, specificity and stability. this created a new trend in applying aunps to overcome the limitations of traditional techniques. aunp-based immunological and serological detection methods are the most common and have been accomplished through multiple scanometric, colorimetric, dls, immuno-pcr, electrochemical, electrical, sers, lspr, fluorometric, and afm-based approaches (table 1) . aunp-based dls and lspr assays are especially novel, simple and among the most sensitive reported assays for the immunodetection of viruses with a detection limit that is hundreds of times lower than that of the standard elisa technique [51, 52] . in contrast, the developed aunp-based molecular assays are not as sensitive as the conventional pcr technique or other pcr-based molecular techniques. however, the addition of aunps greatly simplifies the detection procedures of viral nucleic acids, and largely eliminates the need for complex procedures and expensive thermal cycling machines, which greatly reduces the detection time and total cost. in particular, colorimetric assays based on the intense red color of aunps represent an interesting qualitative shift in nucleic acid detection due to the complete elimination magnetic np (mnp)-dna probes scavenge target hiv sequences, which are then magnetically collected and labeled with aunp-dna probes. after repetitive washing, aunps are released into solution by a dehybridization step and subjected to silver metal enhancement, resulting in particle growth and increased scattering [90] . (b) electrochemical impedance assay of hiv rna. graphene oxide sheets decorated with aunps are applied for the immobilization of dna detection probes. hybridization with specific dna sequence results in increased impedance [73] . (c) atomic force microscopy (afm)-based immunoassay of hiv. aunp-antibody probes form a three-component sandwich immunocomplex with the target virus antigen captured on the surface of a specific antibody nanoarray; eventually, the increase in the surface height of the array is detected by afm [95] . of the pcr amplification step and the possibility of direct and rapid detection of the target virus within a few minutes. other assays rely on well-known analytical techniques, such as icp-ms and qcm, which have been introduced to virus nucleic acid detection for the first time through the application of aunps and have achieved detection sensitivity down to femtomolar concentrations [93, 94] . aside from these immunological and molecular assays, aunps have also been described for viral load testing by detecting intact virus particles, which are of special interest as a substitution to traditional cell culture technique [58, 65, 92] . in this context, the small size of aunps probes that is comparable to virus particles, allows rapid, and highly dynamic interaction with viruses for gaining information about infectivity. the application of aunps, especially in biology and medicine, require specific structural and physicochemical characteristics, and any subtle alterations to the intended characteristics can compromise the function and performance of the whole system. for bioanalytic and diagnostic applications, aunps exhibiting homogeneous morphological and fine structural characteristics are important specifically for effective and accurate response to target detection. therefore, many pre-analytical factors related to np synthesis and surface modification are critical to the development of an efficient virus detection assay. the synthesis of aunps commonly relies on methods that basically employ wet chemistry reduction techniques, as presented in other reviews [9, 32] . although these techniques are generally simple and fast and can support the large-scale production of aunps, the synthesis of uniform aunps with a discrete size and shape remains challenging [215] . recently, some approaches have been reported for the controlled synthesis and modification of aunps. however, these methods remain unsuitable either due to the protocol complexity or difficulty of particle separation, which might require additional steps or specific surface modifications. therefore, the development of more stringent and advanced methods for the facile and controlled synthesis of uniform aunps with the option of performing multiple surface modifications is strongly recommended. because of the direct relationship between aunp size and shape and their function as labels, such characteristics are also of inherent importance to both the performance of au nanoprobes and their efficiency for diagnostic applications. for instance, small aunps are preferred in colorimetric and light scattering-based assays due to their spr band sensitivity. in contrast, large aunps are preferentially employed in assays such as bca, immuno-pcr, and enzymatic-based electrochemical detection techniques, which rely on the surface functionalization of aunps with targeting or signaling biomolecules. additionally, a large np size is highly desirable in assays that depend on aunps as metal labels, such as electrochemical stripping, qcm, and icp-ms detection methods. in addition, spherical aunps are more commonly applied than rod-shaped nps. however, the latter exclusively enable a very interesting class of lspr-based detection methods that have shown an unprecedented detection sensitivity range. since aunps by themselves are "blind" with respect to sensing the target, the addition of specific biomolecules to their surfaces is a key step for detecting viruses [37, 38, 40] . unfortunately, most biomolecules have been reported to be very sensitive to harsh chemical modifications that can adversely affect their reactivity and specificity. thus, the type of targeting biomolecule and the surface conjugation reaction method applied are crucial and require optimization. short oligonucleotides and other dna sequences are physically rigid and more stable than protein biotargeting structures, including antibodies, which usually have several secondary and tertiary conformations that are important for their full, specific interaction with a target molecule. furthermore, the full reactivity of surface biomolecules via a suitable conjugation reaction is critical and should be retained and confirmed with additional assessment steps before use in virus detection. while the application of traditional conjugation techniques, such as electrostatic adsorption, is still widely reported, it is strongly recommended to be avoided due to the incident loss of conjugated biomolecule activity and large inequality in loading efficiency, which directly result in decreased assay sensitivity and reproducibility [216] . in this regard, aunps have an enhanced surface area-to-volume ratio that greatly improves the efficacy of current conjugation techniques, and several directional and controlled conjugation methods based on covalent and noncovalent interactions have been thoroughly described in the literature [9, 32, 35] . although aunps are widely employed in various sensing functions and formats, their use for monitoring of virus infection is challenged by long-term stability and reusability of the developed aunp-based sensors. the reusability of sensing platforms has a crucial role in developing applications that are reliable, economic and sustainable. nonetheless, the reusability of aunps in virus detection is limited to electrical and electrochemical assays, in which the main function of aunps is limited to enhancing the performance of the modified electrodes [68, [70] [71] [72] [73] [75] [76] [77] [78] . the long-term stability of aunps is reported and well investigated in numerous studies. however, their conjugates to different biomolecules (i.e., dna, rna, antibody, enzymes) are sensitive to storage time and conditions and the reusability of these conjugates is usually hampered by the loss of activity of surface molecules and irreversible aggregations. it was demonstrated that exposure to elevated temperatures, high salt concentrations, and reducing agents such as dithiothreitol (dtt), mercaptoethanol, and glutathione (commonly existing in biological fluids) results in the loss of stability and activity of aunp-conjugates [9, 41, 43] . thus, the development of new chemistries and novel conjugation strategies for the synthesis of stable aunp-conjugates becomes very crucial for practical use of aunps in virus detection. considering these limitations and challenges, important analytical parameters, such as the use of positive and negative controls, follow-up patient specimens, and internal reference tests, are recommended to confirm the overall accuracy and practicality of developed schemes. a restricted set of measures related to the assay detection sensitivity, specificity, and performance is required for the quality assurance and assessment of these applications [217] . due to the rapid development rate of aunp-based assays, international data analysis measures and standards are also critical to reflect the true assay sensitivity and specificity and to allow the evaluation of their ability and potential to substitute current detection techniques. furthermore, more efforts are needed to test the practicality and generalizability of these assays through clinical sample testing and in-field evaluation of the newly developed aunp-based methods along with the conventional methods using standard evaluation systems and protocols. aunps are a powerful addition to the range of techniques available for virus testing and control. when used as probes, aunps enable simple, rapid, and sensitive detection with an unprecedented multiplexing capability. importantly, aunp-based probes have matured from tools being added to improve traditional techniques, such as pcr, eliza, and many others, to the basis of completely novel techniques developed and introduced for the first time for virus detection. with aunps, virologists can create an unlimited number of detection assays that match the diagnostic needs of each virus in terms of simplicity, speed and cost with sensitivity levels that reach zeptomolar concentrations. aunps can be further developed to become components of more routine techniques, and they will likely engage the interest of scientists from all the disciplines, whether fundamental, environmental, clinical or industrial, in the future. the challenge of emerging and re-emerging infectious diseases globalization and infectious diseases in women globalization, international law, and emerging infectious diseases globalization and infectious diseases globalization of infectious diseases: the impact of migration gold nanoparticles in biomedical applications: recent advances and perspectives gold nanoparticles: opportunities and challenges in nanomedicine biomedical applications of plasmon resonant metal nanoparticles a review on functionalized gold nanoparticles for biosensing applications optical resonator biosensors: molecular diagnostic and nanoparticle detection on an integrated platform ebola virus disease (the killer virus): another threat to humans and bioterrorism: brief review and recent updates insights from clinical research completed during the west africa ebola virus disease epidemic gold aggregating gold: a novel nanoparticle biosensor approach for the direct quantification of hepatitis c virus rna in clinical samples an improved gold nanoparticle probe-based assay for hcv core antigen ultrasensitive detection a highly sensitive electrochemical immunosensor for hepatitis b virus surface antigen detection based on hemin/g-quadruplex horseradish peroxidase-mimicking dnazyme-signal amplification development of lateral flow assay based on size-controlled gold nanoparticles for detection of hepatitis b surface antigen sers detection of hepatitis b virus dna in a temperature-responsive sandwich-hybridization assay cross-species virus transmission and the emergence of new epidemic diseases genetic variability: the key problem in the prevention and therapy of rna-based virus infections factors in the emergence of infectious-diseases updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic in situ self-assembly of gold nanoparticles on hydrophilic and hydrophobic substrates for influenza virus-sensing platform avian influenza virus (h5n1): a threat to human health re-emergence of zika virus: a review on pathogenesis, clinical manifestations, diagnosis, treatment, and prevention diagnostic virology protocols clinical and diagnostic virology high sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18 the brazilian zika virus strain causes birth defects in experimental models molecular diagnosis of influenza diagnostic virology viral serology and detection functionalized gold nanoparticles: synthesis, properties and applications-a review molecular techniques for clinical diagnostic virology molecular diagnosis of medical viruses nanoparticle-mediated systemic delivery of sirna for treatment of cancers and viral infections there's plenty of room at the bottom engineering nanomaterial surfaces for biomedical applications nanostructures in biodiagnostics sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-nanogold, and silver acetate autometallography: detection of single-copy human papillomavirus the use of nanocrystals in biological detection enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides gold nanoparticles in nanomedicine: preparations, imaging, diagnostics, therapies and toxicity various methods of gold nanoparticles (gnps) conjugation to antibodies chemical synthesis of novel plasmonic nanoparticles a molecular ruler based on plasmon coupling of single gold and silver nanoparticles gold and silver nanoparticles in sensing and imaging: sensitivity of plasmon response to size, shape, and metal composition review: synthesis of fluorescent metallic nanoclusters toward biomedical application: recent progress and present challenges gold nanoparticles for the development of clinical diagnosis methods surface-enhanced raman scattering (sers) detection of multiple viral antigens using magnetic capture of sers-active nanoparticles nanoparticles with raman spectroscopic fingerprints for dna and rna detection detection of hepatitis b surface antigen by target-induced aggregation monitored by dynamic light scattering gold nanorod-based localized surface plasmon resonance biosensor for sensitive detection of hepatitis b virus in buffer, blood serum and plasma one-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles colorimetric detection of dna sequences based on electrostatic interactions with unmodified gold nanoparticles oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for dna analysis by hybridization direct detection of unamplified hepatitis c virus rna using unmodified gold nanoparticles multiplexed colorimetric detection of kaposi's sarcoma associated herpesvirus and bartonella dna using gold and silver nanoparticles a fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin g antibodies in serum and whole blood sizeand distance-dependent nanoparticle surface-energy transfer (nset) method for selective sensing of hepatitis c virus rna a gold nanorods-based fluorescent biosensor for the detection of hepatitis b virus dna based on fluorescence resonance energy transfer multiple homogeneous immunoassays based on a quantum dots-gold nanorods fret nanoplatform detection of swine-origin influenza a (h1n1) viruses using a localized surface plasmon coupled fluorescence fiber-optic biosensor aggregation effects of gold nanoparticles for single-base mismatch detection in influenza a (h1n1) dna sequences using fluorescence and raman measurements hybrid nanocluster plasmonic resonator for immunological detection of hepatitis b virus electrophoresis-enhanced detection of deoxyribonucleic acids on a membrane-based lateral flow strip using avian influenza h5 genetic sequence as the model nanomolecular detection of human influenza virus type a using reverse transcription loop-mediated isothermal amplification assisted with rod-shaped gold nanoparticles gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus dna using disposable microband electrodes ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based dna amplification impedimetric detection of influenza a (h1n1) dna sequence using carbon nanotubes platform and gold nanoparticles amplification electrochemical detection of avian influenza virus h5n1 gene sequence using a dna aptamer immobilized onto a hybrid nanomaterial-modified electrode genosensor for sars virus detection based on gold nanostructured screen-printed carbon electrodes green-synthesized gold nanoparticles decorated graphene sheets for label-free electrochemical impedance dna hybridization biosensing nanoparticle-based electrochemical detection of hepatitis b virus using stripping chronopotentiometry highly sensitive electrochemical stripping detection of hepatitis b surface antigen based on copper-enhanced gold nanoparticle tags and magnetic nanoparticles gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis b surface antigen in human serum electrochemical immunoassay of hepatitis b surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode catalytic signal amplification of gold nanoparticles combining with conformation-switched hairpin dna probe for hepatitis c virus quantification development of array-based technology for detection of hav using gold-dna probes visual gene diagnosis of hbv and hcv based on nanoparticle probe amplification and silver staining enhancement detection of hiv-1 p24 gag in plasma by a nanoparticle-based bio-barcode-amplification method array-based nano-amplification technique was applied in detection of hepatitis e virus scanometric analysis of dna microarrays using dna intercalator-conjugated gold nanoparticles nanoparticle-based immunoassays for sensitive and early detection of hiv-1 capsid (p24) antigen detection of hepatitis b virus deoxyribonucleic acid based on gold nanoparticle probe chip multiplexed, rapid detection of h5n1 using a pcr-free nanoparticle-based genomic microarray assay label-free and naked eye detection of pna/dna hybridization using enhancement of gold nanoparticles rapid and simultaneous detection of human hepatitis b virus and hepatitis c virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus homogeneous detection of nucleic acids based upon the light scattering properties of silver-coated nanoparticle probes a lateral flow assay for quantitative detection of amplified hiv-1 rna influenza virus detection with pentabody-activated nanoparticles a method of layer-by-layer gold nanoparticle hybridization in a quartz crystal microbalance dna sensing system used to detect dengue virus gold nanoparticle-based inductively coupled plasma mass spectrometry amplification and magnetic separation for the sensitive detection of a virus-specific rna sequence the use of nanoarrays for highly sensitive and selective detection of human immunodeficiency virus type 1 in plasma gold nanoparticle enhanced immuno-pcr for ultrasensitive detection of hantaan virus nucleocapsid protein two types of nanoparticle-based bio-barcode amplification assays to detect hiv-1 p24 antigen multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using au nanoparticle probes and quantum dots as labels virus taxonomy: classification and nomenclature of viruses fields virology bunyaviruses and the type i interferon system hantaviruses: a global disease problem hantavirus infections: epidemiology and pathogenesis hantavirus infection: a review and global update isolation of the causative agent of hantavirus pulmonary syndrome rift valley fever virus rift valley fever virus(bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention epidemiologic investigations into outbreaks of rift valley fever in humans outbreak of rift valley fever affecting veterinarians and farmers in south africa an assessment of the regional and national socio-economic impacts of the 2007 rift valley fever outbreak in kenya rift valley fever and a new paradigm of research and development for zoonotic disease control supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron ciyomicroscopy coronaviruses post-sars: update on replication and pathogenesis coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus a previously undescribed coronavirus associated with respiratory disease in humans identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia no novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide codehop-based primers isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease filoviruses in bats: current knowledge and future directions marburg and ebola viruses evolutionary history of ebola virus ebola virus outbreaks in africa: past and present efficacy and effectiveness of an rvsv-vectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial. the lancet virus taxonomy: ninth report of the international committee on taxonomy of viruses opinion -architects of assembly: roles of flaviviridae non-structural proteins in virion morphogenesis phylogeny of the genus flavivirus perspectives for the treatment of infections with flaviviridae a brief review on dengue molecular virology, diagnosis, treatment and prevalence in pakistan world health organization. dengue guidelines for diagnosis, treatment, prevention and control: new edition the global distribution and burden of dengue the burden of hepatitis c in the united states estimating the future health burden of chronic hepatitis c and human immunodeficiency virus infections in the united states histological and clinical outcome after liver transplantation for hepatitis c peginterferon alfa-2a plus ribavirin for chronic hepatitis c virus infection telaprevir for previously untreated chronic hepatitis c virus infection sofosbuvir for previously untreated chronic hepatitis c infection sofosbuvir and ribavirin in hcv genotypes 2 and 3 simeprevir with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients with chronic hepatitis c virus genotype 1 infection (quest-1): a phase 3, randomised, double-blind, placebo-controlled trial quest for a cure for hepatitis c virus: the end is in sight virus taxonomy: classification and nomenclature of viruses (ninth report of the international committee on taxonomy of viruses) hepatitis b virus infection--natural history and clinical consequences natural history and clinical consequences of hepatitis b virus infection world health organization best practice in the treatment of chronic hepatitis b: a summary of the european viral hepatitis educational initiative (evhei) diagnosis and management of chronic hepatitis b in children, young people, and adults: summary of nice guidance virus taxonomy: ninth report of the international committee on taxonomy of viruses hepatitis e virus --an update array-based nano-amplification technique was applied in detection of hepatitis e virus virus taxonomy: ninth report of the international committee on taxonomy of viruses herpesvirus entry: an update herpesviruses remodel host membranes for virus egress genital herpes. the lancet the nine ages of herpes simplex virus an estimate of the global prevalence and incidence of herpes simplex virus type 2 infection epidemiology, clinical presentation, and antibody response to primary infection with herpes simplex virus type 1 and type 2 in young women herpesviruses: latency and reactivation-viral strategies and host response herpes simplex virus 2 infection: molecular association with hiv and novel microbicides to prevent disease hsv-2 vaccine: current status and insight into factors for developing an efficient vaccine immune evasion by herpes simplex virus evasion of early antiviral responses by herpes simplex viruses review of cytomegalovirus seroprevalence and demographic characteristics associated with infection cytomegalovirus: pathogen, paradigm, and puzzle international consensus guidelines on the management of cytomegalovirus in solid organ transplantation neonatal cytomegalovirus blood load and risk of sequelae in symptomatic and asymptomatic congenitally infected newborns is hcmv a tumor promoter? vaccine prevention of maternal cytomegalovirus infection identification of herpesvirus-like dna sequences in aids-associated kaposi's sarcoma kshv infection and the pathogenesis of kaposi's sarcoma kaposi's sarcoma-associated herpesvirus kaposi's sarcoma-associated herpesvirus infection prior to onset of kaposi's sarcoma detection of kaposi sarcoma associated herpesvirus in peripheral blood of hiv-infected individuals and progression to kaposi's sarcoma. the lancet virus taxonomy: ninth report of the international committee on taxonomy of viruses avian influenza viruses and their implication for human health influenza: lessons from past pandemics, warnings from current incidents the viruses and their replication seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america influenza and rsv: know the diagnostic options world health organization. pandemic h1n1 2009 update 112 cost-benefit analysis of a strategy to vaccinate healthy working adults against influenza burden of influenza-like illness and effectiveness of influenza vaccination among working adults aged 50-64 years antiviral drugs for seasonal influenza efficacy of high-dose versus standard-dose influenza vaccine in older adults acip releases updated recommendations on influenza vaccination to include the 2014-2015 season virus taxonomy: ninth report of the international committee on taxonomy of viruses cross-roads in the classification of papillomaviruses the biology and life-cycle of human papillomaviruses chapter 1: hpv in the etiology of human cancer screening to prevent cervical cancer: guidelines for the management of asymptomatic women with screen detected abnormalities: commonwealth of australia epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update multi-site study of hpv type-specific prevalence in women with cervical cancer, intraepithelial neoplasia and normal cytology, in england global burden of human papillomavirus and related diseases efficacy and safety of prophylactic vaccines against cervical hpv infection and diseases among women: a systematic review & meta-analysis safety and persistent immunogenicity of a quadrivalent human papillomavirus types 6, 11, 16, 18 l1 virus-like particle vaccine in preadolescents and adolescents: a randomized controlled trial prophylactic hpv vaccines: prospects for eliminating ano-genital cancer gold nanoparticle mediated method for spatially resolved deposition of dna on nano-gapped interdigitated electrodes, and its application to the detection of the human papillomavirus natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily clinical significance, diagnosis, and treatment of picornavirus infections hepatitis a virus. mandell, douglas, and bennett's principles and practice of infectious diseases hepatitis a acute liver failure: follow-up of paediatric patients in southern brazil centers for disease c, prevention. surveillance for acute viral hepatitis -united states hepatitis a in the era of vaccination virus taxonomy, ninth report of the international committee on taxonomy of viruses historical introduction to the general properties of retroviruses translational control of retroviruses global report: unaids report on the global aids epidemic abc of aids: natural history and management of early hiv infection new paradigms for hiv/aids vaccine development synthetic approaches to metallic nanomaterials aspects of recent development of immunosensors towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative radioimmunossay of australia antigen detecting the aids virus directly with pcr nanoparticle-based electrochemical detection of hepatitis b virus using stripping chronopotentiometry development of array-based technology for detection of hav using gold-dna probes we would like to thank frank fanqing chen the authors have declared that no competing interest exists. key: cord-006856-b1w25ob5 authors: nan title: 19th meeting of the austrian society of transplantation, transfusion, and genetics, october 26–28, 2005 date: 2005 journal: eur surg doi: 10.1007/s10353-005-0216-6 sha: doc_id: 6856 cord_uid: b1w25ob5 nan background. nowadays lung transplantation is an established standard procedure for the treatment of most end-stage respiratory disorders. in the last years, like for all solid-organ transplantations, the demand for donor lungs exceeded significantly the existing organ pool. thus, most specialised centers face a high mortality on the waiting list. this retrospective study compares the outcome of marginal versus ideal donor lungs. methods. we performed a retrospective analysis of 98 consecutive primary lung transplantations from 94 donors from 1/2001 to 12/2002 . recipients were divided in two groups (standard versus "extended") according to the donor lung acceptance criteria (age, >55 years; pao 2 at fio 2 /peep 5, <300 mmhg; positive tobacco anamnesis [>20 packages per year]; inhalation of noxious agents; presence of infiltration on chest x-ray or purulent secretions at bronchoscopy). results. twenty-three donors (24.5%) were extended. twenty-six recipients (26.55%) received organs from extended donors. according to our data, differences in intubation times, icu stay, and hospital stay were not statistically significant. furthermore, postoperative bleeding rates were comparable as well as bronchial anastomotic complications. we encountered no significant statistical difference in the 3-month (standard 88.89% vs. extended 92.31%) and 1-year (standard 81.94% vs. extended 84.62%) survival between the two groups. conclusions. our study suggests that the use of selected marginal donor lungs has no influence on the outcome after transplantation. background. female donor gender has been described to be an independent risk factor for primary graft failure. we performed this study to evaluate the impact of donor gender on outcome and complications after lung transplantation. methods. we retrospectively reviewed the impact of donor gender on outcome of 163 primary lung transplant recipients (93 recipients were male [57%], 70 were female [43%]) from january 2001 to december 2003. recipients were stratified whether they received a female-or male-donor organ. both groups were compared with regard to duration of intubation time, icu stay, postoperative complications and survival. both groups were comparable with regard to mean age, indications, and mean waiting list time. results. mean time until extubation was 8 days in the group receiving organs from male donors and 16 days in the group receiving organs from female donors (p = 0.041). mean icu stay of 12 days for the male-donor recipient group was significantly shorter than that for the female-donor recipient group, 20 days (p = 0.044). 3-month survival rates were comparable in both groups: 89.53% (male-donor recipi-lunge 19th meeting of the austrian society of transplantation, transfusion and genetics background. nowadays lung transplantation is an established therapeutic option for most end-stage respiratory diseases and one of the fastest-growing solid-organ transplantation procedures in the world, reflected by the enormous demand for lungs. this retrospective review of the vienna lung transplantation group demonstrates data about waiting time and mortality rate for various end-stage respiratory diseases for the years 2003 and 2004. methods. . different variables specific for pretransplant period such as probability of transplantation, death on the waiting list, and time till transplantation were analysed for most frequent end-stage respiratory diseases. results. we found no significant changes in the average mortality rate (constantly 8%) on the waiting list during the studied period. however, patients suffering from pph have the highest probability of dying while being listed for lung transplantation (2003, n = 2 [20%] ; 2004, n = 2 [14.3%]), followed by patients with idiopathic fibrosis (2003, n = 3 [15%]; 2004, n = 4 [11.4%] ). subsequently the next subgroup is represented by cystic fibrosis patients, who are characterized by a moderate mortality rate (2003, n = 1 [3.8%] ; 2004, n = 1 [5%]). in contrast, patients who were suffering from copd showed the lowest probability of dying (2003, n = 0 [0%]; 2004, n = 1 [2.9%]). the average waiting time for the observed 2 years amounts to 97.5 days (2003, 99 ± 98 [min, 0; max, 464] days; 2004, 96 ± 96 [min, 0; max, 400] days). regarding the disease-specific waiting time, pph patients had to wait a longer period (133 ± 128 days) for their transplantation than patients with other diagnosis. conclusions. waiting time and mortality on the waiting list are showing remarkable differences within the disease-specific subgroups. v05 increased recipient vegf serum level is a risk factor for severe reperfusion edema after lung transplantation k. krenn, s. taghavi, w. klepetko, s. aharinejad laboratorium für kardiovaskuläre forschung, zentrum für anatomie und zellbiologie, medizinische universität wien, wien, österreich background. primary graft dysfunction (pgd) due to ischemia-reperfusion injury is a severe complication in lung transplantation (ltx) . therapeutic strategies are limited and there exist no preoperative markers to predict the risk for reperfusion-induced edema. vascular endothelial growth factor (vegf) is a key regulator of vascular permeability. methods. preoperative vegf serum levels were measured by elisa for 76 patients undergoing ltx. underlying diseases in ltx patients were copd (n = 22), cystic fibrosis (n = 15), idiopathic pulmonary fibrosis (n = 11), emphysema (n = 6), primary pulmonary hypertension (n = 6), sarcoidosis (n = 4), and others (n = 12). the ischemia time of the grafts and the blood gas parameters in donors were comparable. reperfusion edema was diagnosed and scored by characteristic changes in chest radiographs and deteriorating blood gases according to the guidelines of the international society for heart and lung transplantation, grading pgd from 0 to 3 (0, none; 1, only radiographic evidence; 2, moderate, pao 2 /fio 2 ratio of 200-300; 3, severe, pao 2 /fio 2 ratio of <200 or ecmo support necessary). results. grade 3 pgd occurred in 15%, grade 2 in 23%, grade 1 in 43%, and grade 0 in 19% of the patients. the preoperative vegf serum levels were significantly higher in patients with pgd grade 2 and 3 versus those without clinically relevant pgd (grade 0 and 1) following ltx (p = 0.0007). preoperative recipient vegf serum levels significantly predicted pgd in receiver operating characteristic analysis (p = 0.0002, auc = 0.755, ci = 1.001-1.003). conclusions. preoperative serum vegf levels in patients awaiting ltx could identify those at risk for reperfusion-induced edema following transplantation. background. airway stenoses are well known after lung transplantation although most occur due to surgical problems of the bronchial sutures. we wanted to analyse the use of endobronchial stenting in stenoses not related to the bronchial anastomoses. methods. we performed a retrospective analysis in 12 patients after bilateral lung transplantation with consecutive stent placement aside the bronchial anastomoses. the indication for stent implantation was central bronchial stenoses due to bronchomalacia, granulation tissue with bronchial wall destruction, and endoluminal stenosis with airflow limitation or occurrence of segmental or lobar atelectasis. we used boston ultraflex (23 of 26), rüsch polyflex (2 of 26), rüsch dynamic (1 of 26) stents. results. in 12 lung allograft recipients, we implanted in total 26 stents between 2 and 72 month after bilateral lung transplantation. the predominant locations were the right (20%) and left lower lobe bronchus (25%) and the bronchus intermedius (33%). the patient had granulation tissue proliferation due to ischemia and/or chronic bacterial or fungal bronchitis. only one patient had concomitant anastomoses dehiscence. the stents remained in situ from 1 day to 850 days after placement. in 18 of 26 stent placements no severe complications occurred. stent migration was observed in 6 of 26; severe granulation tissue that revealed further interventional treatment, in 4 patients. one patient died because of necrotizing vasculitis and letal hemoptysis. all other stents are still in place (10 of 26), were in place at the time of death due to other reasons (4 of 26), or were explanted regularly (5 of 26). conclusions. endobronchial stent placement is an effective treatment for bronchial stenoses that are not related to the bronchial anastomoses. complications occurred in about 40% of all stents. v07 preoperative oral corticosteroids predict the risk of late postoperative bleeding and perioperative mortality in lung transplantation conclusions. patients listed for lung transplantation with high-dose preoperative oral cs intake have a significantly increased risk of late postoperative bleeding. the perioperative mortality and the probability of 1-year survival of recipients with late bleeding are severely affected compared with patients with no or early bleeding. background. the efficacy of induction therapy after lung transplantation remains controversial and data on its use are limited. we hypothesized that induction therapy would have an impact on incidence of early rejection after lung transplantation and may cause a higher rate of infectious complications during the first 6 months post transplantation. methods. all patients who underwent lutx between jannuary 2003 and march 2004 and received induction therapy with rabbit antithymocyte globulin (2.5 mg/kg/d for 3-6 days) were analysed retrospectively. basic immunosuppression consisted of tacrolimus, mycophenolate mofetil, and prednisone. primary end point was patient survival after 6 months. secondary end point was histologically proven grade i or higher grade of rejection within the first 6 months, incidence of infectious episodes and bronchiolitis obliterans syndrome (bos). a total of 29 adult patients who underwent single (n = 2), heart-lung (n = 2), or bilateral lung (n = 26) transplantation entered the study. female, 10 (35%); male, 19 (65%). mean age was 37 ± 12.9 years (range, 19.3-59.9 years). results. the 6-month survival using kaplan-meier analysis was 92%; 27 patients are alive, two died 151 and 160 days after transplantation. one patient was retransplanted 3 days after primary transplantation because of graft dysfunction. follow-up ranged from a minimum of 151 days to 492 days (mean, 279 ± 85 days). 14 patients had one histologically proven rejection episode. rejections were graded ai for 10 patients, grade ai-ii for 2, and grade aii for 2 patients. 7 rejection episodes were treated with iv methylprednisone, no recurrent or ongoing rejections were observed. incidence of bacterial infections requiring treatment was 21/100 patients days. 2 cmv infections, 1 non-cmv viral infection, and 4 fungal infections (2 candida, 2 aspergillus) were diagnosed and treated. no patient developed signs of bos of grade ≥1. no lymphoma occurred. conclusions. this retrospective analysis suggests that induction therapy with atg in combination with tacrolimusbased triple immunosuppressive regimen results in excellent survival rates and a low rate of acute early rejections. however, this high immunosuppression efficacy was paralleled by a considerably high rate of bacterial infections during the first 6 postoperative months. rin inhibitor based quadruple drug is. an extensive infectious monitoring was used in this cohort. results. one-year patient survival was 71.4%, perioperative rejection rate 35%. infection incidence during first hospitalization was 79.6% (1.3 episodes per transplant): pneumonia, 74%; sepsis, 13%; wound infection, 17%; hsv/vzv, 28%; uti, 2%. during follow-up, cmv-associated complications were observed in 50% of patients including cmv infection (n = 9), cmv disease (n = 16). there were nine patients with cmv syndrome, ten patients with cmv graft pneumonitis and two patients with cmv gastrointestinal disease. excluding the 3 retransplants and the 2 perioperative deaths, the incidence of aspergillosis was 27%, six patients with aspergillus tracheobronchitis and seven patients with invasive disease. a total of 33 patients died during follow-up, 28 from infectious complications. as part of our centers' microbiological monitoring, 2773 specimens (51/transplant) were collected. these specimens were taken on 1276 observation days. 1426 investigated specimens were sterile and in 993 specimens microorganisms could be isolated (354 normal flora, 639 pathogens). a total of 1155 pathogens were identified: 673 gram-positive cocci, 177 gram-negative rods, 155 pseudomonas/acinetobacter, 124 candida. >60% of staphylococcus aureus and 55% of coagulase-negative staphylococci were methicillin resistant. other multiresistant organisms were e. faecium (no vre), n = 75; corynebacterium jk, n = 2; stenotrophomonas maltophila/burkholderia cepacia, n = 31. conclusions. infection remains the most common complication and the most common cause of death following lung transplantation. further refinement of infectious prophylaxis is required to improve results. background. valgancyclovir is routinely used for prophylaxis against cytomegalovirus (cmv) reactivation in lung transplant recipients. problems may arise due to its myelotoxic properties. it is unclear whether cytotect ® , a human igg cmv antibody preparation, offers similar protection against cmv reactivation without causing neutropenia. methods. we report on a female patient (63 years) who received a double lung transplant in 2002. cmv status was d+r+. four months postoperatively, she developped cmv pneumonitis and gastroduodenitis and was treated with gancyclovir and cytotect. thereafter, valgancyclovir was instituted but had to be stopped repeatedly because of neutropenia; each withdrawal of valgancyclovir prompted further reactivation episodes as detected by pp65 monitoring. in july 2004, she was started on cytotect, 150 mg q.d. over five days, followed by weekly doses of 50 mg for twelve weeks. results. cytotect was tolerated without side effects. of note, leucocyte counts remained within normal limits. since she has been started on cytotect, she has not experienced any further episodes of cmv reactivation until to date. conclusions. the observations made in this patient suggest that long-term therapy with cytotect ® has the potential to prevent cmv reactivation in selected patients who do not tolerate valgancyclovir due to its myelotoxic side effects. v12 die kombinationsprophylaxe verbessert die cmv bedingte morbidität und mortalität und reduziert das risiko der bronchiolitis obliterans (bos) nach lungentransplantation e. ruttmann, c. geltner, b. bucher, h. ulmer, d. höfer, h. b. hangler, s. semsroth, h. bonatti, r. margreiter, g. laufer, l. müller klinische abteilung für herzchirurgie, universitätsklinik für chirurgie, medizinische universität innsbruck, innsbruck, österreich grundlagen. die opportunistische cmv-infektion stellt ein schwerwiegendes problem nach lungentransplantation dar. ziel dieser untersuchung war, den einfluss der cmv-kombinationsprophylaxe mittels ganciclovir und cmv-hyperimmunglobulinen (cmv-ig) bezüglich patientenüberleben, cmv-reaktivierung, klinischer cmv-erkrankung und entwicklung der bronchiolitis obliterans (bos) zu evaluieren. methodik. eine konsekutive serie von 68 cmv-hochrisikopatienten (d+/r-, d+/r+) mit einem minimalen followup von mindestens 1 jahr post-transplant wurden analysiert. dreißig patienten (44,1 %) erhielten eine alleinige ganciclovir-prophylaxe für 3 monate (kontrollgruppe), 38 transplantationsempfänger (55,9 %) erhielten eine zusätzliche prophylaxe mit cmv-ig (cytotect ® , biotest pharma) in 5 dosen während des 1. postoperativen monats (studiengruppe). das mediane follow-up betrug 16,5 monate in der kontrollgruppe und 23,8 monate in der studiengruppe (p = 0,54). ergebnisse. insgesamt 5 cmv-assoziierte todesfälle (16,7 %) ereigneten sich in der kontrollgruppe, jedoch keiner in der studiengruppe (p = 0,014). in der kontrollgruppe wurden 13 fälle mit klinischer cmv-erkrankung beobachtet (43,3 %), in der studiengruppe 5 patienten (13,2 %) (p = 0,007). zusätzlich zeigte sich ein signifikant verbessertes patientenüberleben in der studiengruppe (log-rank, p = 0,01). die 1-jahresfreiheit von cmv-reaktivierungen betrug 52,1 % in der kontrollgruppe und 71,5 % in der studiengruppe (logrank, p = 0,027). die 3-jahresfreiheit von bos war signifikant höher in der studiengruppe (54,3 % vs. 82 %, log-rank, p = 0,024). schlussfolgerungen. eine zusätzliche cmv-hyperimmunglobulinprophylaxe senkt die cmv-assoziierte morbidität und mortalität. weiters kann das auftreten der bos mittels augmentierter cmv-prophylaxe reduziert werden. durch die dadurch reduzierte morbidität ist die kosteneffizienz gegeben. v13 extracorporeal photoimmune therapy (ecp) with uvadex in conjunction with standard therapy compared to standard therapy alone for the prevention of rejection in lung transplantation patients p. jaksch, r. knobler, b. schlechta, s. guth, w. klepetko klinische abteilung für herz-thoraxchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich background. extracorporeal photopheresis has been shown to be beneficial in acute and chronic rejection in heart transplant patients and has also been used in lung transplant recipients with acute rejection or bronchiolitis obliterans. methods. we performed a prospective study to document the efficacy of photoimmune therapy in the prevention of acute rejections in the first 12 months after lung transplantation. 12 lung transplant recipients with copd were randomized in 2 groups. group a (6 pat) received in total 16 ecp treatments (starting 2 weeks after tx) and group b without ecp or other kind of induction therapy, both groups receiving standard triple immunosuppression with tacrolimus, mycophenolate, and steroids. surveillance bronchoscopies with biopsies were performed after 2, 4, 8, 12, 26, and 52 weeks. primary objectives were acute biopsy-proven rejections of ishlt grade >1, secondary objectives were number of infections (cmv, bacterial, fungal, viral non-cmv, tuberculosis, parasitic), patients and graft survival. results. demographics in both groups were similar (gender, age, underlying disease, cmv mismatch, and type of tx). fev 1% and mef 50% values after 1 year were equal (88.3 ± 7.9% vs. 90 ± 28. 1 and 113.5 ± 41.7% vs. 114.5 ± 42.2%, respectively) . the number of rejections in group a (with ecp) was lower than in group b (0.17 ± 0.41 vs. 0.83 ± 0.75, p = 0.094), as well as the median rejection grade (0.16 ± 0.4 for group a vs. 0.66 ± 0.51 for group b, p = 0.092). after one year post tx, all patients are alive, none developed bo(s) within the first year post tx. conclusions. our preliminary data show a clear trend towards reducing the number and severity of acute rejections in lung transplant recipients. the number of infections was similar in both groups. adding ecp to a standard triple-drug immunosuppressive regimen seems to be a safe and efficient tool in reducing rejection rates without increasing the rate of bacterial, fungal, or viral infection. grundlagen. erhöhte natriumspiegel bei organspendern können mit der bildung von reperfusionsödemen und transplantatdysfunktion assoziiert sein. unklarheit besteht allerdings über die klinischen auswirkungen von erhöhtem spender-natrium nach herztransplantation (htx). in dieser studie wurde der einfluss von hohem spender-natrium auf die frühund 1-jahresmortalität nach htx in einem großen patientenkollektiv analysiert. methodik. es wurden 3157 herztransplantationen der eurotransplant-region aus dem zeitraum jänner 1997 bis dezember 2001 analysiert. entsprechend der spender-natrium-spiegel (sns) wurde das kollektiv in drei gruppen unterteilt: gruppe a, <160 mmol na + je liter n = 2903; gruppe b, 160-170 mmol na + je liter n = 218; gruppe c, ≥170 mmol na + je liter n = 54. eine kaplan-meier-überlebensanalyse und eine multivariate analyse bezüglich des einflusses des sns wurden durchgeführt. endpunkte waren die mortalität ein monat und ein jahr nach htx. ergebnisse. der sns hatte in der univariaten analyse keinen einfluss auf die frühmortalität und einen grenzwertig signifikanten einfluss auf die 1-jahres-mortalität (p = 0,06). in der multivariaten analyse war dieser effekt nicht signifikant (p = 0,2). die kombination aus hohem spenderalter mit hohem sns hatte jedoch in der multivariaten analyse einen hochsignifikanten einfluss auf die früh-und 1-jahresmortalität (p = 0,004). schlussfolgerungen. diese daten zeigen, dass hohe sns mit einer erhöhten früh-und 1-jahresmortalität nach htx einhergehen. diese ergebnisse stehen im gegensatz zu früheren arbeiten mit geringeren patientenzahlen. vor allem die kombination aus höherem spenderalter und hohem sns zeigt einen deutlichen risikoanstieg. herzen von spendern mit einem natriumspiegel von >170 mmol/l sollten nicht elektiv transplantiert werden, bei gleichzeitig hohem spenderalter sollte das herz nicht verwendet werden. die organempfänger wurden entsprechend dem spenderalter in 2 gruppen geteilt: gruppe 1, <35 a, n = 12; gruppe 2, >35a, n = 10. die gruppen waren hinsichtlich organischämiezeit, geschlechts-und cmv-mismatch sowie lipidstatus, nierenfunktion und medikamentöse therapie (immunsuppresion, ace-hemmer, statine, ca-antagonisten) 1 jahr post-htx vergleichbar. pv1 war in gruppe 1 tendenziell niedriger als in gruppe 2 (25,2 ± 17,3 mm 3 vs. 34 ± 22 mm 3 ; p > 0,05). umgekehrt wies gruppe 1 im verlauf des 1. jahres nach htx einen zuwachs an plaquevolumen auf, während in gruppe 2 eine abnahme (∆pv, 2,6 ± 17,6 mm 3 vs. 3,0 ± 10,2 mm 3 ; p > 0,05) festgestellt wurde. von den oben angeführten risikoparametern zeigte lediglich der triglyzeridspiegel 1 jahr post htx eine signifikante korrelation mit ∆pv (r = -0,613, p = 0,002). ∆pv und spenderalter waren nicht miteinander korreliert. schlussfolgerungen ergebnisse. die mortalität beträgt 0 %. wegen gastrointestinaler beschwerden (übelkeit, erbrechen) mussten 3 patienten (12%) auf ein anderes immunsuppressives schema umgestellt werden. bei 2 patienten (everolimus-talspiegel, >8 ng/ml) zeigte sich eine schwere pneumonie (pseudomonas), welche stationär behandelt wurde. es gab keine stationäre behandlung wegen cmv-infekten. die nierenfunktion war in allen patienten stabil (mittleres crea, 1,84 ± 0,85), außer in 2 patienten, welche bereits vor der umstellung erhöhte kreatininwerte zeigten und in denen sich eine weitere erhöhung der kreatininwerte (+20,25%) feststellen ließ. eine bei den meisten patienten auftretende hyperlipidämie konnte unter erhöhung der statintherapie eingestellt werden. in den routinemäßig durchgeführten endomyocard-biopsien fanden sich einen monat nach umstellung und danach keine akuten zellulären abstoßungen mit grad von >1b nach dem ishlt-grading. schlussfolgerungen. certican erwies sich als sicher und verträglich, die umstellung auf das neue immunsuppressivum war in allen patienten komplikationslos. everolimus-talspiegel von 8 ng/ml scheinen ausreichend, höhere spiegel könnten das infektionsrisiko erhöhen. bezüglich der nierenfunktion bleibt abzuwarten, wie sich ein cyclosporin-a-talspiegel von 60-80 ng/ml auswirkt. eine aussage bezüglich der cav steht zu diesem zeitpunkt noch aus. v17 20 jahre herztransplantation in wien (eine retrospektive über 1000 transplantationen) a. zuckermann empfänger-und spenderalter sind im laufe der jahre signifikant gestiegen (recipient age: 42,5 ± 11,8 vs. 52,0 ± 13,8, p < 0,05; donor age, 28,8 ± 10,3 vs. 35,2 ± 12,9, p < 0,01) . mehr patienten wurden präoperativ stationär aufgenommen (28 % vs. 44 %), jedoch hat sich die zahl der intensivpflichtigen patienten signifikant reduziert (21 % vs. 7 %). die zahl der patienten, die zur transplantation "gebridged" werden, ist ebenfalls massiv angestiegen (28 vs. 63 %, p < 0,01). innerhalb dieser gruppe hat die gruppe der patienten mit mechanischer herzunterstützung am stärksten zugenommen (7 % vs. 19 %, p < 0,01). pharmakologisches bridging (55 % vs. 42 %) und aicd (38 % vs.40 %) blieben stabil. pharmakologisches bridging wird heutzutage vermehrt mit prostaglandinen und levusimendan als mit dopamin oder dobutamin durchgeführt. mehr patienten sind am herzen voroperiert (28 % vs. 52 %, p < 0,01), patienten warten länger auf die transplantation (75,9 ± 91,6 vs. 289,8 ± 368,8, p < 0,01) . trotzdem hat sich die mortalität auf der warteliste stark verbessert (27,6 % vs. 12,0 %, p < 0,01), was ein klares zeichen der verbesserten überbrückungsmaßnahmen ist. ischämiezeiten sind ebenso angestiegen (129,3 ± 49,8 vs. 190 ,0 ± 49,2, p < 0,01) wie die liegezeiten auf der intensivstation (4,7 ± 5,7 vs. 7,6 ± 7,7, p < 0,01). dies ist ein klares indiz dafür, dass heute ältere, kränkere und komplexere patienten transplantiert werden. diese veränderungen sind international bei allen zentren zu be-obachten. dies hat dazu geführt, dass mit der zunehmenden erfahrung und verbessertem überleben die nachbeobachtungszeit der patienten stark gestiegen ist und damit die behandlung dieser patienten kostenintensiver geworden ist, was aber mit der guten lebensqualität der herztransplantierten patienten zu rechtfertigen ist. v18 neoplastic diseases after heart transplantation: a retrospective study d. kammerstätter, a. aliabadi, j. ankersmit, d. dunkler, g. wieselthaler, e. wolner, m. grimm, a. zuckermann klinische abteilung für herz-thoraxchirurgie, universitätsklinik für chirurgie, medizinische universität wien, wien, österreich background. prolonged immunosuppression after solidorgan transplantation is associated with an increased risk for development of neoplasms. the purpose of this analysis was to investigate neoplasm incidence and outcome in patients with induction therapy. methods. 921 cardiac recipients were included in this retrospective analysis. all patients received antibody induction therapy with either polyclonal-atg or monoclonal antibodies. neoplasms were devided into 3 groups: (1) skin cancer, (2) ptld, (3) other neoplasms. overall incidence of neoplasms, patient survival after diagnosis of neoplasms were analysed by the kaplan-meier method. results. a total of 143 tumors were diagnosed at a mean follow-up of 59.6 ± 51.2 months after cardiac transplantation. freedom from neoplasms was 96.7%, 86.6%, and 71.5% after 1-, 5-, and 10-year respectively. 5-year survival after diagnosis of tumor was 50.3%. 65 patients developed skin cancers after 62 ± 40.6 months. 1-and 5-year survival after diagnosis was 96% and 71% respectively. there were 7 tumor-related deaths in this group. 19 patients developed ptld 40.7 ± 33.7 months after transplantation. 1-and 5-year survival was 63% and 40% with 12 deaths associated with the neoplasm. in the third group, a total of 59 patients were included. this group consisted of lung cancer (n = 17), abdominal cancer (n = 15), urogenital cancer (n = 7), and other tumors (n = 20). neoplasms were diagnosed at an average follow-up of 53.5 ± 42.7 months. 1-year and 5year survival was 57% and 25%. 31 deaths were associated with tumor. conclusions. although all patients received antibody induction therapy, overall incidence of neoplasms was comparable to centers using no induction therapy. especially incidence of ptld was low. as long-term survival after cardiac transplantation increases steadily and the risk of cancer increases continuously, patients in long-term follow-up should be checked for malignant diseases on a routine basis. background. while the predictive value of n-terminal pro-b-type natriuretic peptide (nt pro-bnp) in nontransplant cardiac patients is well recognized, its value in heart transplantation (htx) is incompletely understood. certain graft factors (e.g., isolated diastolic dysfunction) may affect both, nt pro-bnp levels and peak exercise tolerance. we therefore hypothesized a relationship between these variables in long-term htx recipients. methods. we measured nt pro-bnp levels of 27 htx patients before a symptom-limited upright bicycle exercise test was performed. graft function was stable in all patients and there were no signs of rejection. patients were divided according to a cut-off value of 70% exercise tolerance predicted normal into "low" and "normal" htx fitness groups. results. in 12 patients (11 m, 1 f; 57 ± 10 years; 6.5 ± 4 years posthtx; donor age, 36 ± 10 years; bmi, 28.6 ± 2.8 kg/m 2 ; creatinine clearance, 42 ± 10 ml/min) peak exercise tolerance was "low" (93 ± 25 w), while in 15 patients (11 m, 4 f; 57 ± 11 years; 7.4 ± 3.6 years posthtx; donor age, 32 ± 10 years; bmi, 26.6 ± 2.7 kg/m 2 ; creatinine clearance, 42 ± 8 ml/min) it was "normal" (136 ± 37 w). in the "low" htx fitness group, nt pro-bnp levels were 702 ± 778 pg/ml, in the "normal" htx fitness group, 324 ± 250 pg/ml (p = 0.08 between groups). regression analysis of peak exercise tolerance, achieved percentage of exercise tolerance predicted "normal", and renal function with nt pro-bnp levels failed to demonstrate a significant relationship. conclusions. the findings confirm previous studies that nt pro-bnp levels are increased in asymptomatic long-term htx recipients. a larger sample size is warranted, however, to support the hypothesis that nt pro-bnp might be a useful indicator to predict physical fitness in these patients. calcineurin inhibitor therapy is an important cause of renal dysfunction in heart transplant patients. sirolimus (srl) is a novel immunosuppressive (is) drug without nephrotoxic side effects. however, cases of proteinuria associated with srl have been reported following renal transplantation. in cardiac transplantation the potential incidence of proteinuria is not known. 29 long-term cardiac transplant patients (age, 60.9 ± 7.3 years) were switched from cyclosporine-based immunosuppression to a srl-based is 8.8 ± 4.5 years after transplantation. concomitant is consisted of mycophenolate-mofetil with or without steroids. two patients died 14 and 37 months post switch due to infection. both patients were dialysis dependent at time of death. one other patient was switched back to cni-based is due to interstitial nephritis. 24 h collections of urine were performed in all patients before switch and at several time points post switch to measure proteinuria. proteinuria increased significantly from 0.45 ± 1.0 mg/dl (median, 0.17) pre switch to 1.03 ± 2.04 mg/dl (median, 0.21) post switch (p = 0.017). proteinuria of 0.21-1.0 mg/dl was seen in 21% of patients before switch and in 28% after switch. proteinuria of >1.0 mg/dl was seen in 10% of patients before switch and in 24% after switch (n.s.). three patients developed severe proteinuria (>3.5 mg/dl) after switch. one died on dialysis, one was switched back to cni and one still remains on srl. in conclusion, proteinuria may develop in cardiac transplant patients after switch to srl-based is. srl seems to have a potential adverse renal effect in these patients. srl should be used cautiously with close monitoring for proteinuria or increased renal dysfunction. v21 early growth-response factor-1 is involved in cellular injury of transplanted hearts background. we have shown a persistent mitochondrial pathology in patients with idiopathic dilative (dcm) but not ischemic (icm) cardiomyopathy following cardiac transplantation. early growth response factor (egr)-1 that is stimulated by cytokines and hypoxia is suggested to induce inflammation and tissue injury. whether egr-1 mediates the persistent cellular pathology in hearts transplanted to dcm patients is unknown. methods. egr-1 and hypoxia-inducible factor-1 (hif-1) gene expression was examined in left ventricular biopsies of explanted failing hearts in 28 icm and 42 dcm patients, as well as in 12 donor grafts before reperfusion (control), at 10, 30, 60 minutes after reperfusion, and at 1, 2, 3, 4, 6, 12 posttransplant weeks, using real-time rt-pcr. hif-1 binding activity was examined using emsa. results. egr-1 myocardial gene expression was upregulated in dcm and icm (p < 0.01). hif-1 mrna levels were unchanged in both groups, whereas hif-1 binding activity was increased in icm only (p < 0.01) vs. controls. egr-1 and hif-1 myocardial expression increased during reperfusion in donor grafts (p < 0.01) vs. control hearts. in icm group, graft egr-1 and hif-1 expression returned to and remained at the baseline level of control hearts one week after transplantation. in contrast, in dcm group, egr-1 levels remained significantly upregulated during the follow-up period in transplanted hearts (p < 0.01), although hif-1 expression returned to the control baseline level one week after transplantation. conclusions. chronic hypoxia specifically triggers hif-1 binding activity in icm, and reperfusion upregulates egr-1 and hif-1 mrna expression in heart grafts. the persisting egr-1 overexpression in grafts transplanted to dcm patients could mediate mitochondrial impairment. targeting egr-1 overexpression that bypasses hif-1 might be beneficial to counteract acute reperfusion-induced injury, and the chronic cellular pathology in cardiac grafts transplanted to dcm patients. introduction. giant-cell myocarditits (gcm) is a rare and frequently fatal disorder of unknown origin that is defined histopathologically as diffuse myocardial necrosis with multinucleated giant cells in the absence of sarcoid-like granuloma. patients usually have ventricular arrhythmias or congestive heart failure. although a variety of systemic disorders have been seen in association with giant cell mycocarditis, most cases occur in previously healthy adults. conclusive diagnosis requires histologic analysis of myocardial tissue obtained by endomyocardial biopsy (emb). congestive heart failure (chf) is the most common cardiac presentation (75%), with sustained, refractory ventricular tachycardia. the case presented here is that of a 65-old-man suffering of gcm in whom an extracorporeal membrane oxygenation (ecmo) had to be implanted to overcome cardiogenic shock. antithymocyte gobuline (ratg, thymoglobuline, sangstat), respectively ciclosporine (inn: cyclosporine), mycophenolate and steroids were utilized to bridge the time to complete myocardial recovery. we are reporting the first successful implantation and bridging to myocardial regeneration in a patient suffering of gcm by means of ecmo and initiation of immunomodulating drugs including polyclonal ratg. clinical summary. a 65-year-old man was admitted to a public hospital because of thoracic pain, positive heart enzymes, and a highly pathologic electrocardiogramm. echocardiography demonstrated a pericardial effusion and reduced left ventricular function (lvf, ef 15%). performed angiography evidenced and a high-grade stenosis of the left anterior descending (lad) which led to stent implantation. despite stent placement, the clinical condition worsened leading to cardiogenic shock including incipient shock liver. in this clinical condition the patient was transferred to our institution. in addition, the patient developed malign ventricular tachycardia (lowen iv) and had to undergo repetitive defibrillation. in this clinical scenario it was decided to implant a femoral veno-arterial ecmo. the patient's metabolic data improved noticeably; however, due to repeated ventricular tachycardia, the patient had to be defibrillated multiple times (max. 30×/d). to define exact cardiac pathology, we performed a heart biopsy with the pathology of gcm. immunosuppressive therapy with cyclosporine (50 mg/d), mycophenolate and prednisolon (250 mg/d) was initiated. in addition, rabbit atg (ratg, thymoglobuline, sangstat) at the dosage of 75 mg/d was added to the therapy. this drug therapy was continued for 5 days. of note is the fact that with initiation of ratg, cardiac fibrillation episodes abated immediately. routinely performed echo-cardiography (tee) revealed an improvement in ventricular function, and one week after ecmo support, we were able to wean the patient from extracorporeal support. moreover, a routine biopsy after 14 days revealed complete remission of gcm in the heart tissue. an intracardial defibrillator (icd) was additionally implanted. three months after emergency admission to our department the patient was discharged. continuous medication of prednisolon 5 mg/d, mycophenolat mofetil 500 mg/d, and plavix 75 mg/d was prescribed. 12 month after initial event the patient describes nyha class i heart function and echocardiography reveals an moderate impairment of heart function (ef 55%). immunosuppressive with low-dose steroid and mycophenolate drug regimen is continued as the patient describes no side effects. discussion. this report adds to the available knowledge of giant-cell myocarditis by providing that (a) ecmo implantation is feasible if the patient is demonstrating acute haemodynamic deterioration because of biventricular dilation and medically intractable ventricular fibrillation and (b) after verified histological diagnosis of gcm immunemodulation with cyclosporine/mycophenolate with additional application of ratg is feasible with favourable outcome. in various studies, patients with gcm were treated with immunosuppression (cyclosporine, steroids, murine monoclonal antibodies [okt-3]) and even assist device as bridge to transplantation. heart transplantation has shown to be successful as method of treatment. autoimmune diseases and its mechanisms were suggested to be involved in the pathogenesis of gcm. most recently, a novel mechanism of action of immunoglobulin was proposed to be due to anti-inflammatory activities through the inhibitory fc receptos (fcrs). it has been suggested that t-cell-mediated autoimmune diseases is the result of inappropriate antigen presentation of either a self-antigen or an antigen with the capacity to mimic a self-antigen in the peripheral lymphoid tissues. relevant to this novel application of ratg, polyclonal suspensions like igm/g, ivig containing fc receptors and have been demonstrated to be beneficiary in autoimmune myocarditis. in an elegant study by shioji et al. ivig was highly effective in ameliorating experimental myocarditis. however it has to be mentioned that immunoglobulin treatment failed to ameliorate myocarditis. in respect to our patient suffering of rcm fc containing ratg provided remarkable clinical benefit. background. the criteria for liver donation have been widely extended due to the increasing waiting time and waiting list mortality. marginal donors provide an upcoming option to diminish the number of waiting patients. methods. the criteria for marginal were icu stay of >7 days, age of >65 years, bmi of >27, alcohol or oral drug abuse, infection, hypernatremia (na, >150 mg/dl), high liver enzymes (ast, alt 2 times the normal), liver parenchymal damage and metabolic diseases. from 01/00 until 06/05 our center reported 55 donors, who fulfilled at least 3 of the above criteria. results. all livers were transplanted either in standard or in piggyback technique with a cold ischemic period between 130 minutes and 12 hours. the mean recipient age was 45 (13-75) years. 6 livers were used for hu patients, 5 for a retransplantation, 2 were implanted in combination with kidneys, and 37 organs were transplanted electively. primary diseases were cryptogenic cirrhosis, hepatocellular carcinoma, post-hepatitis cirrhosis, polycystic liver disease, scleroting bile ducts, hepatic artery thrombosis, and acute liver failure. 2 month after transplantation, 54 recipients were alive, 1 died 1 month after tx not transplant related. 16 livers were implanted at our center in piggyback technique with retrograde reperfusion. 15 patients were elective patients in good or moderate condition, 1 patient was a hu patient suffering a hepatic artery thrombosis. the initial graft function was good (got, <1000 mg/dl pod 1) in 8, moderate (got, 1000-2000 mg/dl pod 1) in 5, and delayed (got, >2000 mg/dl pod 1) in 3 cases, all patients survived. conclusions. marginal livers are eligible for transplantation. delayed graft function has to be taken into account. hospital between january 1993 and december 2003. we employed the local registry of the department of transplant surgery, where variables of all patients are routinely and prospectively recorded. primary outcome was initial graft function, secondary outcome was patient survival. results. cumulative number of marginal donor criteria was significantly and linearly associated with an increased rate of primary dysfunction (p = 0.005). in patients with more than three cumulative marginal donor criteria the rate of primary dysfunction was 36 percent. patient survival was not influenced by the cumulative number of donor criteria (log-rank test, p = 0.81). independent marginal donor criteria to predict primary dysfunction were cold ischemia time of >10 hours (or, 0.56; 95% ci, 0.32 to 0.98) and donor peak serum sodiumof >155 meq/l (or, 0.44; 95% ci, 0.26 to 0.77), as assessed in a multivariate regression model. conclusions. the use of marginal liver donors with more than three marginal donor criteria shows deleterious effects on initial graft function. noteworthy, patient survival was not associated with marginal donor criteria, which may be explained by early and successful retransplantation of liver recipients with primary nonfunction. ergebnisse. alle transplantate zeigten eine gute initiale organfunktion, der transaminasenverlauf und das gesamtbilirubin, gemessen am 1. postoperativen tag, am 7. postoperativen tag und bei entlassung (mittelwerte mit standardabweichung) waren wie folgt: got 1334 u/l (±848), 43 u/l (±13), 25 u/l (±9); gpt 877 u/l (±245), 420 u/l (±649), 54 u/l (±40); ggt 118 u/l (±86), 355 u/l (±232), 232 u/l (±205); gesamtbilirubin 5,1 mg/dl (±2,2), 3,5 mg/dl (±2,5), 1,9 mg/dl (±0,9) . background. meld score is a useful tool in predicting mortality in patients awaiting liver transplantation. its capacity to predict patient survival seems to be relatively poor and is still discussed controversially. the purpose of the study was to analyse the impact of alterations of the meld score during waiting time on the posttransplant survival rate. additionally, the impact of donor quality on posttransplant survival was investigated. methods. 242 adult patients were transplanted between 1997 and 2003 because of end stage liver disease without malignancy. the meld scores at time of listing (meld on) and of transplantation (meld tx) were gathered. additionally the delta-meld was calculated from listing to transplantation. results. a high meld tx showed only a trend to poorer survival. patients who died within the 1 st year after transplantation showed a significant increase in the meld score during waiting time (p < 0.01). patients with a delta-meld higher than 4 during waiting time had a 50% 1-year mortality after transplantation, patients with a meld increase not higher than 4 had only a 23.1% 1-year mortality (p < 0.01). patients with a meld score higher than 24 who received a marginal graft showed a trend to poorer posttransplant survival. conclusions. patients with a substantial increase of the meld score during waiting time had a significantly poorer 1year posttransplant survival. in contrast, the meld score at time of listing or transplantation had no impact on the posttransplant survival rate. the use of marginal grafts in patients with a higher meld score has to be evaluated carefully. there is no significant difference of serum sodium levels in ltx candidates with or without ascites crease in serum creatinine is a late event in patients with ascites and is not directly reflected within the meld formula. for this purpose we compared patients who died on the waiting list with patients who finally were transplanted. the impact of serum sodium and ascites on death on the waiting list was evaluated. methods. from 1997 to 2005, 621 adult patients with end-stage liver disease were listed for orthotopic liver transplantation (olt). only patients without hepatoma who died on the waiting list (123 patients) or were finally transplanted (300 patients) were evaluated. in addition to the meld score, the serum sodium and the ascites were investigated at time of listing and of coming off the list (transplantation or death). results. transplanted patients had a significantly lower meld on (p < 0.01) than the patients who died while on the waiting list. patients who died while on the waiting list had a significant increase in the meld score during waiting time (p < 0.01). patients who underwent transplantation showed a stable meld score during their waiting time. refractory ascites and spontaneous bacterial infection were evaluated as independent risk factors for death on the waiting list as well as the meld on. 47.2% of the patients (58 of 123 patients) who died on the waiting list were suffering from ascites, in contrast to only 28.7% of the transplanted patients (86 of 300 patients). there was no significant difference in the mean meld on between the patients who were suffering from ascites and those who were not (p = 0.72). nor was any significant difference found in the meld off (p = 0.77). the serum sodium of patients suffering from ascites showed no significant difference to those who showed no signs of ascites. conclusions. ascites was evaluated as independent risk factor for death on the waiting list. no significant difference in the serum sodium levels were found between patients suffering from ascites or not. therefore complications of portal hypertension should be treated adequately and rigorously, especially in patients with lower meld scores. renal failure is an established risk factor for impaired patient outcome after orthotopic liver transplantation (olt). as the endothelin pathway is known to be involved in the development of acute renal failure (arf), we designed a study to clarify its role in arf following olt. 20 consecutive patients with intact kidney function scheduled for their first olt were prospectively studied. plasma big endothelin-1 (et-1) levels were measured before surgery, after graft reperfusion, and on the first and second postoperative days. according to postoperative gfr, patients were assigned to the acute renal dysfunction group (ardf) and the non-ardf group. each patient's gfr was estimated according to the four variable formula used in the modification of diet in renal disease before surgery, daily within the first postoperative week, and at 1, 3, 12 and 24 months after surgery. postoperative mean big et-1 lev-els correlated significantly with the maximum percent decrease of gfr within 3 days after olt (p < 0.01). the proportion of patients who developed ardf was significantly correlated to mean postoperative big et-1 quartiles (p < 0.01). in the ardf group, the percent decrease of gfr within 24 months was significantly higher (p < 0.05) as compared to the non-ardf group. in conclusion, patients who develop acute renal dysfunction immediately after olt do not fully recover to baseline regarding long-term kidney function. short-term gfr was significantly correlated with postoperative big et-1 plasma levels, suggesting renal dysfunction is mediated by the activated endothelin system. background. with improved survival of liver transplant recipients, chronic renal failure has become an important cause of morbidity and is associated with a high mortality. serum creatinine is widely used as marker for renal function, but it depends on various nonrenal factors and major changes will occur late in the course of progressive renal impairment. we evaluated cystatin c and urine microscopy for detection of renal dysfunction in patients after liver transplantation. methods. from november 2003, 70 liver transplant recipients at various intervals from liver transplantation were included to our follow-up. every three months we investigated serum creatinine, urea, renal creatinine clearance and cystatin c as marker for renal function. furthermore urinary sediment was examined by urinary test, automated urinary sediment analyser, and urine microscopy. in patients with reference to renal deterioration we tried to decrease immunosuppressive therapy, to optimize the blood pressure, and to discontinue nephrotoxic medication. infections were detected early by urine microscopy and treated, even when there was no clinical appearance and the urinary test was negative. results. the results of our study showed that concerning the renal function, cystatin c is more sensitive than creatinine and creatinine clearance. the microscopy of the urine sediment showed the highest sensitivity compared with the other methods. concerning damages of the kidney, urine microscopy offered the best possibility to identify the etiology. during the follow-up and after adequate and early therapy, fifteen patients (21.4%) showed an amelioration of renal function after a few months. in 3 patients (4.2%) there was a marked deterioration. two of them had to receive a higher dose of immunosuppressive therapy and one had an infection which was treated with nephrotoxic medication. conclusions. the early identification of renal failure and its etiology are necessary in patients after liver transplantation. the results of our study confirmed cystatin c as early prognostic marker for patients with renal dysfunction. in combination with urine microscopy, renal dysfunction could be detected in time and renal function could be protected with an adequate therapy. background. hcv-infected patients and their grafts have short-term survival rates similiar to other indications. recent data on long-term outcome are contradictory, showing a trend towards poorer outcome in patients transplanted for hcv cirrhosis. in this study we present a retrospective analysis of our experience with patients who underwent liver transplantation (lt) due to hcv-associated end stage liver disease. methods. patients' charts were reviewed for survival, histologically defined hcv recurrence, genotype, presence of cirrhosis, donor and recipient age as well as type of immunosuppression (cyclosporine and tacrolimus; azathioprine and mycophenolate mofetil). survival was analysed by the kaplan-meier procedure, the influence of baseline variables was analysed by binary logistic regression. results. between 1986 and october 2004, 162 patients were transplanted for hcv-related cirrhosis. ten pts. received one and 3 pts. two relts. in 3 (23%) pts. recurrent hepatits c was the cause for relt, in 10 vascular and/or biliary complications. hepatitis b coinfection was present in 6 patients. median follow-up was 44 months (range, 0.6-221). the overall, 1-, 2-, 3-, 5-, and 10-year survival rates were 86%, 81%, 78%, 71%, and 59%, respectively, which were comparable to other indications. the probability of recurrent hcv was 34%, 51%, 62%, 70%, and 83% after 1, 2, 3, 5, and 10 years, respectively, post lt. nine (6.6%) pts. developed cirrhosis after a median of 50 months (28-116). hcv recurrence did not negatively influence patient and graft survival. concerning genotype, cmv status, presence of hcc before lt, rejection episodes, immunosuppression, donor and recipient age only immunosuppression had a significant effect on survival. in cyclosporine-treated patients (lt after 1995) 1-, 2-, and 5-year survival rates were 79%, 72%, 64% compared to 93%, 91%, 77%, respectively, for tacrolimus-based regimens (p = 0.04, log rank test). conclusions. on the basis of our data, the overall survival of hcv transplanted patients were similiar to other indications. recurrent hcv infection did not influence patient and graft survival. immunosuppression may have an impact on survival in hcv-positive recipients, but optimal regimens need to be better defined by prospective studies. the advent of highly sensitive molecular techniques has revealed the possible persistence of hepatitis b virus (hbv) genomes in hbsag-negative patients with or without serologic markers of previous infection, a state called occult hbv infection. the highest prevalence of such infection has been shown in patients infected with hepatitis c virus (hcv). some studies suggested that occult hbv infection might favor or accelerate the progression of hcv infection towards cirrhosis. hcv infection recurs almost in all patients after liver transplantation (lt). about 5-10% of lt patients develop a fibrosing cholestatic hcv recurrence, which is associated with a very poor outcome. no specific risk factor for this pattern of recurrence has been described so far. the aim of this study was to determine the prevalence of occult hbv infection in patients presenting with fibrosing cholestatic hcv recurrence after lt. between 1986 and 2004, 151 hcv patients (104 m; 47 f) underwent lt at our institution. the mean follow-up was 51 months (range, 1-228 months). the diagnosis of recurrence was based on biochemical and histologic parameters. genotype 1 was predominant (75%), followed by 2 (15%), 3 (8%), and 4 (3%). eleven patients (7.3%; 10 m, 1 f) developed a fibrosing cholestatic pattern of recurrence characterized by highly elevated cholestatic parameters and typical histologic findings. also in this group, genotype 1 was predominant (n = 7), three had type 3, and one type 4. serum hbv dna was tested with the taqman test (roche, austria). five patients were hbsag negative, whereas six had serologic markers (antihbc positive). interestingly, hbv dna could not be detected in the sera of any of these patients with fibrosing cholestatic hcv recurrence. the actuarial 1-, 2-, 5-, and 10-year survival rates of all hcv patients were 83%, 79%, 68%, and 60%. hcv recurrence did not show a negative impact on patient and graft survival; however, the outcome of the patients with the fibrosing cholestatic pattern was less favorable. seven out of 11 patients died due to hcv recurrence, one patient had to be retransplanted secondary to recurrent disease. only three patients are alive with a functioning first allograft. this study indicates that occult hbv infection is not associated with fibrosing cholestatic hepatitis c recurrence after lt. background. the use of grafts from hepatitis b core antibody (anti-hbcab)-positive donors for liver transplantation (lt) is associated with the risk of de novo hepatitis b. patients who test positive for anti-hbcab pretransplant are also theoretically at risk to develop graft hepatitis b. methods. the outcome of 467 consecutive lts performed in 402 individuals between 1998 and 2001 was retrospectively analyzed with regard to the presence of anti-hbcab in donors and recipients. patients with hepatitis b and recipients of known anti-hbcab-positive grafts received hbig/lamivudine prophylaxis. results. a total of 111 recipients (28%) tested positive for anti-hbcab including 24 patients (7%) with hbv-associated cirrhosis and three patients with fulminant hepatitis b. a total of 20 allografts from anti-hbcab-positive donors were utilized, 14 of those (70%) were allocated to anti-hbcab-positive recipients. anti-hbcab-positive recipients were significantly more likely to have concomitant hcv (62% vs. 34%, p < 0.0001) and hepatocellular carcinoma (30% vs. 14%, p < 0.002). anti-hbcab-positive donors were more frequently non-caucasian (60 vs. 24%, p < 0.001) and cmv seropositive (85% vs. 65%, p < 0.035). survival of anti-hbcab-positive individuals and recipients of allografts from anti-hbcab-positive donors did not differ from their anti-hbcab-negative counterparts. there were no reported cases of recurrent hepatitis b in anti-hbcab recipients or patients receiving lt for hbv-associated liver disease. three cases of de novo acute posttransplant hepatitis b were identified, one being acquired during unprotected intercourse and two being transmitted through the graft. the two with graft-transmitted disease were anti-hbcab negative, treated initially with lamivudine and were switched to adefovir due to the emergence of ymdd mutants. conclusions. the frequency of anti-hbcab-positive recipients in our series was higher than expected. these individuals seem at minimal risk for posttransplant hepatitis b. recurrence of hbv after lt in the setting of hbig/lamivudine prophylaxis is extremely rare with 5-year median follow-up. the risk of transmission of hbv through anti-hbcab-positive livers despite prophylaxis cannot be neglected and the emergence of an ymdd mutant is of particular concern. anti-hbcab-positive grafts should be preferably given to anti-hbcab-positive recipients. v36 the response to preoperative transarterial chemoembolisation predicts outcome of patients with hepatocellular carcinoma after liver transplantation division of gastroenterology and hepatology, department of internal medicine, medical unversity of innsbruck, innsbruck, austria liver transplantation (lt) is the only curative therapy for patients with early-stage hepatocellular carcinoma (hcc). the impact of prelt chemoembolisation (ce) on patient survival and incidence of hcc recurrence has been controversially discussed and remains undetermined. the aim of this study was to evaluate the efficacy of ce prior to lt in hcc patients with regard to tumor recurrence and patient outcome. between 1984 and 2004, 167 hcc patients (142 m; 25 f) underwent lt at our institution. the underlying liver disease was viral hepatitis in 87 (hcv 69, hbv 18), alcoholic liver cirrhosis in 49, and other diseases in 18 patients. according to child-pugh classification, 75 patients presented with child a, 79 with stage b and 9 with stage c. hccs were diagnosed according to the easl guidelines. on the basis of prelt radiology, 23 patients were diagnosed with stage i, 79 stage ii, 34 stage iii, and 31 stage iv according to the modified uicc criteria. ce was performed in 120 patients prior to lt. in 47 patients no treatment was performed prior to lt. patients underwent multiple cycles of ce (mean, 1.6 ce/patient). ce response prior to ce was assessed by ct scan. on explant histology, complete response with no evidence of vital tumor was found in 38 of 120 (32%) patients, 55 (46%) patients showed a partial remission (tumor necrosis, >50%), and 27 (22%) patients showed a poor response or even progression. the intention-totreat analysis showed that patients with early-stage hcc showed an excellent survival with a 1-, 5-, and 10-year rate of 98%, 69% and 69%, respectively. ce prior to lt had no positive effect on overall patient outcome and rate of recurrence. however, patients with complete response to ce, on the basis of both pre lt and post lt histology, had a significantly bet-ter long-term survival (1-, 5-, and 10-year rates of 98%, 91%, and 91%) and rate of recurrence compared to those with partial or no response, but only in patients within milan and not san francisco criteria. the 1-, 5-, and 10-years survival rates of patients with advanced hcc were 95%, 67%, and 35%. hcc recurrence was found in 24 patients, 11 of 24 presented with advanced stage (iii and iv). only 13 had undergone ce prior to lt, and 9 of those were nonresponders to ce. this study indicates that response to prelt ce may predict the outcome of hcc patients after lt. patients with early-stage hcc, who responded to ce pre lt, showed an excellent outcome with 5-and 10-year survival rates around 90%. patients with early-stage hcc and only partial or no response to ce had a higher risk of recurrence of hcc after lt, but outcome was still favorable compared with advanced-stage tumors regardless of ce response. the roles of the regenerative factors hepatocyte growth factor (hgf), transforming growth factor a (tgf-a), and of vascular endothelial growth factor (vegf) were described in the context of hypertrophy and regeneration after liver resection but not well known in the transplantation situation. in the recipients of 63 consecutive liver transplantations with a graft survival of >2 weeks, the factors hgf, tgf-a and vegf were determined postoperatively (day 1, 3, 5, 7, 10, 14) by an el-isa in the serum and correlated to graft survival (kaplan-meier). the median concentrations of hgf were constant in total during the observation period (day 1, 2591 pg/ml; day 7, 2434 pg/ml; day 14, 2490 pg/ml). an individual increase to levels above 4000 pg/ml in the middle of the observation period correlated to a significantly decreased one-year graft survival (54% vs. 85%). similar was the course of tgfa. an increase from the median concentration of 39 pg/ml to levels above 80 pg/ml was observed in the context of a decreased primary function. regarding vegf, an almost linear increase of the concentration from 60 pg/ml via 177 pg/ml to 424 pg/ml (day 1, 7, and 14) was observed. here it became obvious, that an extensive increase of the vegf concentration correlated to a good transplant function. under the premise that the systemically determined concentrations were in relevant correlation to the secretion and thus to the local concentration in the liver, it can be concluded that vascular regeneration induced by vegf substantially contributes to graft survival, whereas a temporal increase of hgf and tgfa rather has to be interpreted as an indicator of an injured graft with a decreased functional prognosis. in total there are about 1650 patients on dialysis in bh and percentage of transplanted is 9.24. the aim of the paper is to analyse survival of grafts, patients, and grafts and patients over a 5-year period of living-related kidney transplantation history at university medical center tuzla. the results obtained were compared with the results of the centers with great number of transplants. methods. recipients and donors as family members were admitted after informative discussions and after the test results had been obtained from primary health care laboratories. the protocol was the standard one, in accordance with the recommendations of esot and eurotransplant. donors were tested first and then recipients. lumbotomy was done for nephrectomy. colins solution was used for kidney rinsing and perfusion, reconstruction of blood vessels was done as well as kidney biopsy. then, kidneys were stored at +4°c and grafts implantations were done on the contralateral iliac fossa. terminolateral anastomosis between external iliac artery and vein was done. implantation of ureters was done by modified lich-gregore technic, with or without dj stent. all patients received basic immunosuppressive protocol. the peculiarity was introduction of basiliximab (simulect) in therapy on the first and fourth postoperative day. descriptive statistics was done using spss software for windows 8.0. survival is presented by kaplan-meier curve. results are shown as means with standard deviations (sd). results. the first living-related kidney transplantation was done at university medical center tuzla on 15. 09.1999 09. . until 15.09.2004 . as many as 52 transplants have been done. donors were related to recipients as follows (in parentheses, mean age [years] with sd): mother, n = 24 (55.0 ± 11.3); father, n = 15 (61.4 ± 6.9); sister, 5 (44.5 ± 6.4); brother, n = 6 (46.0 ± 2.9); others, n = 2 (38.5 ± 5.5). table 1 shows mean dtpa clearance rate in donors. average separate dtpa clearance rates were within normal limits. as a rule, left-side donor nephrectomy was done; and in five cases, right-side nephrectomy. three donors showed borderline dtpa clearance rates of about 40 ml/min. bladder neck sclerosis was found in one patient and the air expansion of the bladder had to be done in the other one, at least to achieve its minimal capacity. two renal arteries were found in 5 patients (3 mothers, 1 father, 1 sister), two arteries were found with left kidney transplantation. termino-lateral anastomosis was done in 2 cases. the average age of the donors was 51.08 ± 6.58 years. as many as 16 donors were over 60 years old in our transplants, which reflected in our results. the average donor age of 36 males and 16 females was 32.4 ± 8.1 years. the data on hypertension before transplants were not reliable. the average hemodialysis time was 21.48 ± 10.36 months. the most common recipients' diseases were chronic glomerulonephritis, pyelonephritis, interstitial nephritis, nondefined renal disease, diabetes mellitus, systemic lupus, vesico-ureteral reflux. there were only 8 biopsies done before transplantation so that their histological background was known. the average serum creatinine after 5 years is 153.56 ± 24.65 µmol/l. cumulative graft survival after 5 years is shown by kaplan-meier curve: graft survival, 75.1%; patients survival, 83.9%; graft and patients survival, 67.1%. conclusions. the results on 5year living-related kidney transplantation at university medical center tuzla, bosnia and herzegovina, are similar or identical to the results of the developed centers in the world. the existing program has got to be improved, especially with respect to the donor selection from the standpoint of biological and chronological age. the experience gained so far is the basis for the development of cadaveric kidney transplantation in bosnia and herzegovina. background. shortage of available organs has increased the demand for living kidney donation. whereas donation for relatives is well accepted, there remains some controversy in the setting of emotionally related donation. there might be a survival benefit for grafts donated by relatives due to better hla matching. a retrospective analysis of 162 living donor kidney transplants which were performed at the university hospital of innsbruck was made. we divided the transplants in two groups according to the date of transplant: group 1 (1975-1993), 41 transplants; group 2 (1994-2005) , 89 transplants. the time period from 1994 until 2004 was analyzed in detail and data additionally compared to a cohort of 940 cadaveric renal transplants. during these eleven years, five liv-ing donations were carried out in patients who were not eligible to receive a cadaveric graft within eurotransplant. results. overall there were 128 lrt (53 siblings, 70 parents, 5 off-springs) and 34 ert (22 spouses, 12 others). mean donor age was 45 years (range, 21-70); mean recipient age was 40 (range, 2-72) years. in group 1 only 2 transplants were emotionally related (5%), whereas in group 2 the proportion increased to 25%. graft survival of living donated kidneys was better when compared with cadaveric kidneys (95% versus 92% at one and 89% and 83% at five years), but the difference did not reach statistical significance (p = 0.06). lrts and erts produced equal outcome. overall graft loss rate after a median follow-up of six (range, 0.5-11.5) years was 11% (lrts) vs. 16% (erts). the rejection rate was slightly higher in the lrt group with 31% vs. 22% (p > 0.05, n.s.). ten living donated grafts were lost and seven recipients of a living donated graft died. causes of death were cardiac ischemia (n = 1), pulmonary embolism (n = 1), fungal infections (n = 1), suicide (n = 1), and 3 causes were not specified. conclusions. the frequency of living-related and unrelated kidney donation has increased during the past two decades at our institution. equal results for cadaveric grafts were achieved when compared to lrt and erl. background. the 20s proteasome plays an important role in the nonlysosomal pathway of intracellular protein degradation and apoptosis, thus being a possible marker for ischemic and reperfusion injuries. the aim of this study was to monitor the proteasome levels in patients receiving kidney transplants to detect a relationship with the return of renal function. methods. we examined 12 patients with end stage renal disease, receiving kidney transplants: blood samples were collected intraoperatively and postoperatively on 5 consecutive days and 20s proteasome levels were measured for each sample with a sandwich elisa as described by dutaud et al. anesthesia and immunosuppressive medications were standardized, creatinine clearance and urine output were assessed daily on a routine basis. results. patients who had no adequate urine output (430 ± 300 ml) after the 4th postoperative day had significantly higher proteasome levels intraoperatively than patients with high urine output (4032 ± 1076 ml; 1.7 ± 1.5 µg/ml low vs. 0.5 ± 0.4 µg/ml high urine output, means with standard deviations, p = 0.02). this difference in proteasome levels seemed to even out during the follow-up period. conclusions. patients with impaired renal function after kidney transplant had significantly higher proteasome levels intraoperatively. a higher plasma level of proteasomes intraoperatively may therefore be a negative prognostic marker for postoperative return of renal function of the transplanted kidney. background. dendritic cells (dcs) are the most potent antigen-presenting cells and are pivotal for initiating allograft immunity. recently, however, particular dc subsets have also been implicated in allogeneic tolerance induction. campath-1h (alemtuzumab) is a novel t-cell-depleting antibody that is currently under investigation for the use in allogeneic organ transplantation and may confer tolerogenic properties. here we study the effect of alemtuzumab on peripheral dc subsets in kidney transplant patients under fk506 monotherapy in comparison to patients under conventional triple therapy. methods. patients receiving their first renal allograft were recruited within the tacam trial and randomly assigned to receive either alemtuzumab as induction agent followed by fk506 monotherapy (n = 7) or to receive conventional immunosupression consisting of fk506, mycophenolate mofetil and steroids (n = 7). absolute numbers of peripheral blood dcs and their different subpopulations were assessed by four-colour, single-platform fluorocytometry at the day before and 1, 4, 12, and 24 weeks after the transplant procedure, respectively. peripheral dcs were identified as hla-dr + and lineage-negative cell population. the respective dc subpopulations were cd11c + dcs (myeloid dcs or dc1), cd123 + dcs (plasmacytoid dcs or dc2), cd11c + and further, bdca1 + and bdca3 + dcs. results. induction with campath-1h led to a strong and sustained reduction of the total number of peripheral dcs compared to controls. while the absolute number of peripheral dcs in control patients recovered within 6 months after transplantation, campath-1h-treated patients exhibited a profound reduction of their circulating dcs. interestingly, a prominent shift of the ratio of myeloid to plasmacytoid dc subsets (dc1/dc2 ratio) was observed as early as one month after transplantation in campath-1h-treated patients. conclusions. employment of campath-1h as induction therapy in renal transplant patients is associated with a peculiar alteration of the peripheral dc repertoire. since plasmacytoid dcs have been linked to tolerance induction, the presented data suggest that the use of campath-1h in solidorgan transplantation creates an opportunity to safely introduce novel strategies to achieve alloantigen-specific hyporesponsiveness. background. detection of c4 complement split product c4d in peritubular capillaries represents a valuable diagnostic marker for antibody-mediated rejection (amr). numerous studies have demonstrated inferior allograft function and survival in c4d-positive as compared with c4d-negative kidney allograft recipients. however, anecdotal data implicate that in selected cases stable long-term function can be maintained despite detectable c4d deposits. as recently dicussed in the literature, c4d deposits could also reflect a state of graft acceptance (accommodation), rather than rejection. aim. the objective of this study was to investigate individual clinical outcomes in a large cohort of c4d-positive kidney transplant recipients. our emphasis was thereby to identify and characterize a subpopulation of c4d-positive recipients with only mild graft dysfunction and stable long-term graft function. methods. this retrospective analysis focused on 74 out of 878 adult kidney transplant recipients (transplantation between between 1999 and 2003) included on the basis of a positive c4d result early after transplantation. results. at the time of c4d-positive biopsy (median, 12 days post-tx) median serum creatinine was 6 mg/dl (range, 1.1 to 7 mg/dl) with 49% of the patients dialysis-dependent. according to our local standard, patients with severe graft dysfunction (n = 15) were subject to immunoadsorption treatment (ia). in another 20 highly sensitized recipients, c4d-positive graft dysfunction was diagnosed during or after pre-emptive peritransplant ia. furthermore, intense treatment included antilymphocyte antibody therapy (32%) and/or high-dose steroids in a high proportion of c4d-positive recipients (46%). analyzing all 74 c4d-positive recipients, 1-month post-biopsy se-rum creatinine was 2.1 mg/dl (30% of the patients dialysis-dependent). 1-year graft and patient survival was 73% (serum creatinine, 1.84 mg/dl). in a subsequent subanalysis we focused on twelve patients (two were biopsied under ia/atg induction) with only mild to moderate graft dysfunction (serum creatinine, 1.4 to 2.45 mg/dl) at the time of c4d-positive biopsy (banff borderline lesion in five, and banff i rejection in two biopsies). within this patient subgroup, we were able to identify five recipients in whom stable long-term graft function could be achieved following steroid bolus therapy only, without further therapeutic measures. in this particular subgroup excellent 1-year allograft function (serum creatinine levels between 1.0 to 1.8 mg/dl) was observed. conclusions. our results demonstrate that in the majority of patients peritubular capillary c4d deposits are associated with severe graft dysfunction necessitating aggressive treatment. nevertheless, in a small subgroup of recipients stable graft function for a long period of time can be achieved without intense therapy despite capillary c4d deposition in biopsy. background. combined kidney pancreas transplantation (ptx) evolved as excellent treatment for diabetic nephropathy with infections remaining common and serious complications. methods. 217 consecutive enteric drained ptxs performed from 1997 to 2004 were retrospectively analyzed with regard to bloodstream infection. immunosuppression consisted of antithymocyteglobuline induction, tacrolimus, mycophenolic acid, and steroids for the majority of cases. standard perioperative antimicrobial prophylaxis consisted of pipercillin/tazobactam in combination with ciprofloxacin and fluconazole. results. one-year patient, pancreas and kidney graft survival were 96.4%, 88.5%, and 94.8%, surgical complication rate was 35%, rejection rate 30%, and rate of infection 59%. in total, 46 sepsis episodes were diagnosed in 35 patients (16%) with a median onset on day 12 (range, 1-45) post transplant. sepsis source was intra-abdominal infection (iai) (n = 21), a contaminated central venous line (n = 10), wound infection (n = 5), urinary tract infection (n = 2), and graft transmitted (n = 2). nine patients (4%) experienced multiple sepsis episodes. overall, 65 pathogens (iai sepsis, 39; line sepsis, 15; others, 11) were isolated from blood. gram-positive cocci accounted for 50 isolates (77%): coagulase negative staphylococci (n = 28 [43%]) (nine multiresistant), staphylococcus aureus (n = 11 [17%]) (four multiresistant), enterococci (n = 9 [14%]) (one e. faecium). gram-negative rods were cultured in twelve cases (18%). patients with blood borne infection had a two-year pancreas graft survival of 76.5% versus 89.4% for those without sepsis (p = 0.036), patient survival was not affected. conclusions. sepsis remains a serious complication after ptx with significantly reduced graft but not patient survival. the most common source is iai. background. mobilized allogeneic peripheral blood stem cells (pbsc) are increasingly used as graft source instead of bone marrow. although the short-term safety profile of recombinant human granulocyte colony-stimulated factor (rhg-csf) seems acceptable, minimal data exist regarding long-term safety. methods. we reviewed data of 196 allogeneic pbsc donors (siblings, n = 159; unrelated donors, n = 37) with respect to side effects of rhg-csf administration, adverse events of leukapheresis and late effects. written informed consent was signed before pbsc mobilization and collection. donors (m/f, 119/77) had a median age of 40 years (range, 11-72). they received rhg-csf at a dose of 2 times 5 µg/kg of body weight a day for 4 consecutive days starting on day 1. routinely, pbsc collection was started on day 5. on condition that donors' white cell count exceeded 70.000/µl or cd34-positive cells exceeded 50/µl, pbsc harvest was started on day 4. rhg-csf was administered until end of the apheresis period unless white blood cell count did not exceed 70.000/µl. pbsc were collected with a continuous-flow blood cell separator processing 3-3.5 times total blood volume. citrate was used as anticoagulation and in the majority of donors a continuous calcium infusion (2.25 mmol [89.4 mg] of calcium per h) was given. reinfusion of autologous platelet-rich plasma from pbsc collection was performed in donors with post-donation thrombocytopenia of <100 g/liter if further collection were necessary or in donors with platelet counts of <80 g/liter, respectively. pbsc harvest procedures were repeated until the predicted cd34 + cell yield of 4 × 10 6 /kg of body weight of recipient was collected. however, not more than 3 consecutive collections were performed. flowcytometric analyses were performed using a becton-dickinson facs-scan or facs-calibur, respectively. for follow-up we assessed peripheral blood counts, electrolytes, lactic dehydrogenase (ldh), alkaline phosphatase (ap), total protein, albumin, on days 1, 7, 30, 100, 365 and then yearly for 5 years. donors received a questionnaire for evaluation of medical history and quality of life, results and evaluation are pending. results. the main adverse events related to rhg-csf administration were bone pain (63 of 196, 32%), myalgia (61 of 196, 31%), headache (44 of 196, 22%), fatigue (36 of 196, 18%), sleep disturbance (30 of 196, 15%) and were rated as moderate to severe by 35% of the donors. due to continuous calcium infusion, incidence of citrate toxcicity was low (34 of 196, 17%) and consisted only of mild paraesthesia. in 10 of 196 (5%) donors post donation platelet count decreased below threshold and required reinfusion of autologous rich plasma. eighty-eight of 196 donors (44%) were lost for follow-up. eighty-two were sibling (82 of 159, 52%) and 6 (6 of 37, 16%) were matched unrelated donors, respectively. from the remaining 108 donors, unrelated donors (31 of 37, 84%) had a median of 4 check-ups (range, 1-8) during the first 100 days (median; range, 1 day to 3 years) after donation, whereas siblings (77 of 159, 48%) had a median of 2 check-ups (range, 1-6) during the first 30 days (median; range, 1 day to 5 years), respectively. two donors (1%), both siblings, were hospitalized within 2 weeks after donation due to severe enteritis and subarachnoidal haemorrhage. the latter donor never had platelet counts below 100 g/liter after pbsc donation and recovered without neurological deficit. follow-up of peripheral blood counts showed a loss of platelets during donation and early post-donation period and a decrease of white blood cells 7 days after donation, both returning to normal within 30 days after, respectively. ldh and ap showed a significant increase during pbsc mobilization, they returned to normal within 1 week after donation, other blood parameters remained unaffected. conclusions. due to the fact that we observed hospitalization of 2 donors within 14 days after pbsc collection, a close monitoring of donors in the early post-donation period seems advisable. although reported anomalies in medical history of donors beyond 30 days after pbsc harvest could not be associated with rhg-csf administration, a regular followup for at least 5 years should be considered. with the particular focus on donor safety, a standardized approach to data collection of follow-ups to monitor short-and long-term effects in all centers should be established. background. donor-recipient sex mismatch is an established risk factor for poor outcome after allogeneic myeloablative hematopoietic stem cell transplantation (hsct). the risk of transplant-related mortality (trm) due to graft-versushost disease (gvhd) is higher in male recipients of female stem cells compared with female patients receiving a graft from a female donor. with longer follow-up, however, the graft-versus-leukemia (gvl) effect due to hy minor histocompatibility antigen mismatch may predominate. the contribution of donor-patient sex on outcome after nonmyeloablative hsct, however, has not been examined in detail yet. methods. we therefore analyzed a single-center cohort of 72 high-risk patients transplanted with a related or unrelated stem cell graft after nonmyeloablative conditioning for outcome (acute and chronic gvhd, trm, relapse, and survival). results. of the 72 patients, 19 male patients received a graft from a female donor, 21 males a graft from a male donor. sixteen female donors were transplanted with a male donor and 16 with a female donor. around 30% of the patients with a sex-mismatched donor received stem cells with an hla mismatch compared to 10% of the patients without sex-mis-stammzellen matched donor, the other clinical differences were similar between all groups. the highest cumulative incidence for acute and chronic gvhd was detected in male patients receiving a stem cell graft from a male donor (52.4% and 53.3%, respectively). the highest relapse incidence (55.6%) was detected in male patients transplanted with a female donor. this was borderline significant (p = 0.0845) to female patients receiving a female graft (20% relapse incidence) and argues against an effective anti-hy response in this patient cohort. the mean cumulative incidence for trm was 57.3%; however, female recipients receiving a graft from a female donor displayed an unexpectedly high incidence for trm (87.5%) which could not be explained by clinical characteristics. the overall survival of 41% 2.5 years after transplant in this group, however, was not different from male patients receiving a female graft (43.7% at 1.4 years after transplant). the overall survival from male patients with a male donor was slightly lower (33% at 2.5 years after transplant) compared with female patients transplanted with male stem cells (47.4% at 1.4 years after transplant). conclusions. these data, analyzed in a small cohort of patients, show that a sex mismatch between patient and donor may have a negative impact also on outcome after nonmyeloablative hsct. however, studies with larger and homogeneous cohorts have to be performed to prove these findings. between 1995 and 2004, 20 patients with chronic lymphoid leukemia (cll), binet stage b or c (n = 19) or a with risk factors (n = 1) with a median age of 53 (range, 27-62) years underwent autologous (n = 11) or allogeneic (n = 9) hematopoietic stem cell transplantation (hsct) at the medical university of vienna. the median time from diagnosis to hsct was 29 (range, 6-77) months. eleven patients underwent autologous stem cell transplantation (asct) in partial remission (n = 9) or complete remission (cr) (n = 2) and received bcell-depleted peripheral blood stem cell (pbsc) grafts. nine patients with refractory disease (n = 6) or chemosensitive relapse (n = 3) underwent allogeneic hsct with unmanipulated bone marrow (n = 3) or pbsc (n = 6) from sibling (n = 8) or unrelated (n = 1) donors. in the majority, conditioning therapy consisted of total-body irradiation (tbi) of 12-13.2 gy and cyclophosphamide. three patients underwent reduced-intensity conditioning (ric) with fludarabine and tbi of 2 gy. graft-versus-host disease (gvhd) prophylaxis consisted mainly of cyclosporine (csa) and methotrexate for myeloablative and csa and mycophenolate mofetil for ric-hsct. complete clinical remission was attained in 10 patients (91%) after asct and in 6 (67%) after allogeneic hsct. molecular remission assessed by immunoglobulin heavy chain gene (igh) rearrangement pcr was attained in 8 patients after asct and 5 after allogeneic hsct. in seven patients we noticed persistence of the igh rearrangement, six of these patients died of disease progression or relapse 1-29 months after asct (n = 2) or allogeneic hsct (n = 4). after a median follow-up of 58 (range, 21-122) months, nine autologous (82%) and four allogeneic (44%) graft recipients are alive and 10 patients (asct, n = 6; allogeneic hsct, n = 4) are in clinical and molecular remission. the median time to clinical relapse was 20 (range, 1-102) months. treatment-related death occurred only in one patient 27 months after myeloablative hsct. probability of overall survival is 82% after asct and 42% after allogeneic hsct. in summary, all cll patients with long-term cr after asct and allogeneic hsct also attained sustained molecular remission of the igh rearrangement. . the incidences of the observed genotypes in this small group of patients and donors tested were in the range as published: il10 with 54% c/c, 41% c/a, 5% a/a; nod2 with 12% recipient or donor; 5% recipient and donor, and mpo with 56% g/g, 43% g/a, 1% a/a. since the patient population was very heterogeneous concerning diagnosis (34 aml, 12 all, 9 nhl, 6 cml, 5 mds, 4 saa, 4 solid tumors, 3 mm, 2 omf, 1 cll, and 1 et), course of disease, and condition regimen, patients were divided into four groups for evaluation: 23 with aml and identical high-dose induction therapy (bu/cy), 23 with reduced condition regimen, 6 with unrelated donors, and 29 others. concerning all 81 patients, 19 of them (23%) died through trm (3 aml, 6 reduced, 10 others), but none of them was at high risk determined by nod2 polymorphism (mutated donor and recipient) and only one determined by mpo a/a genotype (aml). four patients (3 reduced, 1 others) with nod2 mutations in donor and recipient dna did not die from trm. twenty-three patients developed severe (grade 3 or 4) acute gvhd (7 aml, 5 reduced, 11 others), 10 of them had il10 c/c genotype (5 aml, 2 reduced, 3 others) and in three patients nod2 was mutated in donor and/or recipient dna (1 aml, 2 others). on the other hand, 34 patients with il10 c/c genotype and 13 patients with nod2 mutations in donor and/or recipient dna developed no or mild gvhd only. in conclusion, so far we were not able to find a correlation between gvhd/trm risks and polymorphisms of il10, nod2, and mpo. this might be due to the small number and the heterogeneity of the patients; however, a panel of additional snps may increase the accuracy of risk assessment prior to allogeneic sct. background. allogeneic stem cell transplantation is a curative therapy for patients with lymphoproliferative disorders as a result of the intensity of the conditioning regimen and the application of a graft-versus-lymphoma effect. however, conventional conditioning regimens have been associated with a 24-61% risk of transplant-related mortality (trm) in advanced hodgkin's lymphoma (hl). in an attempt to reduce the high trm reported with allografting in lymphoma, reduced-intensity regimens have been investigated. methods. four patients between the age of 34 and 44 years underwent allogeneic peripheral blood stem cell (pbsc) transplantation (sct) from hla-identical sibling or unrelated donors at our institution. age, sex, manifestation of disease, and donor were as follows: patient 1-37 years, female, pulmonary bulk, sibling; patient 2-34 years, male, pulmonary bulk, unrelated donor; patient 3-42 years, male, pulmonary bulk, sibling; patient 4-44 years, male, abdominal bulk, sibling. all patients had received multiple courses of polychemotherapy (table 1 ) and local radiotherapy prior to sct. patients 1 and 4 had undergone autologous stem cell transplantation. we administered a reduced-intensity conditioning consisting of beam (bcnu, etoposide, cytarabine and melphalan) over 6 days. to permit durable engraftment in the allogeneic setting, patients received additional pretransplant immunosuppression with an anti-cd52 antibody (campath) at a total dose of 50 mg over 5 days and graft-versus-host disease (gvhd) prophylaxis of cyclosporine a. pbsc of donors were collected after g-csf stimulation at 10 µg/kg of body weight given for 4 days. results. patient 2 rejected his unrelated-donor graft and received an autologous stem cell infusion 44 days thereafter resulting in sustained hematologic recovery (anc, >0.5 g/l on day 10). all other patients engrafted uneventfully (anc, >0.5 g/l on days 14-16; platelets, >20 g/l on days 10-13). none of the patients showed evidence of acute or chronic gvhd. two patients achieved complete donor chimerism in myeloid and lymphoid cell populations, another one had prolonged mixed chimerism and was given a donor leukocyte infusion of 1 × 10 6 cd3-positive cells per kg. one patient experienced a cmv reactivation on day 51 after sct and a sarcoidosis on day +150. three months after sct, all patients showed marked regression of disease. after a follow-up of 5 to 11 months (median, 8 months), 2 patients experienced progression undergoing salvage chemotherapy. time to progres-sion was 5 and 6 months, respectively. two patients remained progression free for 7 and 11 months, respectively. overall survival is 5 to 11 months. conclusions. our data demonstrate that this regimen was well tolerated with a low risk of gvhd and transplant-associated morbidity. an increased dosage of campath could be considered to prevent rejection in unrelated-donor transplantation. longer follow-up and larger patient numbers are warranted for assessment of the efficacy of campath-beam with regards to durable remissions. background. as long-term survivors of hematopoietic cell transplantation (hct) become more numerous, studies addressing the issue of long-term follow-up are necessary. in this study, we report on the quality of life (qol) of hct patients who were alive at least at 5 years after transplantation in comparison with an age-and sex-matched sample of healthy controls assessed in the same time period and the same geographical region. methods. the eortc-quality of life questionnaire (eortc-qlq c30) was sent by post to 39 hct survivors. thirty-four patients answered the questionnaire. patients were compared with 68 healthy controls from the same geographical region. patients and controls completed the eortc in the same time period. results. mann-whitney u tests identified significantly lower qol on the dimensions of physical and social functioning and on the financial impact symptom scale. conclusions. patients who had survived their hct for more than 5 years did generally well in terms of global qol. this is consistent with kiss et al. (2002) who found that cml patients who were alive at least 10 years after hct report lower physical functioning in comparison with healthy controls. problems in the areas of social functioning and financial difficulty can possibly be addressed by intensive rehabilitation processes integrating patients, family members, and significant others. interdisciplinary (medical, psychological, and social) treatment of patients should not come to an end after the acute phase of the illness but should continue during checkups following transplantation. background. bone marrow transplantation (bmt) together with costimulation blockade can reliably induce mixed chimerism and tolerance in certain experimental models. natural killer t cells play an important role in the induction of tolerance in several transplantation and autoimmunity models. it has been reported, for instance, that activation of nk t cells by α-galactosylceramide (α-gal) can prevent the onset and recurrence of autoimmune type i diabetes. in recent experiments we wanted to investigate the role of nk t cells in a model of tolerance induction through bmt with costimulation blockade. to delineate the role of donor and recipient nk t cells, we used nk-t-cell-deficient mice (jα281-/-c57bl/6 and jα281-/-balb/c) as recipient and/or donor strain. we also evaluated whether in vivo stimulation of nk t cells with α-gal has a beneficial effect. methods. c57bl/6 mice and c57bl/6 jα281-/-received a total-body irradiation (tbi) of 3 gy or 1 gy (day -1), approximately 20 × 10 6 fully mismatched balb/c or balb/c jα281-/-bone marrow cells (day 0) and costimulation blockade consisting of anti-cd154 mab (mr1, day 0) and ctla4ig (day +2). groups were additionally treated with α-gal or a vehicle (5 µg on day -1, +2, +7, +14, and +21). multilinage chimerism and skin graft survival were followed for more than 120 days. results. with our standard protocol, 12 of 18 mice developed long-term multilinage chimerism and permanent donor skin graft survival. when using recipients deficient in nkt cells, 5 of 5 became chimeric and showed long-term skin graft survival; using nkt knockouts as donors 6 of 6 and using nkt knockouts both as recipients and donors 5 of 6 became chimeric (p = n.s. for all comparisons). unexpectedly, stimulation of nk t cells by α-gal (using wild-type recipients and donors) prevented chimerism induction after 3 gy tbi (0 of 8 vs. 4 of 6 and 4 of 4 in the vehicle group, p < 0.01) and did not promote engraftment after 1 gy tbi (0 of 8 vs. 0 of 8). conclusions. nk t cells do not play a critical role in tolerance induction after bmt with costimulation blockade. their stimulation with α-gal even prevents induction of mixed chimerism and tolerance. background. phospholamban (plb) is a critical regulator of sarcoplasmic reticulum ca 2+ -atpase activity and myocardial contractility. in this study we investigated the role of plb phosphorylation in ischemia and reperfusion following cardiac transplantation. methods. gene expression of plb was investigated in a syngeneic heterotopic cardiac transplant model in mice. grafts underwent 10 h of cold ischemia or were tranplanted immediately after harvest. gene expression was analysed at various time points employing dna microarray and rt-pcr. for in vitro experiments, hl-1 cardiomyocytes were submitted to a protocol of global normothermic hypoxia for 6 h and reoxygenation in the absence or presence of the ca 2+ /calmodulin kinase ii inhibitor aip (1 µm) or the beta-adrenergic blocker dl-propranolol (1 µm) vs. beta-adrenergic stimulator isoproterenol (1 µm). results. at 24 h, gene expression of plb was diminished by 14.1 and 3.6-fold in groups with and without ischemia, respectively. basal phosphorylation of plb at ser16 (protein kinase a site) and at thr17 (ca 2+ /calmodulin kinase ii site) was present in cultured cardiomyocytes and heart lysates. in the mouse system, increase in plb phosphorylation is observed during early (up to 10 min) reperfusion. thereafter, plb phosphorylation drops below that of control levels. addition of aip diminishes reperfusion-induced thr17 phosphorylation; propranolol significantly decreases ischemia-induced ser16 phosphorylation. in contrast, isoproterenol enhances plb-ser16 and thr17 phosphorylation. conclusions. ischemia regulates phospholamban by two different mechanisms, decrease in expression levels and alterations in the phosphorylation of critical regulatory sites. modulation by aip and dl-propranolol will help for investigation of the role of pbl phosphorylation in ischemia and reperfusion in cardiac transplantation in the future. background. the p38 mitogen-activated protein kinase (p38-mapk) pathway plays a crucial role in pathological events like oxidative stress, inflammation, and abnormal cellular proliferation, resulting in activation of several signalling cascades, involving overexpression of tumor necrosis factor alpha (tnf-α). tnf-α is known to play an important role in chronic rejection. currently, pharmacological inhibitors of p38-mapk are being tested clinically for the treatment of experimentelle transplantation chronic inflammatory diseases such as rheumatoid arthritis, morbus crohn and psoriasis as well as inhibition of vascular smooth muscle cell (vsmc) proliferation after balloon angioplasty. to date, there are no findings that address the role of p38-mapk in the context of chronic allograft vasculopathy, which is characterized by vascular lesions in the graft that consist of concentric myointimal proliferation, resulting in deterioration of allograft function and organ loss. the purpose of this study was to understand the role of p38-mapk in abnormal vsmc proliferation associated with chronic rejection and to investigate the potential therapeutic role of a specific inhibitor of p38-mapk activation in chronic allograft vasculopathy. methods. in vivo, a mouse model of heterotopic aorta transplantation in an allogenic setting has been used. aortas were allografted into recipient mice by a carotid artery cuff technique, using c57bl/6 (h-2 b ) mice as donors and balb/c (h-2 b ) mice as recipients. four weeks after transplantation, the aortic segments were harvested and immunofluorescence was performed using anti-vsmc-α-actin and anti-phospho-p38 antibodies. tnf-α serum levels were measured by elisa. in vitro, vsmcs were isolated from c57bl/6 aortas. expression levels of total and phosphorylated forms of p38-mapk as well as key cell cycle regulators were detected by western blot. immunocytochemistry was performed with primary antibodies directed against phospho-p38-mapk. proliferation of vsmcs was measured by [ 3 h]thymidine incorporation in the presence or absence of sb 203580, a specific inhibitor of p38-mapk activation. cell cycle progression was monitored by dna content analysis; apoptosis by the annexin v binding assay. cell lysates were probed with antibodies directed against cyclindependent kinase 2 (cdk2) and yin yang 1 (yy1). results. in vivo, 4 weeks after the transplant aortic segments were significantly narrowed due to neointimal hyperplasia. the neointimal lesions mainly consisted of vsmcs and showed profound activation of p38-mapk as demonstrated by immunofluorescence. further, serum tnf-α levels were significantly increased even 4 weeks after allogeneic aortic transplantation, suggesting an important role of p38 activation in this model. in vitro, stimulation of vsmcs with 10% fcs resulted in a rapid increase of phosphorylated forms of p38-mapk (8.0 ± 0.7 fold increase) when compared with the nonstimulated quiescent state. immunocytochemistry showed higher levels of phospho-p38-mapk in the nuclei as well as in the cytosol after stimulation. sb 203580 in a dose-dependent manner significantly inhibited vsmc proliferation, which was due to inhibition of cell cycle progression at the g 0 /g 1 phase. we did not observe apoptosis in the sb 203580-treated vsmcs at 20 µm, the highest dose being tested. blockade of p38-mapk activation decreased protein levels of cdk2 and the transcription factor yy1, which plays an important role in vsmc dna replication and protein synthesis. conclusions. p38 mapk activation appears to play an important role in an in vivo allogeneic model of aorta transplantation as well as in vsmc proliferation in vitro, blocking of which prevents the cells from entering the s phase of the cell cycle, thus abrogating cell proliferation. targeting p38-mapk might become a potent strategy in the treatment of vascular proliferative diseases like chronic allograft vasculopathy. background. ctla4ig, a costimulation blocker which is currently under advanced clinical development, has been used for years as part of mixed chimerism protocols. recent data suggest that ctla4ig also functions via modulation of tryptophan metabolism by upregulating indoleamine 2,3-dioxygenase (ido) through b7 signals. we thus investigated the role of ido in our ctla4ig-based mixed chimerism protocol. methods. c57bl/6 mice received a total body irradiation (tbi, d -1) of 3 gy, approx. 20 × 10 6 fully allogeneic balb/c bone marrow cells (d 0) and costimulation blockade consisting of anti-cd154 mab (1 mg, d 0) and ctla4ig (0.5 mg, d 2). different groups of mice were additionally implanted with 1-methyltryptophan pellets on d -1 (1-mt, which is a competitive inhibitor of ido, 7-day release at 0.9 mg/h) or on d -1 and d 6 (14-day release) or placebo pellets. macrochimerism and deletion of donor-reactive cells were followed by flow cytometry. levels of tryptophan, kynurenine, and 1-mt were measured at several timepoints in serum by hplc. results. 8 of 10 mice which received bmt, tbi, mr1 plus ctla4ig but only 2 of 10 mice without ctla4ig developed lasting multilineage chimerism (p < 0.05, measured at week 32 post bmt). 8 of 10 mice with ctla4ig treatment accepted donor skin grafts for more than 150 days, whereas only 1 of 10 mice, which got no ctla4ig injection accepted donor skin long-term (p < 0.05; representative data from two separate experiments), demonstrating that ctla4ig is critical for our protocol. 3rd-party grafts were promptly rejected in all groups. long-term multilineage chimerism developed in 6 of 8 mice with 1-mt treatment, which is not significantly different from treatment with placebo pellets or standard protocol alone (7 of 11, pooled data from both groups). 2-week treatment with 1-mt also did not lead to a significant difference in the rate of multilineage chimerism (4 of 5 with 1-mt vs. 4 of 7 without 1-mt). deletion of donor-reactive t cells was neither blocked nor enhanced by treatment with 1-mt. the kynurenine-to-tryptophan ratio in serum was similar in groups with (11.8 ± 2.7) and without (12.1 ± 3.3) ctla4ig (p = n.s., measured on d 3). substantial serum levels of 1-mt were detected in mice with 1-mt treatment but not in untreated mice, indicating that ido was indeed inhibited in the 1-mt groups. conclusions. ctla4ig plays an essential role for tolerance induction in this model but its mechanisms of action does not critically depend on ido. background. induction of antigen-specific tolerance remains the ultimate goal in clinical organ and cell transplantation, as it would eliminate the need for continuous immunosuppression. one strategy leading to tolerance induction against a transplanted organ consists of infusing blood from the organ donor, an approach known as donor-specific transfusion (dst). although the mechanisms underlying tolerance induction by dst are not fully understood, clonal deletion of alloreactive t cells and generation of immunoregulatory cd25 + cd4 + t cells are important in the process. it is well established that expression of heme oxygenase-1 (ho-1) can promote the survival of transplanted organs. however, the mechanisms underlying this effect remain to be elucidated. we hypostesized that ho-1 is required for tolerance induction involving dsts and that ho-1 can magnify the tolerogenic effect of dsts. methods. allograft survival has been tested by using a well established model of costimulatory blockade (anti-cd40l-ab) plus dst (day -7) in c57bl/6 (h-2 b ) heart allografted balb/c (h-2 d ) wt and ho-1 ko mice. further, ho-1 activity has been induced (by copp) or suppressed (by znpp) in b6af1 (h-2 k/d,b ) mice receiving dba/2 (h-2 d ) allografts plus dst on day 0 or day -7. donor-specific tolerance was tested by challenging the mice with a second dba/2 or third-party fvb (h-2 q ) allograft. leukocytes (depleted or undepleted of cd25 + cd4 + t cells) from mice carrying allografts for >100 days were adoptively transferred to sublethally irradiated b6af1 mice receiving dba2 heart allografts. rna levels of foxp3, tgf-β, il-10 and ctla-4 have been assesed by using rt-pcr. cd25 + cd4 + t cells have been enumerated by flow cytomerty. results. anti-cd40l-ab plus dst treatment resulted in balb/c recipients tolerizing fully mismatched c57bl/6 allografts (n = 6). however, by using ho-1-deficient recipients, this effect was abrogated, in that c57bl/6 allografts have been rejected in a similar manner as in untreated wt recipients (mst = 17.5 d, n = 4). further, dst plus copp (in contrast to dst plus znpp) treatment resulted in donor-specific tolerance of dba/2 allografts in b6af1 recipients (n = 7). tolerant animals showed significantly increased percentage of cd25 + cd4 + t cells and increased levels of foxp3, tgf-β, il-10, and ctla-4 mrna. adoptively transferred b6af1 leukocytes retrieved from the lymph nodes and spleens transferred to sublethally irradiated b6af1 recipients of dba2 allografts led to subsequent allograft loss (mst = 16.7 d, n = 6); in contrast, transfer of leukocytes of tolerant (copp plus dst treated) mice led to indefinite allograft survival in this model. however, when those leukocytes were depleted of cd25 + cd4 + t cells, allografts were immediately rejected (mst = 17.7 d, n = 5). conclusions. ho-1 in a graft recipient can be critical for long-term graft survival and for induction of tolerance. this mainly is mediated by generation of cd25 + cd4 + t cells (t regs). modulation of ho-1 expression and activity may be used therapeutically to promote graft tolerance. background. human immunoglobuline (ivig) has been advocated in the treatment of acute rejection in allograft transplantation and treatment of sepsis. mechanisms describing the immune modulatory activity are however scarce. we sought to investigate immune suppressive properties of pooled human immuneglobuline (ig), unspecific fc and fab fragments and their respective ligands by allogeneic blastogenesis assays. methods. human mixed lymphocyte reactions (mlr) were performed. in detail peripheral blood mononuclear cells (pbmcs) were purified from 5 donors by ficoll density gradient centrifugation (amersham biosciences, buckinghamshire, england). 100,000 cells per well were stimulated with 100,000 radiated pbmc (60 gy) and incubated for 5 days at 37°c together with unspecific igg, igm (both sigma-alderich, st. louis, mo), anti-cd32 (lab vision, fremont, ca) and anti-cd64 (chemicon, temecula, ca) in a dose-dependent fashion. before harvesting cells were pulsed for 18 h with [ 3 h]thymidine (3.7 × 10 4 bq/well) and the [ 3 h]thymidine uptake was measured in a liquid scintillation counter. results. the addition of pooled human igg and igm to allogeneic stimulated cells both resulted in a significant and dose dependent decrease of proliferation, although the suppressive properties of igm was greater as compared to igg (both, p < 0.0001). to investigate whether this effect was partly related to fc receptor involvement we blocked cd32 and cd64 on antigen presenting cells (apcs), known receptors in the activation of mlr. surprisingly, this assay demonstrated that sole fc blocking (high-and low affinity fc receptors) resulted in a significant downregulation of allogeneic response in vitro (both, p < 0.001). conclusions. our data evidence that the addition of unspecific immune globulines results in a reduction of proliferation in in vitro allogeneic blastogenesis assays and is partly fc receptor dependent. these results corroborate the clinical success of ivig and pooled igm in the treatment of solid organ allograft rejection and cautions the utilization of immune globulines in immune suppressed septic patients. die interaktion professioneller antigen-präsentierender zellen mit allogenen t-zellen resultiert in der ausbildung der sog. immunologischen synapse (is), welche für die effiziente aktivierung von t-zellen und damit letztlich für die transplantatabstoßung essentiell ist. im rahmen der is werden der t-zellrezeptor/cd3-komplex, kostimulatorische wie adhäsionsmoleküle und komponenten des zytoskeletts in die kontaktzone des mhc-tcr/cd3-komplexes der is rekrutiert. der einfluss von gängigen immunsuppressiva, die zur prävention der allogenen organabstoßung klinisch verwendet werden, auf die is-evolution ist bislang unbekannt. in dieser studie zeigen wir erstmals, dass kalzineurinhemmer wie csa oder fk506, aber nicht mtor-inhibitoren, selektiv die rekrutierung des tcr/cd3-komplexes in die is blockieren. ein ähnlicher effekt wurde für kortikosteroide, aber nicht mycophenolat beobachtet, was auf eine essentielle rolle des calcineurin-nf-κb-signalweges für die tcr/cd3-regulation im rahmen der t-zellaktivierung hinweist. interessanterweise blockierte das neue immunsuppressivum fk778 nicht nur die tcr/cd3-, sondern auch die lfa-1-und f-aktin-rekrutierung in die is. die bedeutung dieser globalen interferenz mit der is-ausbildung für die spezifische immunantwort muss in weiterführenden studien geklärt werden. diese daten zeigen, dass klassiche immunsuppressiva nicht nur simple blocker der zytokinsynthese sind, sondern schon sehr früh die t-zell-apz-interaktion stören und damit einen weiteren immunologischen mechanismus besitzen, der ihre klinische potenz erklärt. background. ischemia (i) and reperfusion (r) trigger a series of events, which culminate in severe injuries to the affected organs in organ transplantation. cell death, metabolic alterations, and inflammation result in impairment of shortand long-term function. the group of mitogen-activated protein kinases (mapks) are central regulators of these events, they have been implicated through aberrant activation in many pathophysiological settings ranging from autoimmune diseases, cancer, to i/r-associated organ damage. methods. intracellular signaling pathways were analysed in vivo and in vitro employing a cardiomyocyte cell line and a murine heart transplant model. hl-1 cardiomyocytes are a cardiac muscle cell line derived from at-1 mouse atrial cardiomyocytes. syngeneic cardiac transplants were carried out us-ing male inbred balb/c mice, hearts were transplanted heterotopically into the neck of recipients. results. in summary, (i) reoxygenation was characterized by a dramatic increase in the activity of all mapks at the end of the observation period; (ii) growth factor abrogation together with hypoxia (h) and reoxygenation (r) had a substantial effect on the course of signaling events; (iii) signaling processes in response to ischemia and reperfusion in vivo are in line with observations made in cardiomyocytes. conclusions. data obtained so far in our study demonstrate the suitability of the chosen experimental approaches for investigation of i/r-associated alterations in intracellular signaling and cellular responses. preliminary data suggest that h/r treatment of hl-1 results in significant apoptotic cell death, the intracellular signaling pathways involved are therefore currently analyzed. background. ischemia and reperfusion (i/r) in cardiac transplantation results in inflammation and cell death. to gain further insight into the regulation of these processes, we investigated the role of lipocalin-2 (24p3, ngal, uterocalin), a potential regulator of the acute inflammatory response and cellular apoptosis in vitro and in vivo. methods. c57bl/6 hearts were heterotopically transplanted to syngeneic recipients immediately and after 10 hours of prolonged cold ischemia with the grafts harvested at various timepoints (2 min, 2 h, 12 h, 24 h, 48 h, 10 d) after transplantation. gene expression analysis on mrna extracts was performed employing cdna microarray and rt-pcr. protein synthesis was investigated by western blotting and apoptosis by using tunel assay. for the identification of the cellular source of lipocalin-2 and its function in i/r-associated cell death, the effect of recombinant lipocalin-2 was investigated in the hl-1 cardiomyocyte cell line. hl-1 cardiomyocytes undergoing ischemia/reoxygenation as well as purified mononuclear cells and granulocytes were analyzed for lipocalin-2 expression by rt-pcr. results. in the cardiac transplants, high levels of lipocalin-2 gene and protein expression were detected at 12 h of reperfusion, whereby transcription was higher in groups without cold ischemia (22-versus 8.8-fold). he staining demonstrated dense mononuclear infiltrates, cellular edema, and small focal necrosis in groups with and without prolonged cold ischemia. upregulation of gene transcription was confirmed by pcr. apoptotic cells were first detectable at day 2 and peaked 10 days after transplantation. expression of lipocalin-2 was also detected in hl-1 cells by rt-pcr and western blotting following i/r, demonstrating for the first time the presence of lipocalin-2 in this cell lineage. lipocalin-2-transfected hl-1 cardiomyocytes showed a higher cell viability especially under ischemic condition. megalin, known as the lipocalin-2 receptor, was detected in hl-1 cells by rt-pcr. conclusions. this study demonstrates the time-dependent expression of lipocalin-2 in a cardiomyocyte culture as well as in transplanted hearts in response to ischemia and reperfusion/reoxygenation. lipocalin-2 is suggested to target cardiomyocytes with ameliorating effects on cell viability during ischemia. obvious alterations in lipocalin-2 expression at the protein level suggest a possible involvement of lipocalin-2 during i/r-induced cell death probably as a self-limiting process for inflammation. background. protease activation as well as inflammatory responses contribute to organ damage in response to ischemia and reperfusion. in this study we investigated the role of the protease inhibitor slpi in ischemia and cardiac transplantation. methods. hearts from slpi knockout mice (slpi-/-) were heterotopically transplanted to slpi-/-recipients. grafts underwent 10 hours of cold ischemia (ci) prior to transplantation or were transplanted immediately. c57bl/6 wild-type isografts (wt) undergoing the same procedure served as controls. in selected groups, 200 µg of recombinant slpi (rslpi) were added to the preservation solution or given i.v. after reperfusion. after evaluation of graft function, hearts were removed at 15 min, 12 h, 24 h, and 10 days. morphology was investigated by histology, slpi gene expression was analysed using quantitative rt-pcr. slpi protein expression was studied by immunohistochemistry (ihc). slpi, tnf-α, tgf-β, and nf-κb, cathepsin g, and elastase activity were analysed employing elisa and western blot. results. at 15 min, recovery of graft function was normal in wt and slpi-/-mice transplanted without ci (4.0 ± 0.0). in contrast, slpi-/-hearts transplanted after 10 h of ci showed no or marginal recovery of organ function (0.6 ± 0.65). at 24 h, cardiac function in slpi-/-(2.8 ± 0.89) was less when compared with wt (3.36 ± 0.55). single administration of rslpi i.v. had no effect; however, when slpi was added to the preservation solution, organ function comparable to wt mice was observed (3.36 ± 0.55). a mild mononuclear cell infiltrate and small focal necrosis where found in all groups at 24 h. at 10 days, postischemic inflammation as well as myocyte necrosis were significantly higher in the slpi-/group (2.5 ± 0.5 vs. 1.8 ± 0.4 and 1.6 ± 0.5 vs. 0.2 ± 0.4). myocyte vacuolisation as a sign of sublethal ischemic injury was present at high level in slpi-/-mice undergoing ci only. slpi gene expression was detected in wt mice at 12 and 24 h after reperfusion. gene transcription at 12 h was significantly higher after prolonged ci (7.99 vs. 1.57 orders of magnitude) and was associated with significantly decreased nf-κb, tgfβ, and tnf-α activity. slpi protein was first observed at 24 h, high levels of slpi protein were found at 10 days. slpi-positive cells were mainly identified as macrophages (ihc). high intragraft levels of slpi activity were found early as well as 10 days after application of recombinant protein. high slpi levels correlate with decreased cathepsin g early and decreased nf-κb, tgf-β, and elastase activity late after reperfusion. conclusion. herein we demonstrate that slpi has a substantial effect in prevention of inflammation and myocyte damage in response to ischemia and reperfusion of the heart via inhibition of nf-κb, tgf-β, and elastase. in addition, slpi seems to be crucial for recovery of organ function early after heart transplantation -inhibition of protease activity seems to be the underlying mechanism. perfusion with rslpi ex vivo represents a promising therapeutic option for modulating the destructive processes of postischemic inflammation while preserving its restorative nature. methodik. die studie wurde an 5 hausschweinen durchgeführt. die tiere wurden in allgemeinnarkose versetzt und intubiert. alle hämodynamischen daten wurden invasiv gemessen. die parameter (abp, pap, zvd und lap) wurden wie in der klinischen praxis mit einem hp-patientenmonitor registriert und gespeichert. das herzzeitvolumen (co) wurde mit hilfe eines flowmeters der firma transsonic systems inc. gemessen. die hämodynamischen ausgangswerte von 4 patienten vor ecmo wurden retrospektiv mit den experimentellen daten verglichen. ergebnisse. in der frühphase des tierversuchs nach erfolgter abklemmung blieb ein eindeutiger anstieg des pap bei 4 von 5 versuchstieren aus. auch ein rascher abfall des co war nicht zu beobachten. auch der pvr zeigte keine signifikante veränderung. die pap/ap-druckratio reagierte sehr rasch und in allen fällen mit einem anstieg auf das ereignis. von 4 patienten, die mittels ecmo erfolgreich therapiert worden waren, hatten 3 eine pap/ap-ratio über 0,3, ein patient hatte eine pap/ap-ratio von 0,26. die patienten mit pap/ap über 0,3 wurden mittels va-ecmo therapiert, der patient mit pap/ap von 0,26 mittels vv-ecmo. schlussfolgerungen. isolierte veränderungen der pulmonalen hämodynamik repräsentieren offenbar nur bedingt den schweregrad der vorhandenen beeinträchtigung. als wichtiger parameter für eine rasche einschätzung des grades der kompromittierung kann die druckratio zwischen pulmonalem und systemischem kreislauf angesehen werden. als leicht zu erhebender parameter könnte die pap/ap-ratio eine entscheidungsgrundlage für die zu wählende ecmo-konfiguration darstellen. nach den bisherigen erfahrungen wäre die indikationsgrenze bei einer pap/ap-ratio von etwa 0,3 zu ziehen. background. pancreas transplantation in the mouse is an extremely demanding procedure and severe technical problems have limited its widespread use. since the mouse, however, would be a good model for the study of various transplantation-related problems, such as ischemia-reperfusion injury or graft pancreatitis, we designed a new surgical strategy for cervical heterotopic vascularized pancreas transplantation using a cuff technique. methods. male syngeneic c57bl6 (h-2 b ) mice (n = 27) 10-to 12-week-old were used as size-matched donor and recipient pairs. recipients were intraperitoneally injected with 312.5 mg of streptozotocin per kg in order to become hyperglycemic (blood glucose, >300 mg/dl) and transplantation was performed 4 days later. recipient operation: the right external jugular vein (ejv) and common carotid artery were dissected free. by using a polyethylen cuff (od, 0.63 mm) it became possible to evert the artery over the cuff body and finally fix the vessel with 8-0 silk ligatures. similarly, the ejv could be everted over a 0.94 mm cuff. donor operation: after a complete midline incision the pancreas was isolated using a no-touch technique on a segment of the aorta, including the celiac axis and the superior mesenteric artery. the venous outflow was provided by the portal vein. all grafts were flushed with 4 °c saline solution. implantation: the graft was placed in the right cervical region and vascular anastomoses completed by pulling the pv over the ejv cuff and the donor aortic segment over the carotid cuff and held in place with a 8-0 silk ligature. after releasing venous and arterial clamps, all grafts immediately returned to their normal pink color with the arterial stump pulsating. results. out of 27 recipients, surviving over 50 days, 2 animals died from haemorrhage (survival rate, 93%). donor operation lasted 40 ± 5 min and dissection of recipient vessels took 20 ± 4 min. implantation time was 4 to 6 min, resulting in a total pancreas ischemia time of 33 ± 6 min. no thromboembolic complications at the cuff side were observed. preoperative glucose levels were 518 ± 59 mg/dl and could all be normalized by po day 1 (88 ± 13 mg/dl). histopathological examination on po day 10 and 30 showed almost normal islet cell and acinar architecture of all grafts. conclusions. for the first time a method of cervical heterotopic pancreas transplantation using a non-suture cuff technique in the mouse is described. major advantages are a short ischemia time, lack of arterial thrombosis or venous stenosis, and short operation time, and thus a very high survival rate. this model is especially applicable for investigating preservation, reperfusion injury, and graft pancreatitis. background. as human islet transplantation is limited by the lack of sufficient numbers of human donor organs, xenotransplantation with the use of porcine islet cells seems to be a promising therapeutic option to cure diabetes. in order to achieve sufficient numbers of viable islet cells, better protocols for organ preservation, isolation, and purification are needed. recent studies showed that the two-layer method (tlm) of pancreas preservation prior to isolation significantly improved islet yield. the tlm oxygenates pancreata and activates metabolism to generate atp and leads to resuscitation of ischemically damaged organs. another possibility to achieve a higher partial pressure of oxygen levels in fluids is the use of hyperbaric oxygenation (hbo). the aim of this study was to assess the influence of preoxygenated preservation solutions on the porcine pancreas. methods. university of wisconsin solution (uw), celsior, perfadex, custodiol, and a preservation solution especially designed for this study on the basis of ketoglutarate are oxygenated with 100% oxygen for 50 minutes at 1.5 bar using a hyperbaric chamber. porcine pancreata are harvested at a local slaughter house and stored in preoxygenated and not oxygenated preservation solutions at 4 °c for 4 hours. tissue cuts are performed to assess the occurrence of apoptosis and to determine the oxidative stress. atp-to-adp ratio is measured and immunohistochemistry is performed. results. it is feasible to preoxygenate preservation solutions. the oxygen levels can be raised up to 100 times in the preservation solutions using hbo and can be maintained for at least 12 hours after hbo treatment. mda and carbonylated protein levels are not significantly elevated in organs stored in preoxygenated solutions. atp-to-adp ratio as a sign of viability is significantly higher in organs stored in preoxygenated solutions. conclusions. preoxygenation of preservation solutions using 100% oxygen and a hyperbaric chamber is feasible. hbo has a positive impact on porcine organ preservation. as ischemically damaged islet cells are likely to undergo cell death or lose functionality due to hypoxia, the use of preoxygenated preservation solutions is a promising method to achieve better yields after islet isolation and transplantation. alternative zur humanen pankreastransplantation und inselzelltransplantation stellt die xenotransplantation von porcinen inselzellen dar. um xenogene zellen erfolgreich transplantieren zu können, müssen sie vom immunsystem des empfängers abgeschirmt werden. die verwendung von mikrokapseln scheint eine vielversprechende methode dazu zu sein, wobei natrium zellulose sulfat (nacs) in graz verwendet wird. darüber hinaus muss eine ausreichende anzahl an vitalen zellen mit hoher reinheit aus dem porcinen pankreas isoliert werden. methodik. porcine pankreata werden von einem lokalen schlachthof erhalten. die organe werden kanüliert, und ein enzymgemisch aus neutraler protease und kollagenase nb wird infundiert. die digestion erfolgt in einer modifizierten ricordi-kammer mechanisch und enzymatisch, und die anschließende purifikation wird mit einem ficoll-dichtegradienten durchgeführt. vitalität der zellen wird mit fluorescein-diacetat/propidium jodid, mtt und der bestimmung der atp/adp-ratio überprüft. die reinheit wird mit einer dithizon-färbung festgestellt. insulinkonzentrationen werden mittels elisa detektiert, der dna-gehalt der inselzellen mit dem dneasy-kit festgestellt. inselzellen werden in kooperation mit der firma austrianova mit nacs mikroverkapselt. ergebnisse. die organe wurden durchschnittlich 35 min digestiert, es wurde eine reinheit von 96% und eine zellzahl von durchschnittlich 2 × 10 5 zellen isoliert. beste resultate wurden mit dem lymphoprep tm -dichtegradienten erzielt. isolierte porcine inselzellen überleben bei 37 °c im durchschnitt 12 tage in vivo und produzieren glucose-abhängig insulin. die funktionalität der zellen bleibt über den gesamten zeitraum erhalten. mikroverkapselung mit nacs ist machbar. schlussfolgerungen. die isolation porciner inselzellen ist eine viel versprechende methode den mangel an humanen spenderorganen in der pankreastransplantation und humanen inselzelltransplantation zu umgehen. nacs als verkapselungsmaterial ist weniger immunogen und weitaus biokompatibler als alle bisher verwendeten materialien und die mikroverkapselung xenogener inselzellen mit nacs scheint eine innovative methode zur therapie des diabetes mellitus zu sein. background. to achieve its full potential, transplantation of pancreatic islets has to overcome a number of obstacles. one of the obstacles are the still lacking read-out parameters to assess the quality of human islets after the isolation procedure and prior to transplantation. being able to predict the functional potential of the pancreatic islets after isolation or even short-term culture would greatly enhance the success of islet transplantation. therefore one of the primary challenges in islet transplantation is to identify and understand the changes taking place in islets after isolation or culture. life confocal microscopy is a powerful tool to identify such changes in living islets not achievable by use of fixed-cell techniques. methods. islets were isolated according to the method of ricordi et al., using a continuous ficoll gradient. 3 fluorescent dyes, dichlorodihydrofluorescein diacetate (dcf), tetramethylrhodamine methyl ester (tmrm), and fluorescent wheat germ agglutinin were used to assess either overall oxidative stress, time-dependent mitochondrial membrane potentials, or localisation of oligosaccharides. confocal microscopy was performed with an microlens-enhanced nipkow disk-based confocal system ultraview rs (perkin elmer, wellesey, ma, usa) mounted on an olympus ix-70 inverse microscope (olympus, nagano, japan). results. with the above described confocal system we were able to identify differences in the localisation and amount of oligosaccharides in endocrine vs. exocrine cells of freshly isolated pancreatic islets. the staining pattern changed during the course of culture, suggesting a remodeling of cell surface oligosaccharides. the study of the mitochondrial membrane potential proved to be very useful in order to early identify damaged or stressed cells and thereby gain insights into the vitality of the isolated islets. conclusions. this makes us believe that, especially in the light of the many other fluorescent dyes which can be used as subcellular markers, a combination of these with a powerful live confocal imaging system will be of great value for a better islet assessment after isolation and culture. background. therapeutic drug monitoring (tdm) of immunosuppressants is a well established concept supporting the work of clinicians from the historical onset of the use of these drugs. within the last years the combination of hplc (highperformance liquid chromatography) with tandem mass spectrometry (ms/ms) provides an alternative to antibody-based immunoassays. this new analytical method to quantify immunosuppressants affords a robust and rapid separation technique with high selectivity. up to now, the quantification of wholeblood levels of cyclosporin a, fk 506 (tacrolimus), and 42-o-(2-hydroxyethyl)-rapamycin (everolimus) at our institute have relied upon three different, indirect assays from different companies. however, these systems have drawbacks caused by cross reactions with active and inactive drug metabolites, fluctuations in assay performance, and comparatively high prices. in contrast, hplc-ms/ms platforms are now becoming frequently used to measure circulating drug levels and their metabolites with lower associated costs and higher specificity, accuracy, and precision. methods. the setup chosen at our laboratories consists of two independent mass spectrometers (one machine at the zimcl, a backup machine at biocrates) and allows a sample throughput of about 25 samples per hour. all immunosup-varia pressants currently monitored in whole blood can be quantified within one analysis from a sample volume of less than 100 µl. the sample workup (cell lysis and protein precipitation) is performed in a bar-code-supported parallel setup, minimizing the risk of sample mix-up. an online solid-phase extraction (spe) strategy has been chosen to reduce matrix interferences and ion suppression. daily calibrations and quality control samples performed with certified reference materials assure a high level of accuracy and precision. results. the hplc-ms/ms assay established for cyclosporin a, tacrolimus, everolimus, and sirolimus covers the analyte concentration range needed for tdm (e.g., up to 2000 mg/ml for cyclosporin a). intraday and interday repeats of the assay and data from quality control measurements were sufficiently accurate and precise (all cvs, <15%; bias, <15%). comparison of the quantitative results did show a linear relationship between antibody-based immunoassays and the hplc-ms/ms-derived data with bias values (ca. 10% for cyclosporin a and ca. 30% for tacrolimus on the basis of bland-altmann plots) in agreement with the literature. conclusions. the established hplc-ms/ms platform will allow replacement of the current antibody-based assays. the major advantage of this technique is the ability to simultaneously acquire absolute quantitative concentrations of each of the therapeutic drugs administered from one sample. therefore, the requirement for different sample preparation schemes or parallel measurements on different analytical instruments are no longer needed. the high sample throughput also assures timely tdm data report to the ward. a significant reduction of costs is now expected due to the lower consumable expenses in hplc-ms/ms assays. background. a clinical trial towards improved safety of the application of everolimus (certican) in heart transplantations (crad001a2403, data collection started in august 2004 and is ongoing) allowed us to compare therapeutic drug monitoring (tdm) data (e0 and e2 levels) measured on two different platforms -an lc-ms/ms method in an external laboratory and a immunochemical method recently established at our institute. methods. lc-ms/ms measurements of everolimus were performed in a reference laboratory. the immunochemical measurements were performed with a fluorescence polarization immunoassay (innofluor certican immunoassay by seradyn) measured on a tdx instrument from abbott. method comparison included analysis of the time series of the patients (n = 7, 65 observation pairs) and the quality control samples of the immunochemical assay gathered over a year as well as inter-and intra-assay data comparisons based on bland-altmann plots and regression data. results. comparison of the quantitative results obtained from the lc-ms/ms and the immunochemical assay showed good agreement between these methods. the bias between the methods (bland-altmann plot) was found to be rather small, with the immunochemical method measuring in average approximately 5% (±25%) lower values as the lc-ms/ms method, which is in good agreement with current reports. the coefficient of variation (cv) (measured over several months) of the innofluor quality control measurements was <7% for the medium (ca. 12 ng/ml) and high (ca. 25 ng/ml) quality control samples, whereas the lower quality control level (ca. 4.5 ng/ml) showed an increased cv (<13%). however, especially during the phase of method establishment, higher cv values and bias deviations have been found conclusions. the method comparison did show that the immunochemical everolimus assay provided by seradyn is a good alternative to hplc-ms/ms measurements. the assay bias was found to be rather low and the assay uncertainty was within acceptable ranges. however, a stringent quality control network must be provided to assure stable assay performance over time. grundlagen. die inzidenz der endokarditis beträgt ca. 6/100 000 einwohner pro jahr in der gesamtbevölkerung. obwohl die immunsuppression das auftreten systemischer infektionen begünstigt, sind endokarditisstudien an transplantationsempfängern nicht verfügbar. ziel dieser epidemiologischen studie war, das auftreten der endokarditis und ihrer risikofaktoren nach organtransplantation zu evaluieren. methodik. insgesamt 2556 patienten, welche sich zwischen 1989 und 2004 einer soliden organtransplantation an unserem zentrum unterzogen, wurden untersucht. der mbds unserer klinik wurde zum patientenscreening herangezogen. ergebnisse. insgesamt wurden im beobachtungszeitraum 27 endokarditisfälle beobachtet. neun endokarditisfälle (33,3 %) konnten erst post mortem mittels autopsie diagnostiziert werden, 8 patienten (29,6 %) konnten durch alleinige antibiotische therapie geheilt werden. insgesamt 10 transplantationsempfänger (37,1 %) mussten sich einem kardiochirurgischen eingriff unterziehen. die gesamtmortalität betrug 44,4 % (12 patienten). staphylococcus aureus konnte in 16 fällen (59,3 %) und pilze konnten in 4 fällen als ursächliche keime isoliert werden. die inzidenz der endokarditis in transplantationsempfängern beträgt 1 % (95 % ci, 0.67-1.49) und zeigt ein 171-fach erhöhtes risiko verglichen mit der gesamtbevölkerung. schlussfolgerungen. die endokarditis stellt ein signifikantes problem nach organtransplantation dar und ist mit einer exzessiv hohen mortalität assoziiert. eine erhöhte aufmerksamkeit ist daher indiziert, da wir durch den einsatz der transösophagealen echokardiographie vermehrt derartige fälle in der zukunft diagnostizieren werden. ein 47-jähriger patient erhält 1995 eine herztransplantation aufgrund einer dilatativen kardiomyopathie bei altersentsprechender nierenfunktion. die immunsuppression besteht aus cyclosporin a und mycophenolat mofetil. innerhalb der ersten drei monate nach der operation steigt das kreatinin auf 2,0 mg/dl an, bleibt dann aber konstant. ab 2003 nimmt die nierenfunktion weiter ab, weshalb im august 2004 wegen des verdachtes auf eine chronische calcineurininhibitor-nephropathie auf sirolimus und mmf umgestellt wird. das serum-kreatinin beträgt zu diesem zeitpunkt 3,0 mg/dl. zwei monate später ist das serum-kreatinin auf 7,0 angestiegen, der inzwischen 57-jährige patient leidet unter einer renalen anämie, hyperphosphatämie und hypokalziämie. im harn finden sich leukozyten und erythrozyten, die allerdings nicht deformiert sind (keine dysmorphen erythrozyten und akanthozyten), sowie eine geringgradige proteinurie (300 mg/24 h). in der harn-polyacrylamidgel-elektrophorese zeigt sich vor allem alpha-1-mikroglobulin als indikator einer tubulointerstitiellen schädigung. nach erneuter umstellung der immunsuppression von sirolimus/mmf auf niedrigdosis-cyclosporin/mmf kommt es zum raschen rückgang des serum-kreatinins, der harnbefund sowie die elektrolyte normalisieren sich, einzig die erythropoetin-substitution muss beibehalten werden, um den hämoglobin-zielwert von 11 g/dl zu erreichen. acht wochen nach der zweiten therapieumstellung führen wir eine nierenbiopsie durch. es findet sich eine akute interstitielle nephritis ohne glomeruläre schädigung. unseres wissens ist dies der erste dokumentierte fall einer akuten interstitiellen nephritis auf sirolimus. background. group milleri streptococci (gms) are a heterogeneous group of streptococci including the species streptococcus intermedius, s. constellatus, and s. anginosus. due to their ability of producing toxins, they tend to cause chronic intra-abdominal and intrathoracic abscesses, which are difficult to treat, as gms are able to escape conventional antibiotic therapy. aim. evaluation of epidemiology, clinical significance, and impact on the outcome in all solid-organ recipients with gsm infections during a 4-year period. patients and methods. retrospective analysis comprising 45 solid-organ recipients with gms. results. between 2001 and 2004, 45 solid organ recipients (88 isolates) including 34 liver, four kidney and pancreas, one kidney, two small bowel, three combined liver and kidney, and one combined kidney and small bowel transplant re-cipients developed infection with gms. in 42 cases, gms caused intra-abdominal infection; in two cases, pleural empyema; and in one case, soft tissue infection. in only one case, gms were cultured from blood. in 54 of the 88 specimens (61%) which grew gms, also other pathogens could be isolated. gms frequently caused recurrent cholangitis (n = 17) associated with anastomotic and anastomotic biliary strictures. these cases were managed by repeated stenting or surgical intervention and prolonged antibiotic therapy. no patient died directly related to gms infection. all responded to combined surgical and antibiotic treatment. one pancreas graft was lost due to erosion haemorrhage associated with an abscess. all isolated strains of gms were susceptible to penicillin g, carbapenems, and clindamycin, whereas cephalosporins and quinolones showed intermediate activity or resistance in some cases and gms in general were found resistant to aminoglycosides. conclusions. gms are frequent pathogens in transplant surgery and are capable of causing difficult to treat infections. their prevalence in transplant surgical site infection thus far might have been underestimated. therefore, we recommend empiric antibiotic treatment for sufficiently long time in combination with surgical intervention when necessary. background. by using intensified immunosuppressive protocols the incidence of immunological complications after solid-organ transplantation has constantly declined. however, the incidence of some infections, in particular complicated fungal infections, seems to increase. candida krusei (ck) is resistant to fluconazole and recently this pathogen has been more commonly isolated in severe infections, particularly in the immunocompromised host. methods. between 1. 1. 2004 and 30. 6. 2005 , a total of 400 solid-organ transplants were performed at the innsbruck medical university. this included 150 renal, 70 liver, 50 pancreas, 5 intestinal, 30 cardiac, 20 lung, and 10 islet transplants. prophylactic immunosuppression consisted of calcineurin inhibitor-based triple drug therapy for the majority of cases. antifungal prophylaxis was given to all pancreatic and intestinal recipients (fluconazole 400 mg per day) and to all patients considered at high risk for filamentous-fungal infections (ambisome 3 mg/kg). results. a total of five patients with ck infection were identified within this series (2%). five patients developed infection with candida tropicalis and three with candida glabrata. within this time period, another two patients with ck infection were identified, one had a pleural empyema following esophageal perforation and the other was treated with a ventricular assist device and developed pulmonary infection with ck. both patients were treated on the transplant intensive care unit. the transplant patients were three pancreas, one liver, and one lung recipient. all three pancreas recipients were diagnosed intra-abdominal infection, the liver recipient had an ischemic cholangiopathy with ck cholangitis and required retransplantation, and the lung recipient developed postoperative hemorrhage and subsequently ck pleural empyema. all patients presented also with other infectious complications. treatment of ck infection consisted in four cases of caspofungin or voriconazole or combination of the two, and in one case, ck was isolated from infected hematoma and did not require antifungal therapy after surgical removal. all infected collections were evacuated either surgically (n = 3) or through pig-tail drainage (n = 2). ck infection was successfully managed in all cases and patients are currently alive, only one pancreas graft was lost. conclusions. candida krusei infections now represent frequent severe complications in solid-organ recipients. however, rapid diagnosis and treatment with new antifungal agents such as voriconazole, caspofungin, or ambisome allow successful therapy of these infections. solid-organ recipients seem to be at increasing risk to acquire non-albicans candida infections. background. after solid-organ transplantation immunosuppression (is) and concomitant infection with cmv, ebv, hhv-6, -8, or papillomavirus put patients at risk for developing malignant diseases (14% cumulative risk at 10 years; adami et al. 2003) . posttransplant lymphoproliferative disorders (ptld) and skin cancers are known to have the highest incidence in immunocompromised patients. reports on colorectal cancer after different types of organ transplantation are rare and the incidence was 0.01% to 3.9% and did not differ from that of the normal population. we analysed our database with regard to the incidence and course of colorectal malignancies following treatment and introduction of tor inhibitor-based is. methods. medical records of solid-organ transplants performed between 1986 and 2005 at our center were analysed retrospectively. in a total of 3568 patients 247 heart, 118 lung, 2074 kidney, 757 liver, 367 pancreas, and 9 combined heart-lung transplantations were performed (27 small bowel and 4 hand transplantations not included). immunosuppressive therapy consisted of triple therapy comprising steroids, azathioprine/mmf, and cya/tac. some of them received induction therapy with antithymocyte globulin. results. a total of 206 patients (5.72%) developed malignancies. of them, 9 patients (1 female, 8 male; median age at diagnosis, 66.1 years; 2 kidney, 3 heart, 4 liver recipients) had colorectal malignancies (0.25%) during a mean follow-up period of 7.3 years. on average, diagnosis was made 5.8 years after transplantation. four carcinomas were located in the rectum or at the rectosigmoid junction and five were colon cancers (five pt3, one pt2, and three pt1 stages). r0 resection was performed in all 9 patients plus radio-and/or chemotherapy in all t3 stages. five patients (55%) died 7.2 years post transplant due to cardiovascular disease (n = 4) and recurrent tumor disease (n = 1). the 1-year survival rate was 67% for t3 and 100% for t1 rectal cancers, 50% for t3 and t1 each and 100% for the only t2 colon cancer. three anal neoplasms (one ain iii°, two anal cancers, pt1 and pt2; median age at diagnosis, 50 years) developed on average 7.2 years after transplantation (0.08% vs. 0.001% in the general population) with a 100% 1-year survival rate. all patients were switched to rapamycin or everolimus after completion of primary therapy. conclusions. the incidence of anal but not of colorectal cancers in our transplant patient population differed from that of immunocompetent patients of corresponding age (0.08% vs. 0.001% and 0.25% vs. 0.2% tyrol tumor registry and 0.3% seer). the 1-year survival rate was significantly decreased in the transplant group with t3 tumors (67% [rectum] and 50% [colon] vs. 80%). potential antineoplastic effects of rapamycin and overall less immunosuppression long-term may improve prognosis of colorectal malignancies following transplantation. background. solid-organ transplantation is the treatment of choice for terminal organ failure. due to the scarcity of organs, the mandate to utilize extended-criteria donors has dramatically increased over time and achievable results are good. this harbors the risk of transmission of infections from the donor to the recipient. aim. an overview on the magnitude of possible transmittable diseases that could accompany donor organs is given. possible preventive strategies are discussed. overview. the faith of an organism that is transmitted by a donor allograft depends on the virulence and the quantity of the transmitted pathogen and the site of infection and furthermore is impacted by prophylactic steps undertaken during donor procurement, organ perfusion, implantation, and post transplant course and lastly depends on the ability of the recipient to control the infection by the immune system. in the best case, the microorganism is cleared before it can do any harm; however, in the worst case scenario, transmitted pathogens can lead to fatality. there is a multitude of pathogens that can potentially be transmitted by an allograft including viruses, bacteria, fungi, and protozoa. there are microorganisms that can be transmitted by any type of graft and there are pathogens which due to a certain tropism can be transmitted by certain organs only. there are common pathogens such as cmv and ebv as opposed to extremely rare pathogens such as lyssa, west nile fever, or lymphochoriomenigitis virus. intracellular pathogens are preferably targeted by mhc restricted cytotoxic t-cell reaction. as in organ transplantation hla matching is in general not performed, donor-derived antigen-presenting cells cannot be recognised by recipient-specific cd8 + lymphocytes. therefore, either donor-specific cytotoxic t cells must function as counterparts or alternative tar-gets must be defined by the immune system. bacterial and/or fungal contamination must be considered in lung transplantation (bronchial stump), pancreas transplantation (duodenal segment), and small bowel allografts. cardiac, renal, and liver grafts can be considered sterile but can become contaminated during procurement. rare transmitted organisms are toxoplasma gondii, trypanosoma cruzii, and schistosoma mansoni. conclusions. when looking at allocation of latently virus-infected organs, they should be preferably given to recipients who have antibodies against the particular agent. in terms of bacterial or fungal contamination of graft and/or preservation solution, routine monitoring seems mandatory in order to assure quality. keeping blood and serum samples from donors should be carried out from an epidemiological and legal standpoint. bacterial and fungal prophylaxis should be used to prevent not only recipient-but also donor-derived pathogens. antiviral prophylaxis is standard for cmv and hbv, for all other viruses no final recommendations are available. methods. the aim of the current review was to investigate the impact of several risk factors on ptld. therefore, patients developing ptld after heart transplantation at our institution were screened. results. 8 patients have been identified with ptld, all cases occurring during the last decade. mean age at ptld onset was 46.4 ± 20.0 years and time between transplant and development of ptld was 26.4 ± 30.4 months. there were 5 ebv-associated ptlds, 2 non-ebv-associated, cd20-negative b-cell lymphomas, and 1 t-cell lymphoma. immunosuppression at ptld onset was calcineurin inhibitor based (cyclosporine a, 5 patients; tacrolimus, 3 patients). initial immunosuppression included atg induction. six patients received perioperative antiviral prophylaxis with either valgancyclovir/gancyclovir (n = 4) or acyclovir (n = 2) in combination with anti-cmv hyperimmunoglobulin (n = 1). two patients experienced a total of five episodes of acute rejection (ishlt ii°), all were treated with bolused steroids. four patients are still alive (50%), three of them in current remission of ptld, one patient is under therapy recently. median survival was 27 months in survivors and 3.4 months in nonsurvivors. conclusions. these data show that ptld is associated with a high mortality rate. the majority of ptlds are ebvassociated. therefore, screening for ebv infection and prophylactic treatment may help to prevent a potentially fatal consequence of heart transplantation. background. late acute cellular rejection is associated with a decrease of survival and the development of cav. new immunosuppressice drugs have been introduced into clinical practice. everolimus, as one of these, has shown to be safe in cardiac transplantation. we report of our experience with everolimus in heart transplant recipients who developed late cardiac rejection. methods. patients with a history of previous rejection episodes who experienced cardiac rejection after at least 2 months postoperative were switched to an everolimus, cyclosporine a, and corticosteroid-based immunosuppressive regime. all patients already received statins and antihypertensive medication before. everolimus, cyclosporine a trough levels and laboratory values were controlled monthly. drug administration was adapted to an everolimus trough level between 3 and 8 ng/ml, mean maintenance dosage was at 0.25 to 1.5 mg/day. death, safety, side effects, biopsy-proven acute rejection, laboratory values and blood levels were evaluated retrospectively. results. 4 cardiac allograft recipients (2 male, 2 female), at a median of 1473.25 days post orthotopic heart transplantation (ohtx) (65-3045), received 1 mg to 1.5 mg everolimus per day. in a follow-up period of at least 2 month (2-10) mortality was 0%. the drug was well tolerated and no acute cellular rejection greater than grade 1a (ishlt grading) was observed after two month. in one patient raised cholesterol values and in two others elevated triglyceride levels were seen but were controlled with higher statine therapy. no obvious raised creatinine values were seen with certican ® . conclusions. in conclusion, conversion to an everolimus-based immunosuppressive regimen after late cardiac rejection is safe and effective. no major side effects were observed. p02 collagen iii in transplanted heartdonor or recipient derived m. pichler, b. tessaro, d. kniepeiss, r. kleinert, g. hoefler institute of pathology, medical university of graz, graz, austria background. transplantation of donor hearts is often associated with progressive development of interstitial myocardial fibrosis and alterations in composition and organisation of the extracellular matrix. changes in cardiac interstitial collagen network are thought to contribute to abnormal stiffness and loss of function of the myocardium. fibroblasts are the main producers of type i and type iii collagens, the major in-poster terstitial collagens found in the heart. in transplanted hearts, intragraft fibroblasts may consist of two cell populations. donor-derived fibroblasts preexist in donor organs, whereas hostderived fibroblasts may progressively immigrate as mesenchymal progenitors from the circulation to the allograft. purpose of the study. to determine the contribution of these two distinct fibroblast populations to progression of myocardial fibrosis, we studied endomyocardial biopsies over a time period of some years from a male patient who had received a heart from a female donor. methods. in these sex-mismatched patients two frequent genetic polymorphisms at the collagen iii locus were determined by polymerase chain reaction-based restriction enzyme digestion, both in donor allograft and in the corresponding explanted recipient heart. on the basis of differences of the collagen genotype in donor and recipient tissue, we selected 10 endomyocardial biopsies by hematoxylin eosin staining covering an overall follow-up period of nine years since the transplantation event. immunohistochemistry, chromogene in situ hybridisation, and population-specific collagen expression using single nucleotide polymorphisms were used to determine the course of fibrosis. results. we developed an analytical system generally applicable to measure population-specific differences of collagen iii synthesis in transplanted organs. the amounts of interstitial collagen type i and type iii increased in a time-dependent manner within cardiac allograft. years after transplantation, a number of y-chromosome-positive recipient-derived cells consisted of noninflammatory spindle-shaped fibroblast-like cell types. conclusions. our data confirm the existence of a substantial number of fibrosis-mediating immigrated recipient-derived fibroblasts in cardiac allografts. furthermore, this suggests a potential, future therapeutic approach for reduction the cardiac fibrosis process. methods. between 1993 and 1998 a total of 32 lung transplants (31 patients) and six combined heart-lung transplants were performed at our center. immunosuppression consisted of atg induction, cyclosporine a, azathioprine, and steroids. all patients with rti underwent a meticulous micro-biological screening and underwent bronchoscopy with bronchiolo-alveolar lavage (bal) and transbronchial biopsies (tbb). rsv was detected from bal specimens by an immunoassay. results. a total of five lung recipients (31% of 19 survivors) and one of the two alive heart-lung recipients developed rsv infection of the lung allograft. all cases were observed during an rsv epidemic in children in the area between october 1998 and may 1999. the patients were 47-55 years old, all were females. one patient experienced two episodes. onset of rsv was median 3 (range, 4-29) months post transplant. clinical symptoms included cough (6 of 7), rhinitis (6 of 7), and fever (4 of 7). deterioration of lung function occurred in six of the seven episodes with one deteriorating to respiratory failure requiring ventilator support. two individuals developed pulmonary infiltrates. in all patients, immunosuppression was significantly tapered, 86% required hospitalization and antibiotic therapy. the patient with rsv recurrence received inhalative ribavirin therapy during the second episode. all patients recovered and survival of this cohort was 86% after 1 year and 72% at 4 years after rsv infection. none of the patients developed bronchiolitis obliterans syndrome (bos) or rejection during follow-up. conclusions. rsv is a severe but benign complication following lung transplantation with a wide range of clinical presentations. routine use of antiviral treatment is not necessary; however, reduction in the level of immunosuppression is required. no long-term effects were observed in this cohort. a. stamatelopoulos 1,2 , s. guth 2 , a. abrahim 2 , g. m. marta 2 , l. tsourelis 3 , p. jaksch 2 , c. konaris 4 , s.taghavi 2 , w. klepetko 2 due to cni nephrotoxicity in a substantial number of ntx patients. we therefore studied the effects of a switch from a cni regimen (cni + mpa + p) to a dual regimen of sirolimus (srl) plus prednisolon (p) in 26 pts with moderately impaired kidney function (s-crea, 1.5+0.4 mg%) due to either cni toxicity or clinical evidence for chronic allograft disease. 13 pts received regimen 1 consisting of srl 12 mg dosage on day 1 and from days 2 to 5 srl 4 mg plus csa at half of maintenance dosage. from day 6 on, pts received srl 8 mg and csa was withdrawn. mpa or aza dose was continued halfed for 4 to 6 weeks. p was kept constant. target level for srl was 12-20 ng/ml. another 13 pats received regimen 2 consisting of srl 12 mg and csa withdrawal on day 1 and from days 2 to 6 srl 6 mg was administered for a target level of 7 to 10 ng/ml. mpa or aza were continued halfed for 4 to 6 weeks, p was kept constant. with regimen 1 there were 6 dropouts due to adverse events, whereas with regimen 2 only 2 dropouts occurred. 45% of the patients showed a decrease of s-crea after 2-year observation period, 20% were unchanged and only 25% showed an increase. overall, there was a slight but significant increase of cholesterol and triglycerides, whereas other parameters were unchanged. conclusions. switch from cni containing immunosuppression to a dual regimen of sirolimus plus prednisolon results in an improved kidney function after 2 years in the majority of pts. a regimen of sirolimus with target levels of 12 to 20 ng/ml and an overlap of immunosuppression shows a high rate of adverse events and is associated with a 3-fold dropout rate as compared to a regimen with a target level of 7-10 ng/ml. background. certain anatomical variations mainly concerning the portal system preclude living donor liver transplantation (ldlt). to the best of our knowledge, two left lateral segments with two arteries have never been transplanted so far. case report. a 6-month-old girl was diagnosed with endstage liver cirrhosis secondary to biliary atresia and therefore scheduled for ldlt. preoperative evaluation of the donor including ct with 3-d reconstruction revealed normal vascular supply of the liver with a left and a right hepatic artery. during donor operation, two tiny arteries with a diameter of 2 mm for segments ii and iii were identified. they were found to arise from the left hepatic artery right behind the bifurcation of the proper hepatic artery, which made it impossible to preserve a common trunk. venous and portal venous reconstruction was performed in an end-to-end fashion. the left graft artery was directly anastomosed to the proper hepatic artery with 8/0 pds interrupted sutures using a microscope. the 2nd graft artery was revascularised with the help of saphenous vein from the recipient as interposition graft to the gastroduodenal artery but failed. therefore, the trunk of the inferior mesenteric vein was used for reconstruction with 8/0 pds with excellent outcome. under fk-based immunosuppression, postoperative course was uneventful with both arteries patent. conclusions. multiple arteries for the left lateral segments are not a contraindication for paediatric ldlt. the inferior mesenteric vein can be used as an interposition graft. combined renal-pancreas transplantation is an established treatment for patients with diabetic nephropathy, producing excellent results. we report on a patient who suffered from long-standing multiple sclerosis and underwent successful combined renal-pancreas transplantation. post transplant course was complicated by yeast intra-abdominal infection, which was treated with antifungal agents. using tacrolimus and sirolimus, both grafts are well functioning at two years and the patient is without any symptoms of disseminated encephalitis and does not require specific medication for multiple sclerosis. for diabetic patients suffering from multiple sclerosis, pancreas transplantation offers an excellent treatment option. whether normalisation of carbohydrate metabolism or chronic immunosuppression or both lead to complete response of multiple sclerosis is not clear. background. until recently, the peripheral blood stem cell (pbsc) donation procedure was used only infrequently among unrelated allogeneic donors. nowadays, both related and unrelated donors are expected to consider this alternative donation. to promote pbsc donation both safety and well-being of healthy unrelated volunteer donors must be protected and data are to be collected to establish the long-term safety of g-csf stimulation. methods. from 2000 to 2004, 16 pbsc aphereses on unrelated allogeneic donors have been carried out in our center. all pbsc donors were treated with 5 µg of g-csf per kg twice a day from day -4 to day -1. aphereses were performed using peripheral venous access on day 0 and -if indicatedon day 1. since 2003, all donors have annually received a questionnaire about their actual state of health and medication, as well as their physical and mental conditions. detailed questions concerned donors' anamnesis for epistaxis, bruises, thrombosis or embolia, as well as infections and fever, night sweat, and weight loss of unclear origin. results. 11 male and 2 female donors (81%) with an average age of 44.59 (29-56) years responded to the questionnaire. the observation periods were between 3 and 53 months (mean, 28.75 months) after g-csf stimulation. 5 pbsc donors (38%, all male) reported that they had been severely ill during the observation period: one donor developed an exostosis of the 5th rib, one was operated on an umbilical hernia, one suffers from recurrent articular and muscle pain accompanied by night sweat and weight loss, one has a chronic compensated renal failure, one had diarrhoe and a common cold and suffered from fatigue, nausea, sleeping problems, and circulatory disorders. one female donor recognized dizziness and an increased tendency for bruises as well as paraesthesia in both arms. the disorders these donors reported occurred between 3 and 22 months after g-scf stimulation. all the other pbsc donors (62%) have never been severely ill or under medical observation. no donor had fever of unclear origin, phlebitis, thrombosis, or embolia, no donor recognized an increased tendency for epistaxis. three donors need medication they did not have before g-csf stimulation, which are to lower blood lipids and anti-inflammatory ones. one donor estimates that he is getting ill more easily than others, all the other donors feel themselves in best physical and mental condition. background. hematopoietic stem cell transplantation (hsct) has been successfully performed in patients with otherwise incurable malignant diseases. however, relapse after hsct is one of the main reasons for treatment failure and further therapeutic strategies with acceptable toxicity are warranted. since myeloablative (ma) conditioning after prior hsct has been associated with high treatment-related mortality (trm), reduced-intensity conditioning (ric) regimens have been developed as salvage therapies for these patients. so far, encouraging results have been achieved with ric; however, a direct comparison with standard conditioning has never been performed. therefore, we retrospectively analysed these two conditioning strategies in patients experiencing relapse after prior stem cell grafting. methods. we analyzed 45 patients with relapsed disease (acute myeloid leukemia, n = 16; indolent lymphoma, n = 9; multiple myeloma, n = 7; chronic myeloid leukemia, n = 4; myelodysplastic syndrome, n = 2; chronic lymphocytic leukemia, n = 2; acute lymphocytic leukaemia, n = 2; aggressive lymphoma, n = 1; hodgkin's disease, n = 1; ovarian cancer, n = 1) after prior hsct who received either reduced-intensity or myeloablative conditioning for allogeneic hsct between 1986 and 2005. ric consisted of fludarabine 90 mg/m 2 and total-body irradiation (tbi) of 2 gy according to the seattle protocol (n = 18) or alemtuzumab in combination with the beam regimen (n = 1). myeloablative therapy consisted of cyclophosphamide (cy) and tbi of 12 to 13 gy (n = 13), cy plus busulfan (bu) (n = 4), c plus antithymocyte globulin plus bu (n = 2), or bu alone (n = 7). donors were syngeneic in 4, related in 21, and unrelated in 20 patients. stem cell source was bone marrow in 13 (29%) and peripheral blood in 32 (71%) patients. for graft-versus-host disease (gvhd) prophylaxis, 22 patients received cyclosporine a (csa) plus mycophenolate mofetil, 16 csa plus methotrexate, 2 mtx alone, and 1 csa alone. results. all patients achieved complete hematopoietic engraftment by day 28 after stem cell transplantation with complete donor chimerism. all patients conditioned with ric presented complete donor chimerism of t cells, myeloid progenitor cells, and nk cells 28 to 81 days after hsct. the in-cidence of acute gvhd was 37% and comparable in both groups consisting of grade i in 6 patients, grade ii in 3, grade iii in 6, and grade iv in 2. fourteen patients died after ma conditioning of acute gvhd (n = 2), infections (n = 5), or severe toxicity (n = 7), while only one patient died due to infection after ric. probability of transplant-related mortality (trm) at 1 year after hsct was with 54% significantly (p = 0.001) higher in patients given myeloablative conditioning compared to 5% after ric. incidence of therapy requiring chronic gvhd was with 64% versus 33% significantly (p = 0.001) higher in patients who received ric. response rates were comparable between patients who received ric or ma conditioning (90% versus 100%). relapses occurred in 37% of patients after ric and 32% after ma conditioning. after a median follow-up of 44.5 (range, 8-204) months 3 (11%) of patients of the ma group and 11 (58%) of the ric group are currently alive. probability of survival at 1 and 4 years after hsct is with 73% versus 24% and 48% versus 12% significantly (p = 0.008) higher after ric than after myeloablative conditioning. conclusions. allogeneic stem cell transplantation is a highly effective treatment option in patients relapsing after prior hsct. durable hematologic engraftment and sustained complete remissions can be achieved in patients with otherwise poor prognosis. since transplant-related mortality of dose-reduced conditioning is in comparison to myeloablative hsct considerably lower, overall survival can be significantly improved with this new treatment modality. background. patients with comorbidities such as organ dysfunctions or preexisting infections experience a high treatment-related mortality which makes them ineligible for conventional conditioning therapy. for these patients, reduced-intensity conditioning (ric) is an option which offers the benefits of an allogeneic transplant with lower extramedullary toxicity. we report on 8 pediatric and young adult patients who were considered for ric because of severe cumulative pretreatment or substantial comorbidities treated at our institution between 2001 and 2005. methods. eight patients (median age, 11 years; range, 1-28 years; m, 4; f, 4) diagnosed with aml (cr 1, n = 4; cr 2, n = 1), all (cr 3, n = 1), alps (n = 1), and relapsed ewing's sarcoma (n = 1). 7 of 8 patients were planned to receive ric, in one patient the regime was changed during conditioning due to an acute viral infection. all patients received fludarabine (n = 7, 150 mg/m 2 ; n = 1, 60 mg/m 2 ) combined with melphalan (n = 6, 100-140 mg/m 2 ), busulfan (n = 1, 13 mg/kg), or total-body irradiation (n = 1, 2 gy). 3 patients received atg (45-60 mg/kg), two patients campath (40 mg/m 2 ). post-transplant immunosuppression consisted of cyclosporine a in all patients combined with mycophenolate mofetil (n = 1) or methotrexate (n = 2). 7 of 8 patients received hla identical grafts (sibling, n = 3; hla identical mother, n = 2; mud, n = 2), one patient received a c-locus mismatched graft. stem cell sources were bone marrow in 6 patients and peripheral blood stem cells in 2 containing a median of 5.13 × 10 6 cd34 + cells per kg of body weight of the recipient. results. all patients had primary engraftment; the median time to neutrophil recovery (n > 1.0 × 10 9 /l) was 15 days. complete donor chimerism as evidenced by vntr was achieved in median on day +22 (+11 to +28). acute mild graftversus-host disease (gvhd) of the skin occurred in 5 of 8 patients and responded to prednisolone; one patient required additional immunosuppression with mmf. one patient progressed to extensive chronic gvhd; one patient developed chronic gvhd of the skin, another patient shows clinical evidence of chronic gvhd of liver and gut. the remaining 3 patients showed no gvhd at all. 5 of 8 patients were positive for one or multiple viruses on routine viral pcr monitoring, requiring virostatic treatment. no treatment-related mortality occurred. the patient with ewing's sarcoma died 4 months posttransplant from tumor progression; the patient with extensive chronic gvhd died of sepsis on day +400. all 5 patients with aml are in remission. the patient with alps is still positive for various autoantibodies. 3 patients developed a posttransplant macrophage-activation syndrome (mas). all of them required a stem cell boost because of pancytopenia. conclusions. ric followed by allogeneic hsct was successful in terms of achieving primary engraftment and complete donor chimerism as well as avoiding transplant-related mortality in all patients; in our patients with myeloid malignancies it was also successful in terms of maintaining stable remission. as for posttransplant problems, we encountered acute gvhd of the skin in 5 of 8 patients, three of whom developed chronic gvhd; and viral infections in 5 of 8 patients, three of whom developed mas and eventually required a stem cell boost. background. due to an aggressive course mantle cell lymphoma is characterized by poor prognosis with a median survival of 3 years and only 10-15% long-time survivors. recent improvements in therapy have been made by combined immunochemotherapy and intensification with high-dose chemotherapy. methods und results. ten patients (4 female, 6 male) with a median age of 56 (49-65) years were treated with rituximab plus chop for four or six cycles. furtheron, patients received 2 or 3 cycles of claeg-d (3 days cladribine 0.2 mg/kg, ara-c 1.5 g/m 2 , etoposid 60 mg/m 2 , and on day 1 daunoxome 80 mg/m 2 ) including stem cell collection. as a result, 5 out of 7 newly diagnosed patients reached cr1 and 2 pr. out of 3 patients treated after first relapse, 2 reached again cr and 1 pr. autologous transplantation was performed at a median of 9 (5-11) months after diagnosis. high-dose conditioning consisted of beam chemotherapy plus the addition of 4 doses of rituximab (days -9 and -1 of the conditioning regimen and days +48 and +55 after transplantation). four patients could not receive the rituximab therapy at days +48 and +55 due to complications. a median number of 5.71 × 10 6 cd34+ cells (1.65-21.21 ) per kg were reinfused. all patients had a short haematologic regeneration time (granulocytes, <0.5 g/l day +10 [9-11]; platelets, day +13 [10-17]) and received a median number of 3 erythrocyte (2-6) and 2 platelet (2-5) concentrates. all patients suffered from mucositis grade i-ii, 3 of 10 had emesis grade i-ii. infectious complication of short duration appeared in 7 patients (4 fuo, 2 pneumonia, and 1 sepsis) early after transplantation. one patient developed late complications (hypothyreosis on day +88 and sarcoidosis on day +105). following transplantation, all patients reached clinical and molecular cr lasting for a median time of 35 (1-53) months. two patients relapsed 17 and 26 months after transplantation. despite continuous salvage immunochemotherapy, one of these patients died 34 months after transplantation, the other one is still in pr (+48 months). median observation periode after diagnosis is 46 (11-105) months. conclusions. treatment intensification with immunochemotherapy and high-dose consolidation is accompanied by acceptable toxicity and seems to be an effective treatment procedure for mantle cell lymphoma. background. bone marrow transplantation (bmt) together with costimulation blockade can reliably induce mixed chimerism and tolerance. in recent studies, we showed that regulation by cd4 + cd25 + t cells plays an important role in this model. stimulation of cd4 + cd25 + t regs by an anti-cd3 mab can inhibit and reverse the outbreak of certain autoimmune diseases, and the maintenance and function of t regs was demonstrated to critically depend on il-2. we thus investigated if stimulation of regulatory cells by anti-cd3 or interleukin-2 facilitates bm engraftment and reduction of tbi in our model. methods. c57bl/6 mice received a total-body irradiation (day -1) of 3 gy or 1.5 gy, approximately 20 × 10 6 fully mismatched balb/c bone marrow cells (day 0) and costimulation blockade consisting of anti-cd154 mab (mr1, day 0) and ctla4ig (day +2). additional groups received further treatment with (n = 4-8 per group): (1) anti-cd3 mab (5 µg, day 0 to +4), (2) il-2 (4000 ie/day, day 0-27), (3) rapamycin (0.3 mg/kg/day, day 0-27), and (4) rapamycin and il-2 (day 0-27). multilinage chimerism and skin graft survival were followed for more than 120 days. results. with our standard protocol, 10 of 10 (3 gy) and 8 of 14 (1.5 gy) mice developed long-term multilinage chimerism and accepted donor skin grafts permanently. while the majority of mice treated with anti-cd3 and 3 gy (7 of 8), rapamycin plus 1.5 gy (4 of 6), and il-2 and rapamycin plus 1.5 gy (4 of 5) developed multilinage chimerism and longterm skin graft survival, groups treated with anti-cd3 plus 1.5 gy tbi (0 of 8, p < 0.01) and il-2 plus 1.5 gy (2 of 6, p = 0.1) showed markedly reduced rates of chimerism and tolerance. we investigated if these negative effects might be correlated with a cytokine shift caused by anti-cd3 treatment, but we did not observe such a shift towards th1 or th2 (as measured on day 13 post-bmt). conclusions. unexpectedly neither anti-cd3 nor il-2 had a positive effect in this model, in some groups anti-cd3 treatment even showed a negative effect. thus, other strategies for augmenting the effect of regulatory cells need to be developed. the role of minor antigen disparities for the induction of mixed chimerism and tolerance through bmt plus costimulation blockade s. bigenzahn 1 , i. pree 1 , p. nierlich 1 , e. selzer 2 , f. mühlbacher 1 , t. wekerle 1 long-term donor skin grafts looked macroscopically intact in the b10.d2 group but showed shrinking and scabs in the balb/c group. conclusions. minor antigen disparities pose a substantial barrier for the induction of mixed chimerism and tolerance. for increased clinical relevance, tolerance models should preferably use strain combinations including major plus minor mismatches. p20 immunohistochemical and confocal analysis of pancreatic tetranectin d. pirkebner 1 , m. hermann 1 , a. draxl 1 , w. mark 2 , r. margreiter 2 , p. hengster 2 background. islet transplantation is not yet widely used in part because of the shortage of human islet cells. gaining detailed knowledge of the physiological basis governing the processes of differentiation of pancreatic stem or progenitor cells that have the capacity to self-renewal and to generate differentiated beta cells is instrumental for the ambitious goal of engineering new pancreatic islets in order to cure type i diabetes. the aim of our study was to cultivate and characterize a pancreatic cell population expressing tetranectin (tn). the ability of tn to bind plasminogen indicates that it may have a role in regulating pericellular proteolysis and proteolytic activation of latent forms of metalloproteinases and growth factors. methods. islets were isolated according to the method of ricordi et al. (1992) using a continuous ficoll gradient. immunohistochemistry and immunofluorescence were performed with a monoclonal antibody against human tetranectin (amino acids 17-181 of human tetranectin monomer; antibodyshop, grusbakken, denmark). to determine in vitro cell proliferation, cells were labeled with brdu (roche, basel, switzerland). results. we describe the localization of tn-positive cells in the human pancreas and their growth in vitro. interestingly, individual positive cells are present within the exocrine acini. we were able to isolate human and murine islets and cultivate these tetranectin-positive cells under adherent and nonadherent conditions as shown by immunohistochemistry and confocal immunofluorescence. the possibility to culture these cells is a first step towards their better characterisation. conclusions. together with its above mentioned ability to bind plasminogen-like hepatocyte growth factor and tissuetype activator, tn may thereby play an important role in the survival of islets after islet transplantation. as we could show, tn positive cells can be isolated and maintained in culture after human islet isolation, thereby providing the possibility to further clarify their role and function in vivo as well as in the course of islet transplantation. p21 fty720 interferes with effector functions and downregulates protein expression of s1p1 and s1p4 in human dendritic cells tures with fty720/fty720-p-treated dc illustrated a cytokine production profile with a lower ifn-? (22% vs. 25% relative reduction) and a higher il-4 (64% vs. 92% relative increase), indicating a shift from th1 toward th2 differentiation as previously evinced for s1p. dc yields, phenotypic differentiation into idc and mdc (besides a minor reduction in cd18 surface expression), as well as the investigated mechanisms of idc antigen uptake (bacterial phagocytosis, mannose receptor-mediated, and fluid-phase endocytosis), were not affected at therapeutically relevant concentrations. conclusions. we conclude that treatment of human dc with fty720 and fty720-p interferes with dc effector functions that are essential for dc to serve their pivotal duty as professional antigen-presenting cells and that dc can therefore be added to the potential list of target cells of fty720. impairment of dc migration and th1-priming capacity due to downmodulation of dc-expressed s1p 1 and s1p 4 might represent a new aspect in the overall mechanism of action and hence contribute, in part, to fty720-mediated immunosuppression. the ibal-fresenius: bioartificial liver innsbruck (ibal) utilizing fresenius standalone, rotating bioreactor m. wurm, v. lubei, p. hengster in order to establish a suitable environment for cultivation of hepatocytes serving as bioartificial liver (bal), we were testing and further developing fresenius standalone, rotating bioreactor. to create optimal conditions for cultivation of hepatocytes, a special environment of nearly gravity-free, low shear force and high mass transfer is needed. furthermore, basic parameters like stability of heating, gas exchange, and sufficiency of nutrition have to be evaluated allowing utilization of various breeding chambers. in summary, we have established a standalone bioreactor capable of quick mass transfer between small and big chambers and external media supply, in nearly gravity-free environment minimizing shear force thus allowing for cultivation of various cell types. background. laparoscopic donor nephrectomy is a less invasive alternative to open nephrectomy for living kidney donation. this study presents the results of the first 58 laparoscopic donor nephrectomies in our center. methods. from june 2000 to may 2005, 58 patients underwent laparoscopic donor nephrectomy for living-related renal transplantation. patient demographic, intraoperative, and postoperative parameters and complications, as well as renal allograft outcome, were evaluated. results. 58 patients (40 female and 18 male) donated their left kidney. the mean donor age was 45 years (24-69 years). the mean surgical time was 233 ± 54 minutes. mean warm ischemia time was 179 ± 63 seconds. patients could be discharged from hospital after a mean time of 5.8 ± 1.5 days. in four patients (6.9%) conversion to open surgery had to be performed. reasons for conversions were lack of operative progression in two cases, in one case venous bleeding, and in one case lesion of the renal artery. there were no reoperations required in the donors. in the recipients, 3 (5.2%) delayed graft functions and 1 (1.7%) primary nonfunction were observed. mean serum creatinine level in the recipients was 1.3 mg/dl 3 months after transplantation. conclusions. laparoscopic live donor nephrectomy is safe for the donor and the transplant kidney. we believe that offering this technique for living renal donation can safely and effectively increase the pool of donor organs available to patients with end-stage renal disease. background. liver transplantation is the treatment of choice for end-stage acute and chronic liver failure. some liver diseases are associated with diseases of the intestinal tract such as primary sclerosing cholangitis and inflammatory bowel disease. moreover, post transplant immunosuppressive agents might cause colonic diseases and there is an abundance of opportunistic pathogens that can manifest in the large intestine. aim. we retrospectively analyzed the incidence and spectrum of colonic disorders in a cohort of 402 liver recipients and determined the impact of these complications on survival. methods. a total of 467 consecutive lts in 402 individuals were performed between 1998 and 2001 at the mayo clinic, jacksonville, florida. standard immunosuppression consisted of tacrolimus, mycophenolic acid, and rapidly tapered steroids. results. there were 29 patients transplanted for psc and 19 were also diagnosed with inflammatory bowel disease (ulcerative colitis, n = 16; crohn's disease, n = 3), with eight having undergone colonic resection prior to lt. in four patients, colitis persisted post transplant. six patients had a history of colonic resection for malignant (n = 3) or infectious diseases. five patients had pre-transplant endoscopic polypectomy. combined colon resection and transplantation were done in 2 patients; one with peritonitis and multiple colonic perforations during retransplantation and the other for ischemic colitis leading to fulminant liver failure. in another case a preexisting transverse colostomy had to be reinforced. there were 32 cases of clostridium difficile-associated enterocolitis. nine patients developed cmv gastrointestinal complications with three cases of colitis, one leading to perforation, intra-abdominal sepsis and death. two patients developed sigmoid diverticulitis and one appendicitis. colonic polyps were endoscopically removed in seven patients and three patients were diagnosed with colorectal cancer (one cecal, two rectal cancers), which all were surgically treated. chronic unexplained diarrhea was observed in fifteen patients, which led to withdrawal of mycophenolic acid. one patient developed a hemorrhage of the terminal ileum/cecal region in the course of intra-abdominal sepsis and was treated by endovascular embolization of the ileocolic artery. four patients had ongoing ulcerative colitis. one herpetic rectal ulcer and two perianal hsv-associated lesions were diagnosed. two patients developed hemorrhoids requiring surgical interventions, and two patients had perianal fistulas. conclusions. the frequency of colonic disorders in our series was higher than expected, with infections accounting for majority of cases. the high incidence of clostridial colitis warrants improvement in screening and preventive measurements. screening for polyps pre-transplant and annually post-transplant might be recommended. background. bartonella has been identified as causative agent of cat scratch disease but is also inflicted in other diseases in the immunocompromised host. case reports. we describe two cases of bartonella henselae-associated diseases in liver transplant recipients who both had contact with cats. the first recipient developed localized skin manifestation of bacillary angiomatosis in association with granulomatous hepatitis. he tested positive for igg antibodies against bartonella henselae. the second patient developed axillary lymphadenopathy, with biopsy showing necrotizing granulomatous inflammation and pcr studies were positive for bartonella henselae dna. her serology for bartonellosis showed a fourfold rise in antibody titers during her hospitalization. both patients responded to treatment with azithromycin in combination with doxycyclin. these were the only cases within a series of 467 liver transplants in 402 patients performed during a four-year period. conclusions. although bartonellosis is a rare infection in lt recipients, one should consider this disease in patients presenting with fever, cns symptoms, skin lesions, lymphadenopathy or hepatitis in particular if contact with cats is reported. background. new immunosuppressive protocols and advanced surgical technique resulted in a major improvement in the outcome of pancreatic transplantation. by reducing the incidence of immunological complications using intensified immunosuppressive protocols, the incidence of some infections, in particular complicated fungal infections, might increase. methods. 217 enteric drained whole-pancreas transplants (ptx) performed at the innsbruck university hospital between march 1997 and october 2004 were retrospectively analysed. prophylactic immunosuppression consisted of atg induction, tacrolimus, mmf, and steroids for the majority of cases. perioperative antimicrobial prophylaxis consisted of amoxicillin/clavulanic (30 ptx), pipercillin/tazobactam (157 ptx), and others (30 ptx). 168 patients additionally received fluconazole. results. actuarial patient, pancreas and kidney graft survival at one year were 96.4%, 88.5% and 94.8%, rejection rate was 30%. within this series, a total of 13 patients developed invasive fungal infections. of those, four had aspergillosis, one zygomycosis, and the remaining ten were caused by yeast. two patients had aspergillosis and later pulmonary infection with candida albicans and candida glabrata. the zygomycosis was the only fungal infection that was diagnosed post mortem and this patient had received pretreatment with caspofungin for non-albicans candida wound infection. one patient died due to aspergillosis following his second pancreas retransplant. three cases of aspergillosis were successfully treated using liposomal amphotericinb in one and a combination of caspofungin and voriconazol in two cases. this combination was also used in a patient who developed intra-abdominal infection with candida krusei. the remaining infections were due to candida albicans including six cases of intra-abdominal infection, one urinary tract infection, and one mucocutaneous candidiasis. type ii diabetics were found at increased risk for fungal infection. conclusions. fungal infections represent frequent and life-threatening complications after ptx. they are amongst the most common causes of graft loss and death. non-albicans candida strains are increasingly isolated and the incidence of filamentous fungal infections has increased during the study period parallel to a decreasing rejection rate. c57bl/6 (h-2 b ) mice received a total-body irradiation (tbi, d-1) of 3 gy and costimulation blockade consisting of anti-cd154 mab (1 mg, d0) vß11 + cd4-cells, p < 0.05). donor skin acceptance fty720 is the first agent in a new class of immunomodulators termed sphingosine-1-phosphate (s1p) human dc, which are known to express mrna for s1p 1 , s1p 2 , s1p 3 and s1p 4 , have not been described so far. methods. to elucidate for the first time the influence of s1p receptor agonists on human monocyte-derived dc (mo-dc), we used therapeutically relevant concentrations (2-200 ng/ml) of fty720 and its phosphorylated metabolite fty720-p and investigated their effects on dc surface marker expression (lineage markers, costimulatory and adhesion molecules, chemokine receptors), protein levels of s1p receptors and dc effector functions: antigen uptake ccl19/elc; or fty720-p-treated mdc (53% vs. 49%; untreated dc, 72%) 24-25 datenkonvertierung und umbruch: manz crossmedia druckerei ferdinand berger & söhne gesellschaft m. b. h., 3580 horn, österreich. -verlagsort: wien. -herstellungsort: horn. printed in austria p. b. b. / erscheinungsort: wien / verlagspostamt 1201 wien -fachkurzinformation rapamune 1mg bzw. 2mg überzogene tablette; rapamune 1 mg/ml bzw. 1mg/ 1ml bzw. 2 mg/2ml lösung zum einnehmen wirkstoff: sirolimus zusammensetzung: 1 tablette enthält 1 mg bzw. 2 mg sirolimus. 1 ml lösung enthält 1 mg sirolimus. 1beutel zu 1 ml bzw. 2 ml enthält: 1 mg bzw. 2 mg sirolimus. sonstige bestandteile: tablettenkern: laktose-monohydrat, macrogol, magnesiumstearat, talkum, tablettenüberzug: macrogol engmaschige überwachung der sirolimus-talspiegel im vollblut bei: leichter bis mittelgradiger leberfunktionsstörung; gleichzeitiger verabreichung starker cyp3a4-induktoren oder -inhibitoren sowie nach deren absetzen; bei absetzen oder deutlicher dosisreduktion von ciclosporin. begrenzte exposition gegenüber sonnen-und uv-strahlung bei patienten mit einem erhöhten risiko für hautkrebs. antimikrobielle prophylaxe gegen pneumocystis carinii pneumonie während der ersten 12 monate nach der transplantation sowie eine zytomegalievirus (cmv)-prophylaxe über 3 monate nach der transplantation ( insbesondere für patienten mit einem erhöhten risiko für eine cmv-erkrankung ) empfohlen. in kombination mit einem hmg-coa-reduktaseinhibitor oder fibrat überwachung auf entwicklung einer rhabdomyolyse und anderen nebenwirkungen dieser präparate. bei kombinierter gabe mit ciclosporin nierenfunktion überwachen, ggf. bei erhöhten serumkreatininspiegeln eine angemessene dosisanpassung erwägen. vorsicht bei gleichzeitiger anwendung von anderen substanzen, die bekanntermaßen eine schädigende wirkung auf die nierenfunktion haben erhöhte laktat-dehydrogenase (ldh), arthralgie, akne, infektion des harntraktes gelegentlich: pankreatitis, lymphom/lymphoproliferative erkrankung nach transplantation, panzytopenie. die immunsuppression erhöht die anfälligkeit, lymphome oder andere bösartige neubildungen, vor allem der haut, zu entwickeln fachkurzinformation zu inserat von umschlagseite 2 p08 liver transplantation for patients with hepatitis b-related liver disease: a single-center experience l. hinterhuber, i. w. graziadei background. liver transplantation (lt) is the only effective therapy for end-stage liver disease due to hepatitis b (hbv). before introduction of passive immunoprophylaxis with hepatitis b immunoglobulin (hbig) and new antiviral nucleoside analogues, hepatitis b recurrence was seen in the majority of the patients resulting in an inferior graft survival. in this study we analyzed the different clinical courses of hbv recurrence, the impact of hbv recurrence on patient survival, and potentially contributing factors for long-term outcome of hbv patients after lt.methods. between 1983 and 2003, 57 out of 692 patients (50 m, 7 f; mean age, 51 years) were transplanted secondary hbv cirrhosis at our center. the mean follow-up was 44 months (range, 1-246 months). immunosuppression (is) consisted of cya/fk 506, prednisolone and/or azathioprine/mmf. fourteen patients received no hbig (prior to 1993), 14 patients received hbig alone, and 29 patients hbig in combination with lamivudine (lam).results. the actuarial overall 1-, 5-, 10-year survival rates were 83%, 69%, and 65%, comparable to those of other indications. patients with combined prophylaxis showed the best survival rates (88%, 77%, 77%), compared to patients treated with hbig (82%, 51%, 51%) and patients without treatment (76%, 70%, 63%). five patients required reltx, one patient two reltx. in total, 17 patients (33.5%) developed recurrent hbv infection after lt: 50% (6 of 12) in the non-hbig group, 42% (6 of 14) in the hbig mono, and 18% (5 of 28) in the combined prophylaxis group. four of the five patients in the hbig/lam group were hbv dna positive prior to lt, two presented with lam mutants. hbv recurrence, however, did not negatively impact patient outcome. all patients with recurrent disease were treated with antivirals (famcyclovir, lam, adefovir). forty-seven percent of patients responded to the treatment and remained hbv dna negative. only one patient was retransplantated due to hbv recurrence. no possible risk factors for overall survival were found to be significant.conclusions. our study showed that patients with combined hbig/lam prophylaxis had excellent long-term survival. recurrent hbv in the allograft could be effectively treated in the majority of patients and did not influence longterm survival. background. liver failure is associated with reduced synthesis of clotting factors, consumptive coagulopathy, and platelet dysfunction. the aim of the study was to evaluate the effects of liver support using the molecular adsorbent recirculating system (mars) on the coagulation system in patients at high risk of bleeding.methods. 61 mars treatments in 33 patients with acute liver failure (n = 15), acute on chronic liver failure (n = 8), sepsis (n = 5), liver graft dysfunction (n = 3), and cholestasis (n = 2) have been studied. standard coagulation tests, standard thromboelastography (teg), heparinase-modified and abciximab-fab-modified teg were performed immediately before and 30 minutes after commencement of mars and after the end of mars treatment. to all patients, prostaglandin i2 was administered extracorporeally. 17 patients additionally received unfractioned heparin.results. three moderate bleeding complications in three patients, requiring 3-4 units of packed red blood cells, were observed. all were sufficiently managed without interrupting mars treatment. although there was a significant decrease in platelet counts (median, 9 g/l; range, -40 to 145 g/l) and fibrinogen concentration (median, 15 mg/dl; range, -119 to 185 mg/dl) with a consecutive increase in thrombin time, the platelet function, as assessed by abciximab-fab-modified teg, remained stable. mars did not enhance fibrinolysis.conclusions. mars treatment appears to be well tolerated in patients with marked coagulopathy due to liver failure. although mars leads to a further decrease in platelet count and fibrinogen concentration, platelet function, measured as contribution of the platelets to the clot firmness in teg, remains stable. according to teg-based results, mars does not enhance fibrinolysis. methodik. ein 69 jahre alter patient war vor 13 jahren aufgrund einer post-hepatitischen zirrhose (phcc) lebertransplantiert worden. nun war es im verlauf der letzten zeit zu einer deutlichen gewichtszunahme gekommen, sodass der patikey: cord-274080-884x48on authors: rumlová, michaela; ruml, tomáš title: in vitro methods for testing antiviral drugs date: 2018-06-30 journal: biotechnology advances doi: 10.1016/j.biotechadv.2017.12.016 sha: doc_id: 274080 cord_uid: 884x48on abstract despite successful vaccination programs and effective treatments for some viral infections, humans are still losing the battle with viruses. persisting human pandemics, emerging and re-emerging viruses, and evolution of drug-resistant strains impose continuous search for new antiviral drugs. a combination of detailed information about the molecular organization of viruses and progress in molecular biology and computer technologies has enabled rational antivirals design. initial step in establishing efficacy of new antivirals is based on simple methods assessing inhibition of the intended target. we provide here an overview of biochemical and cell-based assays evaluating the activity of inhibitors of clinically important viruses. viral pandemics remain serious threats to humankind. every year, known viruses such as hiv-1 and hepatitis b virus newly infect millions of people across the globe. in addition, recent outbreaks of ebola virus, influenza virus, severe acute respiratory syndrome (sars) coronavirus and middle east respiratory syndrome-coronavirus (mers-cov) serve as a reminder of the silent danger. the world health organization reported in 2015 that over 1.34 million causalities per year are connected with hepatitis b and almost 500,000 with hepatitis c. occasional epidemic outbreaks of other viruses are also striking. these include outbreaks of noroviruses, flaviviruses (zika and dengue viruses), new strains of influenza viruses, the re-emergence of west nile virus in italy and the united states. despite relatively long pauses, viruses such as influenza virus can re-emerge and cause global pandemic health problems. unlike cellular genomes, which consist of double-stranded (ds) dna, viral genomes can be formed by a broad variety of nucleic acids (nas), including ds or single-stranded (ss) circular or linear dna or positive-, negative-or sometimes ambi-sense rna. as viral life cycles are dependent on cellular factors and cellular metabolic and signaling pathways, the number of possible antiviral drug targets is limited. however, almost all viruses encode unique proteins and enzymes that may serve as specific targets for antiviral therapy. the overarching goal of modern drug development efforts is to design compounds that specifically inhibit viral targets or cellular targets essential for virus replication. the purpose of this review is to provide insight into the broad variety of cell-based and biochemical assays used for identification and evaluation of antivirotics, including high-throughput screening (hts) methods. an overview of available inhibitors and vaccines, which has been reviewed elsewhere [e.g. in (de clercq and li, 2016) ], is beyond the scope of this paper. to package their genomes into particles of very limited size, viruses encode few genes. virions consist of an rna or dna genome that is protected by an outer shell called a capsid (also nucleocapsid) formed by a lattice of capsid proteins. in enveloped viruses, the capsid is additionally surrounded by a lipid bilayer spiked with viral proteins. the size of animal viruses ranges from approximately 25 nm to over 300 nm (cohen et al., 2011) . the capsids and virions of rna viruses adopt various shapes. viral capsids may be icosahedral (e.g. picornaviridae, astroviridae, reoviridae, togaviridae), bullet-shaped (rhabdoviridae), helical (coronaviridae), helical filamentous (filoviridae, orthomyxoviridae, paramyxoviridae), or filamentous (arenaviridae, bunyaviridae). the retroviral capsid core may be conical, spherical, or rod-shaped. the morphology of dna viruses is similarly diverse, ranging from icosahedral (enveloped -hepadnaviridae, herpesvridae; nonenveloped -adenoviridae, parvovirdae, polyomaviridae and papillomaviridae) to rodshaped (baculoviridae) or pleomorphic (poxviridae). the viral life cycle is the process of viral replication in a host cell. first, viruses enter the host cell and replicate their genomes. following translation of viral proteins by the host cell machinery, viruses package their genomic material into protective proteinaceous capsids and exit the cell to infect another host cell. nonenveloped viruses consist only of a protein capsid shell enclosing the viral genome and enzymes, while the capsid shell of enveloped viruses is enclosed in a lipid envelope derived from the host cell membrane. regardless of whether the virus is enveloped, its surface must display (glyco)proteins suitable for specific interactions with host cell receptors. in contrast to the surface proteins of nonenveloped viruses, those of enveloped viruses usually serve another function in addition to host cell recognition and attachment. for example, they may enable fusion of the viral and cellular membranes, usually through an interaction with a secondary receptor(s) or co-receptor(s). the fusion domains of viral surface glycoproteins thus can lower the kinetic barrier for the energetically demanding membrane fusion. the viral particle must be sufficiently stable to protect the genome until its delivery into the host cell. simultaneously, it must be metastable, to allow its disassembly and release of the genome for replication in the infected cell at the appropriate time. the energetic barrier that prevents viral disassembly outside the cell is lowered upon infection by structural changes in the viral components. these changes may be induced by binding of a cellular ligand or by changes in the environment, such as ph change upon entering a specific cellular compartment. numerous viruses preassemble immature particles that undergo irreversible (usually proteolytic) transitions into mature structures of fully infectious virions. mutual interactions of viral capsid proteins are typically different from those of cellular proteins, which predominantly create binary interactions. viral structural proteins interact with multiple neighboring partners to form multicomponent macromolecular structures [reviewed in (cheng and brooks, 2015; jayaraman et al., 2016) ]. the economy of the packaged viral genomes due to the limited capsid size implies formation of only a few types of structural proteins, which are usually symmetrically organized [reviewed in (prasad and schmid, 2012; raguram et al., 2017) ]. in the initial stage of infection, viruses must overcome several obstacles. the first is the cellular plasma membrane with an actin cortex. then, they must traffic through dense cytoplasm to reach their final destination for replication and assembly. these pathways are specific for different viruses and often are dictated by the size of the particle and its structure. the viral life cycle can be divided into several common stages, including entry, uncoating, genome replication, genome packaging and assembly, release, and maturation ( fig. 1) . in general, the first phase of viral infection is specific recognition of the target host cell and binding to a surface receptor displayed on the cell membrane. this process is common to both enveloped and nonenveloped viruses. in enveloped viruses, the binding is mediated by viral surface components, typically oligomers of integral glycoproteins. nonenveloped viruses bind receptors through sites or projections on the capsid surface. viruses can use either a single receptor (e.g. tim-1 for hepatitis a virus, gm1 for sv40, cd155 for poliovirus, low-density lipoprotein receptor for human rhinovirus) or multiple receptors with equivalent roles (e.g., nectin-1/2 or hvem for herpes simplex virus 1/ 2, ace or l-sign for sars coronavirus). for other viruses (e.g. hiv, hcv, adenoviruses, rotaviruses, picornaviruses, and some herpesviruses), the presence of at least two cytoplasmic membrane components is required. for example, hiv-1 binds to the primary receptor cd4 and one of two co-receptors (ccr5 or cxcr4) [reviewed in (cossart and helenius, 2014; grove and marsh, 2011) ]. to infect a cell, viruses must overcome the plasma membrane to deliver their genetic material into the cytosol. viruses enter cells by two main mechanisms. a majority of animal viruses, both enveloped and nonenveloped, enter cells by one or two types of endocytosis, such as clathrin-mediated endocytosis (e.g. vsv, influenza a virus, rhinovirus), caveolin-mediated uptake (echovirus, polyoma virus), clathrin/caveolin-independent endocytosis such as caveolar or lipid raft-mediated (e.g. sv40, polyomavirus mouse), or macropinocytosis (e.g. vaccinia virus, respiratory syncytial virus, ebola virus, hiv-1) (blaas, 2016; cossart and helenius, 2014; fields and knipe, 2013; kirkham and parton, 2005; marechal et al., 2001; mayor and pagano, 2007; mercer and helenius, 2009; parton and simons, 2007; rasmussen and vilhardt, 2015; saeed et al., 2010) . these endocytic mechanisms enable the virus to be transported by the cell's machinery through the plasma membrane and to pass through the dense actin cortex. cellular entry of some viruses is coupled with receptor-mediated signaling, resulting in activation of molecules that facilitate virus entry by cytoskeleton reorganization, induction of long-distance transport of the virus-containing vesicles, or actin cortex disassembly. the second type of entry is used by some enveloped viruses (paramyxo-, herpes-, and retroviruses), which upon cell surface receptor binding, infect the cell by direct fusion of viral and plasma membranes at neutral ph. upon internalization, most viruses become trapped and enclosed in vesicles that pinch off the inner side of the plasma membrane and transport their cargo into the cytoplasm. on their way through the cell, these transport vesicles undergo maturation by fusing with other vesicles. to fulfill their task of genome replication, viruses have to escape from these endosomes. the "great escape" is triggered by activation of a fusion or penetration mechanism, such as changes in conditions in the endosomal interior [e.g. ph, ionic environment (e.g. calcium ion concentration), oxido-reducing conditions], changes in membrane composition, and other physico-chemical cues (blaas, 2016; cossart and helenius, 2014; inoue and tsai, 2013) . depending on the requirements for a particular virus, these events can occur in early endosomes (ph 6.5-6.0; e.g. hepatitis c virus, vesicular stomatitis virus), late endosomes (ph 6.0-5.0; e.g. influenza a virus, dengue virus, sars coronavirus), recycling endosomes, macropinosomes, the endoplasmic reticulum, the golgi apparatus, or lysosomes (ph 5.0-4.5) (blaas, 2016; cossart and helenius, 2014; grove and marsh, 2011; inoue and tsai, 2013) . for the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. structural rearrangements in enveloped viruses usually mediate fusion of viral and endosomal membranes. in nonenveloped viruses, the structural changes uncover amphipathic or hydrophobic domains that may induce pore formation or disruption of endosomal membranes. to deliver genetic material to the replication site, these mechanisms ultimately release viral capsid structures from endosomal vesicles into the cytosol either by fusing with the endosomal membrane (enveloped viruses) or by penetrating the endosomal membrane (nonenveloped viruses). uncoating is the partial or complete disassembly of the protective capsid shell and/or lipid envelope to liberate the viral genome. for many viruses, this process is closely connected with conformational changes induced by the virus binding to the cell surface receptor; a low ph environment; or changes in oxido-reducing conditions, ion concentrations, or other factors. for enveloped viruses, uncoating involves a loss of the viral membrane by fusion either with the plasma membrane or with intracellular vesicles, followed by stepwise uncoating of the protective capsid shell. in nonenveloped viruses, the uncoating process typically involves conformational changes that result in the weakening of intermolecular interactions, loss of structural proteins, proteolytic cleavage, and so on (fields and knipe, 2013) . depending on the virus, uncoating can take place at the plasma membrane, in the cytoplasm, during endocytosis in early or late endosomes, in lysosomes, in the nucleus, or at the nuclear pore complex (npc). the ssrna genome of retroviruses, which is fully enclosed inside a protective capsid shell, must be reverse transcribed into dsdna and released from the capsid. although it is accepted that hiv-1 uncoating is linked to reverse transcription and nuclear import (ambrose and aiken, 2014) and is controlled by host factors (e.g. cyclophilin a, trim5α), the precise molecular mechanisms that trigger the uncoating remain unknown. several hypotheses have been proposed, including breakage of the capsid shell due to increased inner pressure caused by accumulation of the reverse transcription product (rankovic et al., 2017) , the requirement of intact microtubules and dynein and kinesin motors (lukic et al., 2014) , and phosphorylation of capsid shell protein by the host cell kinase melk (takeuchi et al., 2017) . in contrast to the majority of rna viruses, which replicate in the cytoplasm, most dna viruses (with the exception of large dna viruses including poxviridae, asfarviridae, and mimiviridae) and several negative stranded rna viruses enter the nucleus to replicate their genomes (kobiler et al., 2012; koonin and yutin, 2010) . passive diffusion into the nucleus is suitable for molecules smaller than 9 nm in diameter, but larger structures must enter through the nuclear pore complex (npc), which can accommodate molecules of up to 39 nm (pante and kann, 2002) . translocation through the npc is tightly regulated and requires the presence of a nuclear localization signal (nls) on the passing molecule and nuclear import receptors (importins or karyopherins) (cautain et al., 2015) . due to the diversity of viral particle sizes (from 25 nm to over 300 nm) and structures, viruses have evolved several strategies to export their dna or rna genome into the nucleoplasm. with regard to the npc, these pathways can be divided into npc-dependent and npcindependent mechanisms. due to size limitations, only a few, very small simplified scheme of common stages of viral life cycle targeted by antiviral drugs. these stages including: 1) attachment and entry, 2) uncoating, 3) genome replication, 4) genome packaging and assembly of viral particle and 5) virus release and maturation. m. rumlová, t. ruml biotechnology advances 36 (2018) 557-576 viral capsid particles, such as hepatitis b virus (hbv; 32-36 nm diameter), can pass through the npc (cohen et al., 2011; rabe et al., 2003) in an importin-dependent manner. hbv then disassembles at the nuclear side of the npc (the nuclear basket), releasing its genome into the nucleoplasm (fay and panté, 2015) . the capsid shells or ribonucleoprotein complexes (rnps) of some larger viruses, such as influenza a virus and hiv-1, disassemble within the cytoplasm. the viral genome, released from the shell and complexed with nls-containing components, is then translocated through the npc (ambrose and aiken, 2014; campbell and hope, 2015; cohen et al., 2011; fay and panté, 2015; hutchinson and fodor, 2013) . another mechanism, used by herpes simplex virus (hsv) and adenoviruses, involves cellular (importins, nup) or viral protein-mediated attachment (docking) of the capsid shell at the cytoplasmic side of the npc. this facilitates the passage of genomic dna into the npc either by ejection from an almost intact particle (through the capsid portal in hsv-1) or upon complete disassembly of the capsid shell (in adenoviruses) (kremer and nemerow, 2015; ojala et al., 2000; pasdeloup et al., 2009) . npc-independent mechanisms are used by some retroviruses (e.g. mlv) that enter the nucleus during the mitotic phase of the cell division cycle when the nuclear envelope is dissolved (matreyek and engelman, 2013; roe et al., 1993) . another mechanism, described for parvoviruses, involves partial disruption of the nuclear envelope (cohen and pante, 2005; fay and panté, 2015) . viral genomes can be encoded by various types of nas, as summarized in table 1 for dna viruses and table 2 for rna viruses. by convention, ssdna of equivalent polarity to mrna is designated as the positive (+) strand. the complementary ssnas are of minus polarity (−). the majority of dna viruses replicate in the nucleus, where cellular dna replication and transcription also occur. in contrast, rna viruses usually replicate in the cytoplasm. rna viruses are the only "organisms" that store their genetic information in the form of rna. replication of their genomes is accomplished either by rna-dependent rna synthesis ( fig. 2a , b) [reviewed in (ferron et al., 2017; lu and gong, 2017; menéndez-arias and andino, 2017; pietilä et al., 2017; tao and ye, 2010) ] or through rnadependent dna synthesis (reverse transcription) followed by dna integration, replication, and transcription ( fig. 2c ). as some of these enzymatic activities are not commonly found in uninfected host cells, rna viruses must encode enzymes to aid replication (table 2 ). rna viruses are divided according to the baltimore classification into dsrna viruses (birna-and reoviridae); positive-sense ssrna viruses (corona-, flavi-, and picornaviriade); negative-sense ssrna viruses (filo-, rhabdo-, paramyxo-, and orthomyxoviriade), and ambisense rna viruses (arena-and some members of bunyaviridae) with both positive-and negativesense rnas (nguyen and haenni, 2003 ) (see table 2 ). the nature of the genome not only dictates the mechanism of replication, but also has other important consequences. the genome of (+)rna viruses may serve directly as mrna for production of viral proteins. therefore, the mere introduction of genetic material (e.g. in exocytic vesicles) may result in productive infection. the rna polymerases that copy the genetic material of rna viruses are error-prone, which provides considerable genetic flexibility and the propensity to evolve drug-resistant mutants. these features are amplified in viruses with segmented genomes that undergo reassortment. in viruses with segmented genomes (orthomyxoviridae, arenaviridae and bunyaviridae), each segment is transcribed in an autonomous transcription-replication unit by viral rna polymerase that binds to the 5′ end cap structure. of note, the genomic rna (grna) is capped by a unique mechanism called cap-snatching (ferron et al., 2017) , in which the cap is cleaved from cellular mrna and transferred onto the viral grna by a subunit of viral rna polymerase (pflug et al., 2017) . some dsrna viruses (birnaviridae and reoviridae) also contain segmented genomes. upon replication, these viruses must ensure stoichiometric incorporation of single copies of each grna segment into new particles. this is guided by specific packaging signals on each segment of grna that interact with positively charged domains in the capsid proteins [reviewed in (isel et al., 2016; pohl et al., 2016) ]. although different models of packaging have been proposed for various segmented genome viruses, a common feature is co-assembly of the capsid proteins with the grna and rna polymerase. the genomes of dna viruses come in a considerable variety of sizes and shapes, from small ss to large ds molecules that may be linear or circular. the size range of these genomes (from 1.8 kb to 1200 kb) reflects the necessity for some viruses to encode specific proteins required for viral replication. small-genome dna viruses (polyoma-, papilloma-, and parvoviruses) use only host cell enzymes for replication and transcription. the only exceptions are some hepadnaviruses (e.g. hepatitis b virus) that despite having small genomes (approximately 3 kb), encode their own specific dna polymerase/reverse transcriptase that reverse transcribes pregenomic rna (pgrna) into genomic viral dna (fig. 2d , ii) (beck and nassal, 2007) . viruses with intermediate-size genomes (up to 35 kb) (e.g. adenoviruses) encode their own dna polymerase for genome replication, but they usually utilize cellular rna polymerases ii and iii for transcription (fields and knipe, 2013) . viruses with large genomes (larger than 100 kb) (e.g. herpesviruses) encode proteins for replication, including dna polymerase and dna helicase-primase, as well as some enzymes necessary for biosynthesis of deoxyribonucleotide triphosphates (dntps) and several transcription factors (boehmer and nimonkar, 2003) . poxviruses (e.g. vaccinia virus), are another type of virus with large dsdna genomes, and their replication, transcription, and translation take place entirely in the cytoplasm within discrete juxtanuclear sites called virus factories (moss, 2013) , (fig. 2d , iii). these viruses encode all enzymes and specific factors necessary for genomic replication and transcription. with their own replicative machinery, large-genome dna viruses can replicate in nondividing cells. in contrast, the replication of small-genome dna viruses, which depends on cellular dna polymerases, must occur simultaneously with the s-phase of the cell cycle (e.g. parvoviruses), or must express some viral protein/ oncogene to re-program the host cell-cycle regulatory proteins p53 or retinoblastoma protein (prb), triggering entry into the s-phase (e.g. polyomaviruses and papillomaviruses). by affecting the g1/s checkpoint (controlled by p53 and prb), the viruses ensure the production of host enzymes required for viral replication (fields and knipe, 2013) . production of viral proteins of dna viruses with intermediate and large size genomes is divided into early and late phases. in the early (prereplicative) phase, nonstructural proteins required for dna replication are translated. the late phase, during which the late structural proteins needed for assembly are translated, begins after viral dna replication ( fig. 2d , i). depending on the species, viruses assemble either in contact with the cellular membrane or independent of the membrane in either the nucleus or cytoplasm. the membrane-independent route is used by nonenveloped viruses and a few enveloped viruses (e.g. orthomyxoviruses, herpesviruses, and some retroviruses) that acquire the membrane envelope after intracellular assembly during budding from the cell. the limiting step for nuclear assembly is the size of npcs, which are large enough to transport rna and import proteins required for assembly into the nucleus; however, npc size limits the transport of larger assemblies. therefore, some viruses form assembly intermediates in the nucleus. these structures are then exported to the cytoplasm, where they come together to form viral particles. nuclear export is specific and depends on the presence of nuclear export signals (nes) in the transported proteins. one example of a nonenveloped icosahedral virus that assembles in the nucleus is adenovirus, the assembly of which has been studied intensely due to its potential use in gene therapies [reviewed in (ahi and mittal, 2016) ]. recent data suggest that upon accumulation of multiple copies of adenoviral dsdna genomes, coordinated assembly and packaging occur by two interlinked mechanisms that involve both capsid proteins and core components (condezo and san martín, 2017) . the assembly occurs in the so-called peripheral replicative zone with the assistance of scaffolding proteins that facilitate formation of adenoviral particles but are excluded from mature viruses. adenoviruses are finally released upon lysis of the infected host cell. orthomyxoviruses and herpesviruses are enveloped viruses that assemble their nucleoproteins in the nucleus. herpesviruses package their dsdna genome as head-to-tail concatemers and assemble icosahedral procapsids on scaffold proteins in the nucleus [reviewed in (heming et al., 2017) ]. however, subsequent steps of herpesvirus assembly proceed in the cytoplasm. the preassembled procapsid is too large to pass through the npc, but it exits the nucleus by viral proteindriven vesicular transport across the nuclear inner membrane leaflet. thus, the herpesvirus acquires an initial envelope from the inner nuclear membrane [for review see (fields and knipe, 2013; hellberg et al., 2016) ]. next, the herpesviral membrane fuses with the outer nuclear membrane, and the naked particle is released from the nucleus into the cytosol. here, the virus acquires tegument (a protein layer between the capsid and the envelope) and other proteins. final herpesvirus envelopment occurs at the golgi membrane containing the viral glycoproteins (fields and knipe, 2013) . preassembled orthomyxoviral ribonucleoproteins, upon their export from the nucleus, are driven to the plasma membrane to which they attach through electrostatic interactions of the matrix protein m1 with membrane phosphatidylserine. the virions then assemble simultaneously with budding during which they also acquire ha, na and m2 membrane proteins. poxviruses undergo an even more complicated pathway. they are enveloped with multiple membranes acquired from er/intermediate compartments and golgi or early endosomes (moss, 2015 (moss, , 2016 . these membranes also provide the poxviruses with their membrane proteins. most viruses assemble upon interaction of their structural proteins with cellular membranes. the target membrane for assembly differs according to the virus type. flaviviruses assemble at the surface of the er and then upon their budding into the er lumen, the immature particles are then transported into the tgn. some viruses, such as coronaviruses, assemble at the er-golgi intermediate compartment [reviewed in (ujike and taguchi, 2015) ]. the assembly of bunyaviruses occurs concurrently with replication of grna segments in virus factories located along the golgi (guu et al., 2012; strandin et al., 2013) . the presence of membrane glycoproteins at the golgi membrane determines the sites where assembling bunyavirus particles bud into the golgi lumen, similar to other enveloped viruses. numerous viruses, including paramyxoviruses (cox and plemper, 2017) , orthomyxoviruses (pohl et al., 2016) , alphaviruses (jose et al., 2009) , rhabdoviruses (okumura and harty, 2011) , and most retroviruses (freed, 2015) , assemble underneath the cytoplasmic membrane, which facilitates assembly by providing a scaffolding function. the virus then acquires a lipid envelope through budding across the plasma membrane. during this process, it also gains the envelope glycoproteins (env) that are anchored in the plasma membrane by hydrophobic transmembrane domains. env reaches the plasma membrane by a cellular secretory pathway upon synthesis in the er and subsequent glycosylation in the golgi. usually, a specific interaction between viral structural proteins and glycoproteins is required. this may be either direct or mediated through the interaction with matrix protein (e.g. in (-)rna viruses such as ortho-and paramyxoviruses or rhabdoviruses). most retroviruses, including hiv (so-called morphogenetic c-type), also assemble at the plasma membrane of the host cell. the interaction of the structural polyprotein precursor (gag) with the plasma membrane is usually facilitated by a bipartite signal in the n-terminal domain of gag (i.e. matrix protein) comprising both the basic patch and n-terminally linked myristoyl residue (added co-translationally to gag). in contrast to c-type assembly at the membrane, morphogenetic b/d-type retroviruses assemble at pericentriolar sites where gag polyproteins are transported along microtubules by dynein molecular motors (sfakianos et al., 2003; vlach et al., 2008) . for both morphogenetic pathways, the packaging of grna facilitates assembly. (+)rna viruses have adopted a mechanism of extensive rearrangement of intracellular membranes to provide a milieu for virus replication and assembly. this mechanism effectively protects dsrna intermediates from degradation by the host cell machinery (delgui and colombo, 2017; jackson, 2014) . this process has been well-documented for poliovirus, in which the newly formed membranous structures exclusively serve for virus production. various types of vesicles that are formed upon viral infection have different roles in viral replication (rossignol et al., 2015) . some nonenveloped mammalian viruses, such as reoviruses, assemble in the cytosol in so-called viroplasms or viral factories into icosahedral particles consisting of three concentric layers encircling segments of genomic dsdna (benavente and martinez-costas, 2006; jones, 2000; shah et al., 2017) . rotavirus seems to be the only exemption in the reovirus family, as it enters the endoplasmic reticulum to gain its outer protein shell (trask et al., 2012) . numerous viruses assemble from polyprotein precursors that must be specifically cleaved by a viral protease to generate infectious particles. this mechanism, which is an irreversible step in the virus life cycle, ensures equimolar packaging of structural proteins and proportional co-assembly of viral enzymes in the form of precursors. upon proteolytic release, the liberated proteins may undergo different trafficking pathways or fulfill various functions in the virus. maturation changes the energy of interaction forces among those interfaces required for intracellular assembly of immature particles to those suitable for viral stability in the environment and for disassembly and uncoating of genetic material for replication [reviewed in (veesler and johnson, 2012) ]. in poliovirus, the autocatalytic and subsequent viral proteasemediated cleavage of p1 precursor protein allows formation of pentamers that interact with grna. additional cleavage of vp0, yielding vp2 and vp4, is required to form infectious poliovirus particles. in retroviruses, proteolytic processing is initiated by the autocatalytic liberation of viral protease, which subsequently cleaves the polyproteins to trigger major structural rearrangements in the virus and release of other viral enzymes (reverse transcriptase and integrase) and structural proteins. in herpesviruses, maturation involves proteolytic processing of the scaffolding protein and recruitment of tegument proteins that stabilize the particle and mediate interactions with the membrane during envelopment (gibson, 2008; tandon and mocarski, 2012; tandon et al., 2015) . adenoviruses undergo a maturation process that involves processing of six structural components of the core and one non-structural precursor that initiates replication of gdna (gaba et al., 2017) . one interesting feature of adenoviral maturation is that dna is required as a co-factor for protease activity. this means that maturation occurs only in particles that have packaged gdna (mangel and san martín, 2014) . flaviviruses form icosahedral particles upon budding into the neutral milieu of the er. the particles then translocate to the more acidic environment of the trans-golgi network. this ph shift results in disassembly of the immature lattice and extensive rearrangement of the flaviviral particle. this is connected with the exposure of a viral structural glycoprotein (prm) that is specifically processed by the , retroviruses (c) and dna viruses (d) (the schemes a-c were modified from: (ahlquist, 2006) . a: following endocytosis, the genome of (+)rna viruses may directly serve as mrna for translation of virus encoded proteins. among them, there are proteins of rna-replication machinery that recruit (+)rna into a membrane-associated replication complex. the genomic rna is replicated by using (-)rna template, which is transcribed in a low copy number amplified (+)rna is then packaged into newly assembled virions that exit the host cell either through secretion or cell lysis. b: upon the virus attachment, a core containing both grna and virus encoded rna polymerase is delivered into the cytoplasm by endocytosis. cytoplasmic transcription of the (-)rna template provides (+)rna that serves as mrna for translation of viral proteins. in dsrna viruses, the (+)rna is packaged into new cores which undergo maturation by synthesizing (-) rna (dotted strand) and acquiring surface proteins. in the (-)rna viruses, the (+)rna strand is transcribed into genomic (-)rna in the cytoplasm and then packaged. the new virions egress the cell either through secretion or cell lysis. c: following fusion of viral and cell plasma membrane, the retroviral core is released into cytosol, rna genome is transcribed into dsdna by viral reverse transcriptase concomitantly with uncoating of the viral core, ds dna is transferred into nucleus where it is integrated into host cell genome by viral integrase, following translation of retroviral structural and enzymatic polyproteins, the unspliced grna is packaged into the immature particles that usually assemble at the plasma membrane and the viral particles bud from the cell. d: three mechanisms (i.-iii.) of dna viruses replication are shown: (i): following entry and uncoating, the dna genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. dna polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. herpesviruses). (ii) unique replication cycle of hepatitis b virus (hbv): following entry, the viral particle is internalized by endocytosis and the nucleocapsid is released into the cytoplasm; the genome (relaxed circular rcdna) is transported into the nucleus, where it is converted to a covalently closed circular form (cccdna); which serves as a template for transcription of pregenomic rna (pgrna) for translation of the core protein and the viral polymerase and three subgenomic mrnas used for translation of regulatory and envelope proteins; following viral transcription and translation, the hbv core proteins self-assemble in the cytoplasm into viral nucleocapsid with concurrent incorporation of pgrna and hbv polymerase, pgrna is reversely transcribed into a rcdna within the nucleocapsid; nucleocapsid containing rcdna can either re-enter the nucleus to amplify cccdna, or can be enveloped by hbv envelope proteins in the endoplasmic reticulum. the particles are then secreted from the infected hepatocyte by exocytosis. (iii) upon entering the cell, the replication, transcription and translation take place entirely in the cytoplasm, within discrete juxtanuclear sites called virus factories (e.g. poxviruses) adapted from: ahlquist, p., 2006. parallels among positive-strand rna viruses, reverse-transcribing viruses and double-stranded rna viruses. nat rev microbiol 4(5), 371-382. cellular protease furin in the trans-golgi network. the liberated globular heads of prm remain attached at the low ph, but are released when the virus enters neutral body fluids (rey et al., 2017) . one frequently used mechanism of release of nonenveloped and some enveloped viruses is lysis of the infected cell. this is the simplest release mechanism, although the transition to lysis from latent infection is delicately regulated (aneja and yuan, 2017; levings and roth, 2013; schmiedel et al., 2016) . however, viruses that are usually lysogenic may also use alternative release pathways (bird and kirkegaard, 2015a) . these include non-lytic spread of viruses mediated by vesicles, which has been observed for poliovirus (bird and kirkegaard, 2015b; jackson et al., 2005) , coxsackievirus (alirezaei et al., 2012) , and hepatitis a virus . another possibility is that vesicles released from a cell infected with (+)rna viruses contain naked viral grna. this (+)grna-containing vesicle functions itself as an infectious agent as it is transferred to another cell (bird and kirkegaard, 2015a) . the standard way for enveloped viruses to leave the host cell is budding, which includes membrane extrusion and separation of the viral and cellular membranes (so-called pinching off). the lipid envelope layer acquired during viral particle budding through the plasma membrane protects the virus particle. there are three basic mechanisms of budding: i) mediated by envelope glycoproteins, such as the e protein of coronaviruses; ii) independent of glycoproteins, in which the viral structural proteins trigger the extrusion of the plasma membrane, such as retroviral budding in which strong interactions between the gag n-terminal domain (matrix protein) and the plasma membrane induce bulging of the membrane; and iii) requiring interaction between the viral glycoproteins and the capsid for membrane extrusion, as in alphaviruses. the final step that results in the separation of virus from the cell surface is pinching off the particle. this is a controlled process that involves both viral protein domains and cellular factors. during this process, viruses apparently use cellular machinery similar to that used during the last step of cell division (the release of the daughter cell) called endosomal sorting complex required for transport (escrt) [reviewed in (campsteijn et al., 2016; hurley, 2015; olmos and carlton, 2016; scourfield and martin-serrano, 2017) ]. direct interactions of numerous viral domains with escrt complex subunits have been identified (bieniasz, 2006) . in retroviruses, short specific amino acid sequences (ptap or psap) interact with the escrt components, and the interaction of hiv with the escrt proteins nedd4 and alix is wellknown (freed, 2002; fujii et al., 2007; gomez and hope, 2005) . however, this mechanism has also been adopted by other viruses that interact with the same escrt components, such as filoviruses (han et al., 2015; jasenosky and kawaoka, 2004; liu and harty, 2010) . interactions with escrt proteins have also been reported for vesicular stomatitis virus luyet et al., 2008 ), rhabdovirus (taylor et al., 2007 , arenaviruses (ziegler et al., 2016) , picornaviruses , paramyxoviruses (park et al., 2016) , and hepatitis c virus, a representative of the flavivirus family (falcon et al., 2017) . the typical release pathways used by viruses may vary under some conditions. for example, during chronic infection, numerous viruses use alternative cell-to-cell transmission that may help the virus avoid host neutralization (hulo et al., 2017) . syncytium formation, an hivinduced cell fusion, was recognized in the early 1990s (callahan, 1994) . another type of cell-to-cell transmission through tight junctions was shown for hiv (hübner et al., 2009; wang et al., 2017) and murine leukemia virus (sherer et al., 2010) . receptors on tight junctions that specifically recognize hepatitis c virus (carloni et al., 2012; ploss et al., 2009 ) and reovirus (barton et al., 2001) have been identified. some viruses are able to modify cellular pathways to reprogram both the synthesis and metabolism of lipids and membrane compartmentalization for their transmission and to evade cellular defense mechanisms (mazzon and mercer, 2014) . despite a general understanding that poliovirus spreads through cellular lysis, it was recently found that it may also be transferred between cells by an autophagy-dependent mechanism, called autophagosome-mediated exit without lysis (bird and kirkegaard, 2015b; bird et al., 2014; lai et al., 2016; richards and jackson, 2012) . similar mechanisms have been described for varicellazoster virus and human cytomegalovirus (grose et al., 2016; meier and grose, 2017) . poxviruses encode several proteins that block the apoptotic cellular response to the presence of their dsdna in the cytoplasm. this allows cell-to-cell passage of poxviruses by a mechanism known as apoptotic mimicry (amara and mercer, 2015; nichols et al., 2017) . in this process, enveloped viruses expose surface phosphatidylserine to mimic apoptotic bodies. these cells are then macropinocytosed by either dendritic cells or macrophages (mercer and helenius, 2010) . among the plethora of compounds designed to inhibit infectious viruses, only a few (< 100) have been approved for clinical use (de clercq, 2004; de clercq and li, 2016) . nevertheless, some effective antiviral drugs have been on the market for several decades, such as the anti-influenza a virus drug amantadine, marketed under the trade name symmetrel (by dupont), which was approved in 1966. in 1982, burroughs-wellcome introduced acyclovir as an inhibitor of herpesviruses. its remarkable specificity is connected with its activation by viral thymidine kinase-catalyzed phosphorylation. however, due to development of drug resistance in a number of viruses, especially rna viruses, there is a continuous need to design and test new inhibitors, preferably targeting different steps of viral life cycles. here, we provide insights into the world of biochemical and cell-based assays that were developed to test antivirotics targeting various steps in the viral life cycle. different types of assays, including cell-cell fusion assays, cell-virus fusion assays with pseudotyped viral particles, and in vitro biochemical assays have been developed to screen inhibitors of viral entry. in enveloped viruses, entry is initiated by fusion of the viral envelope with the target cellular membrane. the entry mechanism has been well-described for hiv-1, in which it is mediated by env glycoprotein, consisting of transmembrane gp41 and surface gp120 subunits. binding of gp120 to its cellular receptor, cd4, and to one of two co-receptors, cxcr4 or ccr5, triggers a refolding of gp41 that promotes fusion of the viral and cellular membranes. the refolding involves oligomerization of the extracellular n-and c-terminal heptad repeat (hr) domains of gp41, which leads to the formation of a 6-helical bundle [reviewed in (melikyan, 2008) ]. jiang et al. established an in vitro system to quantify formation of the hiv-1 gp41 6-helical bundle (jiang et al., 1999 ). in their system, the bundle is formed by mixing equimolar concentrations of peptides derived from the n-and c-hr regions of gp41. elisa using the monoclonal antibody nc-1, which specifically recognizes and binds an epitope formed on the gp41 6-helix bundle but not the individual peptides, enables screening for compounds that prevent formation of fusion-active gp41. for hts of hiv-1 fusion inhibitors, this method was modified to use a fluorescence-linked immunosorbent assay (flisa), in which the c-hr peptide was replaced with fitc-conjugated c-hr peptide (boyer-chatenet et al., 2003) . cell-based assays are routinely used to screen viral entry inhibitors. high throughput formats have been developed for screening of hiv-1 fusion inhibitors. cell-cell fusion assays involve two types of cells: effector cells that stably (bradley et al., 2004) or inducibly (herschhorn et al., 2011; ji et al., 2006) express hiv-env glycoprotein and target cells that express cd4 and either cxcr4 or ccr5. co-cultivation of these cells leads to hiv-1 env-mediated cell membrane fusion, resulting in formation of multinucleated syncytia. membrane fusion induces expression of a reporter protein such as luciferase (herschhorn et al., 2011; ji et al., 2006) or β-galactosidase (bradley et al., 2004) . one such assay enables determination of both the efficiency and specificity of fusion inhibitors (herschhorn et al., 2011) . this approach uses effector cells that express both hiv-1 env and the renilla luciferase (r-luc) reporter protein using inducible tetracycline-controlled transactivator (tta) and target cells that express the hiv-1 receptor (cd4) and coreceptor (ccr5) and contain the firefly luciferase (f-luc) reporter gene under the control of a tta-responsive promoter. upon fusion of the effector and target cells, tta enters the target cells and activates the expression of the f-luc reporter. the inhibition of fusion of cellular membranes is determined as a decrease in f-luc luminescence, and the inhibitor specificity is measured as the r-luc activity (herschhorn et al., 2011) . based on an hiv-1 cell-cell fusion method that uses a computercontrolled digital image analysis system for automatic quantitation , a modified method was developed to screen inhibitors targeting mers-cov s protein (lu et al., 2014) . to test potential fusion inhibitors, effector cells stably expressing the mers-cov spike protein s2s and egfp were used to mediate fusion with target cells expressing the dpp4 receptor (lu et al., 2014) . virion-based fusion assays are another category of cell-based fusion assays. one such approach is based on production of chimeric hiv-1 virions carrying β-lactamase-vpr chimeric protein (blam-vpr). chimeric hiv released into the cell culture media is isolated by ultracentrifugation and used to infect target cells. entry of the virions into the cytoplasm is detected by cleavage of a fluorescent substrate by βlactamase. the fluorescence shift corresponds to the fusion efficacy and is measurable by fluorescence microscopy, flow cytometry, or fluorometric plate reader (cavrois et al., 2002; cavrois et al., 2004 cavrois et al., , 2014 . modification of this assay by constructing pseudotyped hiv-1 virions in which hiv-1 env was replaced with ebola virus glycoprotein (gp) has also been described (yonezawa et al., 2005) . tscherne et al. developed an approach to monitor viral entry using the blam reporter (tscherne et al., 2010) . their assay uses influenza virus-like particles (vlps) bearing the influenza membrane proteins hemagglutinin and neuraminidase. blam tagged with influenza matrix protein (m1) is incorporated into the vlps and delivered into target cells. upon release, blam can be detected by flow cytometry, microscopically, or fluorometrically. a rapid cell-based hts method was developed to assess sars-cov entry inhibitors (zhou et al., 2011) . this dual envelope pseudovirion (dep) assay employs two hiv pseudoviruses: one encodes an envelope protein from the target virus and firefly luciferase and the second encodes a control, unrelated viral envelope protein and renilla luciferase. reporter expression is determined with the dual-glo luciferase assay system (promega). inclusion of the unrelated envelope protein greatly reduced false positive hits (zhou et al., 2011) . the method was further used to screen compounds that inhibit entry of filoviruses, including ebola virus . a similar approach employing four pseudotyped hiv viruses, carrying marburg virus glycoprotein, hemagglutinin and neuraminidase isolated from a/goose/qinghai/59/05 (h5n1) influenza virus, ebolavirus zaire envelope glycoprotein, and lassa virus envelope glycoprotein, has been used for entry inhibitor screening . this screening identified multiple compounds with potent inhibitory activity against entry of both marburg and ebola viruses in human cancer cell lines, and confirmed their anti-ebola activity in human primary cells . other pseudotyped viral assays have been used to screen entry inhibitors of sars-cov, ebola, hendra, and nipah viruses. to establish infection, the glycoproteins of these viruses must be processed by the host intracellular lysosomal protease cathepsin l (catl). hts resulted in identification of several inhibitors that block the cleavage of viral glycoprotein but not catl itself (elshabrawy et al., 2014) . until recently, the development of anti-hbv therapeutics had been limited by the lack of an in vitro infection system. several aspects of the hbv life cycle have been elucidated using in vitro production of hbv particles after transfection of human hepatoma cell lines (hepg2) with recombinant hbv dna and by establishment of hepatoma cell lines with the entire hbv genome integrated, such as the hepg2.2.15 cell line (ladner et al., 1997; sells et al., 1987; sureau et al., 1986) . in addition to differentiated human (phhs) (gripon et al., 1988) and tupaia belangeri (glebe et al., 2003) hepatocytes, the heparg cell line, which exhibits hepatocyte-like characteristics, also supports hbv replication (gripon et al., 2002) . the identification of sodium taurocholate cotransporting polypeptide (ntcp) as an hbv receptor by yan et al. opened possibilities to use ntcp-complemented hepg2 cells not only for studies of hbv replication mechanisms but also for hts of inhibitors (yan et al., 2012) . in an infection competition assay, hbv particles were isolated and used to infect heparg and phhs cells that had been pre-incubated with hbv envelope protein-derived peptides to test their potential activity to block hbv infection. twelve days after infection, viral rnas were quantified by northern blot (gripon et al., 2002 (gripon et al., , 2005 . using this assay, researchers identified a peptide that specifically prevents hbv and hdv entry into heparg and phhs cell lines (gripon et al., 2005) , primary cultures of tupaia hepatocytes (glebe et al., 2005) , and cells in vivo (lütgehetmann et al., 2012; petersen et al., 2008; volz et al., 2013) . recently, a phase iia clinical trial of a first-in-class entry inhibitor (myrcludex-b) that functions as an ntcp inhibitor was promisingly completed (bogomolov et al., 2016) . development of cell culture systems producing hcv pseudoparticles (hcvpp) and infectious hcv particles (hcvcc) has shed light on hcv interactions and enabled discovery of antiviral drugs and vaccines (colpitts et al., 2016) . hcvpp consist of hcv e1 and e2 glycoproteins enveloping a retroviral particle that packages gfp mrna during assembly (bartosch et al., 2003) . the entry efficiency of hcvpp can be quantified by facs analysis as the number of gfp-positive cells. use of this screening system led to discovery of a triazine derivative that blocks the entry of hcv (baldick et al., 2010) . production of hcvcc is a robust system to generate infectious hcv in naïve cells (kato et al., 2006; zhong et al., 2005) . the anti-hcv activity of hundreds of compounds approved for a wide variety of indications was determined immunochemically with anti-hcv e2 antibody in 96-well plate format. over thirty compounds displayed anti-hcv activities, most of which were directed against hcv entry (gastaminza et al., 2010) . the majority of viruses enter cells by endocytosis. unfortunately, there are no suitable experimental techniques for endosome handling, making it difficult to study early steps in the viral life cycle such as uncoating and capsid disassembly. viruses that enter cells by direct membrane fusion, such as hiv-1, are an exception. there are several methods available to monitor and quantify hiv uncoating. as some of these were reviewed recently by campbell and hope (campbell and hope, 2015) , we will discuss them very briefly. two main techniques are used in these assays: ultracentrifugation or utilization of hiv-1 specific cellular restriction factors. the "in vitro core-stability assay" is based on ultracentrifugation of released hiv-1 virions through a detergent layer, where the viral membrane dissolves, into a sucrose gradient, where the viral cores are concentrated (shah and aiken, 2011) . the "fate of capsid" assay uses ultracentrifugation through a sucrose cushion to separate the hiv-1 core from a whole cell lysate prepared shortly after infection (stremlau et al., 2006) . the "csa-washout assay" involves specific cellular factors (trim-cyclophilin) that restrict hiv-1 infection by binding to the capsid core and cyclosporine a (csa), which can effectively turn off restriction (hulme et al., 2011) . recently, a novel entry/uncoating assay (eurt), an alternative to blam-vpr (described in section 3.1), was reported (da silva santos et al., 2016). it quantifies the protein product of a virion-packaged mrna reporter upon uncoating. a method to monitor the uncoating/disassembly of the capsid of influenza a virus, which enters the cell by endocytosis, also is based on ultracentrifugation (stauffer et al., 2016) . purified virions are separated using velocity gradient centrifugation through a two-layer glycerol gradient. similar to the "in vitro core stability" assay for hiv, the sedimenting viruses migrate through the detergent-containing layer of the gradient, which dissolves the membrane to release the viral core. moreover, modification of the detergent-containing glycerol layer-for example, by lowering ph, changing ionic strength, or adding putative viral uncoating factors-enables study of the factors or compounds that affect viral uncoating in vitro. depending on the virus type, either dna or rna polymerases replicate the viral genome. thus, these enzymes play a key role in viral life cycles. reverse transcriptase, an rna-dependent dna polymerase of retro-and hepadnaviruses, is unique among nucleic acid polymerases. despite the different mechanisms of viral replication, polymerases, which are essential for all viruses, are excellent targets for antiviral therapies. polymerase inhibitors represent the vast majority of clinically approved antiviral drugs, followed by protease inhibitors, immunostimulators, entry inhibitors, and integrase inhibitors [reviewed in (de clercq and li, 2016) ]. polymerases continue to be a preferred target for newly designed inhibitors (clercq, 2008; de clercq and li, 2016) . polymerase inhibitors may be nucleoside and nucleotide analogs, pyrophosphate analogs, or nonnucleosides, such as allosteric inhibitors (de clercq and li, 2016; öberg, 2006) . non-specific approaches such as plaque assays were initially used to monitor the effectiveness of polymerase inhibitors (tino et al., 1993; tisdale et al., 1993) . the activity of these inhibitors also can be evaluated based on their ability to prevent the cytopathic effects of the virus (zhang et al., 2017) . more straightforward assays involve measurement of incorporated radio-labeled nucleotides, which directly reflects the polymerase activity (coates et al., 1992; hirashima et al., 2006; joyce, 2010) ; these include hts methods using homopolymeric polycytidylic acid and [ 3 h]-gtp (amraiz et al., 2016) . alternatively, the pyrophosphate released during the polymerase reaction can be measured luminometrically by combining the primer extension with the commercial piper assay (malvezzi et al., 2015) . the pyrophosphate anion also can be detected with dna-attached magnetic nanoparticles (tong et al., 2015) or quantum dots (chai et al., 2015) . frequently used fluorescence methods avoid the use of radiochemicals. these methods exploit a fluorescent label, such as dsdna binding dyes like sybr green or pi-cogreen (driscoll et al., 2014; holden et al., 2009; zipper et al., 2004) , or fret between two nucleotides (schwartz and quake, 2009; weiss, 2000) . numerous kits for quantification of products of both dna and rna polymerases, including reverse transcriptase, are commercially available (bustin, 2000) . quantitative real-time pcr is a preferred method to monitor the activity of dna polymerases (cellular as well as viral) in the presence of inhibitors (beadle et al., 2016; zweitzig et al., 2012) , and quantitative real-time reverse-transcription pcr has become standard for screening inhibitors of viral rna polymerases okon et al., 2017; pelliccia et al., 2017; zhang et al., 2017) . white et al. described a hts method using a microfluidic apparatus combined with digital pcr for single-cell analysis (white et al., 2013) . in their method, a fluorescently labeled template also serves as a primer, due to stem formation. it emits a measurable polarization signal both upon binding of the polymerase and extension (mestas et al., 2007) . the assay has been used for hts of inhibitors of poliovirus rna polymerase (campagnola et al., 2011) . when available, viral genomes modified with reporter genes can be used for cell-based luminescent or fluorescent screening of viral inhibitors (beadle et al., 2016; edwards et al., 2015; fenaux et al., 2016; feng et al., 2014; lo et al., 2017; madhvi et al., 2017; wang et al., 2015c) . this approach can be extended to screen inhibitors of other enzymes. in the aptamer-based approach, a dna template encodes rna of interest joined to a fluorescence module and ribozyme sequence. when transcribed, the fluorescence module is released and detected (höfer et al., 2013) . hcv uses an rna-dependent rna polymerase (rdrp). a unique cell-based assay for monitoring hcv rna polymerase (ns5b) activity, based on the innate immune signaling molecule retionic acid-inducible gene i product (rig-i), has been developed (ranjith-kumar et al., 2011) . the rig-i-like receptors are cytosolic proteins that recognize viral rna and induce production of proinflammatory cytokines and interferon-activated genes (jensen and thomsen, 2012) . rig-i triggers cytokine production stimulated by various viral rnas from different families, including flaviviridae, paramyxoviridae, rhabdoviridae, orthomyxoviridae, and arenaviridae, as well as ebola virus rna (jensen and thomsen, 2012) . the assay is based on recognition of hcv rna produced by active ns5b by rig-i, followed by rig-i-mediated activation of firefly luciferase expression controlled by the interferon b promoter. renilla luciferase expression is used for normalization of transfection efficacy. the viral grnas of some rna viruses are modified at the 5′ ends with cap structures, which may be either acquired from the host cell mrna (e.g. in influenza virus) or newly synthesized (e.g. flaviviruses). the methylation of viral grna mimics that of cellular mrna cap structures to enhance the chances of the virus to escape from cellular defense mechanisms and also to increase the efficiency of translation of viral proteins. virus-specific methyltransferases are thus a promising therapeutic target. virus-encoded methyltransferases have been identified and characterized in flaviviruses such as zika virus coutard et al., 2017; duan et al., 2017; munjal et al., 2017; zhao et al., 2017) , west nile virus, and dengue virus (dong et al., 2012) ; rhabdoviruses such as vesicular stomatitis virus (vsv) (rahmeh et al., 2009 ); coronaviruses such as sars (wang et al., 2015b) ; and roniviruses (zeng et al., 2016) . in flaviviruses, the n-terminal part of ns5 methyltransferase catalyzes cap formation via both guanine n-7 and ribose 2′-oh methylations at the 5′ end of grna, and the c-terminus of the enzyme is responsible for the rna polymerase activity (ray et al., 2006; zhao et al., 2015) . the c-terminal part of the sars nsp14 protein exhibits guanine n7-methyltransferase activity, forming the grna cap, while the 3′-5′ exoribonuclease activity of the n-terminus enhances the fidelity of viral replication (case et al., 2016; minskaia et al., 2006) . in vsv, the methyltransferase activity resides in the conserved region vi of the multifunctional large polymerase protein (li et al., 2005; ma et al., 2014) . some cellular methyltransferases regulate viral infections, as shown for herpes simplex virus, for which epigenetic control is involved in viral latency (cliffe and wilson, 2017) . inhibition of human histone methyltransferases such as histone-lysine n-methyltransferase (ezh2/ 1) (arbuckle et al., 2017) or lysine-specific demethylase-1 (lsd1) induces antiviral signaling pathways (liang et al., 2009) . the inhibitory effect of histone demethylase activity has been demonstrated for human cytomegalovirus (gan et al., 2017) and herpes simplex virus (hill et al., 2014; liang et al., 2013) . the activity of purified recombinant methyltransferases can be determined by measuring the methylation by-product s-adenosyl homocysteine (sah) using commercially available kits. in one such assay, conversion of s-adenosyl methionine (sam) to sah is monitored luminometrically via luciferase reaction, in which measurable atp is generated through a sequence of reactions with mtase-glo™ reagent (promega). the epigeneous™ methyltransferase kit (cisbio bioassays) is based on competition of produced sah with fluorescently labeled sah (sah-d2 tracer) for binding to a terbium cryptate-labeled anti-sah antibody. the decrease in fret between the tracer and antibody is then evaluated. elisa using anti-5-methylcytosine antibody can be used to quantify methylation of immobilized cytosine-rich dna substrate (e.g. epiquik™ dna methyltransferase activity/inhibition assay kit; epigentek). this method was modified with homogenous time resolved fret (degorce et al., 2009) to screen a library of inhibitors against sars-cov nsp14 . flaviviral and human cap n7-mtases have been screened with radioactive assays using 3 h-labeled sam and gpppac4 or 7m gpppac4 synthetic rnas. the 3 h methyl transferred onto deae filter-bound rna can be measured by scintillation after multiple washings to remove unincorporated 3 h-sam . alternatively, in vitro transcription can be carried out using 3 h-sam. the radioactively labeled products are then resolved by thin-layer chromatography and developed using a phosphorimager (li et al., 2007) . a yeast cell-based method was established based on the finding that coronavirus methyltransferase can functionally replace a methyltransferase essential for yeast viability sun et al., 2014) . in this method, sun et al. constructed recombinant yeasts producing the viral methyltransferase instead of the yeast one (sun et al., 2014) . this strain was used for a hts of methyltransferase inhibitor activity that negatively correlated with the cell density after 20 h incubation. virus-encoded atpase-driven helicases have been identified in numerous human pathogens. helicases from several (+)rna viruses have been characterized, including ns3 helicases from flaviviruses such as dengue virus, hcv, west nile virus, yellow fever virus, and japanese encephalitis virus (cao et al., 2016; gu and rice, 2016; jain et al., 2016; lin et al., 2017; nedjadi et al., 2015; wu et al., 2005) . sars and mers coronaviruses encode nsp13 with helicase activity (adedeji and lazarus, 2016; hao et al., 2017; seybert et al., 2000) . in semliki forest virus, a representative member of the togavirus family, helicase activity is encoded by the n-terminal domain of nsp2 protein. helicases are also common in some dna viruses, including poxviruses. these include the vaccinia virus helicase-primase d5 (bayliss and smith, 1996; hutin et al., 2016) , e1 protein of bovine papilloma virus 1 (yang et al., 1993) , and the large tumor antigen of sv40 (stahl et al., 1986) . helicases appear to be attractive targets for antiviral drugs (briguglio et al., 2011; frick, 2003; reynolds et al., 2015) , but development of such compounds is challenged by cytotoxicity and bioavailability issues (kwong et al., 2005) . traditional methods for monitoring the activity of rna helicases use radioactively labeled dsrna substrates and follow the unwinding reaction by electrophoretic separation (nondenaturing page) of the ss reaction products, which are detected autoradiographically (adedeji et al., 2012; utama et al., 2000) . to determine whether the inhibitor affects binding of helicase to nucleic acid, a standard gel mobility shift assay is usually used (adedeji et al., 2012) . helicase activity is fueled by atp hydrolysis; thus, inhibition of atpase activity became another possible antiviral strategy. atpase activity can be determined either by the decrease in atp or formation of adp (using commercially available fluorescent anti-adp antibodies) or inorganic pyrophosphate. phosphates released by atp hydrolysis can form a molybdophosphate complex that can be measured colorimetrically using malachite green, quinaldine red, or rhodamine b (baykov et al., 1988; debruyne, 1983; miyata et al., 2010) or by lightscattering (oshima et al., 1996) . the colorimetric methods can be miniaturized for hts (zuck et al., 2005) . the absorbance-based assay was also converted into a hts method based on fluorescence quenching by a colored quinaldine red complex ). an alternative method employs a europium-tetracycline complex for luminescent determination of inorganic phosphate (schäferling and wolfbeis, 2007) . a more complex coupled enzyme colorimetric assay (with maltose phosphorylase, glucose oxidase, and horseradish peroxidase) was used for hts of atpase inhibitors (avila et al., 2006) . assays for luminescent and fluorescent screening of atpase activity, including an immunochemical method using fluorescently labeled anti-adp antibodies, have been reviewed by shadrick and colleagues (shadrick et al., 2013) . in a radioactive method using [γ-32 p]atp, the amp product was separated from unreacted atp by thin-layer chromatography on polyethyleneimine-cellulose and visualized autoradiographically (adedeji et al., 2012) . several research groups have described fluorescence assays to identify inhibitors targeting sars-cov helicase (nsp13) (adedeji et al., 2012; cho et al., 2015; özeş et al., 2014) . the substrate is usually a dsdna oligonucleotide consisting of a fluorescently labeled strand annealed to the complementary strand carrying a quencher. this approach was also adapted for real-time determination of the rna helicase activity of hcv (tani et al., 2010) . in this assay, an ssdna capture strand complementary to the strand carrying the quencher was used to prevent reannealing of the unwound duplex. recently, a colorimetric assay for monitoring helicase activity using dna-conjugated gold nanoparticles was developed (deka et al., 2017) . this method is based on shifts in the optical properties of nanoparticles due to dna unwinding and allows simple screening of inhibitor activity. the dsdna melting curves can be determined spectrophotometrically (at 524 nm and 260 nm) or even by the naked eye. another fluorescence hts of dengue virus ns3 helicase inhibitors measures the unwinding of a double-labeled molecular beacon (basavannacharya and vasudevan, 2014) . other approaches involve graphene oxide-based fluorescence monitoring of viral helicase activity [reviewed in (jang et al., 2013) ]. a gquadruplex-based method for label-free determination of hcv helicase ns3 activity measures changes in the luminescence of transition metal complexes with dna upon helicase-mediated quadruplex melting (leung et al., 2015) . both protein tyrosine kinases and protein serine/threonine (s/t) kinases have been found in viruses. tyrosine kinase function is wellunderstood in connection with oncogenic retroviruses, [for review on retroviral oncogenes, see (vogt, 2012) ]. in contrast to tyrosine kinases from oncogenic viruses, such as rous sarcoma virus src tyrosine kinase, viral s/t kinases share little homology with cellular enzymes. they are exclusively encoded by large dna viruses (e.g. herpesviruses), in which they play important roles in viral virulence, helping the virus to escape defense mechanisms such as those regulated by cytokine signaling pathways (jacob et al., 2011; sato et al., 2017) . their autophosphorylation and transphosphorylation activities mimic those of cellular cyclin-dependent kinases such as cdc2. for example, viral kinases can phosphorylate translation elongation factor 1 delta (ef-1δ) (jacob et al., 2011; kawaguchi and kato, 2003) . all herpesviruses encode the s/t protein kinase ul13, and us3 s/t kinases have been described in the alphaherpesvirus subfamily (kato, 2016; kawaguchi and kato, 2003) . in addition to protein kinases, hsv encodes a thymidine kinase. unlike cellular thymidine kinase, hsv thymidine kinase has a wide substrate specificity that includes pyrimidine and purine phosphonate analogs (e.g. acyclovir, ganciclovir, penciclovir) (de clercq and li, 2016) . in the body, ganciclovir is phosphorylated by cellular kinases and penciclovir and acyclovir by virus-encoded thymidine kinases to the active nucleoside triphosphate forms of the drugs that inhibit viral dna synthesis (de clercq and li, 2016; kokoris and black, 2002) . some cellular protein kinases appear to support viral replication. for example, polo-like kinases induce early stages in the influenza virus life cycle (pohl et al., 2017) , and human protein kinase c regulates the assembly of the ribonucleoprotein complexes in influenza virus (mondal et al., 2017) . inhibitors of abelson tyrosine-protein kinase 2 are active against sars and mers coronaviruses (coleman et al., 2016) . several in vitro approaches can be used to determine kinase activity as well as the activity of kinase inhibitors. analyses of the cellular phosphoproteome, sometimes accompanied by phosphopeptide enrichment, have become standard to determine kinase activity (lea and simeonov, 2011; meyer et al., 2017; vyse et al., 2017) . these assays can be used to assess the impact of inhibitors on the overall phosphoproteome in mammalian cells. although these methods provide complex information about the overall array of kinases and phosphatases in the cell (olsen et al., 2006) , they are not applicable to screening inhibitors of a single viral kinase. for these purposes, in vitro assays with recombinant kinases have been developed [for review see ], including some hts fluorescence methods (zegzouti et al., 2009 ) that can replace radioactive techniques using [γ 32 p]atp (sanghera et al., 2009) . mass spectrometric analysis also can be used to identify in vitro kinase inhibitors. for these analyses, synthetic peptides, proteins, or phosphatase-and heat-treated tissue samples (to dephosphorylate the proteins and inactivate all enzymes, respectively) are subjected to kinase treatment in the presence of inhibitors (huang et al., 2007; meyer et al., 2017; xue et al., 2012) . recombinant kinase activity in the presence of inhibitors can be quantified as atp consumption or adp production in the phosphorylation reaction by numerous commercial kits. other methods monitor binding of inhibitors to phage-displayed kinases in ligand competition assays (fabian et al., 2005) . fluorescence methods including fret, fluorescence polarization or intensity endpoint measurement, and lifetime imaging of fluorescence including fluorescence biosensors (zhang and allen, 2007) have been reviewed elsewhere. sulfonamido-oxine labeled peptides can be used as chromophores that bind mg 2 + upon phosphorylation and emit chelation-enhanced fluorescence (devkota et al., 2013; luković et al., 2008) . kinase-catalyzed phosphorylation of fluorescent peptides promotes their binding to metal-coated nanoparticles, which decreases their mobility and enhances measurable fluorescence polarization (lea and simeonov, 2011; sportsman et al., 2004) . tbiii complexes, in which phosphotyrosine induces fluorescence emission (wang et al., 2015a) , may be used to evaluate protein tyrosine kinase activity (akiba et al., 2015; sumaoka et al., 2016) . fluorescence polarization methods also can be useful for drug screening [reviewed in (hall et al., 2016) ]. other alternatives are immunochemical methods that use antibodies specific to the phosphorylated amino acids, such as phosphotyrosine (li et al., 2001; youngren et al., 1997) or phosphoserine/ phosphothreonine. these antibodies can be used to detect protein/ peptide phosphorylation by western blotting, elisa, or immunoprecipitation of phosphorylated proteins for further mass spectrometry-based analysis (grønborg et al., 2002; zhang et al., 2002) . an elegant approach that limits the false-positive hits in screening of specific kinase inhibitors is based on an in situ proximity ligation assay using both an antibody against the target protein and an anti-phosphotyrosine antibody (leuchowius et al., 2010) . both antibodies are coupled with oligonucleotides, which when brought together due to antibody binding, can be enzymatically ligated and replicated through rolling circle amplification to form a long linear tandem repeat of sequences detectable by a complementary fluorescent oligonucleotide. in addition to protein kinases, some lipid kinases have been targeted by antivirals. one example is sphingosine kinase 1, which affects replication of dengue virus (aloia et al., 2017) . its activity can be determined by measuring the production of 32 p-labeled sphingosine-1phosphate from sphingosine and [γ 32 p]atp (clarke et al., 2016; pitman et al., 2012) . retroviral integrase inhibitors are a new type approved new type of inhibitors imposed by the emergence of drug-resistant mutants. hiv integrase activities, integrase inhibitors, and drug resistance have been discussed in detail elsewhere (andrake and skalka, 2015; anstett et al., 2017; hajimahdi and zarghi, 2016; liao et al., 2010; podany et al., 2017; thierry et al., 2016) . methods to assess the two major activities of integrase-end processing of the reverse transcription product and its joining to target chromosomal dna-have been reviewed in detail by several groups (engelman and cherepanov, 2014; marchand et al., 2001; merkel et al., 2009) . initial methods used radioactively labeled dna oligonucleotides comprising the terminal cis-acting sequences of linear viral dna required for integration. the joining of the processed strand to the other strand (self-integration) or to supplemented target dnas can be analyzed by page (katz et al., 1990; katzman et al., 1989) . a less time-consuming, non-radioactive method involves timeresolved fluorescence anisotropy measurement using a 21-meric oligonucleotide fluorescently labeled on the terminal gt dinucleotide. this assay monitors the binding of integrase to the substrate as well as the subsequent 3′-processing reaction, which both change the anisotropy (guiot et al., 2006) . alternatively, the yields of both the processing and joining reactions can be measured upon separation of the radioactively labeled product from the rest of the dna molecule using adsorption to pei-cellulose (muller et al., 1993) . a real-time hts method measures fluorescence emission resulting from removal of the 3′-terminal dinucleotide, labeled with a quencher, by integrase (he et al., 2007) . han et al. described a fluorescence method to screen molecules that inhibit binding of integrase to viral dna . methods evaluating the integrase strand transfer reaction have been modified to a high-throughput format using magnetic beads (he et al., 2008) or streptavidin-coated microplates (john et al., 2005) . a method to assess strand transfer by time-resolved fret with a europium-streptavidin-labeled substrate has been optimized for 384-and 1536-well plate formats (wang et al., 2005) . the hbv capsid protein is the building block of the viral core, surrounding the viral nucleic acid (pre-genomic rna, pgrna) and reverse transcriptase. the hbv core is icosahedral, formed by 240 copies of capsid protein dimers. in vitro hts of hbv core assembly inhibitors using a modified hbv capsid protein has been described (stray et al., 2006) . the capsid protein was modified by deleting the nucleic acid binding domain, which is dispensable for capsid assembly, and the nterminal assembly domain alone was used in the assay. to fluorescently label the hbv capsid protein, all cysteine residues dispensable for assembly were replaced with alanines. a unique cysteine residue (c150) was c-terminally joined to the assembly domain and labeled with fluorescent bodipy-fl dye (c150bo). during assembly, capsid proteins dimerize, bringing c150bo residues close together and resulting in c150bo fluorescence self-quenching. following incubation of the labeled hbv protein with inhibitors, fluorescence was measured in black 96-well microtiter plates. development of in vitro assembly systems has contributed greatly to current understanding of the structure of retroviral particles and mechanisms of virion formation. these systems also became the base for several high throughput assays for screening assembly inhibitors. during the last 20 years, a number of in vitro assembly assays have been established, mainly for hiv-1 (campbell et al., 2001; campbell and rein, 1999; ehrlich et al., 1992; gross et al., 2000; lanman et al., 2002) , mason-pfizer monkey virus (bohmova et al., 2010; klikova et al., 1995; rumlova-klikova et al., 2000; rumlova-klikova et al., 1999; ulbrich et al., 2006) , rous sarcoma virus vogt, 1995, 1997; purdy et al., 2008 purdy et al., , 2009 , and murine leukemia virus (dolezal et al., 2016; hadravova et al., 2012; cheslock et al., 2003) . hts assays for inhibitors of hiv-1 assembly include several methods using purified hiv-1 ca or ca-nc proteins. one of them, the turbidimetric assay, is based on the observation that direct dilution of the hiv-1 capsid protein (ca) into a high-salt solution (1.6-2.2 m nacl) leads to the formation of tubular structures. as the tube formation is accompanied by an increase in light scattering, assembly can be detected as an increase in turbidity, and the rate of turbidity change is proportional to the rate of the assembly (lanman et al., 2002) . other method published by lemke et al., exploits the affinity of nucleocapsid (nc) to a short tg-rich deoxyribooligonucleotide, d(tg25), which is used as a scaffold (lemke et al., 2012) . this arrangement enables ca to assemble at much lower protein and salt concentrations than in the turbidimetric assay (lanman et al., 2002) . biotin-labeled d(tg25) bound on the surface of neutravidin-coated microtiter well plates nucleates assembly of complexes of ca-nc and soluble fluorescein-labeled d(tg25). fluorescence is measured after washing to remove the unbound and unassembled material from the captured assembly products (lemke et al., 2012) . similarly, the faith assay uses a dually labeled oligonucleotide (tqon). however, in this case, the ssdna oligonucleotide tqon is labeled with the reporter dye fluorescein (fam) as well as black hole quencher (bhq); thus, it does not emit any fluorescence. the assembly reaction is triggered by mixing hiv-1 ca-nc or a gagtruncated assembly-competent version with tqon. following incubation, during which tqon is incorporated into the particles, exonuclease is added to degrade unbound tqon, while co-assembled tqon is protected from cleavage. degradation of free unbound tqon with exoi results in separation of fam from its quencher, and the emitted fluorescence is measured (hadravova et al., 2015) . phage display has been employed to screen peptide inhibitors of hiv-1 assembly (sticht et al., 2005) . a commercial library of m13-derived phages presenting random 12-amino-acid peptides was analyzed for specific binding to purified ca or ca-nc proteins. the specifically bound phages were sequenced, and corresponding peptides were chemically synthesized and re-tested in an in vitro assembly assay (gross et al., 2000) . late in the retroviral life cycle, grna is incorporated into the nascent particle during assembly at the plasma membrane. nc contains two zinc-finger domains that are responsible for specific binding of grna. to screen for compounds that would prevent nc-rna/dna interactions, a hts system consisting of two sequential screens was developed (breuer et al., 2012) . the primary screen uses fluorescence polarization (fp), while the secondary one uses differential scanning fluorimetry (dsf). the combination and order of these two techniques were selected to first identify compounds that disrupt interactions between dna and nc, and then identify the compounds binding to nc during the secondary screen. a rapid and simple turbidimetric method was developed to screen inhibitors of assembly of hcv core protein (fromentin et al., 2007) . for the in vitro assembly reaction, two components, an n-terminal part of the hcv core protein corresponding to the minimal assembly competent domain and rna corresponding to the full-length 5′utr of hcv, were used. assembly of hcv nucleocapsid-like particles was initiated by mixing the purified protein and nucleic acid, and the assembly process was monitored by measuring turbidity at 350 nm. numerous viruses, including picornaviruses, retroviruses, alphaviruses, and flaviviruses, encode proteases that are essential for their virulence. the majority of viral proteases specifically cleave viral polyprotein precursors to liberate the functional proteins of the virion. some viral proteases, such as the papain-like proteases of coronaviruses, also reprogram cellular signaling pathways, including ubiquitination mechanisms and interferon controlled responses, to prevent degradation of viral components (clementz et al., 2010; frieman et al., 2009; randow and lehner, 2009; xing et al., 2013) . numerous viruses, including papillomaviruses (bronnimann et al., 2016; buck et al., 2005) and retroviruses (hallenberger et al., 1992) , use also host cell proteases, mainly furin, to trim their envelope and surface proteins. this triggers conformational changes required for interaction with cellular receptors and membrane fusion in enveloped viruses. some viruses use proteases other than furin to modulate their infectivity, as shown for viruses entering airway epithelial cells [reviewed in (laporte and naesens, 2017) ] such as influenza virus (böttcher-friebertshäuser et al., 2010; kühn et al., 2016) , newcastle disease virus (gotoh et al., 1992) , and respiratory syncytial virus (sugrue et al., 2001) . extensive research efforts have yielded detailed information about hiv-1 protease and its inhibitors [reviewed in (de clercq, 2004; konvalinka et al., 2015; midde et al., 2016) ]. large amounts of data also are available for hcv (foote et al., 2011; pawlotsky et al., 2007; razonable, 2011) and sars-cov 3cl protease inhibitors (pillaiyar et al., 2016) , although there is not yet an inhibitor of the latter target approved for clinical use. numerous approved drugs are synthetic peptides derived from the natural proteolytic substrates of target viruses modified to improve the in vivo effects related to bioavailability, stability, and so on. numerous in vitro assays to monitor the activity of proteases and their inhibitors, including commercial kits, have been developed. classical methods use synthetic peptides that mimic the target sites of the protease. although some of the methods described here were not originally designed to screen the activity of viral proteases in the presence of inhibitors, they can be adapted for this purpose by simply changing the peptide sequence to the target site of the protease of interest. the cleavage yield is usually monitored either colorimetrically (ding and yang, 2015; zhou et al., 2014) or as a change in fluorescence triggered by the release of fluorescent labels such as 7-amido-4-methylcoumarin (amc) or rhodamine (grant et al., 2002) . fluorogenic substrates have been used to determine the activity of coronavirus proteases and screen inhibitors (kuo et al., 2004; lee et al., 2014; park et al., 2017; song et al., 2014; tomar et al., 2015; wang et al., 2016; yang et al., 2005; zhao et al., 2008) . some recently described arrangements employ nanoparticles (feltrup and singh, 2012; khalilzadeh et al., 2016; udukala et al., 2016; wang et al., 2014; zeng et al., 2015) or quantum dot bioconjugates (lee and kim, 2015; li et al., 2014; medintz et al., 2006) with immobilized fluorescently or luminescently labeled peptide substrates. alternatively, cleavage products may be monitored by analysis of proteolytic products by mass spectrometric methods (hu et al., 2015; joshi et al., 2017; lathia et al., 2011; rumlová et al., 2003) , analytical hplc (teruya et al., 2016) , or electrochemical methods based on the difference in penetration of substrate and cleavage products through the membrane of a polyionselective sensor (gemene and meyerhoff, 2011; han et al., 1996) . to study the specificity of inhibitor binding and to extend the research to rational design of inhibitors, x-ray or nmr structures of proteases in complex with the inhibitor may be determined, as reported in numerous cases for the proteases of hiv-1 [reviewed in (ghosh et al., 2016) ], hcv (yilmaz et al., 2016) , and mers . cell-based assays can provide additional information, including the capability of the inhibitor to pass through the cell membrane and its stability in the cytoplasm. a general determination of infectivity, such as a plaque cytotoxicity assay in the presence of protease inhibitor, may be used for confirmation. one elegant example exploits the cytotoxicity of hiv protease. cells are transfected with a protease precursor fused to gfp. in the absence of inhibitor, hiv-1 protease is autocatalytically activated and cleaves a broad variety of cellular proteins, resulting in activation of apoptosis and cell death (cummins and badley, 2010; rumlova et al., 2014) . this toxic effect, when suppressed by active inhibitors, results in production of a gfp signal in surviving cells (lindsten et al., 2001) . another elegant approach for hiv and coxsackievirus b3 proteases, which both undergo autocatalytic cleavage, employs constructs in which the protease gene is inserted between sequences encoding the dna-binding domain and the domain that activates transcription of the gal1-lacz reporter gene (dasmahapatra et al., 1992; murray et al., 1993) . the protease-mediated cleavage separates the dna-binding domain from the trans-activating domain and results in failure of reporter gene transcription. this approach has also been modified with gfp as a reporter gene (hilton and wolkowicz, 2010) . co-expression of cleavage-activated luciferase substrate and mers-cov protease permits both live-cell imaging and quantification of the enzyme activity (kilianski et al., 2013) . the need to develop new antiviral compounds will likely persist over the long term, although there has been enormous progress in molecular biology methods, especially rna silencing, bioinformatics, imaging, and structural biology techniques. viruses present challenging targets for drug development due their flexibility and adaptability caused by the error-prone copying of their genomes, which can result in emergence of drug-resistant mutants. viral integration into the host genome and inhibitor toxicity are other obstacles. here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or cell-encoded proteins required for the infectivity. biochemical characterization of middle east respiratory syndrome coronavirus helicase severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase components of adenovirus genome packaging click conjugation of a binuclear terbium(iii) complex for real-time detection of tyrosine phosphorylation pancreatic acinar cell-specific autophagy disruption reduces coxsackievirus replication and pathogenesis in vivo investigation of sphingosine kinase 1 in interferon responses during dengue virus infection viral apoptotic mimicry hiv-1 uncoating: connection to nuclear entry and regulation by host proteins development of robust in vitro rnadependent rna polymerase assay as a possible platform for antiviral drug testing against dengue retroviral integrase: then and now reactivation and lytic replication of kaposi's sarcoma-associated herpesvirus: an update hiv drug resistance against strand transfer integrase inhibitors toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay inhibitors of the histone methyltransferases ezh2/1 induce a potent antiviral state and suppress infection by diverse viral pathogens highthroughput screening for hsp90 atpase inhibitors a novel small molecule inhibitor of hepatitis c virus entry junction adhesion molecule is a receptor for reovirus infectious hepatitis c virus pseudoparticles containing functional e1-e2 envelope protein complexes suramin inhibits helicase activity of ns3 protein of dengue virus in a fluorescence-based high throughput assay format a malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay vaccinia virion protein i8r has both dna and rna helicase activities: implications for vaccinia virus transcription -phosphonomethoxy)ethyl]guanine (ode-bn-pmeg), a potent inhibitor of transient hpv dna amplification hepatitis b virus replication early steps in avian reovirus morphogenesis late budding domains and host proteins in enveloped virus release escape of non-enveloped virus from intact cells nonlytic spread of naked viruses nonlytic viral spread enhanced by autophagy components viral entry pathways: the example of common cold viruses herpes virus replication treatment of chronic hepatitis d with the entry inhibitor myrcludex b: first results of a phase ib/iia study effect of dimerizing domains and basic residues on in vitro and in vivo assembly of mason-pfizer monkey virus and human immunodeficiency virus cleavage of influenza virus hemagglutinin by airway proteases tmprss2 and hat differs in subcellular localization and susceptibility to protease inhibitors development and automation of a 384-well cell fusion assay to identify inhibitors of ccr5/cd4-mediated hiv virus entry identification of hiv-1 inhibitors targeting the nucleocapsid protein inhibition of rna helicases of ssrna(+) virus belonging to flaviviridae, coronaviridae and picornaviridae families furin cleavage of l2 during papillomavirus infection: minimal dependence on cyclophilins maturation of papillomavirus capsids absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays hiv-1 virion-cell interactions: an electrostatic model of pathogenicity and syncytium formation high-throughput screening identification of poliovirus rna-dependent rna polymerase inhibitors hiv-1 capsid: the multifaceted key player in hiv-1 infection in vitro assembly properties of human immunodeficiency virus type 1 gag protein lacking the p6 domain self-assembly in-vitro of purified ca-nc proteins from rous-sarcoma virus and human-immunodeficiency-virus type-1 in vitro assembly of virus-like particles with rous sarcoma virus gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles modulation of hiv-like particle assembly in vitro by inositol phosphates novel escrt functions in cell biology: spiraling out of control? molecular mechanism of divalentmetal-induced activation of ns3 helicase and insights into zika virus inhibitor design hcv infection by cell-to-cell transmission: choice or necessity? mutagenesis of s-adenosyl-l-methionine-binding residues in coronavirus nsp14 n7-methyltransferase m demonstrates differing requirements for genome translation and resistance to innate immunity components and regulation of nuclear transport processes a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes fluorescence resonance energy transfer-based hiv-1 virion fusion assay hiv-1 fusion assay a reversible fluorescence nanoswitch based on carbon quantum dots nanoassembly for detection of pyrophosphate ion functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase tsg101: a novel anti-hiv-1 drug target protein-protein interfaces in viral capsids are structurally unique identification of a coumarin-based antihistamine as an anti-filoviral entry inhibitor charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination identification of a novel small molecule inhibitor against sars coronavirus helicase reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases emerging antiviral drugs restarting lytic gene transcription at the onset of herpes simplex virus reactivation (-)-2'-deoxy-3'-thiacytidine is a potent, highly selective inhibitor of human immunodeficiency virus type 1 and type 2 replication in vitro pushing the envelope: microinjection of minute virus of mice into xenopus oocytes causes damage to the nuclear envelope how viruses access the nucleus abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion structures of ns5 methyltransferase from zika virus highthroughput approaches to unravel hepatitis c virus-host interactions localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center endocytosis of viruses and bacteria zika virus methyltransferase: structure and functions for drug design perspectives structure and organization of paramyxovirus particles mechanisms of hiv-associated lymphocyte apoptosis: 2010 a novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized hiv-1 infection a genetic system for studying the activity of a proteolytic enzyme antiviral drugs in current clinical use approved antiviral drugs over the past 50 years inorganic phosphate determination: colorimetric assay based on the formation of a rhodamine b-phosphomolybdate complex htrf: a technology tailored for drug discovery -a review of theoretical aspects and recent applications dna-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors a novel mechanism underlying the innate immune response induction upon viral-dependent replication of host cell mrna: a mistake of + srna viruses' replicases. front high-throughput screens for eef-2 kinase quantitative serine protease assays based on formation of copper(ii)-oligopeptide complexes functional and structural characterization of novel type of linker connecting capsid and nucleocapsid protein domains in murine leukemia virus 2′-o methylation of internal adenosine by flavivirus ns5 methyltransferase a quantitative fluorescence-based steadystate assay of dna polymerase the crystal structure of zika virus ns5 reveals conserved drug targets high-throughput minigenome system for identifying small-molecule inhibitors of ebola virus replication assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay retroviral integrase structure and dna recombination mechanism a small molecule-kinase interaction map for clinical kinase inhibitors ultrastructural and biochemical basis for hepatitis c virus morphogenesis nuclear entry of dna viruses development of a fluorescence internal quenching correction factor to correct bont/a endopeptidase kinetics using snaptide antiviral nucleotide incorporation by recombinant human mitochondrial rna polymerase is predictive of increased in vivo mitochondrial toxicity risk a pathogenic picornavirus acquires an envelope by hijacking cellular membranes inhibition of hepatitis c virus replication by gs-6620, a potent c-nucleoside monophosphate prodrug transcription and replication mechanisms of bunyaviridae and arenaviridae l proteins fields virology boceprevir: a protease inhibitor for the treatment of chronic hepatitis c viral late domains hiv-1 assembly, release and maturation helicases as antiviral drug targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling a method for in vitro assembly of hepatitis c virus core protein and for screening of inhibitors beyond tsg101: the role of alix in 'escrting' hiv-1 proteolytic cleavage of bovine m adenovirus 3-encoded pviii epigenetically repressing human cytomegalovirus lytic infection and reactivation from latency in thp-1 model by targeting h3k9 and h3k27 histone demethylases unbiased probing of the entire hepatitis c virus life cycle identifies clinical compounds that target multiple aspects of the infection detection of protease activities by flash chronopotentiometry using a reversible polycation-sensitive polymeric membrane electrode recent progress in the development of hiv-1 protease inhibitors for the treatment of hiv/aids structure and formation of the cytomegalovirus virion pre-s1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis b virus mapping of the hepatitis b virus attachment site by use of infection-inhibiting pres1 lipopeptides and tupaia hepatocytes the ins and outs of hiv replication mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/pace to pc2 or pc1/ pc3 development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates hepatitis b virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide infection of a human hepatoma cell line by hepatitis b virus efficient inhibition of hepatitis b virus infection by acylated peptides derived from the large viral surface protein a mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, frigg, as a protein kinase a substrate varicella-zoster virus infectious cycle: er stress, autophagic flux, and amphisome-mediated trafficking a conformational switch controlling hiv-1 morphogenesis the cell biology of receptor-mediated virus entry the spring α-helix coordinates multiple modes of hcv (hepatitis c virus) ns3 helicase action relationship between the oligomeric status of hiv-1 integrase on dna and enzymatic activity bunyavirus: structure and replication in vitro assembly of virus-like particles of a gammaretrovirus, the murine leukemia virus xmrv faith -fast assembly inhibitor test for hiv progress in hiv-1 integrase inhibitors: a review of their chemical structure diversity fluorescence polarization assays in high-throughput screening and drug discovery: a review inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gpl60 selective monitoring of peptidase activities with synthetic polypeptide substrates and polyion-sensitive membrane electrode detection development of a fluorescence-based hiv-1 integrase dna binding assay for identification of novel hiv-1 integrase inhibitors alix rescues budding of a double ptap/ppey l-domain deletion mutant of ebola vp40: a role for alix in ebola virus egress crystal structure of middle east respiratory syndrome coronavirus helicase highthroughput real-time assay based on molecular beacons for hiv-1 integrase 3'-processing reaction a novel highthroughput format assay for hiv-1 integrase strand transfer reaction using magnetic beads nuclear egress of herpesviruses: the prototypic vesicular nucleocytoplasmic transport herpesvirus capsid assembly and dna packaging an inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of hiv-1 entry inhibitors inhibition of lsd1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes an assay to monitor hiv-1 protease activity for the identification of novel inhibitors in t-cells benzimidazole derivatives bearing substituted biphenyls as hepatitis c virus ns5b rna-dependent rna polymerase inhibitors: structure − -activity relationship studies and identification of a potent and highly selective inhibitor jtk-109 universal aptamer-based real-time monitoring of enzymatic rna synthesis factors affecting quantification of total dna by uv spectroscopy and picogreen fluorescence peptide code-on-a-microplate for protease activity analysis via maldi-tof mass spectrometric quantitation a systematic ms-based approach for identifying in vitro substrates of pka and pkg in rat uteri quantitative 3d video microscopy of hiv transfer across t cell virological synapses complementary assays reveal a relationship between hiv-1 uncoating and reverse transcription the ins and outs of eukaryotic viruses: knowledge base and ontology of a viral infection escrts are everywhere transport of the influenza virus genome from nucleus to nucleus domain organization of vaccinia virus helicase-primase d5 how viruses use the endoplasmic reticulum for entry, replication, and assembly experimental approaches to study genome packaging of influenza a viruses poliovirus-induced changes in cellular membranes throughout infection subversion of cellular autophagosomal machinery by rna viruses viral serine/threonine protein kinases structure of the ns3 helicase from zika virus a new helicase assay based on graphene oxide for anti-viral drug development filovirus budding oligomeric viral proteins: small in size, large in presence sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion development of a novel dual ccr5-dependent and cxcr4-dependent cell-cell fusion assay system with inducible gp160 expression a screening assay for antiviral compounds targeted to the hiv-1 gp41 core structure using a conformation-specific monoclonal antibody development and application of a highthroughput screening assay for hiv-1 integrase enzyme activities avian reovirus infections. revue scientifique et technique a structural and functional perspective of m. rumlová alphavirus replication and assembly the rational design of therapeutic peptides for aminopeptidase n using a substrate-based approach techniques used to study the dna polymerase reaction pathway molecular mechanism by which us3 protein kinase regulates the pathogenicity of herpes simplex virus type-1 cell culture and infection system for hepatitis c virus the avian retroviral in protein is both necessary and sufficient for integrative recombination in vitro the avian retroviral integration protein cleaves the terminal sequences of linear viral dna at the in vivo sites of integration protein kinases conserved in herpesviruses potentially share a function mimicking the cellular protein kinase cdc2 reduced graphene oxide decorated with gold nanoparticle as signal amplification element on ultra-sensitive electrochemiluminescence determination of caspase-3 activity and apoptosis using peptide based biosensor assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers efficient in-vivo and in-vitro assembly of retroviral capsids from gag precursor proteins expressed in bacteria virus strategies for passing the nuclear envelope barrier characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity retroviral proteases and their roles in virion maturation origin and evolution of eukaryotic large nucleo-cytoplasmic dna viruses adenovirus tales: from the cell surface to the nuclear pore complex the proteolytic activation of (h3n2) influenza a virus hemagglutinin is facilitated by different type ii transmembrane serine proteases characterization of sars main protease and inhibitor assay using a fluorogenic substrate viral and cellular rna helicases as antiviral targets inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication the autophagic machinery in enterovirus infection kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro airway proteases: an emerging drug target for influenza and other respiratory virus infections multiplexed protease assays using element-tagged substrates fluorescence polarization assays in small molecule screening fluorescent and bioluminescent nanoprobes for in vitro and in vivo detection of matrix metalloproteinase activity identification of novel drug scaffolds for inhibition of sars-cov 3-chymotrypsin-like protease using virtual and high-throughput screenings characterization of the activity of 2′-c-methylcytidine against dengue virus replication distinct effects of two hiv-1 capsid assembly inhibitor families that bind the same site within the n-terminal domain of the viral ca protein high content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation label-free luminescence switch-on detection of hepatitis c virus ns3 helicase activity using a g-quadruplex-selective probe †electronic supplementary information (esi) available: compound characterisation and supplementary data immunity to bovine herpesvirus 1: i. viral lifecycle and innate immunity small molecule insulin receptor activators potentiate insulin action in insulin-resistant cells amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity vesicular stomatitis viruses resistant to the methylase inhibitor sinefungin upregulate rna synthesis and reveal mutations that affect mrna cap methylation fluorescence detection techniques for protein kinase assay quantum dots based molecular beacons for in vitro and in vivo detection of mmp-2 on tumor inhibition of the histone demethylase lsd1 blocks α-herpesvirus lytic replication and reactivation from latency a novel selective lsd1/kdm1a inhibitor epigenetically blocks herpes simplex virus lytic replication and reactivation from latency authentic hiv-1 integrase inhibitors single-molecule imaging reveals the translocation and dna looping dynamics of hepatitis c virus ns3 helicase cellbased fluorescence assay for human immunodeficiency virus type 1 protease activity viral and host proteins that modulate filovirus budding rapid and automated fluorescencelinked immunosorbent assay for high-throughput screening of hiv-1 fusion inhibitors targeting gp41 gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. sci. rep. 7 a structural view of the rna-dependent rna polymerases from the flavivirus genus automatic quantitation of hiv-1 mediated cell-tocell fusion with a digital image analysis system (dias): application for rapid screening of hiv-1 fusion inhibitors structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor hiv-1 uncoating is facilitated by dynein and kinesin 1 recognition-domain focused (rdf) chemosensors: versatile and efficient reporters of protein kinase activity humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation the escrt-i subunit tsg101 controls endosome-to-cytosol release of viral rna the challenge of selecting protein kinase assays for lead discovery optimization mrna cap methylation influences pathogenesis of vesicular stomatitis virus in vivo a screen for novel hepatitis c virus rdrp inhibitor identifies a broad-spectrum antiviral compound quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors structure, function and dynamics in adenovirus maturation in vitro human immunodeficiency virus type 1 integrase assays human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes pathways of clathrin-independent endocytosis lipid interactions during virus entry and infection proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates variable effects of autophagy induction by trehalose on herpesviruses depending on conditions of infection common principles and intermediates of viral protein-mediated fusion: the hiv-1 paradigm viral polymerases virus entry by macropinocytosis apoptotic mimicry: phosphatidylserine-mediated macropinocytosis of vaccinia virus oligonucleotide-based assays for integrase activity a fluorescence polarization based screening assay for nucleic acid polymerase elongation activity multiplex substrate profiling by mass spectrometry for kinases as a method for revealing quantitative substrate motifs investigational protease inhibitors as antiretroviral therapies discovery of an rna virus 3′ → 5′ exoribonuclease that is critically involved in coronavirus rna synthesis high-throughput screen for escherichia coli heat shock protein 70 (hsp70/dnak): atpase assay in low volume by exploiting energy transfer influenza virus recruits host protein kinase c to control assembly and activity of its replication machinery poxvirus dna replication poxvirus membrane biogenesis. virology 479-480 membrane fusion during poxvirus entry rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant rous sarcoma virus integrases advances in developing therapies to combat zika virus: current knowledge and future perspectives inactivation of a yeast transactivator by the fused hiv-1 proteinase: a simple assay for inhibitors of the viral enzyme activity tackling dengue fever: current status and challenges expression strategies of ambisense viruses poxviruses utilize multiple strategies to inhibit apoptosis rational design of polymerase inhibitors as antiviral drugs herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro anchimerically activatable antiviral protides rabies virus assembly and budding the escrt machinery: new roles at new holes global, in vivo, and site-specific phosphorylation dynamics in signaling networks determination of phosphate as aggregates of ion associates by light-scattering detection and application to flow injection real-time fluorescence assays to monitor duplex unwinding and atpase activities of helicases nuclear pore complex is able to transport macromolecules with diameters of about 39 nm nipah virus c protein recruits tsg101 to promote the efficient release of virus in an escrt-dependent pathway evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors the multiple faces of caveolae herpesvirus capsid association with the nuclear pore complex and viral dna release involve the nucleoporin can/nup214 and the capsid protein pul25 the hepatitis c virus life cycle as a target for new antiviral therapies inhibition of dengue virus replication by novel inhibitors of rna-dependent rna polymerase and protease activities prevention of hepatitis b virus infection in vivo by entry inhibitors derived from the large envelope protein structural insights into rna synthesis by the influenza virus transcription-replication machine alphavirus polymerase and rna replication an overview of severe acute respiratory syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy isoform-selective assays for sphingosine kinase activity human occludin is a hepatitis c virus entry factor required for infection of mouse cells comparative clinical pharmacokinetics and pharmacodynamics of hiv-1 integrase strand transfer inhibitors late stages of the influenza a virus replication cyclea tight interplay between virus and host identification of pololike kinases as potential novel drug targets for influenza a virus principles of virus structural organization critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly retroviral capsid assembly: a role for the ca dimer in initiation nuclear import of hepatitis b virus capsids and release of the viral genome a chiral pentagonal polyhedral framework for characterizing virus capsid structures ribose 2'-o methylation of the vesicular stomatitis virus mrna cap precedes and facilitates subsequent guanine-n-7 methylation by the large polymerase protein viral avoidance and exploitation of the ubiquitin system a cell-based assay for rna synthesis by the hcv polymerase reveals new insights on mechanism of polymerase inhibitors and modulation by ns5a reverse transcription mechanically initiates hiv-1 capsid disassembly macropinocytosis is the entry mechanism of amphotropic murine leukemia virus west nile virus 5′-cap structure is formed by sequential guanine n-7 and ribose 2′-o methylations by nonstructural protein 5 antiviral drugs for viruses other than human immunodeficiency virus flavivirus structural heterogeneity: implications for cell entry melting of duplex dna in the absence of atp by ns3 helicase domain through specific interaction with a single-strand/ double-strand junction intracellular vesicle acidification promotes maturation of infectious poliovirus particles integration of murine leukemia virus dna depends on mitosis the role of electron microscopy in studying the continuum of changes in membranous structures during poliovirus infection specific in vitro cleavage of mason-pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection hiv-1 protease-induced apoptosis conditions resulting in formation of properly assembled retroviral capsids within inclusion bodies of escherichia coli analysis of mason-pfizer monkey virus gag domains required for capsid assembly in bacteria: role of the n-terminal proline residue of ca in directing particle shape cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes comparison of the luminescent adp-glo assay to a standard radiometric assay for measurement of protein kinase activity involvement of herpes simplex virus type 1 ul13 protein kinase in induction of socs genes, the negative regulators of cytokine signaling europium tetracycline as a luminescent probe for nucleoside phosphates and its application to the determination of kinase activity human herpesvirus 6b downregulates expression of activating ligands during lytic infection to escape elimination by natural killer cells single molecule measurement of the "speed limit" of dna polymerase growing functions of the escrt machinery in cell biology and viral replication production of hepatitis b virus particles in hep g2 cells transfected with cloned hepatitis b virus dna the human coronavirus 229e superfamily 1 helicase has rna and dna duplex-unwinding activities with 5′-to-3′ polarity the m-pmv cytoplasmic targeting-retention signal directs nascent gag polypeptides to a pericentriolar region of the cell discovering new medicines targeting helicases: challenges and recent progress in vitro uncoating of hiv-1 cores genome packaging of reovirus is mediated by the scaffolding property of the microtubule network directional spread of surface-associated retroviruses regulated by differential virus-cell interactions papain-like protease (plpro) inhibitory effects of cinnamic amides from < i > tribulus terrestris < /i > fruits immobilized metal ion affinity-based fluorescence polarization (imap): advances in kinase screening targeting zoonotic viruses: structure-based inhibition of the 3c-like protease from bat coronavirus hku4 -the likely reservoir host to the human coronavirus that causes middle east respiratory syndrome (mers) dna helicase activity of sv40 large tumor antigen in vitro disassembly of influenza a virus capsids by gradient centrifugation a peptide inhibitor of hiv-1 assembly in vitro cytoplasmic tails of bunyavirus gn glycoproteins-could they act as matrix protein surrogates an in vitro fluorescence screen to identify antivirals that disrupt hepatitis b virus capsid assembly specific recognition and accelerated uncoating of retroviral capsids by the trim5alpha restriction factor furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells selective sensing of tyrosine phosphorylation in peptides using terbium(iii) complexes yeast-based assays for the high-throughput screening of inhibitors of coronavirus rna cap guanine-n7-methyltransferase production of hepatitis b virus by a differentiated human hepatoma cell line after transfection with cloned circular hbv dna phosphorylation of the hiv-1 capsid by melk triggers uncoating to promote viral cdna synthesis viral and host control of cytomegalovirus maturation the a, b, cs of herpesvirus capsids real-time monitoring of rna helicase activity using fluorescence resonance energy transfer in vitro ubiquitin depletion and dominant-negative vps4 inhibit rhabdovirus budding without affecting alphavirus budding structural basis for the development of sars 3cl protease inhibitors from a peptide mimic to an aza-decaline scaffold different pathways leading to integrase inhibitors resistance synthesis and antiviral activity of novel isonucleoside analogs rapid in vitro selection of human immunodeficiency virus type 1 resistant to 3'-thiacytidine inhibitors due to a mutation in the ymdd region of reverse transcriptase ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl(pro)): implications for nsp5 regulation and the development of antivirals fluorescent sensing of pyrophosphate anion in synovial fluid based on dna-attached magnetic nanoparticles structural insights into the coupling of virion assembly and rotavirus replication an enzymatic virus-like particle assay for sensitive detection of virus entry early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection incorporation of spike and membrane glycoproteins into coronavirus virions distinct roles for nucleic acid in in vitro assembly of purified mason-pfizer monkey virus canc proteins identification and characterization of the rna helicase activity of japanese encephalitis virus ns3 protein virus maturation d-retrovirus morphogenetic switch driven by the targeting signal accessibility to tctex-1 of dynein retroviral oncogenes: a historical primer the entry inhibitor myrcludex-b efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis b virus advances in mass spectrometry based strategies to study receptor tyrosine kinases homogeneous highthroughput screening assays for hiv-1 integrase 3β-processing and strand transfer activities nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases a terbium (iii)-complex-based on-off fluorescent chemosensor for phosphate anions in aqueous solution and its application in molecular logic gates coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis establishment of a high-throughput assay to monitor influenza a virus rna transcription and replication structure of main protease from human coronavirus nl63: insights for wide spectrum anti-coronavirus drug design mathematical analysis of an hiv latent infection model including both virus-to-cell infection and cell-to-cell transmission measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy highthroughput microfluidic single-cell digital polymerase chain reaction structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus the e1 protein of bovine papilloma virus 1 is an atp-dependent dna helicase design of wide-spectrum inhibitors targeting coronavirus main proteases improving viral protease inhibitors to counter drug resistance studies of ebola virus glycoproteinmediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha decreased muscle insulin receptor kinase correlates with insulin resistance in normoglycemic pima indians adp-glo: a bioluminescent and homogeneous adp monitoring assay for kinases compact, programmable, and stable biofunctionalized upconversion nanoparticles prepared through peptide-mediated phase transfer for high-sensitive protease sensing and in vivo apoptosis imaging identification and characterization of a ribose 2′-o-methyltransferase encoded by the ronivirus branch of nidovirales fret-based biosensors for protein kinases: illuminating the kinome phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs cell-based high-throughput screening assay identifies 2′,2′-difluoro-2′-deoxycytidine gemcitabine as a potential antipoliovirus agent structure of the main protease from a global infectious human coronavirus, hcov-hku1 molecular basis for specific viral rna recognition and 2′-oribose methylation by the dengue virus nonstructural protein 5 (ns5) structure and function of the zika virus full-length ns5 protein robust hepatitis c virus infection in vitro inhibitors of sars-cov entryidentification using an internally-controlled dual envelope pseudovirion assay a new colorimetric strategy for monitoring caspase 3 activity by hrp-mimicking dnazyme-peptide conjugates protease inhibitors targeting coronavirus and filovirus entry the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles investigations on dna intercalation and surface binding by sybr green i, its structure determination and methodological implications miniaturization of absorbance assays using the fluorescent properties of white microplates characterization of a novel dna polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes this work was supported by ga čr (cz) ga17-25602s to mr, ga17-24281s to tr and the ministry of education, youth and sport of the czech republic(oppc cz.2.16/3.1.00/24503), through its "national program of sustainability i" npu lo 1601. key: cord-027860-s97hdhh6 authors: zeimet, anthony; mcbride, david r.; basilan, richard; roland, william e.; mccrary, david; hoonmo, koo title: infectious diseases date: 2020-06-22 journal: textbook of family medicine doi: 10.1016/b978-1-4377-1160-8.10016-8 sha: doc_id: 27860 cord_uid: s97hdhh6 nan infections of the upper respiratory tract accounted for more than 36 million ambulatory medical visits in 2005, according to the national ambulatory medical care survey (cherry et al., 2007) . although a large percentage of these infections are viral in origin, antibiotics are still prescribed for more than 50% of patients with acute respiratory tract infection (arti). acute bronchitis, in the arti category, is defined as a respiratory infection in which cough is the predominant symptom and there is no evidence of pneumonia. antibiotics are often prescribed despite limited evidence that they shorten the duration of acute bronchitis. with increasing incidence of antibiotic resistance, bronchitis allows physicians to practice "prescriptive restraint" and to provide supportive therapy. consider using the phrase "chest cold" to help patients understand the viral and benign nature of this infection. chronic bronchitis is one of the manifestations of chronic obstructive pulmonary disease (copd) and is defined clinically as cough and sputum production on most days for 3 months annually for 2 years. chronic bronchitis is thought to be primarily inflammatory in origin, although infection may be associated with acute exacerbations; with increased sputum production and worsening dyspnea, antibiotics have proved effective in acute episodes. however, systemic corticosteroids are the mainstay of copd exacerbation management. the patient with acute bronchitis presents with cough, often productive. patients may report clear or colored mucus in association with the presumed diagnosis of acute bronchitis. despite what many patients believe, the color of sputum, even purulent sputum, is not predictive of bacterial infection. the cough of bronchitis can last up to 4 weeks, sometimes even longer. typically, acute bronchitis is associated with other manifestations of infection, such as malaise and fever. respiratory viruses are thought to cause the majority of cases of acute bronchitis. influenza a and b, parainfluenza, respiratory syncytial virus (rsv), coronavirus, adenovirus, and rhinovirus are common pathogens in the viral category. clues to a specific virus may be found in the patient history; for example, rsv might be considered when there is household exposure to infected children. influenza typically presents with sudden onset of symptoms, including fever, myalgias, cough, and sore throat. neuraminidase inhibitors are modestly effective in shortening the duration of influenza in ambulatory and healthy patients (by about 1 day), if initiated in the first 48 hours of illness. the resistance patterns of influenza a and b have shifted in the last several years and may vary based on yearly viral strains. influenza b has remained, as of 2010, sensitive to zanamivir (relenza) and oseltamivir (tamiflu). currently circulating strains of influenza a, both h1n1 and h3n2, and influenza b have generally remained sensitive to both oseltamivir and zanamivir (fiore et al., 2011) . family physicians are advised to consider restraint in the prescribing of these agents, since resistance is of great concern. yearly influenza immunization and cough etiquette and hygiene are likely the most useful techniques for influenza management. studies have identified other pathogens, such as mycoplasma pneumoniae and chlamydophila pneumoniae, in a small minority of cases of clinical acute upper respiratory illness with cough as the predominant symptom. no significant benefit has been found in treating these infections with antibiotics. an exception in the treatment of acute bronchitis-like illness with antibiotics is when confirmed or probable bordetella pertussis is present. early treatment with a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) . although common upper respiratory bacterial pathogens, such as moraxella (branhamella) catarrhalis, streptococcus pneumoniae, and haemophilus influenzae, may be isolated from patients with acute bronchitis, their relevance is questionable because these bacteria can be present in the respiratory tract of healthy individuals. obtaining sputum for culture when bronchitis is the diagnosis generally is not useful. antibiotics may offer a modest benefit in the treatment of acute bronchitis, with many studies showing no statistical significance in the outcome of treated versus not-treated groups. measures of function, such as duration of illness, loss of work, and limitation of activity, have not shown clinically significant improvement in those with acute bronchitis taking antibiotics. coupled with cost and the potential for side effects, the use of antibiotics for acute bronchitis is not recommended. if a provider decides to use an antibiotic in a specific patient situation, narrow-spectrum respiratory agents are preferred, such as a first-generation macrolide or doxycycline. treating the symptom of cough in acute bronchitis is an important concern for patients. in adults with acute bronchitis with signs of airway obstruction, evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough. these agents are not helpful for children with acute cough or adults with cough and no evidence of airway obstruction. side effects of tremor and an anxious feeling must be weighed against this benefit. patients often are primarily interested in alleviating symptoms caused by respiratory illness. unfortunately, there is mixed evidence for the use of over-the-counter (otc) and prescription cough medications. dextromethorphan and codeine may be somewhat effective, although they have not been evaluated in randomized, double-blinded, placebo-controlled trials for acute bronchitis. combination first-generation antihistamine-decongestant products may be effective for the cough associated with colds. naproxen showed efficacy against cough in one upper respiratory model study (sperber et al., 1992) . guaifenesin acts as an expectorant and may have some effect on cough by its mucus-thinning properties. community-acquired pneumonia (cap) is defined as an acute infection of the pulmonary parenchyma and, along with influenza, is the seventh leading cause of death in the united states. fever, cough, sputum production, pleuritic chest pain, and dyspnea are common symptoms of cap. nausea, vomiting, and diarrhea also may occur, and in elderly patients, cap may present with mental status changes. although its absence usually makes pneumonia less likely, fever can be absent in the elderly patient. other physical examination findings include an elevated respiratory rate, conversational dyspnea, tachycardia, and rales. egophony and dullness to percussion may be noted with focal consolidation. typical laboratory findings include leukocytosis. the diagnosis of pneumonia is based on the presence of symptoms and the presence of an infiltrate on chest radiograph. if infiltrate is not present, consider obtaining a chest tomography scan (which has higher sensitivity) to rule in or rule out cap. if negative, other diagnoses should be considered. the most common microbiologic agent of pneumonia is often not isolated (table 16 -1). furthermore, studies have shown that bacteriologic causes of pneumonia cannot be determined by radiographic appearance (i.e., "typical" vs. "atypical"). in the proper clinical setting, certain clinical microbes should be considered because they can affect treatment considerations and epidemiologic studies. these include legionella spp., influenza a and b, and communityacquired methicillin-resistant staphylococcus aureus (mrsa). certain diagnostic tests are performed based on clinical setting. blood cultures are not routinely done in the outpatient setting but should always be done if the patient is being admitted to the hospital, ideally before antibiotics are given. the use of gram stain and sputum culture remains controversial but can provide more evidence of a bacterial cause (e.g., many pmns). if sputum cultures are being obtained, it is recommended that the physician have the patient expectorate directly into a specimen cup and have it sent immediately for processing. this can increase the yield of isolating streptococcus pneumoniae among antibiotics for the treatment of bronchitis is not recommended because of the cost, potential for side effects, and lack of clinical benefit (braman, 2006; smith et al., 2009 ) (sor: a). in the treatment of bordetella pertussis, early administration of a macrolide antibiotic and patient isolation will likely decrease coughing paroxysms and limit spread of disease (braman, 2006) (sor: a). in adults with acute bronchitis with signs of airway obstruction, as evidenced by wheezing on examination or decreased peak expiratory flow rate, beta-2 agonists may be helpful in alleviating cough (braman, 2006) (sor: b). for acute exacerbation of copd associated with purulent sputum and increased shortness of breath, treatment with antibiotics decreases mortality by 77% and treatment failure by 53% (ram et al., 2009 ) (sor: a). 210 other respiratory pathogens. other tests include urine antigen tests for s. pneumoniae, legionella pneumophila serogroup 1, and nasal swab for influenza a and b. in young children, rsv, adenovirus, and parainfluenza in addition to influenza are common causes. nasal swab for rsv and influenza can be rapidly done, but the other causes can be determined with viral cultures, serology, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr), although results usually are received after resolution of the acute symptoms. perhaps the most important decision for clinicians is to determine the location of treatment. the american thoracic society (ats) and the infectious diseases society of america (idsa) recommend use of the pneumonia severity index (psi), which uses 20 variables to risk-stratify the patient into five mortality classes, or the curb-65, which measures five clinical variables in this decision making. the curb-65 may be the easiest and most convenient to use at the site of decision making. a score of 0 or 1 indicates treatment as an outpatient; a score of 2 requires hospital admission to the general medical ward; and a score of 3 or more indicates admission to an intensive care unit (icu) (box 16-1). treatment of cap should be targeted toward the most likely etiology (table 16 -2). outpatient therapy for patients who have no comorbidities and have not received antibiotics within the last 3 months includes doxycycline or a macrolide antibiotic. use of a fluoroquinolone antibiotic (levofloxacin or moxifloxacin) should be reserved for patients with more complicated pneumonia and those requiring hospitalization. patients who have comorbid conditions or recent antibiotic exposure, or who will be hospitalized, should receive a respiratory fluoroquinolone or combination therapy with a betalactam drug plus a macrolide, for 48 to 72 hours after fever abates (usually 5-7 days' total therapy). if an organism is isolated, therapy may be narrowed to cover the causative agent. the clinician should consider longer therapy and appropriate antibiotics to cover for infection by less common organisms such as staphylococcus aureus or pseudomonas aeruginosa. if the patient has no more than one abnormal value (temperature <37.8° c, heart rate <100, respiratory rate <24, sbp >90, o 2 saturation >90%, po 2 >60 on room air) and the patient is able to maintain oral intake and has a normal mental status, the clinician can safely switch to oral therapy and discharge the patient from the hospital. unless the etiology of the pneumonia is known, the physician should switch to oral antibiotics in the same class as the intravenous antibiotics used. the u.s. preventive services task force (uspstf) along with idsa and ats recommend annual influenza vaccinations to those over 50 years of age, those who are (or who reside with those who are) at high risk for influenza complications, and all health care workers. furthermore, the pneumococcal vaccine should be given to all those over age 65. smoking cessation is also important and should be discussed at each clinic visit. • concerns about development of resistant seasonal and h1n1 swine-derived influenza virus should be considered in the decision to administer antiviral medications to healthy patients with these infections. • the abrupt onset of fever with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season" is sufficient to diagnose influenza. • prevention of influenza is generally with vaccination. influenza deserves special mention because it is an important cause of pneumonitis and can precede a bacterial pneumonia. influenza viruses are medium-sized enveloped ribonucleic acid (rna) viruses that consist of a lipid bilayer with matrix proteins with spiked surface projections of glycoproteins (hemagglutinins, neuraminidase) on the outer surface ( figure 16-1) . both influenza a and influenza b have eight segmented pieces of single-stranded rna. the only difference between influenza a and b is that b does not have an m2 ion channel. hemagglutinins, three types of which typically infect humans (h1, h2, h3), bind to respiratory epithelial cells and allow fusion with the host cell. neuraminidase, consisting of two types (n1, n2), allows release of virus from the infected cells. a unique aspect of influenza is that antigenic variation occurs annually. antigenic shift is caused by a genetic reassortment between animal and human influenza strains, producing a novel virus that generally causes the worldwide pandemics. influenza viruses circulate mostly among humans, birds, and swine. sometimes; a human strain and an animal strain can intermingle and create a new, unique virus. this is what happened during spring 2009, heralding the most recent pandemic and creating "novel h1n1 influenza" (swine influenza). genotype analysis • score 0 or 1: outpatient treatment • score 2 : inpatient treatment on a general medical floor • score >3: inpatient treatment in an intensive care unit bun, blood urea nitrogen. locally adapted guidelines should be implemented to improve the processing of care variables and relevant clinical outcomes in pneumonia (mandell et al., 2007) (sor: b) . objective criteria or scores should always be supplemented with physician determination of subjective factors, including the patient's ability to take oral medication safely and reliably and the availability of outpatient support resources (sor: b). for patients with curb-65 score of 2 or higher, more intensive treatment (i.e., hospitalization or, where appropriate and available, intensive in-home health care services) is usually warranted (sor: c). of this strain determined that components came from an influenza virus circulating among swine herds in north america that combined with a virus circulating among ill swine in eurasia, creating a new influenza strain capable of causing disease in humans. because this virus had not previously infected humans, it had the potential to cause widespread morbidity and mortality worldwide. during pandemics, the u.s. centers for disease control and prevention (cdc) estimates an additional 10,000 to 40,000 deaths caused by influenza. although higher than in nonpandemic years, mortality was significantly less than initially predicted in 2009. no recent antibiotic therapy a respiratory fluoroquinolone alone or an advanced macrolide plus a β-lactam † † an advanced macrolide plus a β-lactam, or a respiratory fluoroquinolone alone (regimen selected will depend on nature of recent antibiotic therapy) intensive care unit (icu) a β-lactam † † plus either an advanced macrolide or a respiratory fluoroquinolone pseudomonas infection is not an issue but patient has a β-lactam allergy a respiratory fluoroquinolone, with or without clindamycin pseudomonas infection is an issue ‡ ‡ (cystic fibrosis, impaired host defenses) either (1) copd, chronic obstructive pulmonary disease; mrsa, methicillin-resistant staphylococcus aureus. * azithromycin or clarithromycin. † that is, the patient was given a course of antibiotic(s) for treatment of any infection within the past 3 months, excluding the current episode of infection. such treatment is a risk factor for drug-resistant streptococcus pneumoniae and possibly for infection with gram-negative bacilli. depending on the class of antibiotics recently given, one or another of the suggested options may be selected. recent use of a fluoroquinolone should dictate selection of a nonfluoroquinolone regimen, and vice versa. ‡moxifloxacin, levofloxacin, or gemifloxacin. § dosage: 1 g orally (po) three times daily (tid). ¶ dosage: 2 g po twice daily (bid). ** high-dose amoxicillin (1 g tid), high-dose amoxicillin-clavulanate (2 g bid), cefpodoxime, cefprozil, or cefuroxime. † † cefotaxime, ceftriaxone, ampicillin-sulbactam, or ertapenem. ‡ ‡the antipseudomonal agents chosen reflect this concern. risk factors for pseudomonas infection include severe structural lung disease (e.g., bronchiectasis) and recent antibiotic therapy, health care-associated exposures or stay in hospital (especially in the icu). for patients with cap in the icu, coverage for s. pneumoniae and legionella species must always be considered. piperacillin-tazobactam, imipenem, meropenem, and cefepime are excellent β-lactams and are adequate for most s. pneumoniae and h. influenzae infections. they may be preferred when there is concern for relatively unusual cap pathogens, such as p. aeruginosa, klebsiella spp., and other gram-negative bacteria. § § piperacillin, piperacillin-tazobactam, imipenem, meropenem, or cefepime. ## data suggest that older adults receiving aminoglycosides have worse outcomes. ¶ ¶ dosage for hospitalized patients, 750 mg/day. the abrupt onset of fever, along with chills, headache, malaise, myalgias, arthralgias, and rigors during "flu season," is sufficient to diagnose influenza. as the fever resolves, a dry cough and nasal discharge predominate. a rapid nasal swab or viral cultures can be used to confirm the diagnosis of influenza but is rarely needed. in fact, the sensitivity of these rapid tests can range from 50% to 70%, so a negative test does not rule out influenza. the primary care physician needs to determine if the patient has influenza or the common cold, because symptoms of both illnesses generally overlap (table 16-3) . treatment of influenza is generally not necessary because it is usually a self-limiting condition. treatment should be reserved for those with comorbidities who present within 48 hours of symptom onset. neuraminidase inhibitors (zanamivir and oseltamivir) prevent the release of virus from the respiratory epithelium and are approved for both influenza a and influenza b. the m2 inhibitors (amantadine and rimantadine) are approved by the u.s. food and drug administration (fda) for the treatment of influenza a because these drugs block the m2 ion protein channel, preventing fusion of the virus to host cell membrane (influenza b has no m2 ion channel). the use of m2 inhibitors is limited because of increasing resistance among influenza a viruses, as well as causing central nervous system (cns) problems that are usually exacerbated in elderly persons, who are more likely to seek treatment for influenza (table 16 -4). the major complication of influenza is a secondary bacterial pneumonia or exacerbation of underlying copd. initial improvement in clinical symptoms followed by deterioration usually suggests a secondary bacterial pneumonia, which can usually be confirmed with a chest radiograph showing an infiltrate. other, less common complications of influenza include myositis, myocarditis, pericarditis, transverse myelitis, encephalitis, and guillain-barré syndrome. prevention of influenza is generally with vaccination. box 16-2 outlines patients at risk for influenza complications who should be vaccinated yearly. although anyone wanting an influenza vaccine should be vaccinated, during periods of vaccine shortage, high-risk groups have priority. a well-matched vaccine can prevent influenza among 70% to 90% of adults and decrease work absenteeism. conversely, a poorly matched vaccine only prevents influenza in 50% of healthy adults. proper hand hygiene and covering one's cough are two additional important components in preventing the spread of influenza virus. • population-based vaccination programs have been highly effective in decreasing the incidence of many viral infections. • acyclovir can be used in adults and children with varicella to decrease symptoms if given in the first 48 hours after rash onset, but its benefit must be weighed against its cost and the possibility of development of viral resistance. • antiviral medications should be considered to decrease the incidence of postherpetic neuralgia, particularly in older patients. early treatment (within 48 hours of onset of symptoms) with oseltamivir or zanamivir is recommended for influenza a (jefferson et al., 2006) (sor: a). use of oseltamivir and zanamivir is not recommended for patients with uncomplicated influenza with symptoms for more than 48 hours (kaiser and hayden, 1999) (sor: a). oseltamivir and zanamivir may be used to reduce viral shedding in hospitalized patients or to treat influenza pneumonia (sor: c). (from treanor jj: influenza viruses, including avian influenza and swine influenza. in mandell gl, bennett je, dolin rd (eds) . mandell, douglas, and bennett's principles and practices of infectious diseases, 7th ed. philadelphia, churchill livingstone, 2010, p 2266.) • measles has had a resurgence in recent years and should be suspected when a patient presents with cough, coryza, conjunctivitis, and head-to-toe rash. • epstein-barr virus and cytomegalovirus infections are generally not clinically distinguishable, and their treatment is primarily supportive. vaccinations have dramatically decreased the incidence of a number of historically common viral infections; smallpox has been eradicated through widespread vaccination. however, recent outbreaks of measles and mumps on college campuses underscore the need to remain vigilant in administering vaccines at the population level, even though no vaccine is available for many common viruses. varicella is one of the classic viral exanthems of childhood. before routine vaccination, having chickenpox was one of childhood's "rites of passage." the virus, a herpesvirus (human herpesvirus 3), is effectively transmitted, causing outbreaks in schools and households. patients with primary varicella present with fever, headache, and sore throat. generally within 1 to 2 days of onset of symptoms, a papulovesicular rash erupts diffusely. the classic description of the chickenpox lesion is "a dewdrop on a rose petal," suggesting a central vesicle on an erythematous base. lesions continue to appear for 5 to 7 days. all lesions going from papule to vesicle to crusted lesion takes about 2 weeks. patients are considered to be infectious, primarily through respiratory secretions, during the 2 days before symptoms appear and until all lesions are crusted. treatment of varicella is generally supportive. control of spread may be a concern in group-living environments such as schools or residence halls. isolation of the infected patient away from those susceptible to varicella infection is standard practice. acyclovir can be started within the first 24 hours after rash eruption to achieve an attenuation of the infectious course. in children, this means a decrease in the duration of fever by about 1 day and a decrease in the number of lesions (swingler, 2010) . in adults, acyclovir decreases rash duration and the number of lesions, although the results are less significant than for children. adult dosing of acyclovir for varicella is 800 mg five times daily. the marginal benefit must be weighed against the possible development of resistance at a population level and the cost of the medication. complications of varicella can include secondary infection of skin lesions, pneumonitis, encephalitis, and dehydration from vomiting and diarrhea. varicella is prevented primarily through administration of vaccine. the vaccine is highly effective in children, with recommended dosing at 12 to 15 months with a second dose at 4 to 6 years. varicella is now included in a measles-mumpsrubella (mmr) vaccine, which can be given between 12 months and 12 years of age. the varicella vaccine is a live, attenuated virus and should not be given to certain immunocompromised patients. the vaccine can also be administered to exposed immunocompetent contacts, although the benefit is clearer for children than adults. severely immunocompromised patients exposed to varicella (particularly those with advanced hiv disease) may be given high-dose acyclovir to prevent development of disease. herpes zoster is a reactivation of the neurotropic varicella virus, typically in a dermatomal distribution. this is more common in elderly or immunocompromised patients but can occur in healthy people as well. patients with zoster may note generalized malaise, hyperesthesia, numbness, tingling, and pain in the skin before development of a rash. the appearance of the rash is the same as for chickenpox, although most often isolated to a unilateral dermatome. the diagnosis of herpes zoster is clinical based on the history and the classic appearance of the rash. in immunocompromised patients, however, the rash may not be dermatomally isolated. when the diagnosis is unclear, viral culture can be obtained from the base of a lesion. antiviral medications are likely to decrease the incidence of postherpetic neuralgia and are recommended, particularly in elderly patients (wareham, 2010) . valacyclovir (1 g three times daily) or famciclovir (500 mg every 8 hours) for 7 days is likely more effective than acyclovir in achieving this result. either drug should be started as soon after the diagnosis as possible, preferably within 48 to 72 hours of rash onset. when patients have established postherpetic neuralgia, gabapentin and tricyclic antidepressants are helpful in alleviating the pain. the rash of zoster is infectious to the touch. patients should be advised to keep the rash covered until all the lesions have crusted. zoster of the trigeminal nerve can extend to the eye and warrants immediate ophthalmologic intervention. a vaccine to prevent herpes zoster in adults was released in 2006. the zoster vaccine differs from the varicella vaccine in that the amount of attenuated virus is 14 times higher in the zoster vaccine. the vaccine decreases the incidence of zoster by 50%. it is recommended for administration by the american academy of family physicians (aafp) to adults over age 60, regardless of prior varicella or zoster history. although generally well tolerated, the vaccine is somewhat costly. in 2008, more measles cases were reported than in any other year since 1997 (cdc, 2010) . measles is the "first disease" of childhood from the history of medicine. in adults, measles infection may be acquired in the face of waning immunity from remote immunization. a booster dose of mmr vaccine is recommended before college entry. clinically, measles presents with cough, coryza (nasal irritation and congestion), and conjunctivitis. fever is common several days before the onset of the rash. the rash of measles typically spreads from head to toe and has an erythematous, papular appearance with a "sandpaper" feeling. koplik's spots are erythematous papules with a bluish center on the oral mucosa and appear early in measles. measles is highly contagious through droplets. lymphopenia and neutropenia are common laboratory findings with measles infection. complications of measles include primary infections such as pneumonia, gastroenteritis, encephalitis, and the rare subacute sclerosing panencephalitis. secondary infections such as otitis media, pneumonia, and adenitis may also occur. treatment is supportive, and the implications of measles infection are primarily in the public health realm. patients with measles should be isolated for at least 4 days after the appearance of the rash. it is important to recognize that patients are contagious for 2 days before the development of symptoms. careful verification of immunization status for close contacts is essential. clinical infectious mononucleosis is a common infection in adolescents and early adults. the clinical syndrome is most often caused by epstein-barr virus (ebv), although cytomegalovirus (cmv) may also be the source in this clinical syndrome, which includes fever, exudative tonsillitis, adenopathy (often including posterior cervical or occipital nodes), and fatigue. ebv is transmitted in oral secretions and may be transmitted sexually as well. b cells are infected with ebv either directly or after contact with epithelial cells, resulting in diffuse lymphoid enlargement. the diagnosis of infectious mononucleosis is made by recognizing the clinical symptoms of fever, pharyngitis, and adenopathy along with the laboratory findings of greater than 50% lymphocytes with 10% or more atypical lymphocytes (hoagland, 1952) . also, a positive serologic test for heterophile antibody assists the family physician in the diagnosis. to differentiate ebv from cmv mononucleosis, serology (igg and igm) may be obtained. results of these tests are generally not available in time to have a significant benefit clinically. splenic enlargement as part of this lymphoid hypertrophy can lead to splenic rupture (0.1% risk) (dommerby et al., 1986) . athletes with infectious mononucleosis must be managed carefully to avoid their participation in sports that could result in abdominal trauma. other risks associated with infectious mononucleosis include upper airway obstruction, asymptomatic transaminase elevation, thrombocytopenia, and rash after the administration of ampicillin or amoxicillin. routinely obtaining transaminase levels in patients without clinical hepatitis is of little value and can increase the overall cost of management. treatment of infectious mononucleosis is largely supportive. patients should be instructed to treat fever with antipyretics, rest, and expect symptom duration of 2 to 4 weeks, although symptoms can last for several months. the use of steroids, such as prednisone, has shown limited benefit. data suggest an initial benefit 12 hours after steroid administration, although this is lost within several days (candy and hotopf, 2006) . combination of steroid and an antiviral (valacyclovir) may have some positive effect on fatigue. • the most common presentation of tuberculosis is pulmonary disease. • tuberculosis is diagnosed by acid-fast bacilli smears and cultures. • standard first-line agents to treat tb are isoniazid, rifampin, pyrazinamide, and ethambutol. • high-risk patients with a positive purified protein derivative skin test or quantiferon-tb gold test should be treated for latent tb infection. • the current recommendation for first-line treatment for latent tb is 9 months of oral isoniazid. tuberculosis skin testing should be interpreted without regard to bacille calmette-guérin (bcg) history, because bcg is administered in areas where tb is endemic and bcg does not provide complete protection from tb infection. tuberculosis (tb) is a disease that has plagued humans since antiquity, with evidence of spinal tb in neolithic and early egyptian remains. at present, tb affects approximately one third of the world's population. tb is the world's second most common cause of death from infectious disease after human immunodeficiency virus or acquired immunodeficiency syndrome (hiv/aids). tuberculosis is caused by mycobacterium tuberculosis, an acid-fast bacillus. tb is acquired by inhalation of respiratory droplets. these respiratory droplets are spread by coughing. brief contact carries little risk for acquiring tb, and infection generally does not occur in open air; open-air sanatoriums were the cornerstone of tb treatment before antimicrobial therapy. in the united states, tb incidence rates have been on the decline since 1992, coinciding with the control of hivinduced aids by antiretroviral therapy. however, tb remains prevalent in certain high-risk groups (i.e. immigrants, iv drug use, homeless persons). most cases of tb are in people age 15 to 49 years. tb in elderly persons is generally caused by a reactivation of latent infection acquired in the remote past, whereas tb in young children indicates ongoing active transmission in the community. infection in children is more likely to progress to active tb and disseminated disease. persons with hiv infection have a disproportionately higher risk for acquiring tb than the general population. tuberculosis is most frequently manifested clinically as pulmonary disease, but it can involve any organ. extrapulmonary tb accounts for about 20% of disease in hiv-seronegative persons but is more common in hiv-seropositive persons. pulmonary tb typically manifest with fever, night sweats, chronic cough, sputum production, hemoptysis, anorexia, and weight loss. chest radiographs in patients with pulmonary tb typically reveal upper-lobe cavitary lesions and can reveal infiltrates or nodular lesions, as well as lymphadenopathy ( figure 16 -2). tb in the setting of advanced hiv co-infection does not generally manifest in the typical manner (table 16-5) . acyclovir started within the first 24 hours after varicella rash eruption can attenuate the infectious course, decreasing duration of fever by 1 day and reducing the number of lesions (sor: a). administration of varicella vaccine to a susceptible child within 3 days of exposure will likely modify or prevent disease (macartney and mcintyre, 2008) the diagnosis of pulmonary tb is made by the demonstration of acid-fast bacilli (afb) in sputum and the growth of m. tuberculosis in culture. these patients typically have an abnormal chest radiograph, as previously described. m. tuberculosis is a slow-growing bacterium, and cultures can take up to 6 weeks to grow. a pcr assay developed for m. tuberculosis can be run on afb smear-positive sputum to hasten the diagnosis of pulmonary tb. a positive pcr on afb-positive sputum is diagnostic of pulmonary tb, but a negative test does not rule out the diagnosis. patients with afb positive smears from sputum samples should be started on anti-tb therapy while awaiting results of pcr and cultures. the treatment of tb always uses multiple agents with anti-tb activity. single agents should never be used. the standard first-line agents are isoniazid (inh), rifampin (rif), pyrazinamide (pza), and ethambutol (emb) (figure 16 -3 and table 16-6). if administered, inh should be given with pyridoxine (vitamin b 6 ; 25-50 mg orally daily) to prevent neuropathy. treatment of active pulmonary tb is generally for 6 months regardless of hiv status, but treatment may need to be extended in certain situations. directly observed therapy (dot) is the preferred mechanism of administration to ensure compliance. many local county and state health departments have systems for dot. treatment of hiv-seropositive patients with tb who are receiving an antiretroviral (arv) regimen that contains a protease inhibitor is complicated by the latter's interaction with rifamycins (particularly rifampin). management of such patients should be coordinated with an infectious diseases specialist, who also should manage drug-resistant tb treatment. in the united states, latent tuberculosis infection (ltbi) is the most prevalent form of tuberculosis. ltbi is the term given to patients with a positive purified protein derivative (ppd) skin test without evidence of active tb. ppd has been used for more than 100 years and relies on delayed-type hypersensitivity (dth) to m. tuberculosis cellular proteins. early late figure 16 -3 treatment algorithm for tuberculosis. patients in whom tb is proved or strongly suspected should have treatment initiated with isoniazid, rifampin, pyrazinamide, and ethambutol for the initial 2 months. a repeat smear and culture should be performed when 2 months of treatment has been completed. if cavities were seen on the initial chest radiograph or the acid-fast smear is positive at completion of 2 months of treatment, the continuation phase of treatment should consist of isoniazid and rifampin daily or twice weekly for 4 months to complete a total of 6 months of treatment. if cavitation was present on the initial chest radiograph and the culture at completion of 2 months' therapy is positive, the continuation phase should be lengthened to 7 months (total of 9 months of treatment). if the patient has hiv infection and the cd4+ cell count is less than 100/mm 3 , the continuation phase should consist of daily or three-times-weekly isoniazid and rifampin. in hiv-uninfected patients having no cavitation on chest radiograph and negative acid-fast smears at completion of 2 months of treatment, the continuation phase may consist of either once-weekly isoniazid and rifapentine, or daily or twice-weekly isoniazid and rifampin, to complete a total of 6 months (bottom). patients receiving isoniazid and rifapentine, and whose 2-month cultures are positive, should have treatment extended by an additional 3 months (total of 9 months). *emb may be discontinued when results of drug susceptibility testing indicate no drug resistance. †pza may be discontinued after it has been taken for 2 months (56 doses). ‡rpt should not be used in hiv-infected patients with tb or in patients with extrapulmonary tb. therapy should be extended to 9 months if 2-month culture is positive. afb, acid-fast bacilli; cxr, chest radiograph (x-ray); emb, ethambutol; inh, isoniazid; pza, pyrazinamide; rif, rifampin; rpt, rifapentine. because ppd relies on dth, any factor that reduces the dth affects the host response to ppd. the most common clinical example is use of corticosteroids, which blunt the dth response and can complicate ppd interpretation. therefore, ppd testing should not be performed while a patient is taking corticosteroids. also, tb testing should be targeted to those with higher risk of infection and should not routinely be done in those with low risk (ats/cdc, 2000) . the ppd can also give false-positive results in patients with previous bacille calmette-guérin (bcg) vaccination or with infection by other mycobacterial infections. in the united states, this may cause difficulties in testing immigrants from countries who routinely use bcg vaccination programs. however, previous bcg vaccination should not change the interpretation of the ppd or willingness to treat such individuals accordingly. ‡when dot is used, drugs may be given 5 days per week and the necessary number of doses adjusted accordingly. although there are no studies that compare five with seven daily doses, extensive experience indicates this would be an effective practice. § patients with cavitation on initial chest radiograph and positive cultures on completion of 2 months of therapy should receive a 7-month (31 weeks, either 217 doses [daily] or 62 doses [twice weekly]) continuation phase. ¶ five-days-a-week administration is always given by dot. rating for 5 day per week regimens is aiii. ¶ ¶ not recommended for hiv-infected patients with cd4+ cell counts <100 cells/μl. ** options 1c and 2b should be used only in hiv-negative patients who have negative sputum smears at completion of 2 months of therapy and who do not have cavitation on initial chest radiograph. for patients started on this regimen and found to have a positive culture from the 2-month specimen, treatment should be extended an extra 3 months. the dth response can wane over time. to overcome this problem, nonreacting patients may undergo repeat ppd 1 week after their initial ppd. the diagnosis of ltbi is made by interpretation of a ppd and by ascertaining the patient's risk factors for progression to active tb if left untreated . interpretation of the ppd should be based on the area of induration and not the area of surrounding erythema. persons whose ppds have converted from negative to positive within 2 years are presumed to have been infected recently. the decision to use ppd means treating the patient for ltbi if the ppd test is positive. patients at increased risk for progression to active tb include those who have been recently infected (recent ppd converters); patients who are hiv seropositive; patients who have silicosis, diabetes, or chronic renal failure (including those receiving hemodialysis); solid-organ transplant recipients; patients with gastrectomy or jejunoileal bypass or head and neck cancer; injection drug users; patients with chest radiograph evidence of prior tb; and patients who weigh at least 5% less than ideal body weight. patients taking chronic corticosteroid therapy and those who are to receive tumor necrosis factor alpha (tnf-α) blockers (e.g., infliximab) are also at risk. patients taking corticosteroids also have higher risk of progression to active tb with larger doses and longer courses of corticosteroids. standard therapy for ltbi is inh, 300 mg orally daily for 9 months, regardless of hiv status. again, inh should always be administered with pyridoxine to prevent neuropathy. to overcome the false-positive results and confusion of ppd testing in certain populations, newer interferon-gamma (ifn-γ) release assays such as the quantiferon-tb gold (qft-g) test have been developed to detect latent m. tuberculosis. qft-g quantifies the release of ifn-γ from lymphocytes of the host's blood in response to three m. tuberculosis target antigens that are absent from bcg and most other nontuberculous mycobacterium spp. the advantages of using qft-g include one-time blood testing without the need for followup visit, no triggering of amnestic responses, and possibly more specific response to m. tuberculosis. however, qtf-g use in immunocompromised or anergic patients is limited, with indeterminate results. some studies also show discordant results in individuals tested with both ppd and qtf-g. in general, qtf-g may be used in all circumstances in which the ppd is used. however, whether the qtf-g is truly more specific or sensitive than the ppd in latent or active tb is yet to be determined. • the u.s. preventive services task force recommends "highintensity" behavioral counseling to at-risk adults and adolescents to prevent sexually transmitted infections. • be specific in addressing patients' sexual practices so as to provide appropriate prevention advice. hiv-positive persons recent contacts of tuberculosis patients fibrotic changes on chest radiography consistent with prior tuberculosis patients with organ transplants and other immunosuppressed patients (receiving equivalent of ≥15 mg/day of prednisone for at least 1 month) development in the primary prevention of stis is immunization against human papillomavirus (hpv). the vaccine can prevent infection with certain strains of hpv that cause cervical cancer and genital warts. trials are ongoing to determine the effectiveness of daily arv therapy in preventing transmission of hiv. vaccination investigation is ongoing for herpes simplex, chlamydia trachomatis, and hiv. this breadth of research effort holds promise for the future in the prevention of stis. the uspstf recommends "high-intensity" behavioral counseling to at-risk adults and adolescents to prevent stis. highintensity counseling involves multiple sessions and often is delivered to groups of patients. unfortunately, this type of intervention has limitations in its practicality for population-based delivery. no risk of harm was discovered in the delivery of counseling for sti prevention. vaccination is the most important form of primary prevention of common infectious diseases. two vaccines are currently on the market for hpv prevention-one that protects against four viral subtypes (6, 11, 16, 18) and is licensed for use in males and females 9 to 26 years of age, and the other against two subtypes (16,18), licensed for females 10 to 25 years of age. hepatitis b is a sexually transmitted infection, and immunization is recommended for adolescents who have not been previously inoculated. this is a requirement in many states for school entry. hepatitis a can be transmitted by oro-anal sexual contact, and vaccination should be offered to patients who are contemplating engaging in this sexual practice. recommendations surrounding the use of barrier methods for sti prevention should be tailored to the sex practices of the client. for example, a percentage of women use anal sex as a method of birth control but may not consider the need for condom use with this practice. the question, "do you regularly use condoms?" has little relevance to infection control for many sexual practices. evidence supports the advice to use barrier methods of latex or other approved material in a manner that prevents the exchange of blood and body fluids in decreasing stis. condoms confer a 30% risk reduction for herpes simplex and up to an 80% risk reduction for hiv, when used correctly (weller and davis-beaty, 2002; martin et al., 2009) . the secondary prevention of stis is achieved through direct and nonjudgmental patient assessment and screening and avoiding assumptions about patient sexual practices. screening is a tool to prevent the inadvertent spread of infection as well as the sequelae of undetected disease. infectious genital ulcers are associated with herpes simplex virus (hsv), syphilis, chancroid, lymphogranuloma venereum, and granuloma inguinale. hsv is by far the most common, affecting 50 million people in the united states. hsv-1 and hsv-2 are chronic, neurotropic viral infections that enter through epithelium and come to rest in the dorsal root ganglia. therefore, infection leads to lifetime presence of the virus, but the clinical manifestation of this condition is variable. a small percentage of those with serologic evidence of hsv-2 (10%-25%) have had symptoms of clinical herpes infection. in addition, patients with hsv infection can shed the virus in the absence of symptoms, creating a prime opportunity for spread. herpes simplex outbreak may be followed by a prodrome of malaise, fever, and regional lymphadenopathy before the appearance of grouped vesicles on an erythematous base. the vesicles are typically quickly broken and become ulcerated in appearance, with each vesicle usually less than several millimeters in size. true first-time infections tend to present more severely than secondary presentations of previously infected individuals, with a prodrome present in 80% of cases. the lesions can be in any location around the genitals or rectum, on the proximal thighs and buttocks, inside the vagina, and in and around the mouth. the lesions are most who to screen? often painful, particularly when on mucosal surfaces, or itchy. in women, herpes simplex can present with cervicitislike symptoms with bleeding and discharge and cervical ulcerations on examination, or simply mucopurulent cervicitis. herpetic lesions around the urethra tend to be extremely painful and can make urination difficult. rectal hsv can be confused with irritation, perianal fissure, and even candidiasis because of its often beefy-red appearance and itching. vesicles typically appear 6 days after infection and can last up to 2 weeks in an initial infection. subsequent outbreaks tend to have a shorter duration and to be less uncomfortable for patients. confirmation of infection is helpful, but the diagnosis can be made primarily on the clinical appearance of the exanthema. vigorous sample collection from an ulcer (which the patient may not appreciate) to be sent for pcr identification and typing is the most readily available method of laboratory diagnosis. serum antibody testing is not useful in the initial hsv diagnosis because antibody levels will not be appreciable early in infection. the appearance of convalescent immunoglobulin g (igg) and igm levels several weeks after a suspected outbreak might help to support the diagnosis of hsv infection. the value of screening for hsv immunity is debatable and should generally not be recommended for asymptomatic individuals. in addition, the uspstf recommends against screening asymptomatic pregnant women for hsv to prevent transmission to the newborn. given that many patients with hsv infection never manifest symptoms, the value of knowing that one is hsv seropositive is questionable. in addition, hsv-1 and hsv-2, although classically oral and genital, respectively, can "mix and match" based on sexual practices. it is often confusing for asymptomatic individuals to know that they have hsv antibody (do i have cold sores? do i have genital herpes? how should this change the way i live my life?). in monogamous couples with one partner known to be hsv positive and the other with unknown status, testing of the latter may indicate suppressive therapy in the seropositive partner if the other is found to be negative. regular barrier method use decreases transmission of herpes in both men and women, with patients using condoms 100% of the time having a 30% reduction in hsv acquisition from those who never use condoms (martin et al., 2009) . serodiscordant couples may also decrease transmission through antiviral suppressive therapy to the hsv-positive partner (table 16-8) . syphilis is a spirochetal infection that has resurged since 2001, the nadir year since 1996. syphilis infection rates are highest in men who have sex with men. syphilis is much less common than the other stis, with an infection rate of 5.6 per 100,000 population in the united states (vs. 496 per 100,000 for chlamydia). syphilis presents in several stages. the primary phase of syphilis is a painless ulcer called a chancre (figure 16-4) . the chancre may be visible on the genitals, although it can also be inside the vagina, mouth, or rectum, making it difficult to find. this lesion will appear within 3 weeks of transmission and will last for several weeks untreated. the secondary phase of infection is disseminated and involves a diffuse macular rash, typically with palm and sole lesions, generalized lymphadenopathy, fever, and condyloma latum (smooth, moist lesions on genitals without cauliflower appearance of condyloma acuminatum). tertiary syphilis is often asymptomatic but affects the heart, eyes, and auditory system and can be associated with gumma formation. gummas are soft, granulomatous growths in organs that can cause mechanical obstruction and weakening of blood vessel walls. latent infection often involves the cns. diagnosis of primary syphilis is challenging. the test of choice is darkfield microscopy, which is not readily available. direct fluorescent (monoclonal) antibody (dfa) testing may be available. antibody tests for syphilis, such as the rapid plasma reagin (rpr) and the less frequently used venereal disease research laboratories (vdrl), are often not positive early in infection and thus cannot be used to rule out primary syphilis based on a single reading. treponemal antigen testing (eia) may be available in some laboratories. the fluorescent treponemal antibody absorption (fta-abs) test may also be negative in the early infection. direct pcr for primary syphilis lesions has been tested but is not yet fda approved. a physician may choose to treat presumptively if a painless chancre and risk factors are present and may then do a convalescent rpr test in 1 to 2 weeks to confirm the infection by the appearance of a positive reaction. one would expect a fourfold change in titer of either test to indicate the presence of disease. primary and secondary syphilis are treated with a single injection of penicillin g, 2.5 million units. other regimens do not have proven effectiveness but can be used in the penicillin-allergic patient, including doxycycline, 100 mg twice daily for 14 days; ceftriaxone, 500 mg to 1 g intramuscularly (im) daily for 8 to 10 days; or azithromycin, 2 g as a single oral dose, although resistance to azithromycin has been observed. patients treated for primary syphilis should have periodic clinical follow-up and serologic testing to determine a fourfold decrease in rpr reactivity within 6 months. latent syphilis can be either early, meaning infection within the last year, or late, meaning infection beyond a year. early latent syphilis is treated with a single injection of penicillin g, 2.4 million units. syphilis of late latency or unknown duration is treated with three injections of penicillin g, 2.4 million units, in 3 consecutive weeks. for penicillin-allergic patients, doxycycline, 100 mg twice daily for 28 days, is required. those with latent syphilis should have ophthalmic examination as well as evaluation for vascular gumma formation. suspected neurologic involvement of latent syphilis must be evaluated with cerebrospinal fluid (csf) examination and treatment with aqueous penicillin g, 3-4 million units intravenously (iv) every 4 hours for 10 to 14 days. partners of patients with newly diagnosed syphilis are at risk for infection. partners within 90 days of a diagnosis of primary syphilis should be tested, but treated presumptively even if serologic testing is negative. for partners prior to 90 days before diagnosis, serology is generally reliable in detecting presence of infection and may guide treatment. patients with secondary syphilis should inform partners within 6 months before diagnosis, or 12 months for those diagnosed with tertiary syphilis (table 16 -9). chancroid may occur in regional outbreaks and presents with a painful genital ulcer and suppurative regional adenopathy. herpes and syphilis should both be ruled out in the patient suspected of having chancroid infection. chancroid is caused by haemophilus ducreyi and there is currently no fda approved test to directly detect this organism. treatment with azithromycin (1 g as single dose), ceftriaxone (250 mg im as a single dose), ciprofloxacin (500 mg twice daily for 3 days), or erythromycin (500 mg three times daily for 7 days) are all alternatives (table 16 -10). it may be necessary to perform incision and drainage on fluctuant inguinal nodes. patients should be reexamined in 1 to 2 weeks to ensure healing of the primary ulcer(s) and resolution of the adenopathy. partners who had contact with the infected patient starting 10 days before development of the patient's symptoms should be treated, regardless of the presence of symptoms. less common ulcerating stis include lymphogranuloma venereum (lgv) and granuloma inguinale ( figure 16 -5). lgv causes regional adenopathy and often an ulcer at the point of entry. rectal lgv may cause a proctocolitis with anal pain, discharge, bleeding, and diarrhea. lgv is caused by chlamydia trachomatis serotypes and can be detected by testing swabbed material from open lesions or aspirates from lymph nodes with culture, dfa, or nucleic acid detection. treatment is noted above (table 16-10) . granuloma inguinale, caused by klebsiella granulomatis, is rare in the united states and causes progressive ulcerative disease of the genitals. a second sti category includes those causing the clinical presentation of vaginal discharge, pelvic pain, dyspareunia, and dysuria in women and penile discharge and dysuria in men, as well as possible rectal pain and discharge in men and women. of this group, chlamydia trachomatis is the most common, causing 1.2 million infections in the united states in 2008 (cdc, 2009 ). in fact, chlamydia is the most frequently reported reportable infection. the majority of women with chlamydia infection are without symptoms. many men are asymptomatic as well. regular screening for chlamydia, as recommended by the uspstf, can significantly reduce the incidence of pelvic inflammatory disease (pid), one of the most serious sequelae of untreated infection. in women with untreated chlamydia infection, in addition to pid, tubo-ovarian abscess, tubal scarring and ectopic pregnancy, and infertility can all result. as previously mentioned, regular screening is currently recommended for all sexually active women under age 24, all pregnant women under 24, and at-risk pregnant and nonpregnant women over 24. chlamydia testing can be performed on several liquid-based papanicolaou (pap) tests. endocervical swabs for nucleic acid amplification are acceptable when a conventional pap smear is being used. given the recent liberalization of recommendations about pap testing for women under 21 years of age, urine nucleic acid amplification is a readily available alternative for chlamydia testing. this can easily be done at a contraceptive counseling clinic. urine testing is also an acceptable method of testing for men, in addition to a urethral swab. rectal chlamydia infection can occur in individuals who practice receptive anal intercourse. an fda-approved method of testing should be used for screening and diagnosis of this infection. asymptomatic chlamydia infection is treated with either a single dose of azithromycin, 1 g orally, the drug of choice, or doxycycline, 100 mg twice daily, for 7 days (table 16-11) . patient-delivered partner therapy (pdpt), the practice of dispensing treatment to diagnosed patients to treat their partner(s), has proved effective in reducing reinfection rates and further spread of infection. ept is legally allowable in 21 states and potentially allowable in another 21. chlamydia infection may present symptomatically in men or women with symptoms of dysuria and with discharge and with pelvic pain and dyspareunia in women. the discharge of c. trachomatis, versus that of neisseria gonorrhoeae, is said to be more mucoid than purulent, although this characteristic is not specific enough to provide diagnostic accuracy. symptomatic chlamydia, without evidence of pid, is treated the same as asymptomatic infection. many practitioners will treat presumptively for chlamydia and gonorrhea in patients who present with the symptoms previously mentioned while they wait for confirmatory testing. neisseria gonorrhoeae infection may be asymptomatic in both men and women. the current uspstf recommendation is for screening women at risk. men with penile gonorrhea typically present with purulent penile discharge and dysuria with n. gonorrhoeae infection. mucopurulent discharge, dysuria, pelvic pain, and dyspareunia are typical symptoms in women. in patients who engage in anal intercourse, anal discharge, rectal pain, and bleeding can be presenting symptoms. gonococcal pharyngitis is within the differential of exudative pharyngitis in sexually active patients. when symptomatic, throat pain, tonsillar exudates, and anterior cervical adenopathy may be present. testing for gonorrhea can be done using liquid-based pap technologies, cervical or urethral swabs, or urine for nucleic acid amplification. in men with visible discharge, a gram stain with white blood cells (wbcs) and gram-positive intracellular diplococci has a high degree of sensitivity. culture testing may be preferred for suspected pharyngeal and rectal specimens pending fda approval of other methods. again, physicians may opt to treat patients with mucopurulent cervicitis or urethritis presumptively for gonorrhea and chlamydia while waiting for confirmatory testing. fluoroquinolone therapy is no longer recommended because of widespread resistance (table 16-11) . because reinfection with gonorrhea is common for several months after treatment, it may be advisable to retest patients with confirmed gonorrhea in the 3 months after treatment. similarly, stis may be an indicator of risk behavior, and a complete risk history and testing for other stis is advisable if not completed at the initial visit. in male patients with symptomatic urethritis, a causative agent may not be identified, a situation often referred to as nongonococcal urethritis (ngu). technically, chlamydia is included in this category. organisms such as ureaplasma urealyticum and mycoplasma genitalium may be the cause and may be difficult to detect. treatment for these infections is the same as for symptomatic chlamydia, with azithromycin or doxycycline (table 16 -11). it is recommended that partners of patients with ngu should be evaluated and treated. in some cases, testing of partners may detect a specific organism as the cause of infection (e.g., chlamydia). trichomonas vaginalis causes vaginitis in women, who may have a stereotypic frothy, green, and foul-smelling discharge. many women are asymptomatic with trichomoniasis. in addition to causing asymptomatic infection in men, t. vaginalis may cause urethritis. this organism may be suspected in men when patients have repeated treatment failures and no other explanation for symptoms. microscopic examination of vaginal discharge is 60% to 70% sensitive in women. a first voided urine specimen or urethral swab for microscopic exam may be helpful in identifying the protozoa. culture for trichomonas, which requires a special medium, may be necessary to identify this infection accurately in men. trichomonas is effectively treated with a single 2-g dose of metronidazole (table 16-11) . for non-sti causes of vaginal discharge, see the online discussion at www.expertconsult.com. pelvic inflammatory disease can be caused by a number of organisms, including chlamydia, and presents with pelvic pain and discharge. findings that contribute to the diagnosis of pid include fever greater than 101° f, cervical or vaginal mucopurulent discharge, abundant wbcs on saline preparation of vaginal discharge, elevated erythrocyte sedimentation rate (esr), elevated c-reactive protein (crp), and evidence of n. gonorrhoeae or c. trachomatis infection. hospitalization with parenteral antibiotics may be necessary in pregnant patients, patients in whom surgical emergency cannot be ruled out, those who do not respond to oral treatment, those who cannot tolerate oral treatment, and patients who have severe illness or tubo-ovarian abscess. when treating pid parenterally, improvement of symptoms for 24 hours may prompt a change to oral therapy (table 16-12) . conversely, if oral therapy is not producing significant improvement within 2 to 3 days, admission for parenteral therapy may be necessary. patient awareness of human papillomavirus infection has greatly increased in recent years, in large part related to the patient-directed advertising of the hpv vaccine. hpv is likely the most common sti. thirty types of hpv can infect the genital area, some causing genital warts, some causing malignancies of the genital organs, and most being asymptomatic. the gross categories most often used are "high risk" (most often types 16 and 18) and "low risk" (types 6 and 11) hpv infection, the former more often associated with genital cancer. prevention of hpv infection and cervical cancer was revolutionized with the release of the hpv vaccine, which is effective in reducing the incidence of hpv-associated disease. currently, two vaccines are licensed in the united states. gardasil (merck), released in 2006, includes protection against viral types 6, 11, 16, and 18. it is approved for the prevention of vulvar and vaginal cancer and for the prevention of cervical cancer, cervical dysplasia, and genital warts in females age 9 to 26. the vaccine was recently approved for males of the same age range for the prevention of genital warts. more recently, cervarix (glaxosmithkline) was approved for the prevention of cervical cancer and cervical dysplasia from hpv types 16 and 18 in women age 10 to 25. ideally, the vaccine should be administered before initiation of sexual activity to prevent initial acquisition of these hpv types. patients who are already sexually active may also receive the vaccine. the transmission of hpv to men decreases with consistent condom use, from 53.9% in men who never use condoms to 37.9% in men who "always" use them. unfortunately, hpv can infect skin that is not covered by the use of traditional barrier methods (nielson et al., 2010) . male circumcision may decrease the transmission of hpv. patients have many questions about hpv, in particular about screening for asymptomatic infection. hpv infection occurs with high frequency in the sexually active population; up to 50% or more of sexually active individuals have hpv at some point in their life. in addition, hpv is effectively transmitted, even if contact does not involve genital-togenital touching (i.e., manual stimulation can transmit the virus). again, most hpv infections are without symptoms and resolve spontaneously through eradication by the intact immune system. for all these reasons, screening for the mere presence of hpv infection has minimal utility. there is no treatment for asymptomatic hpv infection. the most common presentation of hpv infection is in the context of an abnormal pap smear. hpv is directly linked to cervical dysplasia. for women over age 21 and under 35, hpv testing with high-risk viral detection is common. the presence of high-risk hpv informs further management of the pap result. it is currently recommended that women over 35 be automatically tested for high-risk hpv infection at the pap smear. patients may present with visible warts, or these may be detected at routine or sti screening. genital warts are often cosmetically unacceptable to patients, even though they are infrequently functionally problematic. in some circumstances, wart burden can be high enough to cause physical discomfort or relative obstruction of the vagina or rectum. vulvovaginal candidiasis and bacterial vaginosis are generally not thought to be sexually transmitted, although they are often in the differential diagnosis of sexually transmitted infection (sti). both these infections likely are related to changes in the vaginal ph and the normal flora distribution. it is not always clear which of these factors is primary and which is secondary, because at diagnosis, both ph and normal vaginal flora will often be abnormal. vulvovaginal candidiasis is a common infection causing typically white, curdlike discharge, itching, and sometimes dysuria. the causative organism is usually candida albicans but can be other candida spp. antibiotics can alter normal vaginal flora, so the recent use of antibiotics may predispose women to candidiasis. physical examination may reveal erythematous external genitalia as well as external and internal white, clumping discharge. usually, no distinctive odor is associated with vaginal yeast. wet preparation of vaginal specimen or treatment with potassium hydroxide (koh) may reveal branching pseudohyphae and yeast. when ph is performed, it should be directly on the vaginal discharge and not on the saline-diluted specimen because the saline will alter the ph of the specimen. typically, the ph of yeast discharge is less than 4.5 (normal vaginal ph,3.8-4.5). bacterial vaginosis (bv) is the most common cause of infectious vaginal discharge (spence and melville, 2007) . many different organisms are associated with the diagnosis of bv, including gardnerella vaginalis and mycoplasma hominis. women with bv may report discharge, vaginal irritation, vaginal odor, and at times, dysuria. findings of bv are often detected during a normal screening pap smear or pelvic examination. physical findings may reveal signs of vaginal irritation. the discharge is usually thin and gray. an amine (fishy) odor may be produced with the application of koh. the finding of clue cells, or epithelial cells with adherent bacteria, under saline preparation microscopy and a decrease in normal lactobacilli are common findings. the amsel criteria are useful in bv diagnosis; other scoring systems (e.g., nugent criteria) have been used but require gram staining. the specific amsel criteria are (1) milky, homogeneous, adherent discharge; (2) discharge ph greater than 4.5; (3) positive whiff test (fishy smell with addition of koh); and (4) at least 20% clue cells on microscopic examination. if three of the four criteria are present, the likelihood of bv is 90%. in routine vaginal examination and bimanual examination for patients with vaginal discharge, signs and symptoms of vaginitis are poor predictors of the microbiologic cause of infection (schaaf et al., 1990) . the clinical examination and office testing, in fact, are fair predictors of the true cause of infection (lowe et al., 2009 ). many patients with vaginal discharge will use over-the-counter preparations before consulting a physician, which can delay correct diagnosis of the etiology of symptoms. patient-collected, low vaginal swabs may be as useful as provider-collected specimen in making a diagnosis for the patient with vaginal discharge. the purpose of bimanual examination is to evaluate for signs of pelvic inflammatory disease and is not necessary in the low-risk patient with vaginal discharge. treatment of asymptomatic bv or vaginal yeast is not necessary in the nonpregnant patient or usually is not needed to test or treat partners of patients with isolated yeast or bv. when infection is recurrent, particularly when a woman's male partner is uncircumcised, treatment of the male partner for carriage of either infection may be warranted. options for treatment of recurrent infections are presented in etable 16-1. the treatment of warts is destructive and may serve to stimulate an immune response to the hpv-infected cells, which are typically "above" the surveillance mechanisms of the immune system in the epidermis. office methods of treatment include cryotherapy and trichloroacetic acid or podophyllin resin application. patients may apply podofilox 0.5% solution or gel or imiquimod 5% cream (table 16-13) . for more extensive cases of warts or intra-anal or intravaginal infections that are difficult to treat using the previous methods, surgical techniques may be necessary to achieve resolution. untreated, warts may resolve spontaneously, remain the same, or worsen. patients with pediculosis pubis, or pubic lice, most often present with pruritus or with visible nits. pubic lice are visible on inspection of the pubic area, as are nits, which are adherent to the hair shaft. partners of patients with pubic lice should also be treated to prevent reinfection. linens and clothing should be laundered or dry-cleaned or kept in a closed plastic container or bag for 72 hours. scabies diagnosis can be challenging. again, patients present with itching that can be anywhere on the body, although often in the genital area or on the buttocks when infection is sexual in origin. the pruritus associated with sarcoptes scabiei is a result of sensitization to the mite droppings underneath the skin as the mite burrows. the classic "burrow" or linear papular eruption is not always present. scraping of lesions with microscopic examination may be performed to identify the mite. as with pediculosis, close contacts should be treated. linens and clothing should be laundered or dry-cleaned or isolated in plastic containers for 72 hours. the pruritus-associated with scabies can take several weeks to resolve after treatment. patients living in group settings (dormitories or apartments) may reinfect one another as a result of inadequate primary treatment of all contacts ( cryotherapy trichloroacetic acid (tca): small amount applied until wart whitens podophyllin resin, 10% to 25% all these may be repeated every 1 to 2 weeks until warts are resolved. podofilox 0.5% solution or gel applied twice daily for 3 days, followed by 4 days of no therapy. imiquimod 5% cream applied once daily at bedtime three times a week for up to 16 weeks; washed off 6 to 10 hours after application. urinary tract infection (uti) is defined as significant bacteriuria in the presence of symptoms. uti accounts for a significant number of emergency department visits; an estimated 20% of women experience a uti in their lifetime. the urinary tract is normally sterile. uncomplicated uti involves the urinary bladder in a host without underlying renal or neurologic disease. the bladder mucosa is invaded, most often by enteric coliform bacteria (e.g., e. coli) that ascend into the bladder via the urethra. sexual intercourse can promote this migration, and cystitis is common in otherwise healthy young women. frequent and complete voiding has been associated with a reduction in the incidence of uti. complicated uti occurs in the setting of underlying structural, medical, or neurologic disease. signs and symptoms of a uti include dysuria, frequency, urgency, nocturia, enuresis, incontinence, urethral pain, suprapubic pain, low back pain, and hematuria. fever is unusual. up to 30% of patients with symptoms of cystitis have a smoldering pyelonephritis, especially when symptoms have been present for more than 1 week. a patient with pyelonephritis usually appears ill, with fever, sweating, and prostration, along with costovertebral angle (flank) tenderness in most cases. the differential diagnosis of uncomplicated uti includes use of diuretics or caffeine, interstitial cystitis, vaginitis, pregnancy, pelvic mass, pid, and benign prostatic hypertrophy (bph). if a uti is suspected, the initial test of choice is urinalysis, although with classic signs and symptoms of infection in women, this test is not always necessary. pyuria, as indicated by a positive result on the leukocyte esterase dip test, is found in the majority of patients with uti. the presence of urinary nitrites is fairly specific for uti. the combination of positive leukocyte esterase and nitrites improves sensitivity. on urine microscopy, levels of pyuria as low as two to five leukocytes per high-power field (2-5 wbcs/hpf) in a centrifuged specimen are significant in the female patient with appropriate symptoms, as is the presence of bacteriuria. urine culture and sensitivity are not needed in simple utis. cultures should be done in patients with recurrent utis, patients with pyelonephritis, and pregnant patients. antibiotic therapy can be given in a 3-day regimen for young, sexually active women. a 7-to 10-day course of antibiotics should be used in pregnant patients and patients with complicated utis. all the drugs listed in table 16 -15 can be used in a 3-day or 7-to 10-day course. clinical practice guidelines that include telephone assessment and treatment have shown a decrease in unnecessary laboratory utilization while maintaining quality of care (saint et al., 1999) . trimethoprim-sulfamethoxazole (tmp-smx) has been a mainstay of uti therapy, but in some localities, resistance of e. coli to tmp-smx is 20% (mehnert-kay, 2005) . if a urine culture is done and the organism is resistant to the drug prescribed, a change in antibiotics is indicated only if the patient is still symptomatic. for symptomatic treatment, a bladder anesthetic can be used, such as phenazopyridine (pyridium), 200 mg three times daily for 2 days. patients should be warned that this produces an orange tinge in tears and urine. patients should also be instructed to increase fluid intake. pyelonephritis is suggested by a failure of a short course of antibiotics. signs and symptoms of pyelonephritis include shaking chills and fever higher than 38.5° c (101.3° f), flank pain, malaise, urinary frequency and burning, and costover-tebral angle tenderness. the infection can produce septic shock. a patient who is unable to tolerate oral intake should be hospitalized and given empiric iv antibiotics aimed at broad-spectrum gram-negative coverage, such as third-generation cephalosporins, fluoroquinolones, or aminoglycosides, while awaiting results of blood and urine cultures. a 14-day course of antibiotic therapy (iv or po) is recommended. although the most common bacterial infection during pregnancy, the incidence of uti in pregnancy is similar to that reported in sexually active nonpregnant women of childbearing age. up to 40% of pregnant women with tmp-smx, 160/800 mg q12h trimethoprim, 100 mg q12h fluoroquinolones ‡ ciprofloxacin, 100-250 mg q12h ciprofloxacin xr, 500 mg qd gatifloxacin, 200 mg qd levofloxacin, 250 mg qd nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid amoxicillin, 250 mg q8h or 500 mg q12h cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. amoxicillin, 250 mg q8h or 500 mg q12h nitrofurantoin monohydrate/macrocrystals, 100 mg q12h nitrofurantoin macrocrystals, 50-100 mg qid cephalexin, 250 mg q6h, or other cephalosporin tmp-smx, 160/800 mg q12h male gender, diabetes, symptoms for 7 days, recent antimicrobial use, age > 65 tmp-smx, § 160/800 mg q12h fluoroquinolones, as per 3-day regimens cephalexin, 250 mg q6h, or other cephalosporin consider 7-day regimen. from hooton tm, stamm we. diagnosis and treatment of uncomplicated urinary tract infection. infect dis north am 1997;11:551. tmp-smx, trimethoprim-sulfamethoxazole; qd, every day; q12h, every 12 hours; q6h, every 6 hours; q8h, every 8 hours; qid; four times daily. * treatments listed to be prescribed before etiologic agent is known (gram stain may help); therapy can be modified when cause is identified. † characteristic pathogens are escherichia coli (85%-90%) and staphylococcus saprophyticus (5%-15%); other organisms account for less than 5% of cases and include proteus mirabilis, klebsiella pneumoniae, and enterococcus spp. ‡fluoroquinolones should not be used in pregnancy. § although classified as pregnancy category c, tmp-smx is widely used; however, avoid its use in the first and second trimesters. untreated bacteriuria in the first trimester develop acute pyelonephritis later in pregnancy. premature births and perinatal mortality are increased in pregnancies complicated by uti. therefore, in pregnant women, asymptomatic bacteriuria should be actively sought and aggressively treated with at least one urinalysis, preferably toward the end of the first trimester. nitrofurantoin, ampicillin, and the cephalosporins have been used most extensively in pregnancy and are the regimens of choice for treating asymptomatic or minimally symptomatic uti. tmp-smx should be avoided in the first trimester because of possible teratogenic effects and should be avoided near term because of a possible role in the development of kernicterus. fluoroquinolones are avoided because of possible adverse effects on fetal cartilage development. for pregnant women with overt pyelonephritis, admission to the hospital for parenteral therapy should be the standard of care; beta-lactam agents with or without aminoglycosides are the cornerstone of therapy. prevention of uti, including pyelonephritis, can be accomplished during pregnancy with nitrofurantoin or cephalexin taken prophylactically after coitus or at bedtime without relation to coitus. such prophylaxis should be considered for patients who have had acute pyelonephritis during pregnancy, patients with bacteriuria during pregnancy who have had a recurrence after a course of treatment, and patients who had recurrent uti before pregnancy that required prophylaxis. catheter-associated utis are associated with increased mortality and costs. risk factors for catheter-associated utis include the duration of catheterization, lack of systemic antibiotic therapy, female gender, age older than 50 years, and azotemia. to help prevent infection, urinary catheters should be avoided when possible and used only as long as needed. the catheter should be inserted with strict aseptic technique by trained persons, and a closed system should be used at all times. treatment of catheter-associated uti depends on the clinical circumstances. symptomatic patients (e.g., those with fever, chills, dyspnea, and hypotension) require immediate antibiotic therapy along with removal and replacement of the urinary catheter if it has been in place for a week or longer. in an asymptomatic patient, therapy should be postponed until the catheter can be removed. patients with long-term indwelling catheters seldom become symptomatic unless the catheter is obstructed or is eroding through the bladder mucosa. in patients who do become symptomatic, appropriate antibiotics should be administered and the catheter changed. therapy for asymptomatic catheterized patients leads to the selection of increasingly antibiotic-resistant bacteria. recurrence of uncomplicated cystitis in reproductive-age women is common, and some form of preventive strategy is indicated if three or more symptomatic episodes occur in 1 year. however, risk factors specific to women with recurrent cystitis have received little study (sen, 2008) . several antimicrobial strategies are available, but before initiating therapy, the patient should try such simple interventions as voiding immediately after sexual intercourse and using a contraceptive method other than a diaphragm and spermicide. ingestion of cranberry juice has been shown to be effective in decreasing bacteriuria with pyuria, but not bacteriuria alone or symptomatic uti, in an elderly population. cranberry juice may be effective for preventing uti in young, otherwise healthy women. if simple nondrug measures are ineffective, continuous or postcoital-if the infections are temporally related to intercourse-low-dose antimicrobial prophylaxis with tmp-smx, a fluoroquinolone, or nitrofurantoin should be considered. typically, a prophylactic regimen is initially prescribed for 6 months and then discontinued. if the infections recur, the prophylactic program can be instituted for a longer period. an alternative approach to antimicrobial prophylaxis for women with less frequent recurrences (<4 a year) is to supply tmp-smx or a fluoroquinolone and allow the patient to self-medicate with short-course therapy at the first symptoms of infection. a minority of patients have relapsing uti, as evidenced by finding the same bacterial strain within 2 weeks after completion of antimicrobial therapy. two factors can contribute to the pathogenesis of relapsing infection in women: (1) deep tissue infection of the kidney that is suppressed but not eradicated by a 14-day course of antibiotics and (2) structural abnormality of the urinary tract, particularly calculi. patients with true relapsing utis should undergo renal ultrasound, intravenous pyelogram (ivp), or voiding cystourethrogram, and longer-term therapy should be considered. urinary tract infection is one of the most common infections of childhood. factors predisposing to uti include taking broad-spectrum antibiotics (e.g., amoxicillin, cephalexin), which are likely to alter gastrointestinal and periurethral flora; incomplete bladder emptying or infrequent voiding; voiding dysfunction; and constipation. uti in young children serves as a marker for abnormalities of the urinary tract. imaging of the urinary tract is recommended in every febrile infant or young child with a first uti to identify children with abnormalities that predispose to renal damage. imaging should consist of urinary tract ultrasonography to detect dilation of the renal parenchyma. voiding cystourethrography is often ordered but does not appear to improve clinical outcomes in uncomplicated utis (alper and curry, 2005) . a common complication of uti in men is prostatitis. bacterial prostatitis is usually caused by the same gram-negative bacilli that cause uti in female patients; 80% or more of such infections are caused by escherichia coli. the pathogenesis of this condition is poorly understood. antibacterial substances in prostatic secretions probably protect against such infections. a national institutes of health (nih) expert consensus panel has recommended classifying prostatitis into three syndromes: acute bacterial prostatitis, chronic bacterial prostatitis, and chronic pelvic pain syndrome (cpps). acute bacterial prostatitis is a febrile illness characterized by chills, dysuria, urinary frequency and urgency, and pain in the perineum, back, or pelvis. the bladder outlet can be obstructed. on physical examination, the prostate is found to be enlarged, tender, and indurated. pyuria is present, and urine cultures generally grow e. coli or another typical uropathogen. chronic bacterial prostatitis is a clinically more occult disease and may be manifested only as recurrent bacteriuria or variable low-grade fever with back or pelvic discomfort. urinary symptoms usually relate to the reintroduction of infection into the bladder, with both pyuria and bacteriuria. a chronic prostatic focus is the most common cause of recurrent uti in men. cpps is the diagnosis for the large group of men who present with minimal signs on physical examination but have a variety of irritative or obstructive voiding symptoms; perineal, pelvic, or back pain; and sexual dysfunction. these men can be divided into those with and those without inflammation (defined as >10 wbcs/hpf in expressed prostatic secretions). the etiology and appropriate management in these patients, regardless of inflammatory status, is unknown. • laboratory findings in acute tick-borne infection often include a normal or low wbc count, thrombocytopenia, hyponatremia, and elevated liver enzymes. • doxycycline is the drug of choice for patients with rmsf. • appropriate antibiotic treatment should be initiated immediately with strong suspicion of ehrlichiosis. • if left untreated, lyme disease can progress to cognitive disorders, sleep disturbance, fatigue, and personality changes. in the united states, more vector-borne diseases are transmitted by ticks than by any other agent. tick-borne diseases can result from infection with pathogens that include bacteria, rickettsiae, viruses, and protozoa. most tick-borne diseases are transmitted during the spring and summer months when ticks are active. a knowledge of which species of tick is endemic in an area can help narrow the diagnosis (table 16-16) . rocky mountain spotted fever (rmsf) is the most severe and most often reported rickettsial illness in the united states. it is caused by rickettsia rickettsii, a species of bacteria that is spread to humans by ixodid (hard) ticks (figure 16-6) . initial signs and symptoms include sudden onset of fever, headache, and muscle pain, followed by development of rash. the disease can be difficult to diagnose in the early stage. rmsf is most common among males and children. risk factors are frequent exposure to dogs and living near wooded areas or areas with high grass. the presentation of rsmf is nonspecific, following an incubation of about 5 to 10 days after a tick bite. initial symptoms can include fever, nausea, vomiting, severe headache, muscle pain, and lack of appetite. later signs and symptoms include rash, abdominal pain, joint pain, and diarrhea. the rash first appears 2 to 5 days after the onset of fever. most often it begins as small, flat, pink, nonitchy spots on the wrists, forearms, and ankles. the characteristic red spotted rash of rmsf is usually not seen until the sixth day or later after onset of symptoms. as many as 10% to 15% of patients never develop a rash (figure 16-7) . no widely available laboratory assay provides rapid confirmation of early rmsf, although commercial pcr testing is available. therefore, treatment decisions should be based on epidemiologic and clinical clues. treatment should never be delayed while waiting for confirmation by laboratory results. routine clinical laboratory findings suggestive of rmsf include normal wbc count, thrombocytopenia, hyponatremia, and elevated liver enzyme levels. serologic assays are the most often used methods for confirming cases of rmsf. doxycycline is the drug of choice for patients with rmsf. therapy is continued for at least 3 days after fever subsides and until there is unequivocal evidence of clinical improvement, generally for a minimum total course of 5 to 10 days. tetracyclines are usually not the preferred drug for use in pregnant women. whereas chloramphenicol is typically the preferred treatment for rmsf during pregnancy, care must be used when administering chloramphenicol late during the third trimester of pregnancy because of risks associated with gray baby syndrome. three species of ehrlichia in the united states are known to cause disease in humans. ehrlichia chaffeensis, the cause of human monocytic ehrlichiosis, occurs primarily in southeastern and south-central regions and is primarily transmitted by the lone star tick, amblyomma americanum ( figure 16-8) . human granulocytic ehrlichiosis is caused by anaplasma phagocytophila or anaplasma equi and is transmitted by ixodes ticks. ehrlichia ewingii is the most recently recognized human pathogen, with cases reported in immunocompromised patients in missouri, oklahoma, and tennessee. after an incubation period of about 5 to 10 days following the tick bite, initial symptoms generally include fever, pregnant women should be screened for asymptomatic bacteriuria in the first trimester of pregnancy (wadland and plante, 1989) (sor: a). pregnant women who have asymptomatic bacteriuria should be treated with antimicrobial therapy for 3 to 7 days (nicolle et al., 2005) (sor: b) . pyuria accompanying asymptomatic bacteriuria should not be treated with antimicrobial therapy (nicolle, 2003) (sor: c1). a 3-day course of tmp-smx (bactrim, septra) is recommended as empiric therapy of uncomplicated utis in women, in regions where the rate of resistant e. coli is less than 20% (warren et al., 1999) (sor: c). fluoroquinolones are not recommended as first-line treatment of uncomplicated utis, to preserve their effectiveness for complicated utis (warren et al., 1999) (sor: c). a randomized, placebo-controlled trial of 150 women over 12 months found that cranberry juice and cranberry extract tablets significantly decreased the number of patients having at least one symptomatic uti per year (stothers, 2002) appropriate antibiotic treatment should be initiated immediately when there is a strong suspicion of ehrlichiosis on the basis of clinical and epidemiologic findings. the treatment recommendations are the same as for rocky mountain spotted fever. rifampin has been used successfully in a limited number of pregnant women with documented ehrlichiosis. babesiosis is caused by hemoprotozoan parasites of the genus babesia. the white-footed deer mouse is the main reservoir in the united states, and the vector is ixodes ticks. most infections are probably asymptomatic. manifestations of disease include fever, chills, sweating, myalgias, fatigue, hepatosplenomegaly, and hemolytic anemia. symptoms typically occur after an incubation period of 1 to 4 weeks and can last several weeks. the disease is more severe in immunosuppressed, splenectomized, or elderly patients. diagnosis can be made by microscopic examination of thick and thin blood smears stained with giemsa, looking for the parasite in red blood cells (rbcs). options for treatment include clindamycin plus quinine or atovaquone plus azithromycin. lyme disease is caused by the spirochetal bacterium borrelia burgdorferi. ixodes ticks are responsible for transmitting lyme disease bacteria to humans. in the united states, lyme disease is mostly localized to states in the northeastern, mid-atlantic, and upper north-central regions, as well as northwestern california. lyme disease most often manifests with a characteristic bull's-eye rash (erythema migrans) accompanied by nonspecific symptoms such as fever, malaise, fatigue, headache, muscle aches, and joint aches (figure 16-9) . lyme disease spirochetes disseminate from the site of the tick bite, causing multiple (secondary) erythema migrans lesions. other manifestations of dissemination include lymphocytic meningitis, cranial neuropathy (especially facial nerve palsy), radiculoneuritis, migratory joint and muscle pains, myocarditis, and transient atrioventricular blocks of varying degree. if left untreated, the disease can progress to intermittent swelling and pain of one or a few joints (usually large weight-bearing joints such as the knee), cognitive disorders, sleep disturbance, fatigue, and personality changes. the diagnosis is based primarily on clinical findings, and it is often appropriate to treat patients with early disease solely on the basis of objective signs and a known exposure. serologic testing may provide valuable supportive diagnostic information in patients with endemic exposure and objective clinical findings that suggest later-stage disseminated lyme disease. treatment for 3 to 4 weeks with doxycycline or amoxicillin is generally effective in early disease. cefuroxime axetil or erythromycin can be used for persons allergic to penicillin or who cannot take tetracyclines. later disease, particularly with objective neurologic manifestations, can require treatment with intravenous ceftriaxone or penicillin for 4 weeks or more, depending on disease severity. tularemia is caused by francisella tularensis, one of the most infectious pathogenic bacteria known. most cases in the united states occur in south-central and western states. humans can become infected through diverse environmental exposures, including bites by infected arthropods; handling infectious animal tissues or fluids; direct contact with or ingestion of contaminated food, water, or soil; and inhalation of infective aerosols. inhaled f. tularensis causes pleuropneumonitis. some exposures contaminate the eye, resulting in ocular tularemia; penetrate broken skin, resulting in ulceroglandular or glandular disease; or cause oropharyngeal disease with cervical lymphadenitis. untreated, bacilli inoculated into skin or mucous membranes multiply, spread to regional lymph nodes, multiply further, and then can disseminate to organs throughout the body. the onset of tularemia is usually abrupt, with fever, headache, chills and rigors, generalized body aches, coryza, and sore throat. a dry or slightly productive cough and substernal pain or tightness often occur with or without objective signs of pneumonia. nausea, vomiting, and diarrhea can occur. sweats, fever, chills, progressive weakness, malaise, anorexia, and weight loss characterize continuing illness. rapid diagnostic testing for tularemia is not widely available. respiratory secretions and blood for culture should be collected in suspected patients and the laboratory alerted to the need for special diagnostic and safety procedures. streptomycin (1 g im bid for 10 days) is the drug of choice, and gentamicin is an acceptable alternative. tetracyclines and chloramphenicol can also be used. colorado tick fever is an acute viral infection transmitted by the bite of the dermacentor andersoni tick (figure 16-10) . the disease is limited to the western united states and is most prevalent from march to september. symptoms start about 3 to 6 days after the tick bite. fever continues for 3 days, stops, and then recurs 1 to 3 days later for another few days. other symptoms include excessive sweating, muscle aches, joint stiffness, headache, photophobia, nausea, vomiting, weakness, and an occasional faint rash. routine blood tests might show a low wbc count, mildly elevated liver function, and mildly elevated creatine phosphokinase (cpk). diagnosis is confirmed by testing blood for complement fixation immunofluorescent antibody staining to colorado tick virus. treatment is removal of the tick and treatment of symptoms. physicians should advise patients who walk or hike in tickinfested areas to tuck long pants into socks to protect the legs and wear shoes and long-sleeved shirts. ticks show up on white or light colors better than dark colors, making them easier to remove from clothing. if attached, ticks should be removed immediately by using a tweezers, pulling carefully and steadily. insect repellents such as deet, alone or in combination with permethrin, may be helpful. • most cases of cellulitis are caused by staphylococci or streptococci, but other causes should be considered by clinical situation. • physicians must rule out more ominous causes of skin inflammation, such as necrotizing fasciitis and pyomyositis, when considering cellulitis. • edema-associated cellulitis is best treated by mobilizing edema fluid. cellulitis is an acute, spreading inflammation of the derma and subcutaneous issue. patients complain of tenderness, warmth, swelling, and spreading erythema. in contrast to erysipelas, cellulitis usually lacks sharp demarcation at the border. factors that predispose to cellulitis include trauma, an underlying skin lesion (furuncle, ulcer), or a complication arising from a wound, ulcer, or dermatosis. occasionally, cellulitis results from a blood-borne infection that metastasizes to the skin. pain and erythema usually develop within several days and are often associated with malaise, fever, and chills. the area involved is often extensive, red, hot, and swollen. patchy involvement with skip lesions can be seen. regional lymphadenopathy is common, and bacteremia can occur. several clinical entities resemble cellulitis, including pyoderma gangrenosum, gout, and insect bites. necrotizing fasciitis and gas gangrene are surgical emergencies. given that the predominant organism involved in most cases of cellulitis is a grampositive coccus, clinical history and morphology on physical examination usually suffice in the diagnosis and treatment of cellulitis. a history of freshwater exposure may implicate aeromonas hydrophila as the causative organism; saltwater appropriate antibiotic therapy should be initiated immediately when there is suspicion of rocky mountain spotted fever, ehrlichiosis, or relapsing fever rather than waiting for laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). treatment with doxycycline (vibramycin) or tetracycline is recommended for rmsf, lyme disease, ehrlichiosis, and relapsing fever (bratton and corey, 2005; spach et al., 1993) (sor: c). recommended actions to prevent tick-borne disease include avoidance of tick-infested areas; wearing long pants and tucking the pant legs into socks; applying diethyltoluamide (deet) insect repellents; using bed nets when camping; and carefully inspecting oneself frequently while in an at-risk area (bratton and corey, 2005; spach et al., 1993) (sor: c). antibiotic prophylaxis is not routinely recommended for a tick bite to prevent lyme disease, unless the risk of infection is high (wormser et al., 2006) (sor: b). recommended treatment for suspected tularemia is streptomycin or gentamicin given empirically before evidence of laboratory confirmation (bratton and corey, 2005; spach et al., 1993) (sor: c). exposure suggests vibrio spp. cellulitis in a patient with liver disease and shellfish ingestion moves vibrio vulnificans to the top of the differential. patients with soft tissue infection should have blood drawn for laboratory testing if signs and symptoms of systemic toxicity are present (e.g., fever or hypothermia, tachycardia, hypotension). laboratory testing should include blood culture and drug susceptibility tests; wbc count with differential; and measurement of creatinine, bicarbonate, cpk, and crp levels. hospitalization should be considered for patients with hypotension or an elevated creatinine level, low serum bicarbonate level, elevated cpk level (i.e., 2-3 times upper limit of normal), marked left shift, or crp level greater than 13 mg/l (123.8 nmol/l). gram stain with culture and culture of needle aspiration or punch biopsy specimens should be performed to determine a definitive etiology, and a surgical consult should be considered for inspection, exploration, and drainage. findings that may signal potentially severe, deep, soft tissue infection and that may require emergent surgical evaluation include cutaneous hemorrhage, gas in the tissue, pain disproportionate to physical findings, rapid progression, skin anesthesia, skin sloughing, and violaceous bullae. radiologic studies may be helpful if abscess or osteomyelitis is a possibility. ultrasonography is helpful in detecting a subcutaneous collection of fluid. magnetic resonance imaging (mri) is also useful in differentiating cellulitis from necrotizing fasciitis. the diagnosis of necrotizing cellulitis is by direct surgical examination or by frozen pathology sections. empiric antibiotics for immunocompetent patients with cellulitis should be targeted toward gram-positive cocci (table 16-17) . broader coverage should be initiated for diabetic patients to include gram-positive aerobes, gram-negative aerobes, and anaerobes. patients who present with severe infection or whose infection is progressing despite empiric antibiotic therapy should be treated more aggressively; the treatment strategy should be based on results of appropriate gram stain, culture, and drug susceptibility analysis. in the case of staphylococcus aureus, the physician should assume that the organism is resistant, and agents effective against mrsa, such as vancomycin, linezolid (zyvox), or daptomycin (cubicin), should be used. the antibiotic may be switched from an intravenous drug to an oral drug when fever has subsided and the skin lesion begins to resolve, usually in 3 to 5 days. the total duration of therapy should be 7 to 14 days. longer duration may be required if the response is slow or is associated with abscess, tissue necrosis, or underlying skin processes (infected ulcers or wounds). treatment of cellulitis should include elevation and immobilization to decrease swelling. patients with interdigital dermatophytic infections should be treated with a concomitant topical antifungal applied once or twice daily. topical antifungals can also help reduce the risk of recurrence of the cellulitis. support stockings, good skin hygiene, and prompt treatment of tinea pedis helps with prevention of cellulitis in patients with peripheral edema, who are predisposed to recurrence. in patients who continue to have frequent episodes of cellulitis or erysipelas, prophylactic treatment with penicillin v, 250 mg or 500 mg orally twice daily, or erythromycin, 250 mg once or twice daily (for penicillin-allergic patients), may be indicated. • the majority of furuncles and carbuncles are caused by staphylococcus spp., increasingly, community-acquired methicillin-resistant s. aureus. • drainage of pus is of primary importance in treating skin and soft tissue infections. • culture of sstis is important in guiding antibiotic treatment when initial measures of drainage are not effective. • for recurrent boils, consider referral to infectious disease specialist, possibly to eradicate carriage state. furuncles, or boils, are infections of the skin and soft tissue usually associated with a hair follicle. carbuncles are an extension of this skin and soft tissue infection continuum and involve more of the surrounding and subcutaneous tissue. the broad category skin and soft tissue infections (sstis) is used to describe this continuum that includes furuncles and carbuncles. sstis are common in both healthy and immunocompromised patients and likely initiate with some breach of the skin integrity, such as irritation of hair follicles from friction or microscopic trauma to the skin. up to 74% of furuncles and carbuncles are caused by community-acquired methicillin-resistant staphylococcus aureus (ca-mrsa) (cdc, 2010). other potential causative organisms include nonresistant staphylococcus spp. and streptococcus spp. it has become increasingly important to obtain culture of a lesion to direct antibiotic coverage given the increase in ca-mrsa. there is no reliable historical or examination element that will distinguish a ca-mrsa from a methicillin-sensitive staphylococcal skin lesion. stereotypically, patients report ca-mrsa lesions starting like a spider bite. furuncles and carbuncles can occur anywhere on the body, although the axillae, groin, and buttocks are particularly common sites. in addition, practices that cause skin trauma (e.g., shaving, waxing) are often noted in patients with these sstis. fever and malaise are uncommon with milder lesions but become more frequent with the increasing scope of localized infection. of primary importance in the management of carbuncles and furuncles is facilitation of drainage of any purulent material. with smaller lesions, this may be accomplished by heat application by the patient at home. as lesions increase in size and fluctuance, surgical drainage is essential to facilitate resolution of an ssti. it is important to consider culture penicillin, given parenterally or orally depending on clinical severity, is the treatment of choice for erysipelas (sor: a). for cellulitis, a penicillinase-resistant semisynthetic penicillin (amoxicillin/clavulanate) or a first-generation cephalosporin should be selected, unless streptococci or staphylococci resistant to these agents are common in the community (sor: a). for suspected mrsa skin infections, oral treatment options include trimethoprim-sulfamethoxazole, clindamycin, and doxycycline of purulent material when performing incision and drainage in the event that the patient fails to improve and antibiotic coverage becomes necessary. cure rates of lesions with drainage alone exceed 90%. careful follow-up after drainage is essential to ensure clinical improvement; daily dressing changes in the office after surgical drainage is effective. the addition of postdrainage antibiotics has not shown much added benefit. to prevent the spread of infection to others who come into contact with the patient recovering from an ssti, an occlusive dressing to prevent leakage of lesion fluid and careful hygiene are indicated. there is no evidence that extensive cleaning of common spaces (e.g., locker rooms) prevents the spread of ssti-causing bacteria more than routine cleaning measures. towels and soiled clothing should be laundered in hot water, and any common equipment should be cleaned per manufacturer recommendations. when lesions do not respond to heat, or when lesions are larger yet not amenable to drainage, antibiotics may be used. reasonable first-line antibiotic coverage for nonfluctuant lesions may include dicloxacillin, first-or secondgeneration cephalosporins, macrolides, or clindamycin. in patients with suspected ca-mrsa, better choices include tmp-smx, tetracycline, or clindamycin. it is important to note that up to 50% of ca-mrsa species will be resistant to clindamycin, particularly if the patient has been treated with other antibiotics in the previous weeks to months . oral administration of these antibiotics is acceptable in the nontoxic patient. patient signs and symptoms that would warrant hospital admission include fever or hypothermia, tachycardia, or hypotension as signs of sepsis and lesions greater than 5 cm in size (table 16-18) . for patients with recurrent sstis, evaluation for the presence of nasal carriage with a nasal culture is indicated. the value of eradication of bacterial carriage is unclear. referral for infectious disease specialist evaluation may be indicated to guide decision making in the patient with recurrent furuncles and carbuncles. • the existence, severity, and extent of infection, as well as vascular status, neuropathy, and glycemic control, should be assessed in patients with a diabetic foot infection. • visible bone and palpable bone on probing suggest underlying osteomyelitis in patients with a diabetic foot infection. • before an infected wound of a diabetic foot infection is cultured, any overlying necrotic debris should be removed to eliminate surface contamination and to provide more accurate results. patients with diabetes are prone to skin ulcers caused by neuropathy, vascular insufficiency, and diminished neutrophil function. minor wounds can be secondarily infected, leading to ulcer formation. these ulcers often have extensive undermining with necrotic tissues and are often close to the anus, thus promoting an environment suitable for multiple species of microorganisms, including anaerobes. diabetic foot infections range in severity from superficial paronychia to deep infection involving bone. non-limb-threatening infections involve superficial ulcers with minimal cellulitis (<2 cm from portal of entry), no signs of systemic toxicity, and no significant ischemia in the limb. cure rates of fluctuant skin lesions with drainage alone is over 90%. postdrainage antibiotics do not significantly improve outcomes rajendran et al., 2007) (sor: a). trimethoprim-sulfamethoxazole (tmp-smx), clindamycin, and tetracycline are first-choice antibiotics when ca-mrsa is suspected. up to 50% of ca-mrsa species will be resistant to clindamycin, particularly in the patient previously treated with other antibiotics (sor: c). subcutaneous tissues, and prominent ischemia. infection in patients who have recently received antibiotics or who have deep, limb-threatening infection or chronic wounds are usually caused by a mixture of aerobic gram-positive, aerobic gram-negative (e.g., escherichia coli, proteus spp., klebsiella spp.), and anaerobic organisms (e.g., bacteroides, clostridium, peptococcus, and peptostreptococcus spp.) . surgery is necessary to unroof encrusted areas, and the wounds need to be examined and probed to determine the extent of the infection and check for bone involvement (dinh et al., 2008) . debridement or drainage should be promptly performed. deep wound cultures should be obtained if possible. if deep culture is not feasible, gram stain and culture from the curettage of the base of the ulcer or from purulent exudates may be needed to guide antibiotic therapy (figure 16-11) . plain radiography of the foot is indicated for detection of osteomyelitis, foreign bodies, and soft tissue gas. when plain radiography is negative but osteomyelitis is clinically suspected, radionuclide scan or mri should be performed. mri provides more accurate information regarding the extent of the infectious process. the presence of peripheral artery disease and neuropathy should be assessed. the antibiotic regimen should be based on meaningful bacteriologic data. however, the initial regimen for a previously untreated patient with non-limb-threatening infection should focus on s. aureus and streptococci. mild infections may be treated with dicloxacillin or cephalexin for 2 weeks. amoxicillin/clavulanate may be used if polymicrobial infection is suspected. if msra is suspected, oral treatment options include tmp-smx or doxycycline. for limb-threatening infections, broad-spectrum antibiotics are recommended for coverage of group b streptococci, other streptococci, enterobacteriaceae, anaerobic gram-positive cocci, and bacteroides spp. treatment regimens include ampicillin-sulbactam or ertapenem (invanz), clindamycin plus a third-generation cephalosporin, and clindamycin plus ciprofloxacin. intravenous vancomycin should be added if mrsa infection is suspected. ciprofloxacin as a single agent is not recommended. in addition to antibiotic treatment, good glycemic control should be obtained and open wounds gently packed with sterile gauze moistened with ¼-strength povidone-iodine (betadine) solution. edema should be reduced by bed rest, elevation, and diuretic therapy as indicated. for prevention of diabetic foot ulcers, all patients with diabetes should have an annual foot examination that includes assessment for anatomic deformities, skin breaks, nail disorders, loss of protection sensation, diminished arterial supply, and inappropriate footwear. • the use of prophylactic antibiotics may be necessary in the initial management of bite wounds, particularly if the bite is on the hand or face or from a cat. • first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy for bite wounds because of resistance issues. • avoid primary wound closure in the management of bite wounds. it is estimated that bites account for 800,000 medical visits annually in the united states, making up 1% of emergency department visits. bite wounds consist of lacerations, evulsions, punctures, and scratches. the microbiology of bite wounds is generally polymicrobial, with an array of potential bacteria from the environment, the victim's skin flora, and the biter's oral flora. dog bites account for approximately 80% of all animal bites requiring medical attention, in which 85% are provoked attacks. most dog bites occur on the distal extremities, but children tend to sustain facial bites. patients who present for medical attention are often concerned about the care of disfiguring wounds or the need for appropriate vaccination (i.e., tetanus, rabies). however, up to 30% of medically treated wounds may become infected. these wounds are often contaminated with multiple strains of aerobic and anaerobic bacteria. local signs of infection with erythema, edema, pain, and purulent drainage are common with animal bite wounds. although the most frequently isolated pathogen related to dog and cat bite wounds is pasteurella multocida, the array of potential organisms is much greater. anaerobes such as bacteroides tectum, prevotella spp., fusobacteria, and peptostreptococci can be isolated from animal bite wounds 75% of the time, mostly from wounds with abscess formation. capnocytophaga canimorsus has also been associated with fatal infection from fulminant sepsis in asplenic patients. wounds inflicted by cats are often scratches or tiny punctures located on the extremity and are likely to become infected and lead to abscess formation. in the united states, venomous snakes bite approximately 8000 people yearly. envenomation in such snakebites account for the majority of morbidity and mortality associated with such bites. however, infection of soft tissue structures may also occur as a result of oral flora from the snake, which tends to be fecal in nature because live prey usually defecate in the snake's mouth with their ingestion. human bites are not uncommon, especially in children. human bites have a higher complication and infection rate than do animal bites. human bite wounds most often affect the hand and fingers and in some cases may present as "love routine wound swabs and cultures of material from sinus tracts are unreliable and strongly discouraged in the management of diabetic foot infection (pellizzer et al., 2001; senneville et al., 2006) nips" to the breast and genital areas. self-inflicted bites often include wounds of the lip and tissues surrounding the nail, such as paronychia. also included in this are clenched-fist injuries or "fight bites," which result in small lacerations to the knuckles when striking a person in the mouth. normal human oral flora, rather than skin flora, is the source of most bacteria isolated from human bite wound cultures (viridans streptococci, eikenella corrodens). management of bite wounds is the same as for any other wound: good wound care in the form of adequate irrigation and debridement of nonviable tissue as needed (table 1619) . bite wounds in general do not require primary closure, but after adequate irrigation and debridement, wounds may be approximated and closed by delayed primary or secondary intention. an exception to this rule may include bite wounds to the face. general wound management measures such as tetanus toxoid administration should also be employed. bite wounds involving the hands should be evaluated by a hand surgeon, given the risk of adjacent tendon sheath, bone, or joint involvement and the dire consequences if such structures are involved. the transmission of rabies through the bites of domestic pets in the united states and developed countries is rare. in fact, the dog strain of rabies is considered eliminated in the u.s. dog population, and cat bites are often managed through observation of the animal, without the immediate need for rabies postexposure treatment (pet). however, wild mammal exposure, especially bat, skunk, or raccoon, often warrants pet, which involves thorough cleaning of the bite wound, ideally with povidone-iodine solution, along with rabies immune globulin given at the wound site and rabies vaccine given on days 0, 3, 7, and 14. bite wounds should be considered contaminated wounds from presentation, given the oral microbial flora of humans and animals, and most patients should probably receive antibiotics early. empiric antibiotics are used to eradicate oral flora inoculated from the mouth of the biter, whether human or animal, into the wound. all moderate to severe animal bite wounds, or wounds that have an associated crush injury or that are close to a bone or joint, should be considered contaminated with potential pathogens, and these patients should receive 3 to 5 days of "prophylactic" antimicrobial therapy. gram stains with culture of bite wounds are specific but not sensitive indicators of bacterial growth. nonetheless, gram stain can be used to help guide initial empiric antibiotic therapy. amoxicillin-clavulanic acid (amoxicillin-clavulanate; augmentin) or penicillin plus a penicillinase-resistant penicillin are normally first-line agents for empiric therapy directed at bite wounds. first-generation cephalosporins (e.g., cephalexin) are not effective as monotherapy because of resistance of some anaerobic bacteria and e. corrodens. a 5-to 10-day course of antibiotics is usually adequate for infections limited to the soft tissue, and a minimum of 3 weeks of therapy is required for infections involving joints or bones. close follow-up is required in all bites to ensure adequate healing. of special consideration in human bite wounds is the potential for spread of viral pathogens, most notably hepatitis b virus (hbv) and hiv, if the source person is positive. hbv exposure in this setting should be handled in the same manner as other exposures, with administration of hbig and hbv vaccination. with regard to hiv, cdc guidelines for managing nonoccupational hiv exposure recommend handling each case individually in consultation with an infectious diseases specialist. • the diagnosis of osteomyelitis is based on radiographic findings (plain radiograph or mri) showing bony destruction along with histologic analysis and culture results. • chronic osteomyelitis is not an emergency, and antibiotics can be safely withheld until an etiologic diagnosis is established. • diabetic foot infections require a careful evaluation to assess perfusion and vascular supply, and corrective measures should be undertaken to reestablish adequate perfusion if necessary. • in diabetic foot ulcers, if one can probe to bone, the patient most likely has osteomyelitis. • orthopedic hardware infections are best managed in conjunction with an infectious diseases specialist and orthopedic surgeon. osteomyelitis is defined as progressive, inflammation leading to destruction of the bone, usually secondary to an infectious agent. bacteria can enter bone through hematogenous seeding or a contiguous focus after trauma, implantation of a foreign device, or a local soft tissue infection. acute osteomyelitis is defined as infection that evolves over a few weeks. chronic osteomyelitis implies persistent infection of several weeks to months. hematogenous osteomyelitis occurs primarily in children within the metaphyses of long bones (tibia and femur) and vertebrae in adults. in addition to local signs of inflammation and infection, patients generally have various systemic signs, including fever, irritability, and lethargy. physical findings include tenderness over involved area and decreased range of motion in adjacent joints. chronic osteomyelitis usually occurs in adults, caused by an open injury to bone and surrounding soft tissue. erythema, drainage around area, and bone pain are usually present on physical examination. systemic symptoms occur less frequently. the diagnosis of osteomyelitis is based on the clinical picture and supporting laboratory and radiologic findings. leukocytosis and elevations in crp and esr may use of antibiotic prophylactic after bites of the hand reduces the incidence of infection (medeiros and saconato, 2005) (sor: b) . antibiotic prophylaxis after bites by humans reduces incidence of infection (sor: c). animal bite: ascertain the type of animal, whether the bite was provoked or unprovoked, and the situation/environment in which the bite occurred. if the species can be rabid, locate the animal for 10 days' observation or sacrifice. patient: obtain information on antimicrobial allergies, current medications, splenectomy, mastectomy, liver disease, and immunosuppression. record a diagram of the wound with the location, type, and depth of injury; range of motion; possibility of joint penetration; presence of edema or crush injury; nerve and tendon function; signs of infection; and odor of exudate. infected wounds should be cultured and a gram stain performed. anaerobic cultures should be obtained in the presence of abscesses, sepsis, serious cellulitis, devitalized tissue, or foul odor of the exudate. small tears and infected punctures should be cultured with a minitipped (nasopharyngeal) swab. copious amounts of normal saline should be used for irrigation. puncture wounds should be irrigated with a "high-pressure jet" from a 20-ml syringe and an 18-gauge needle or catheter tip. devitalized or necrotic tissue should be cautiously debrided. debris and foreign bodies should be removed. radiographs should be obtained if fracture or bone penetration is possible to provide a baseline for future osteomyelitis. wound closure may be necessary for selected, fresh, uninfected wounds, especially facial wounds, but primary wound closure is not usually indicated. wound edges should be approximated with adhesive strips in selected cases. prophylaxis: consider prophylaxis (1) for moderate to severe injury less than 8 hours old, especially if edema or crush injury is present; (2) if bone or joint penetration is possible; (3) for hand wounds; (4) for immunocompromised patients (including those with mastectomy, liver disease, or steroid therapy); (5) if the wound is adjacent to prosthetic joint; and (6) if the wound is in the genital area. coverage should include pasteurella multocida, staphylococcus aureus, and anaerobes. treatment: cover p. multocida, s. aureus, and anaerobes. use oral medication if the patient is seen early after a bite and only mild to moderate signs of infection are present. the following can be considered for cat or dog bites in adults: • first choice: amoxicillin/clavulanic acid, 875/125 mg bid or 500/125 mg tid with food. • penicillin allergy: no alternative treatment for animal bites has been established for penicillin-allergic patients. the following regimens can be considered for adults: 1. clindamycin (300 mg po qid) plus either levofloxacin (500 mg po daily) or trimethoprim-sulfamethoxazole (2 double-strength tablets po bid). 2. doxycycline, 100 mg po bid. 3. moxifloxacin, 400 mg po daily. 4. in the highly penicillin-allergic pregnant patient, macrolides have been used, but the wounds must be watched carefully. on emergency department discharge, a single starting dose of parenteral antibiotic, such as ertapenem (1 g), may be useful in selected cases. if hospitalization or closely monitored outpatient follow-up is required, intravenous agents should be used. current choices include ampicillin/sulbactam and cefoxitin. the rising incidence of community-acquired s. aureus isolates that are methicillin resistant and therefore resistant to the drugs recommended here emphasizes the importance of susceptibility-testing any s. aureus isolates. indications include fever, sepsis, spread of cellulitis, significant edema or crush injury, loss of function, a compromised host, and patient noncompliance. give tetanus booster (td; tetanus and diphtheria toxoids for adults) if original three-dose series has been given but none in the past 5 years. adults who have not received acellular pertussis vaccine (tdap), should be given this instead of td. give a primary series and tetanus immune globulin if the patient was never immunized. rabies vaccine (on days 0, 3, 7, 14, and 28) with hyperimmune globulin may be required, depending on the type of animal, ability to observe the animal, and locality. elevation may be required if any edema is present. lack of elevation is a common cause of therapeutic failure. be seen but can also be normal. blood cultures may be positive in up to half of children with acute osteomyelitis. if plain radiographs show bone destruction and inflammation; the diagnosis of osteomyelitis is confirmed. typical findings on plain-radiographs will include osteolysis, periosteal reaction, and sequestra (segments of necrotic bone separated from living bone by granulation tissue). findings seen on plain radiographs usually denote a process that has been ongoing for at least 2 weeks. bone scintigraphy (bone scan) is often performed on patients with suspected osteomyelitis; however, sensitivity is quite low, and a negative result can offer false reassurance to the physician, so its routine use is not recommended. if the plain-radiographs are negative but the suspicion for osteomyelitis is still high, an mri scan should be considered. once the diagnosis of osteomyelitis has been made, the next step is to obtain an etiologic diagnosis. histopathologic and microbiologic examination of bone is the "gold standard." cultures of sinus tracts are not reliable for identifying the causative organism. common causative bacteriologic organisms in neonates include staphylococcus aureus, group b streptococci, and escherichia coli. later in life, s. aureus is most common, and in elderly persons, gram-negative organisms such as pseudomonas aeruginosa and serratia spp. have increased incidence. empiric antibiotics are rarely required for chronic disease but are often necessary for acute osteomyelitis. ideally, surgical debridement of all necrotic tissue and inflammatory debris (pus) should be undertaken and multiple surgical cultures with bone histology samples obtained. antimicrobial therapy will be dictated by test results. generally, treatment is for 4 to 6 weeks. with the exception of the fluoroquinolone class of antibiotics, which achieve high serum levels with oral administration, bone antibiotic levels cannot exceed the minimum inhibitory concentration (mic) for the infecting organism; therefore, antibiotics must be given intravenously. this underscores the importance in obtaining a bacterial diagnosis so that the appropriate antibiotic can be used for the duration of treatment. acute osteomyelitis is usually readily curable; however, chronic osteomyelitis is generally more refractory to therapy and requires repeat debridement and antibiotic courses. patients with uncontrolled diabetes are at increased risk for development of osteomyelitis, especially in the presence of neuropathy or venous or arterial insufficiency. s. aureus and beta-hemolytic streptococci are the predominant organisms, although other gram-positive or gram-negative aerobic or anaerobic bacteria may also be seen. plain radiographs should be the initial test to evaluate for the presence of osteomyelitis, followed by mri if negative. if there is a draining sinus, the "probe to bone" test should be performed with a sterile probe; if bone is palpated, the diagnosis of osteomyelitis is highly likely. further evaluation of the diabetic patient should be to assess for vascular insufficiency with the use of ankle-brachial indices and transcutaneous oximetry. if significant compromise is found, arteriography followed by revascularization should be undertaken. surgical debridement is again the cornerstone of treatment, along with antibiotics directed toward the causative microorganism. infections secondary to orthopedic hardware devices have become common problems with the increasing incidence of hip, knee, and shoulder replacement surgeries. also, patients with traumatic injury resulting in a fracture often have hardware implanted to stabilize the bone. these patients present in one of the three following ways: 1. early: symptoms develop less than 3 months after surgery and have an acute presentation with pain, erythema, and warmth, usually caused by s. aureus and gram-negative bacilli. 2. delayed: symptoms develop 3 to 24 months after surgery, generally with subtle signs of infection, including implant loosening and persistent pain, and usually caused by less virulent organisms such as coagulasenegative staphylococci and propionibacterium acnes. 3. late: symptoms develop 24 months after surgery and are usually caused by hematogenous seeding from skin, dental, respiratory, and urinary infections. treatment requires debridement of the surrounding tissue and hardware removal, although this cannot always be done in patients with bone instability. it is recommended that treatment follow-up should occur at 24 hours and perhaps 48 hours for outpatients. reporting the incident to a local health department may be required. from goldstein ejc. bites. in mandell gl, bennett je, dolin rd (eds). mandell, douglas, and bennett's principles and practice of infectious diseases, 7th ed. philadelphia, churchill livingstone--elsevier, 2010. po, orally; bid, twice daily; tid, three times daily; qid, four times daily. of these infections be done in conjunction with an infectious diseases specialist working with the orthopedic surgeon. septic arthritis is defined as infection within the joint space of two bones. the major causative organisms include s. aureus and in the sexually promiscuous individual, neisseria gonorrhoeae. intravenous drug users are likely to develop septic arthritis within unusual joints (e.g., sternoclavicular, sacroiliac). rheumatoid arthritis, presence of joint prostheses, and steroid use are predisposing factors for development of septic arthritis. diagnosis is usually based on clinical presentation of a warm, swollen joint with limitation in range of motion. a joint aspiration should be completed and the synovial fluid sent for gram stain with culture, wbc count with differential, and crystal analysis to rule out gout and pseudogout. blood cultures should also be drawn before initiation of antibiotics. gonococcal arthritis usually presents as an acute arthritis involving one or more joints in a sexually active individual. two thirds of patients have dermatitis with one or multiple, usually asymptomatic, lesions that progress from macular to papular and finally vesicular or pustular. joint fluid, urethral, and rectal cultures should also be obtained. treatment is generally with a third-generation cephalosporin intravenously until improvement, followed by oral therapy to complete a 1-week course of therapy. treatment of nongonococcal arthritis requires proper draining of the infected joint. this is often done surgically, although repeat needle drainage may also be successful if the joint is easily accessible. treatment generally depends on the gram stain and includes a third-generation cephalosporin, with the addition of vancomycin if gram-positive cocci in clusters are seen. duration of therapy is 3 to 4 weeks. • a comprehensive history and physical examination with laboratory and radiologic evaluation are important in the workup for fever of unknown origin (fuo). • if routine information is unrevealing, more specific testing for fuo is undertaken based on the patient's age, travel history, and disease process to develop a differential diagnosis. • the serum ferritin level (often elevated with malignancy) and naproxen test (reduces fever with malignancy) may be helpful in determining an underlying malignant process. • initiation of empiric antibiotics should be done only in specific fuo situations to prevent skewing culture results, thus maximizing isolation of the causative organism. patients who have a persistent fever despite workup are generally classified as having a "fever of unknown origin" (fuo). in 1961, petersdorf and beeson described 100 patients with persistent fever, otherwise known as fever of unknown origin. they introduced the standard, classic definition of fuo: fever higher than 38.3° c (101° f) on several occasions, persisting without diagnosis for at least 3 weeks, with 1 week of investigational study in the hospital setting. with advancing technology, this definition has been revised to allow for more than two outpatient visits, or 3 days if investigation is in the hospital setting. most patients with fuo have chronic or subacute symptoms and can be safely evaluated in the outpatient setting, with a median time to diagnosis of 40 days. the differential diagnosis of fuo is quite broad and extensive. determining an etiologic diagnosis of an fuo depends on generating a differential diagnosis compatible with the patient's history and physical examination. the principal disease categories for fuo include infection (30% overall), neoplasms (18%), collagen vascular diseases (12%), and miscellaneous (14%) (box 16-5). because of this broad differential, a newer classification system divides fuo into four groups: classic, nosocomial, neutropenic, and hiv associated, which helps narrow the differential diagnosis. furthermore, classic fuo can be broken down into three subgroups: infants and children, elderly, and travelers. despite an extensive workup, the etiologic diagnosis usually remains elusive in 7% to 30% of patients (box 16-4) . the diagnostic workup of fuo should begin with a thorough history and physical examination, including documentation of the fever. the patient may provide a diary noting the date and time of fever. routine noninvasive investigations are recommended in all patients before diagnosing fuo (box 16-6). acute febrile illness is never called an fuo. the patient's medication profile is reviewed because numerous drugs can be the cause. if unrevealing, a workup is initiated based on the differential diagnosis for the patient's age, travel history, geographic location, and disease process. dukes criteria for infective endocarditis have 99% specificity in patients with fuo. when the initial investigations are not helpful in identifying a cause, imaging should be considered, such as computed tomography (ct) scans of the chest, abdomen, and pelvis; ct may reveal an abscess or suggest an underlying malignancy. an elevated serum ferritin level can suggest a neoplasm or myeloproliferative disorder and, if normal, greatly decreases the chance that the patient has an underlying malignancy. lower-extremity doppler ultrasound should be considered in the sedentary or obese patient to rule out deep venous thrombosis. a temporal artery biopsy should be considered in the elderly patient to rule out temporal arteritis. liver biopsy has a high diagnostic yield with minimal toxicity, whereas bone marrow cultures usually have a low yield and should be considered only in special situations. empiric therapy with antibiotics is rarely appropriate for the patient with fuo. a diagnosis is essential to guide treatment of osteomyelitis requires surgical debridement followed by a 4-to 6-week course of intravenous antibiotic therapy (sor: c). septic arthritis is usually caused by a gonococcus in a sexually active adult, and use of a third-generation cephalosporin is the mainstay of therapy (sor: a). nongonococcal arthritis should be treated with surgical debridement or repeated needle aspirations, with a third-generation cephalosporin and vancomycin if gram-positive cocci are seen (goldenberg, 1998) (sor: b). treatment, and use of antibiotics may delay determining a causative infectious agent. the naproxen test (naprosyn; 375 mg po every 12 hours for 3 days) is helpful in determining if the fever is secondary to infection or malignancy. a dramatic decrease in the patient's temperature during the test generally indicates a malignant focus, whereas minimal or no response indicates an infectious etiology. the prognosis of fuo depends on the etiologic category. undiagnosed fuo has a very favorable outcome. patients in whom diagnostic investigations fail to identify an etiology should be followed clinically with serial history reviews and physical examinations until the fever resolves or new diagnostic clues are found. connective tissue diseases are more prominent. infections: malaria, hepatitis, pneumonia/bronchitis, uti/pyelonephritis, dysentery, dengue fever, enteric fever, tb, rickettsial infection, acute human immunodeficiency virus (hiv) infection, amebic liver abscess. postoperative urinary and respiratory tract instrumentation; use of intravascular devices; drug therapy; immobilization. septic thrombophlebitis, pulmonary embolus, clostridium difficile colitis, drug fever. fungal: 40% susceptible to empiric antifungals, 5% will be resistant to empiric therapy. bacterial: 10% not responding to empiric antimicrobial therapy and usually with cryptic focus. unusual pathogens: 5% will be toxoplasmosis (toxoplasma gondii) reactivation, atypical mycobacterial, tb, fastidious pathogens (legionella, mycoplasma, chlamydophila). viral: 5% of causes (hsv, cmv, ebv, hhv-6, vzv, rsv, influenza, parainfluenza). other: 10% will be transplant related (e.g., gvhd) following stem cell transplant, 25% will be undefined. infections: mycobacterium avium complex (mac), pneumocystis carinii pneumonia (pcp), cytomegalovirus (cmv), histoplasmosis, viral (hcv, hbv, adenovirus, hsv esophagitis, vzv encephalitis), tb, other fungi, cerebral toxoplasmosis, disseminated cryptosporidiosis. neoplasms: lymphoma, kaposi's sarcoma. other: drug fever, castleman's disease. hsv, herpes simplex virus; ebv, epstein-barr virus; hhv, human herpesvirus; vzv, varicella-zoster virus; rsv, respiratory syncytial virus; gvhd, graft-versus-host disease; hcv, hepatitis c virus; hbv, hepatitis b virus. abscesses: hepatic, subhepatic, gallbladder, subphrenic, splenic, periappendiceal, perinephric, pelvic, and other sites. granulomatous: extrapulmonary and miliary tuberculosis, atypical mycobacterial infection, fungal infection. intravascular: catheter-related endocarditis, meningococcemia, gonococcemia, listeria, brucella, rat-bite fever, relapsing fever. viral, rickettsial, and chlamydial: infectious mononucleosis, cytomegalovirus, human immunodeficiency virus, hepatitis, q fever, psittacosis. parasitic: extraintestinal amebiasis, malaria, toxoplasmosis. collagen vascular diseases: rheumatic fever, systemic lupus erythematosus, rheumatoid arthritis (particularly still's disease), vasculitis (all types). granulomatous: sarcoidosis, granulomatous hepatitis, crohn's disease. tissue injury: pulmonary emboli, sickle cell disease, hemolytic anemia. familial mediterranean fever fabry's disease cyclic neutropenia intra-abdominal infections may either be uncomplicated (limited to the gut lumen, such as gastroenteritis or colitis) or complicated (extending through to the peritoneum) . the clinical presentation of complicated intra-abdominal infections can range from mild symptoms such as nausea, mild abdominal pain, and cramping to lifethreatening septic shock. clinical findings result from local or diffuse inflammation with or without abscess formation. fever and abdominal pain are typically present, with additional symptoms depending on the organ involved. elderly and immunocompromised patients present with atypical, usually milder symptoms. imaging studies form an important adjunct to diagnosis. management involves empiric antibiotic coverage for bowel flora-mainly streptococci, enterococci, enteric gram-negative rods, and anaerobes-as well as controlling the source of infection, usually through surgery. • spontaneous bacterial peritonitis usually occurs in the setting of ascites and chronic liver disease. • spontaneous bacterial peritonitis is a diagnosis of exclusion. • ascitic fluid culture yield improves with inoculation into blood culture bottles at bedside. spontaneous bacterial peritonitis (sbp) is a form of infectious peritonitis without a surgically correctable cause and is therefore a diagnosis of exclusion. the route of infection in sbp is usually not apparent and is often presumed to be hematogenous, lymphogenous, by transmural migration through an intact gut wall from the intestinal lumen, or in women, from the vagina via the fallopian tubes (levison and bush, 2010) . sbp occurs in the setting of ascites in most cases, and it is particularly common in patients with cirrhosis. in pediatric populations, those with postnecrotic cirrhosis or nephrotic syndrome are more often affected. in adults, almost 70% of patients who develop sbp have child-pugh class c liver disease, and 10% to 30% of hospitalized patients with cirrhosis and ascites have sbp (mowat and stanley, 2001) . sbp is almost always caused by a single organism, typically enteric gram-negative rods, most often e. coli, followed by klebsiella pneumoniae. gram-positive cocci account for about 25% of episodes of sbp, and streptococci are isolated most often. sbp caused by anaerobes is rare. growth of more than one organism should raise the suspicion of secondary peritonitis. signs and symptoms of sbp are subtle and require a high index of suspicion. fever greater than 100° f (38° c) is the most common presenting sign, occurring in 50% to 80% of cases. abdominal pain, nausea, vomiting, and diarrhea are usually present. peritoneal signs (abdominal tenderness or rebound tenderness) are common but may be absent in patients with ascites. in adults, mental status changes may also occur. sbp is often confused with acute appendicitis in children. in adults, sbp should be suspected in any patient with previously stable chronic liver disease who undergoes acute decompensation in clinical status. spontaneous bacterial peritonitis is diagnosed by analysis of ascitic fluid obtained by abdominal paracentesis. infection has been typically defined as an ascitic fluid wbc count higher than 250 cells/mm 3 , which is considered diagnostic even when the culture of the ascitic fluid is negative. in cases where bloody fluid is obtained ("traumatic paracentesis"), the wbc count should be corrected by 1 wbc per 250 rbcs/mm 3 . the use of bedside dipstick for leukocyte esterase has a high false-negative rate and is not recommended (nguyen-khac et al., 2008) . ascitic fluid culture yield can be increased by inoculating blood culture bottles with 10 ml of ascitic fluid at the bedside. blood cultures should also be obtained as part of the workup. after the diagnosis of peritonitis is established, secondary peritonitis should be ruled out. ct of the abdomen with oral and intravenous contrast can help direct the surgeon to a particular source of infection, as opposed to doing a full exploratory laparotomy. a high ascitic fluid total protein (>1 g/dl) or amylase level is suggestive of secondary peritonitis. the treatment of choice is generally a third-generation cephalosporin such as cefotaxime (2 g iv every 8-12 hours) or ceftriaxone (2 g iv once daily). patients who have an ascitic fluid wbc count higher than 250 cells/mm 3 should be given empiric intravenous antibiotics without delay. oral amoxicillin-clavulanic acid can be used for mild, uncomplicated cases (navasa et al., 1996) . duration of treatment varies diagnosis of fuo may be assisted by the dukes criteria for endocarditis, ct scan of the abdomen, nuclear scanning with a technetiumbased isotope, and liver biopsy (mourad et al., 2003) (sor: b) . routine bone marrow cultures are not recommended in the fuo workup (mourad et al., 2003) (sor: b) . empiric antibiotics should be initiated only in specific situations, to avoid skewing culture results and thus maximizing potential isolation of the causative organism (mourad et al., 2003) (sor: b). from 5 to 14 days depending on clinical response. patients usually respond to appropriate antibiotic therapy within 48 to 72 hours; otherwise, a repeat paracentesis should be performed. if the ascitic fluid wbc count does not decrease by more than 25%, alternative diagnoses should be considered. prophylaxis with a fluoroquinolone or trimethoprim-sulfamethoxazole should be considered, particularly in high-risk patients (garcia-tsao and lim, 2009 • bacterial meningitis is life threatening and requires urgent medical attention and treatment. • viral encephalitis should be treated with acyclovir until herpes simplex virus is ruled out. • most brain abscesses are caused by streptococci and staphylococcus aureus. • the cns infections most likely to be encountered in clinical practice include meningitis, encephalitis, and abscess. • all cns infections can be difficult to diagnose, and a high index of suspicion by the health care provider is sometimes indicated to ensure patient survival. • mri is the most sensitive neuroimaging test for encephalitis. • acyclovir should be started immediately and continued until hsv pcr testing is obtained. meningitis can be acute, subacute, or chronic. in otherwise healthy children, the three most common organisms causing acute bacterial meningitis are streptococcus pneumoniae, neisseria meningitidis, and haemophilus influenzae type b (hib). isolation of an organism other than these three organisms from the csf of a child older than 2 months always requires an explanation or evaluation for unusual host susceptibility. children with cochlear implants, asplenia, hiv infection, or csf leak from basilar skull or cribriform fracture are at greater risk for pneumococcal meningitis. deficiencies in terminal components of complement lead to greater risk for meningococcal infection (saez-llorens and mccracken, 2008) . in adults, the common etiologic agents of acute meningitis include s. pneumoniae, n. meningitidis, and listeria monocytogenes. patients with acute meningitis most often present with fever, headache, meningismus, and altered mental status. infants can present with nonspecific symptoms such as inconsolable crying, irritability, nausea, vomiting, and diarrhea. lethargy, anorexia, and grunting respirations indicate a critically ill infant. older children may complain of headache, vomiting, back pain, myalgia, and photophobia; may be confused or disoriented; and may verbalize specifically that the neck is stiff or sore. seizures are noted in up to 20% to 30% of children before hospital admission or early in the course of the illness. in contrast, patients with subacute or chronic meningitis may have the same symptoms with a much more gradual onset, lower fever, and associated lethargy and disability. mycobacterium tuberculosis, treponema pallidum (syphilis), borrelia burgdorferi (lyme disease), and fungi (e.g., cryptococcus neoformans, coccidioides spp.) are the most common agents (tunkel et al., 2010) . physical examination should look for papilledema, middle ear and sinus infections, petechiae (common with n. meningitidis), nuchal rigidity, and in infants, a bulging fontanel. blood cultures should be taken. a lumbar puncture (lp) for csf analysis should be done as soon as possible. a brain ct scan before lp is not necessary if the patient has no evidence of immunocompromise, cns disease, new seizure, papilledema, altered consciousness, or focal neurologic deficit, and if a subarachnoid hemorrhage is not suspected. if neuroimaging is necessary, blood cultures should be taken and antibiotics given before the study; a delay in administration of antibiotics leads to a worse outcome. csf should be sent for cell count, wbc differential, glucose, protein, and gram stain with culture. acid-fast bacilli stain and cryptococcal antigen may be obtained when indicated. empiric antibiotics for the initial treatment of bacterial meningitis are listed in table 16 -20, but these should be tailored to the isolated organisms whenever possible. adjunctive dexamethasone is recommended for children and infants with hib meningitis, but not if they have already received antibiotics. in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) . close contacts of patients with n. meningitidis should receive rifampin, 20 mg/kg (not to exceed 600 mg) twice daily for 2 days, or ciprofloxacin, 500 mg as a single dose, or ceftriaxone, 250 mg im as a single dose. unimmunized persons exposed to h. influenzae meningitis should receive rifampin (turkel et al., 2010) . pregnant women should not receive rifampin or doxycycline. a repeat lp should be done if no clinical response is seen after 48 hours of appropriate antibiotic therapy, particularly for patients with resistant pneumococcal disease and those who received dexamethasone. neonates with gram-negative bacilli and patients with ventriculoperitoneal (vp) shunts require documentation of csf sterility. the duration of antimicrobial therapy is 7 days for patients with n. meningitidis or hib, 10 to 14 days for pneumococcal meningitis, and 14 to 21 days for streptococcus agalactiae. spontaneous bacterial peritonitis is treated with third-generation cephalosporins (cefotaxime or ceftriaxone), with ampicillin-sulbactam, fluoroquinolones, or carbapenems as alternative agents (solomkin et al., 2010) (sor: b) . patients with diffuse peritonitis should undergo an emergency surgical procedure as soon as possible, even if ongoing measures to restore physiologic stability need to be continued during the procedure (sor: b). viral meningitis viral meningitis manifests similar to bacterial meningitis, although its course is rarely aggressive. the diagnostic process and examination are similar to those for bacterial meningitis. viral meningitis is usually caused by enteroviruses, hsv, mumps virus, and hiv. along with the signs of meningitis, signs that suggest a viral etiology include genital lesions (hsv-2), diarrhea, or a maculopapular rash (enteroviruses). diagnosis is made by the history, examination, and csf results. early in the course, the csf might show predominantly neutrophils that can resemble bacterial meningitis. treatment is symptomatic. suppressive therapy should be offered to patients with recurrent hsv meningitis. although encephalitis can also be caused by bacteria and fungi, the great majority of cases are caused by viruses. herpes simplex accounts for 10% of cases. patients present with fever, acute decreased level of consciousness, and occasionally, seizures and language, memory, or behavior disturbances. mri is the most sensitive neuroimaging test for encephalitis and might show temporal lobe inflammation in early hsv encephalitis. csf studies and electroencephalography (eeg) are also recommended for all patients with encephalitis. herpes simplex pcr should be done, and acyclovir should be given immediately until hsv encephalitis is ruled out. during late summer and early fall, doxycycline should be considered to cover for tick-borne illnesses, and testing should include the mosquito-borne encephalitides such as west nile, st. louis, eastern equine, and western equine. treatment depends on the suspected etiologic agent but is generally supportive (tunkel et al., 2008) . a brain abscess is a focal, intracerebral infection that develops into a collection of pus surrounded by a well-vascularized capsule. although fungi and protozoa (particularly toxoplasma) can also cause brain abscesses, bacterial causes are much more common. streptococci are found in 70% of bacterial abscesses and are usually from oropharyngeal infection or infective endocarditis, whereas staphylococcus aureus accounts for 10% to 20% of isolates and is more often found after trauma. community-associated mrsa strains have been increasing. enteric gram-negative bacilli (e.g., e. coli; proteus, klebsiella, and pseudomonas spp.) are isolated in 23% to 33% of patients, often in patients with ear infection, septicemia, or immunocompromise and those who have had neurosurgical procedures. most clinical symptoms are caused by the size and location of the abscess rather than the systemic signs of an infection. headache is the most common complaint and may be accompanied by fever, mental status changes, evidence of increased intracranial pressure (nausea, vomiting, papilledema), or focal neurologic deficits. diagnosis is usually made by ct scan with iv contrast showing the characteristic hypodense center with a peripheral uniform ring enhancement, with or without a surrounding area of brain edema. mri is becoming the preferred imaging modality because of increased sensitivity, particularly for detecting satellite lesions. additional testing depends on risk factors and the likely underlying source of infection and may include blood cultures, chest imaging, testing for hiv and antibodies to toxoplasma, and transesophageal echogram. empiric therapy typically involves vancomycin, ceftriaxone, and metronidazole. optimal management also includes surgical drainage for most abscesses, both to find an etiologic microorganism and to improve chances of cure (turkel, 2010) . • most acute diarrheal illness is viral and can be managed symptomatically and with appropriate attention to hydration. • travelers' diarrhea is usually caused by diarrheogenic escherichia coli. • the infection in travelers' diarrhea is usually self-limited. • antibiotics may shorten the duration of diarrhea by 1 to 3 days. • the most common cause of antibiotic-associated diarrhea is clostridium difficile. • treatment of antibiotic-associated diarrhea involves discontinuing the offending agent, if possible. adjunctive dexamethasone is recommended for children and infants with h. influenzae type b meningitis, but not if they have already received antibiotics (tunkel et al., 2004) (sor: a). in adults, adjunctive dexamethasone is recommended for pneumococcal meningitis (tunkel et al., 2004) (sor: b). diarrhea is a common presenting complaint in the primary care physician's office. not all causes of diarrhea are infectious, and not all infectious causes of diarrhea require specific antibiotic therapy. diarrhea remains a major cause of morbidity and mortality, particularly for children in the developing world. diarrhea is an alteration of normal bowel function, characterized by an increase in the water content, volume, or frequency of stools. acute diarrhea is typically defined as present less than 14 days, and diarrhea is considered chronic when symptoms persist longer than 30 days (figure 16-12 ). infectious diarrhea seen in the primary care physician's office is most frequently caused by viruses. a number of viral agents can cause diarrheal illness (box 16-7). rotaviruses are the principal enteric pathogens in children less than 5 years of age and the most important cause of hospitalization and infant mortality related to diarrheal illnesses. noroviruses evaluate severity and duration obtain history and physical examination [1] [2] [3] [4] [5] treat dehydration report suspected outbreaks 6 check all that apply: 7 are the most common cause of food-borne disease worldwide. viral gastroenteritis is usually an acute self-limited illness, referred to as the "stomach flu." enteric viruses are easily spread by fecal-oral transmission, through contamination of food and water, fomites, and person-to-person spread. secondary attack rates can be high. nausea and vomiting are the most prominent symptoms of viral gastroenteritis. diarrhea, fever, headache, and constitutional symptoms may also be experienced. these viral infections can occur at any time during the year, but tend to occur more often in the winter. there is no specific therapy. treatment is supportive, with particular emphasis on adequate replacement of fluids and electrolytes. if rehydration can be accomplished enterally, it is preferred. both the pentavalent bovine-human reassortment (rv5) and the oral, live-attenuated monovalent (rv1) rotavirus vaccines are effective for prevention of severe gastroenteritis. the rv5 vaccine series is recommended for children at ages 2, 4, and 6 months, whereas the rv1 vaccine should be administered to children 2 and 4 months of age. approximately 40% of travelers to developing regions of the world will develop diarrhea. bacteria are responsible for approximately 80% of diarrhea acquired by travelers. other important causes include viruses and parasites. the onset of the majority of cases of travelers' diarrhea is usually within 5 to 15 days after arrival. the presentation is typically a noninflammatory, nonbloody diarrhea associated with abdominal discomfort, fever, nausea, or vomiting. the duration is usually 1 to 5 days. enterotoxigenic e. coli is responsible for approximately 30% of travelers' diarrhea. enteroaggregative e. coli is the second most common bacterial agent and causes 20% of cases. salmonella, shigella, and campylobacter spp. are less often detected but are important causes of dysentery, particularly in asia and africa. dysentery is severe inflammatory diarrhea manifested by fever and bloody stools. most cases of travelers' diarrhea are self-limited, but chronic postinfectious irritable bowel syndrome may occur in up to 10% of those who experience diarrhea. prevention of travelers' diarrhea is an important component of pretravel counseling for high-risk countries. food should be boiled, cooked, or peeled and water boiled to avoid consumption of fecal contamination. if a person develops travelers' diarrhea, a short course of antibiotics with rifaximin, ciprofloxacin, or azithromycin can shorten the duration of illness by 1 to 3 days. antibiotic therapy is recommended for persons with bloody diarrhea or fever. rifaximin, a nonabsorbed antibiotic, is not effective against invasive pathogens and should not be administered for dysentery. ciprofloxacin or azithromycin should be used for dysenteric symptoms based on local antimicrobial susceptibilities. antibiotics are frequently prescribed in the primary care physician's office for a variety of infections. unfortunately, antibiotics can alter the normal host microflora that can be protective against other infections. antibiotic effects on the normal gastrointestinal tract microbiome can lead to antibiotic-associated diarrhea, which causes significant morbidity and mortality. administration of antibiotics usually precedes symptoms of antibiotic-associated diarrhea by about 1 week but can be as distant as 2 or 3 months. strong associations with clindamycin (cleocin), cephalosporins, penicillins, and fluoroquinolones have been demonstrated, but any antibiotic can lead to antibiotic-associated diarrhea. the most important cause of antibiotic-associated diarrhea is clostridium difficile, an anaerobic, gram-positive, spore-forming rod. c. difficile is implicated as the cause in up to 25% of antibiotic-associated diarrhea cases, in 50% to 75% of antibiotic-associated colitis cases, and in more than 90% of antibiotic-associated pseudomembranous colitis cases. risk factors for c. difficile diarrhea include antibiotics, health care exposure (recent stay in hospitals or long-term care facilities), older age (>60), and comorbid conditions. the clinical presentation of c. difficile colitis is usually diarrhea, abdominal pain or cramping, and fever in a patient who recently received antibiotics. leukocytosis is common and may be profound; levels can be consistent with leukemoid reaction. a rare but potentially fatal complication is toxic megacolon. toxic megacolon manifests as acute colonic dilation to a diameter greater than 6 cm, associated with systemic toxicity and the absence of mechanical obstruction. with its high associated mortality, any patient who develops toxic megacolon requires immediate surgical evaluation for possible colectomy. diagnosis of c. difficile diarrhea is achieved by demonstration of c. difficile toxin a or b in the stool by enzyme immunoassay (eia) or cell culture cytotoxicity assay in a symptomatic patient with a previous history of antibiotic use. asymptomatic patients should not be tested. with the improved sensitivities of these diagnostic assays, one stool sample is usually sufficient to test for c. difficile, unless symptoms recur. test of cure after therapy with repeat stool for c. difficile toxin is not recommended because stools may remain positive for c. difficile toxin despite clinical resolution. endoscopy can demonstrate pseudomembranes in the colon. pseudomembranes are diagnostic of c. difficile infection, but are often not present. endoscopy may only reveal the presence of nonspecific colitis. clostridium difficile colitis is treated by discontinuing the offending agent(s) if possible and initiating antibiotic therapy (box 16-8). antimotility agents should be avoided. oral metronidazole (flagyl), 500 mg three times daily for 10 to 14 days, is recommended for mild-moderate c. difficile diarrhea. severe diarrhea should be treated with oral vancomycin. oral vancomycin is currently not recommended for all patients with c. difficile diarrhea because of concerns for the promotion of vancomycin-resistant enterococci (vre) and its expense. about 10% to 20% of patients experience relapse in travelers' diarrhea, in which enterotoxigenic e. coli or other bacterial pathogens are likely causes, prompt treatment with a fluoroquinolone, azithromycin, or rifaximin or, in children, azithromycin 10 mg/kg/day once daily can reduce the duration of an illness from 3 to 5 days to 1 to 2 days (dupont, 2010) (sor: a). after therapy. for relapse, a repeat course of the original c. difficile treatment should be administered. patients who have mild to moderate cases without volume depletion or systemic toxicity can be treated as outpatients. discussions of the following infections can be found online at www.expertconsult.com: • infectious viral hepatitis • endocarditis treat mild-moderate c. difficile diarrhea with metronidazole (zar et al., 2007) evidence-based reviews of the diagnosis and treatment of many common clinical problems. www.mdcalc.com/curb-65-severity-score-community-acquired-pneumonia curb-65 score calculator to determine the severity of communityacquired pneumonia and need for hospitalization. the complete reference list is available online at www.expertconsult.com. anthony zeimet hepatitis is defined as inflammation of the liver that is commonly induced by viruses that include the hepatitis viruses a through e, which will be the focus of this discussion. other viruses that can induce hepatitis include epstein-barr virus (ebv), cytomegalovirus (cmv), herpes simplex virus (hsv), varicella zoster virus (vzv), adenovirus, and coxsackievirus. various medications and alcohol abuse are two important nonviral causes. most infectious causes of hepatitis are self-limiting; however, hepatitis b and c viruses can cause a chronic infection that may lead to cirrhosis and eventual liver failure, as well as hepatocellular carcinoma. hepatitis a virus (hav) and hepatitis e virus (hev) are spread by the fecal-oral route and only cause an acute infection. hepatitis b, c, and d viruses (hbv, hcv, hdv) are spread through the blood and have an acute form of disease that sometimes can become chronic. the clinical presentation of hepatitis is clinically indistinguishable. asymptomatic infections are more common than symptomatic infection. symptoms generally include right upper quadrant (ruq) abdominal pain, anorexia, nausea, vomiting, diarrhea, dark-colored urine, pale stools, and generalized malaise; patients may notice a yellow hue to their skin or eyes. pruritus is common, caused by deposition of bilirubin in the skin. the physical examination generally reveals jaundice and sclera icterus in addition to ruq pain. hepatomegaly is seen in 85% and splenomegaly in 15% of patients with hepatitis. liver function tests reveal elevated levels of aspartate transaminase (ast), alanine transaminase (alt), and bilirubin, and to a lesser extent, alkaline phosphatase (alp). hepatitis a virus is the most common cause of viral hepatitis worldwide. poor hygiene practices in both the industrial and the developing world account for its prevalence. in the united states, hav is common among lower socioeconomic groups, daycare attendants and workers, men who have sex with men (msm), and illicit drug users. hepatitis a is often acquired by travelers to endemic areas. the incubation period is 15 to 45 days (mean, 30 days). hav is highly contagious, and peak fecal shedding generally occurs at the onset of illness in most infected patients. viremia averages 30 to 90 days. hav infection manifests as an acute, self-limited illness, with the prodromal symptoms lasting about a week before the onset of jaundice. jaundice generally resolves after 2 weeks, and most patients recover. fulminant hepatic failure is possible but extremely rare. diagnosis of acute hav infection is made by demonstration of anti-hav immunoglobulin m (igm) in the patient's serum. this may be negative if the patient presents early, and repeat testing may be necessary if hav is strongly suspected. anti-hav igg in the serum indicates remote infection or immunization (efig 16-1). treatment is primarily supportive, except in patients with fulminant liver failure, who may require a liver transplant. vaccination should be administered to all patients who are seronegative and to persons at increased risk for acquiring hav, including those about to travel to endemic areas, patients with chronic liver disease or receiving clotting factor concentrates, msm, hiv-positive patients, and illicit drug users. certain areas of the united states now require mandatory vaccination of children as well as those who work in the restaurant industry. the vaccine is safe and highly efficacious and is given as a two-dose series at 0 and at 6 to 12 months. passive immunization with immune globulin is recommended for those exposed to the virus by a known contact, including household and sexual contacts, and those who are traveling to an endemic area for less than 4 weeks but never vaccinated. any person who receives immune globulin should also start the vaccination series. hepatitis b virus infection can be acute or chronic. about 40,000 people die from acute hbv infection annually, and 500,000 die of cirrhosis and hepatocellular carcinoma caused by chronic infection. about 400 million people worldwide are living with chronic hbv infection. in the united states, an estimated 1.25 million residents have chronic hbv infection, with 4000 to 5500 deaths each year. significant burdens of disease are seen in asia, pacific islands, sub-saharan africa, amazon basin, and eastern europe. most adults with acute hbv will clear the virus, with less than 5% progressing to chronic infection. chronic infection will develop in almost all children infected perinatally and in 50% of those who become infected at 1 to 5 years of age. hbv is transmitted through exchange of body fluids, sexually and perinatally. in the united states, most hbv cases are acquired during adolescence and early adulthood with onset of sexual activity, experimentation with drug use, and sometimes occupational exposure. fever, polyarthralgia, rash, and a serum sickness-like illness are features of hbv infection in addition to jaundice and may be seen in association with polyarteritis nodosa. clinicians have the most difficulty in interpreting the various serologic tests for diagnosis of hepatitis b (etable 16-3) . the mean incubation period is 60 to 70 days, with a range of 30 of 180 days after infection. diagnosis of acute infection can be detected by obtaining hbv surface antigen (hbsag), which can appear as early as 1 week after exposure but generally by day 50. in a patient strongly suspected to have hbv infection, the clinician can consider checking the hbv dna viral load; which can be detected as early as 1 week after exposure. eventually the patient will develop an anti-hbv surface antibody, which indicates recovery from the illness. the other viral serologies for hbv are rarely obtained in acute illness. in chronic hbv infection, there are three major phases of infection: 1. immune tolerant. active viral replication in the liver with high levels of hbv dna levels but essentially normal or minimal elevation of ast and alt. most patients eventually progress to the next stage. 2. immune active. more robust liver inflammation with alt elevation, and liver biopsy shows inflammation with or without fibrosis. hbv early antigen (hbeag) is detected along with hbsag. 3. inactive carrier state. as patients enter this phase, they clear the hbeag and develop anti-hbe antibody and have undetectable or low levels of hbv dna, with normalization of alt and liver inflammation. if patients become hbsag negative, they then develop anti-hbs and have resolved their infection; otherwise, they are considered a chronic carrier. treatment of acute hbv is primarily supportive. in the last decade, however, there have been significant advances in the treatment of chronic hbv infection. the use of interferon has long been the mainstay of treatment and has a defined, limited course but is generally poorly tolerated. with the advent of the hiv/aids epidemic and research into treatment of hiv disease, antiviral medications are now starting to replace interferon as the preferred treatment option for hbv patients. nucleoside/nucleotide analogs such as lamivudine, adefovir, entecavir, tenofovir, and telbivudine are generally given for long-term, indefinite therapy to prevent progression of liver disease and development of hepatocellular carcinoma. any patient with chronic hbv infection should be referred to an infectious diseases specialist or a hepatologist to determine the appropriate treatment course. universal vaccination of newborns and infants is routine in the united states since 1991, and the incidence of hbv infection has declined. during primary care visits, the vaccination status of any adult or adolescent born before 1991 should be reviewed and the vaccine offered. the vaccine requires three doses given at 0, 1, and 6 months. an unvaccinated person or neonate who is exposed to the body fluids of a hbv-infected individual should start the vaccination series in addition to receiving the hepatitis b immune globulin (hbig). hepatitis c virus infection is the most common cause of chronic viral hepatitis in the united states. hcv does have an acute form of infection but is usually subclinical and rarely diagnosed. the cdc estimates that there are more than 2.7 million people with hcv infection. hcv is generally transmitted parenterally, as in injection drug users who share needles. before 1992, those who received a blood transfusion may have contracted hcv. sexual transmission acute hbv has been reported in monogamous couples, with one partner who has hcv infection and the other without infection who eventually acquires the virus. this occurs in 3% to 5% of couples and represents a rare mode of transmission. because the most common mode of acquisition is sharing needles, any patient who is hcv positive should be screened for hiv because these two infections often occur together (ebox 16-1). the diagnosis of acute hcv infection can be made by obtaining a hcv rna viral load; although this is rarely done because the initial infection is subclinical. chronic disease is generally discovered by a positive anti-hcv antibody along with an elevated hcv rna viral load. hcv genotype should also be obtained in any positive individual, because this has important prognostic factors with regard to therapy, with genotype 1a and 1b the predominant type in the united states and unfortunately having a poor response to therapy. as with hbv, chronic hcv infection can lead to cirrhosis and the development of hepatocellular carcinoma. treatment consists of 24 to 48 weeks of interferon and ribavirin therapy. any patient being considered for therapy should be referred to an infectious diseases specialist or hepatologist. a liver biopsy is often needed to determine appropriate treatment candidates. also known as the hepatitis delta antigen virus, hdv is a defective virus that requires the presence of hbv to be infectious. hdv should be suspected in any patient with chronic hbv who develops acute hepatitis. hepatitis d is endemic in the mediterranean, balkans, africa, middle east, and amazon basin. diagnosis is made through an anti-hdv antibody in the presence of someone with positive hbsag or anti-hb core antibody igm or igg. treatment is supportive. any person vaccinated against hbv cannot become infected with hdv. similar to hav infection, hev is spread by the fecal-oral route. hev only has an acute form and does not progress to chronic infection. most reported epidemics have been related to consumption of contaminated drinking water. hev is endemic to southeast and central asia, north africa, middle east, mexico, brazil, venezuela, and cuba. hepatitis e can be considered a cause of infectious hepatitis in the united states in the traveler returning from an endemic area. the incubation period is 40 days. infection is of major concern during pregnancy, which can cause death in late pregnancy. diagnosis is made by demonstration of anti-hev antibody in serum. treatment is supportive. • endocarditis prophylaxis is now recommended solely for patients at high risk of a complicated course with a more narrow range of cardiac conditions. • routine prophylaxis for gi and gu procedures is no longer recommended. • the duke criteria represent a reliable scoring system for diagnosing endocarditis. • echocardiography is indicated to confirm suspected endocarditis. bacterial endocarditis is one of the most feared infections; although uncommon, it carries high morbidity and mortality. increase in antibiotic resistance among bacteria causing this infection has created challenges for effective treatment. the fundamental view of the american heart association (aha) in preventing infective endocarditis has shifted in recent years. views on pathophysiology have not changed substantially, but it is now recognized ebox 16-1 persons for whom hepatitis c virus (hcv) screening is recommended persons who have injected illicit drugs in the recent and remote past, including those who injected only once and do not consider themselves to be drug users. persons with conditions associated with a high prevalence of hcv infection, including: persons with human immunodeficiency virus (hiv) infection persons with hemophilia who received clotting factor concentrates before 1987 persons who have ever received hemodialysis persons with unexplained abnormal transaminase (aminotransferase) levels prior recipients of transfusions or organ transplants before july 1992, including: persons who were notified that they had received blood from a donor who later tested positive for hcv infection persons who received a transfusion of blood or blood products persons who received an organ transplant children born to hcv-infected mothers health care, emergency medical, and public safety workers after a needle stick injury or mucosal exposure to hcv-positive blood current sexual partners of hcv-infected persons * modified from centers for disease control and prevention. recommendations for prevention and control of hepatitis c virus (hcv) infection and hcv-related chronic disease. mmwr 1998;47(rr):1-39. *although the prevalence of infection is low, a negative test in the partner provides reassurance, making testing of sexual partners of benefit in clinical practice. • universal vaccination of infants with hepatitis b vaccine reduces the risk of acute hepatitis, chronic carrier state, and complications of chronic infection and may be more effective than selective vaccination of high-risk individuals (lee et al., 2006) (sor: a). • as part of a comprehensive health evaluation, all persons should be screened for behaviors that place them at high risk for hepatitis c infection (ghany et al., 2009 ) (sor: b). • liver biopsy may be considered in patients with chronic hcv infection to determine fibrosis stage for prognostic purposes or to make a treatment decision (ghany et al., 2009 ) (sor: b). that cumulative daily episodes of bacteremia likely carry more risk than the transient bacteremia caused by dental procedures. infective endocarditis likely begins with turbulent flow and damaged endothelium around heart valves, which allow platelet aggregation and thrombus formation, causing a "nonbacterial thrombotic endocarditis" (wilson et al., 2007) . the presence of bacteremia then allows this vegetation to become seeded with infection. bacterial "adhesins" are present to a greater degree in some species and allow for more effective attachment to the injured area of endothelium. with high concentrations of bacteria in the mouth, vagina, gi tract, and perhaps gu system, antibiotic prophylaxis was initiated when these anatomic locations were manipulated. recommendations for infective endocarditis prevention changed in 2007-2008, with aha recognizing more likely benefit from providing adequate population-based dental care and good oral hygiene, and thus less significant ongoing bacteremia at home in brushing, flossing, and "toothpicking," than in providing antibiotic prophylaxis to patients undergoing a dental procedure. no prospective rct has shown that dental prophylaxis prevents infective endocarditis. with recognition of the risk associated with administration of antibiotics (gi upset, diarrhea, rash, anaphylaxis) and the risk of contributing to increasing antibiotic resistance, versus the likely negligible benefit, aha has substantially changed its advice on this long-held practice. a preexisting cardiac condition produces a predisposition to the development of infective endocarditis (ebox 16-2). for example, those who have valve replacement for infection of an infected native valve carry a lifetime risk of 630 per 100,000 patient-years. the risk in the general population without known heart disease is 5 per 100,000 patient-years. more concerning, however, is the risk to a given patient of poor outcome if the patient develops endocarditis, which drives current aha recommendations. those with an infected mechanical valve have a mortality rate of about 20%, versus 5% or less for patients with an infected native valve (wilson et al., 2007) . a summary of current recommendations for endocarditis prophylaxis is provided in etable 16-4. of note, gi and gu procedures have been removed from those for which antibiotics are recommended, unless those systems are actively infected at the time of the procedure. the same is true for skin and soft tissue procedures, in that only infected tissue would warrant antibiotics to prevent infective endocarditis. it is still recommended to provide prophylaxis for respiratory tract procedures, if the respiratory wall will be invaded through biopsy or the procedure. in addition, respiratory procedures to treat infections (e.g., empyema) should be combined with antibiotic administration (nishimura et al., 2008) . antibiotic regimens for prophylaxis for dental procedures are still based primarily on synthetic penicillins as their cornerstone. this is with recognition that streptococcus viridans is both a mouth floral inhabitant and a common agent causing infective endocarditis. with other procedures, antibiotics should be targeted to bacterial pathogens causing any active infection in the system being manipulated. ebox 16-2 cardiac conditions associated with the highest risk of adverse outcome from endocarditis for which prophylaxis with dental procedures is reasonable prosthetic cardiac valve or prosthetic material used for cardiac valve repair previous ie congenital heart disease (chd) * unrepaired cyanotic chd, including palliative shunts and conduits completely repaired congenital heart defect with prosthetic material or device, whether placed by surgery or by catheter intervention, during the first 6 months of the procedure † repaired congenital heart defect with residual defects at the site or adjacent to the site of a prosthetic patch or prosthetic device (which inhibit endothelialization) cardiac transplantation recipients who develop cardiac valvulopathy *except for the conditions listed above, antibiotic prophylaxis is no longer recommended for any other form of chd. †prophylaxis is reasonable because endothelialization of prosthetic material occurs within 6 months after the procedure oral antibiotic william osler discussed "malignant endocarditis" in 1885 and its great diagnostic challenge. in 2009 the modified duke criteria remains a reliable tool for assessing patients with endocarditis. endocarditis is suspected in febrile patients without an obvious source, in those with recent bacteremia (including iv drug use), in those with underlying cardiac predisposition, and perhaps in patients with the clinical finding of a new cardiac murmur. in establishing a diagnosis of infective endocarditis, a patient is considered to have definite disease if two major or one major and three minor or five minor criteria are present. possible disease is defined as one major and one minor or three minor criteria (ebox 16-3). pathologic specimens showing changes consistent with endocarditis would make a definitive diagnosis. echocardiography is indicated in making the diagnosis of infective endocarditis. transthoracic echocardiography (tte) is helpful if vegetations are seen, although size of the patient and other disease (e.g., copd) may limit the ability of tte to view the cardiac valves adequately. if tte is negative and suspicion remains, transesophageal echocardiography (tee) is indicated. tte may be more widely available, depending on regional and institutional variation, and should be used rather than delaying this diagnostic test. bacteria present within valvular vegetations are often less metabolically active, which partly explains the requirement for longer courses of antibiotics for this type of infection. clearly, therapy for endocarditis should be targeted at the organism identified on blood culture, if any. the counting of antibiotic days should begin when the blood culture becomes negative and not at the start of the particular agent. recommendations for antibiotic use in infectious endocarditis are highly variable and based on the presence or absence of synthetic valvular material and the infectious agent (etable 16-5). generally speaking, a minimum of 4 weeks of iv antibiotics is indicated. in cases of resistant organisms, up to 8 weeks may be required. in either case, synergistic use of agents such as gentamicin may be indicated for the first several weeks of treatment, which then can be discontinued. the ability of a given patient to complete this course at home versus in a health care facility is dependent on the dosing frequency of the antibiotic, availability of inhome nursing services, and the type of intravenous access through which the antibiotic will be delivered. at the completion of endocarditis therapy, echocardiography should be repeated to re-assess the function of the valve(s) in question. valvular dysfunction at the completion of therapy is a good indication that the patient will need valve replacement in the future. there are circumstances, like the development of congestive heart failure in the face of endocarditis, in which primary surgery is indicated. typical microorganisms consistent with ie from 2 separate blood cultures: viridans streptococci, streptococcus bovis, hacek group, staphylococcus aureus; or community-acquired enterococci in the absence of a primary focus; or microorganisms consistent with ie from persistently positive blood cultures, defined as follows: at least 2 positive cultures of blood samples drawn >12 hours apart; or all of 3 or a majority of ≥4 separate cultures of blood (with first and last sample drawn at least 1 hour apart). single positive blood culture for coxiella burnetii or anti-phase 1 igg antibody titer >1:800 echocardiogram positive for ie (tee recommended for patients with prosthetic valves, rated at least "possible ie" by clinical criteria, or complicated ie [paravalvular abscess]; tte as first test in other patients) defined as follows: oscillating intracardiac mass on valve or supporting structures, in the path of regurgitant jets, or on implanted material in the absence of an alternative anatomic explanation; or abscess; or new partial dehiscence of prosthetic valve; new valvular regurgitation (worsening or changing or preexisting murmur not sufficient) predisposition, predisposing heart condition, or idu fever, temperature >38° c vascular phenomena, major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial hemorrhage, conjunctival hemorrhages, and janeway's lesions immunologic phenomena: glomerulonephritis, osler's nodes, roth's spots, and rheumatoid factor microbiologic evidence: positive blood culture but does not meet a major criterion as noted above * or serological evidence of active infection with organism consistent with ie echocardiographic minor criteria eliminated echocardiography should be performed in all patients with suspected infective endocarditis (baddour et al., 2005) there is no evidence that antibiotic prophylaxis is effective or ineffective for preventing infectious endocarditis after dental procedures in patients at risk (chung, 2009 ) (sor: c). regimen dosage * and route duration (wk) chronic cough due to acute bronchitis: accp evidencebased clinical practice guidelines interim recommendations for the use of influenza antiviral medications in the setting of oseltamivir resistance among circulating influenza a (h1n1) viruses, 2008-09 influenza season national ambulatory medical care survey: 2005 summary short-course antibiotic treatment in acute exacerbations of chronic bronchitis and copd: meta-analysis of doubleblind studies antibiotics for exacerbations of chronic obstructive pulmonary disease antibiotics for acute bronchitis effects of naproxen on experimental rhinovirus colds: a randomized, double-blind, controlled trial infectious diseases society-american thoracic society consensus guidelines on management of community-acquired pneumonia in adults emergence of a novel swineorigin influenza a (h1n1) virus in humans acute pneumonia influenza. in: schlossberg d, ed. clinical infectious disease seasonal influenza in adults and children: diagnosis, treatment, chemoprophylaxis, and institutional outbreak management. clinical practice guidelines of the infectious diseases society of america antivirals for influenza in healthy adults: systematic review hospitalizing influenza in adults infectious diseases society-american thoracic society consensus guidelines on management of community-acquired pneumonia in adults influenza viruses, including avian influenza and swine influenza steroids for symptom control in infectious mononucleosis hepatosplenomegaly in infectious mononucleosis, assessed by ultrasonic scanning infectious mononucleosis vaccines for post-exposure prophylaxis against varicella (chickenpox) in children and adults centers for disease control and prevention (ats/cdc). targeted tuberculin testing and treatment of latent tuberculosis infection centers for disease control and prevention. extensively drug-resistant tuber culosis-united states short-course therapy with rifampin plus isoniazid, compared with standard therapy with isoniazid, for latent tuberculosis infection: a meta-analysis genital warts expedited partner therapy in the management of sexually transmitted diseases. atlanta: us department of health and human services center for disease control and prevention. sexually transmitted disease surveillance. division of std prevention accuracy of the clinical diagnosis of vaginitis compared with a dna probe laboratory standard a pooled analysis of the effect of condoms in preventing hsv-2 acquisition consistent condom use is associated with lower prevalence of human papillomavirus infection in men the limited value of signs and symptoms in the diagnosis of vaginal infections cervical cancer condom effectiveness in reducing heterosexual hiv transmission sexually transmitted diseases treatment guidelines genitourinary infections urinary tract infection in children diagnosis and management of uncomplicated urinary tract infections american geriatric society. guidelines for the diagnosis and treatment of asymptomatic bacteriuria in adults asymptomatic bacteriuria: when to screen and when to treat the effectiveness of a clinical practice guideline for the management of presumed uncomplicated urinary tract infection in women recurrent cystitis in non-pregnant women a randomized trial to evaluate effectiveness and cost effectiveness of naturopathic cranberry products as prophylaxis against urinary tract infection in women screening for asymptomatic bacteriuria in pregnancy: a decision and cost analysis guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women tick-borne infections tick-borne diseases tick-borne diseases in the united states the clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis practice guidelines for the diagnosis and management of skin and soft tissue infections furuncles and carbuncles centers for disease control and prevention. health-care-associated methicillin-resistant staphylococcus aureus (mrsa) double-blind, placebo-controlled trial of cephalexin for treatment of uncomplicated skin abscesses in a population at risk for community-acquired methicillin-resistant staphylococcus aureus infection practice guidelines for the diagnosis and management of skin and soft-tissue infections bacteriological study of diabetic foot infections diabetic foot infection diagnostic accuracy of the physical examination and imaging tests for osteomyelitis underlying diabetic foot ulcers: meta-analysis diagnosis and treatment of diabetic foot infections deep tissue biopsy vs. superficial swab culture monitoring in the microbiological assessment of limb-threatening diabetic foot infection culture of percutaneous bone biopsy specimens for diagnosis of diabetic foot osteomyelitis: concordance with ulcer swab cultures bite infections antibiotic prophylaxis for mammalian bites (cochrane review) sexually transmitted diseases treatment guidelines fever of unknown origin a comprehensive evidence-based approach to fever of unknown origin fever of unexplained origin: report on 100 cases complicated intra-abdominal infections; spontaneous bacterial peritonitis; and secondary bacterial peritonitis and intra-abdominal abscesses members of veterans affairs hepatitis c resource center program. management and treatment of patients with cirrhosis and portal hypertension: recommendations from the department of veterans affairs hepatitis c resource center program and the national hepatitis c program mandell, douglas, and b ennett's principles and practices of infectious diseases spontaneous bacterial peritonitis: diagnosis, treatment and prevention review article: the utility of reagent strips in the diagnosis of infected ascites in cirrhotic patients randomized, comparative study of oral ofloxacin versus intravenous cefotaxime in spontaneous bacterial peritonitis diagnosis and management of complicated intra-abdominal infection in adults and children. guidelines by the surgical infection society and the infectious diseases society of america acute bacterial meningitis beyond the neonatal period brain abscess practice guidelines for the management of bacterial meningitis the management of encephalitis: clinical practice guidelines of the infectious disease society of america acute meningitis guidelines on acute infectious diarrhea in adults. the practice parameters committee of the american college of gastroenterology a comparison of vancomycin and metronidazole for the treatment of clostridium difficileassociated diarrhea, stratified by disease severity acute cholecystitis nett's principles and practices of infectious diseases diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the surgical infection society and the infectious diseases society of america infectious viral hepatitis national institutes of health consensus development conference statement: management of hepatitis b acute viral hepatitis hepatitis b virus infection chronic viral hepatitis chronic hepatitis diagnosis, management and treatment of hepatitis c: an update acute viral hepatitis hepatitis c virus infection effect of hepatitis b immunisation in newborn infants of mothers positive for hepatitis b surface antigen: systematic review and meta-analysis chronic hepatitis b: update infective endocarditis: diagnosis, antimicrobial therapy, and complications prescription of antibiotics for prophylaxis to prevent bacterial endocarditis experience with a oncedaily aminoglycoside program administered to 2184 adult patients acc/aha 2008 guideline update on valvular heart disease: focused update on infective endocarditis prevention of infective endocarditis: guidelines from the american health association. circulation key: cord-275795-ee7qyw5h authors: monette, anne; mouland, andrew j. title: t lymphocytes as measurable targets of protection and vaccination against viral disorders date: 2018-10-24 journal: int rev cell mol biol doi: 10.1016/bs.ircmb.2018.07.006 sha: doc_id: 275795 cord_uid: ee7qyw5h continuous epidemiological surveillance of existing and emerging viruses and their associated disorders is gaining importance in light of their abilities to cause unpredictable outbreaks as a result of increased travel and vaccination choices by steadily growing and aging populations. close surveillance of outbreaks and herd immunity are also at the forefront, even in industrialized countries, where previously eradicated viruses are now at risk of re-emergence due to instances of strain recombination, contractions in viral vector geographies, and from their potential use as agents of bioterrorism. there is a great need for the rational design of current and future vaccines targeting viruses, with a strong focus on vaccine targeting of adaptive immune effector memory t cells as the gold standard of immunity conferring long-lived protection against a wide variety of pathogens and malignancies. here, we review viruses that have historically caused large outbreaks and severe lethal disorders, including respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic fevers. to observe trends in vaccinology against these viral disorders, we describe viral genetic, replication, transmission, and tropism, host-immune evasion strategies, and the epidemiology and health risks of their associated syndromes. we focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory t cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates. the world was forever changed by the introduction of vaccine against smallpox in the late 1700s, at the time protecting its first 100,000 individuals. this was the first demonstration that a vaccine could successfully eradicate viruses causing disorders and diseases that even when not lethal, still had the potential to cripple both surviving populations and their surrounding geographical economies. since that time, the occurrence of epidemics and outbreaks are now at lower risk, following the introduction of massive vaccination programs able to induce immune system targeting of viruses causing severe disorders affecting distinct geographical locations, and with many epidemiological reports demonstrating long-term efficacy of viral control of non-naïve populations. outbreaks of existing and emerging viral diseases or disorders vary widely in duration and frequency across geographical populations. some can be predicted annually, while others may see decades between outbreaks, therefore driving the continuous epidemiological surveillance of associated infectious disorders, the development and implementation of targeting vaccines, and development of immune-monitoring strategies measuring vaccine efficiency in target populations. despite vaccination-mediated protection of numerous nations documenting complete eradication of the causative agents of viral disorders in endemic populations, there is threat of re-emergence of pandemic proportions of these agents. threats to virus re-emergence are caused by contemporary choices made to not vaccinate children, by strain recombination, by viral spread to naïve populations by world-travelers, migrants, or climate change, causing redistribution of viral vectors, by potential use of these agents in acts of bioterrorism or war upheaval, or by depletion of vaccination stocks required for protection against pandemics. other factors include the increasing and aging global population and increased number of immunocompromised individualsd factors strongly supporting the maintenance of herd immunity against existing viruses, despite lower incidence of outbreaks in industrialized countries. in the event of a re-emergence of previously eradicated viruses or the acquisition of increased pathogenicity by existing viral strains, there is an urgent need for vaccine development strategies that can rapidly and effectively arrest global spread. important advances are continuously being made in vaccine development strategies toward the control of viruses and associated disorders. vaccine design has been modified from the use of attenuated viruses to use of more precise viral protein subunits specifically targeted by t cells. historically, vaccination immunogenicity was documented by measures of serum immunoglobulin (ig) classes and antigen-specific antibodies produced by humoral immunity. more recently, quantification of cellular components of innate immunity at the interface between innate and adaptive immunity are made, in addition to more precise measurements of adaptive immunity. long-term protection achieved by adaptive immunity can be quantified by measuring levels of circulating cytokines, along with specific phenotypic profiles of effector memory, antigen-specific t cells. though both humoral and cellular arms of immunity are integrally linked during the initial induction of immunity against pathogens, these can become disconnected with developing pathology due to their individual needs for survival factors, unequal declines in immune function, and differential cellular lifespans. this loss of correlation between memory t cells and neutralizing antibody responses varies according to different viruses, suggesting that independent time course measures of these separate immune responses are required over time for adequate recording of biomarkers of natural infection and vaccine efficacy or suggesting that t cell status may be most crucial measure of conferred long-term immunity. from the standpoint of fundamental or clinical research, it has become established that the targeted induction of specific pathogen-and tumorclearing effector memory t cell subsets is our endgame armor toward long-term human survival against infectious diseases and cancers. this chapter provides an overview of viruses that have historically caused severe lethal disorders, including those of the respiratory, gastric, skin, hepatic, neurologic, and hemorrhagic types. the features of viruses and associated disorders that we herein describe include viral genetics and replication cycles, transmission modalities, cell and organ tropism, host-immune evasion strategies, associated viral disorders and diseases, and epidemiology. we also report on well-accepted and other important documented instances of viral control by t cells, currently available and successful vaccines, and recorded measures of vaccination immunogenicity. we focus on quantification of vaccine-induced effector memory t cellemediated immunity, representing the gold standard of successful vaccination. just as it is for advances in vaccinology, investigations into the biology of t cells are currently at the forefront of many research fields examining various disorders, diseases, and malignancies not formerly considered to be controlled by immunity. although many viral infections are limited to the upper respiratory tract, it is lower respiratory tract infections (lrti) that most predominantly cause enormous disease burden in children and immunocompromised adults suffering from human immunodeficiency virus (hiv) infection or in patients having received stem cell or solid organ transplants for which immunosuppressive therapies were administered (henrickson et al., 2004; pavia, 2011; kim et al., 2007) . acute lower respiratory illnesses (alris) are a major cause of morbidity and mortality, accounting for approximately 1.6 million deaths, globally, per year (black et al., 2010) . frequently overlapping lrti syndromes include bronchiolitis, asthma exacerbation, wheezing, croup, and pneumonia. although certain specific syndromes can be more precisely associated with infection by specific viruses, syndromes overlaps can complicate diagnosis of these numerous viruses, and quite often, difficulties in differentiating between viral and bacterial pneumonias symptoms can also result in antibiotics being mistakenly prescribed during viral disorders. several viruses are normally, however, considered to be primarily responsible for lrtis, beginning with upper respiratory tract infections, most commonly caused by respiratory viruses that are typically spread from person-to-person by contact with infected respiratory droplets, and including respiratory syncytial virus (rsv), epidemic influenza a and b, h5n1 and h7n9 avian influenza a viruses (iavs), parainfluenza viruses 1 through 4, adenovirus, human metapneumovirus (hmpv), severe acute respiratory syndrome coronavirus, human coronaviruses nl63 and hku1, rhinoviruses, and bocaviruses (pavia, 2011; mahony, 2008; nichols et al., 2008) (table 1) . currently, vaccines for human influenza viruses, human parainfluenza viruses (hpivs), and adenoviruses causing upper and lower respiratory infections are used to control these infections and the resulting propagation of their morbid symptom derivations. human influenza viruses make up three of the five genera of the family orthomyxoviridae and are classified as a, b, and c types, based on their highly conserved matrix protein 1 (m1), membrane matrix protein (m2), and nucleoprotein (np). type a influenza viruses can be further sub-subtyped by the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins (gps). antigenic drift, caused by point mutations in ha and na and recombination of the ha genes, results in the generation of new strains that can escape pre-existing immunity, causing both the prediction of circulating strains difficult and antigenic mismatch by existing vaccines. approximately 18 ha and 9 na subtypes of influenza a are documented in aquatic birds, representing their natural hosts (i.e., vectors). influenza a h1 and h3 subtypes cocirculate seasonally, and influenza b viruses can only infect humans, via two distinct, seasonally cocirculating, lineages. type c influenza viruses are more rarely documented to infect humans and pigs (berlanda scorza et al., 2016) . influenza viruses cause acute upper and lower respiratory infections, and due to their rapid and unpredictable genetic drift, represent the most likely of pathogens to cause a human pandemics. annually, human influenza viruses have the potential to cause up to 5 million cases of severe illness, with an associated 500,000 deaths worldwide (who_influenza_(seasonal), 2018), causing great economic burden. four influenza pandemics have occurred over the past century, as a consequence of the h1n1 (1918), h2n2 (1957), h3n2 (1968), and h1n1 (1977) variants (palese, 2004) . since the most recent outbreak in 2009, an estimated 200,000 people globally have succumbed to the h1n1 variant of swine origin (dawood et al., 2012) . epithelial cells that are infected with influenza virus produce inflammatory cytokines acting as chemoattractants for homing macrophages and dendritic cells (dc). dcs take up influenza viral particles to trigger their maturation and pursuant migration to the lymph, where they initiate antigen-specific t cell maturation. these influenza-specific effector t cells then enter the respiratory tract to counteract viral titres through cytokine expression and the direct lysis of infected cells, with activated cd8 þ effector cytotoxic t cells (ctls) representing the main constituents of this response by their release of perforins and granzymes, and the engagement of tumor necrosis factor (tnf) receptors (spitaels et al., 2016) . influenza-specific cd4 þ t helper cells can act directly and indirectly in viral clearance, primarily by producing cytokines that induce the functions of b cells and cd8 þ t cells and which have also been reported to directly eliminate infected cells themselves (topham and doherty, 1998; hua et al., 2013) . while preexisting cd8 þ t cell immunity has not yet been demonstrated to prevent infection from occurring, it is hypothesized to be the result of the loss of granzyme expression by memory cd8 þ t cells and populations of iavspecific cd8 þ t cells are still importantly correlated with the control of spread and recovery in healthy populations (grant et al., 2016) . the most currently administered influenza vaccines are inactivated (iv) trivalent (tiv) or quadrivalent formulations containing equal amounts of ha of two influenza a strains (h1n1 and h3n2) and one of two influenza b strains (yamagata and victoria lineage). these are derived from viruses typically grown in fertilized chicken eggs, are mainly focused on eliciting a strainmatched humoral immune responsedrequiring yearly updatesdand are unable to provide protection to all vaccinated individuals. the requirement of memory t cell immunity for long-term protection against influenza virus promotes the development of vaccines that elicit both humoral and cellular immunity: a strategy expected to overcome the inadequacies of current vaccines against influenza and other viruses (spitaels et al., 2016) . there is broad interest in the development of a universal influenza vaccine, considered to be the "holy grail" of influenza vaccine research. this approach is being developed to use virus-infected cell-killing antibodies that produce an antiviral environment; these termed antibody-dependent cellular cytotoxicity (adcc)-mediating antibodies, which are predicted to link innate and adaptive immune responses, and is becoming possible due to new technologies for rapid isolation and characterization of monoclonal antibodies targeting conserved regions of influenza virus, reviewed in jegaskanda et al. (2017) . this approach has been postulated to work, in part, from reports of iavspecific cd8 þ t cells, promoting viral clearance in the absence of neutralizing antibodies, and can also mediate cross-reactive immunity against distinct iavs to drive a rapid recovery from severe influenza disorders (grant et al., 2016) . the induction of infection-permissive immunity is both protective and allows virus-induced cross-reactive immune responses. vaccines targeting the conserved ectodomain of m2 deliver this kind of non-neutralizing immunity since these antibodies rely on fc receptors and innate immune components (el bakkouri et al., 2011) . antagonizing antibodies inhibiting na activity represents another promising strategy, not by blocking viral entry and eliciting sterilizing immunity, but by contributing to immunity against a virus possessing a similar na type (wan et al., 2013) . there is also progress being made in the development of recombinant t celleinducing vaccines, with the most advanced version of this strategy demonstrated by modified vaccinia ankara (mva) viruses expressing influenza virus np and m1 antigens (berthoud et al., 2011) , with vaccinated individuals demonstrating increases in interferon-gamma (ifn-g) expressing cd8 þ t cells and increased protection against influenza infection (antrobus et al., 2012; powell et al., 2013) . co-administration of this mva-based vaccine with tiv formulations results in increased influenza strainespecific antibody responses and the generation of memory t cells that recognize a range of influenza a subtypes (antrobus et al., 2014) . additional research demonstrates production of antigen-specific t cell responses using alternate prime/boost regiments of combinations of vaccination regimens employing recombinant replication-deficient adenovirus or mva, expressing iav np and matrix protein 1 (lambe et al., 2013) . quality and clonal t cell receptor (tcr) characteristics of influenza-specific cd8 þ t cells, in addition to in silico predicted and peptide-based approaches for pools of minimal iav epitopes, are investigated for their induction of cellular immunity and recognition by cd8 þ t cells (reviewed in grant et al., 2016) . hpiv are enveloped negative-sense rna genome viruses of 150e250 nm, belonging to the large and rapidly growing paramyxoviridae family, causing significant human and veterinary disorders (henrickson, 2003) . hpiv is divided into serotypes 1 to 4, with hpiv-1 representing the most common etiologic agent of associated disease, but with hpiv-1, -2, and -3 representing common causative agents of respiratory illness in pediatric, geriatric, and immunocompromised populations (schmidt et al., 2011) . hpivs are a common cause of acute respiratory illness throughout all stages of human life (schomacker et al., 2012) , causing acute respiratory infections in children (cooney et al., 1975) . second, only to human respiratory syncytial virus, hpivs are the major contributors to hospitalization due to alris and global pneumonia mortalities in young children, and up to 80% of children are seropositive for hpivs by the age of 5 years (murphy, 1988; weinberg et al., 2009) . hpiv infections can induce potent humoral and cellular immune responses, including innate immune responses, local and systemic igg and iga responses, and adaptive cd8 þ and cd4 þ t cell responses (gitlin et al., 2010; hou et al., 1992) . though cellular responses can restrict hpiv replication dynamics and clear primary infections, neutralizing antibodies against virus envelope hemagglutininneuraminidase (hn) and fusion (f) gps are required for early infection (suzuki et al., 2001; zhang et al., 2005) and confer long-term protection against hpiv-related disorders (murphy, 1988; spriggs et al., 1987; schmidt et al., 2011) . currently, there is no vaccine to protect against human hpiv infection. progress in the development of hpiv vaccines using reverse genetics for serotypes à1 to à3 has generated several live-attenuated, intranasal hpiv vaccines evaluation in adults and in children, two of which, hpiv-3 are well tolerated in hpiv3-seronegative pediatric populations (schmidt et al., 2011) . ongoing pediatric trials testing live-attenuated hpiv vaccines for hpiv-1 and hpiv-2 predict these will replicate in the upper respiratory tract of infants to induce the full spectrum of humoral and cellular immune responses (karron et al., 2015) . heterologous (i.e., jennerian) vaccine design strategies using the sendai virus (sev) to control infections by hpivs and rsv are discussed in the following section. human respiratory syncytial virus (rsv) types a and b are found within the genus orthopneumovirus, family pneumoviridae, of the order mononegavirales. rsv is an enveloped, spherical virus of w150 nm in diameter, and reaching up to several micrometers in length (gower et al., 2005) . the negative-sense rna genome encodes for outer structural, np, polymerase, ns, transmembrane, and regulatory proteins (griffiths et al., 2017) . rsv is a major cause of alris, resulting in numerous pediatric hospitalizations globally, where by 3 years of age, most children have been exposed and are at risk of developing life-threatening bronchiolitis and pneumonia (glezen et al., 1986; hall et al., 2009; henrickson, 1994) . up to 120,000 infants are hospitalized due to rsv infection in the united states (marks, 1992) , and 34 million episodes of rsv-associated alri in children globally represent at least 3 million cases resulting in hospitalization, and approximately 199,000 associated deaths per year (nair et al., 2010) . a balance of adaptive immune ctls and neutralizing antibodies of the humoral immune response mediate protection and clearance of rsv infection (griffiths et al., 2017) . though neutrophils are the highest proportion of leukocytes found in the airways of those infected with rsv (everard et al., 1994) , and despite observations that natural killer (nk) cells are first to attain infected airways (hussell and openshaw, 1998) , it is cd4 þ helper and cd8 þ ctls that correlate with the early clearance of rsv-infected cells (anderson et al., 1990) . in infants and immunocompromised populations, fatalities resulting from rsv infection are associated with deficiencies in cd4 þ and cytotoxic cd8 þ t cells (hall et al., 1986; welliver et al., 2008) . later in infection, increases in neutralizing antibodies prevent reinfection by opsonizing viral epitopes required for rsv entry and infection, and rsv clearance can be associated with rsv-neutralizing nasal immunoglobulin a (iga) (mcintosh et al., 1978) . more recently, enhanced rsv clearance with reduced disease severity has been associated with vaccine-elicited memory cd8 þ t cells (lee et al., 2012) . while memory cd8 þ t cells can mediate protection against rsv infection, in the absence of antibodies and memory cd4 þ t cells, these cause mortality via systemic proinflammatory cytokine storms and local ifn-g production (stoley et al., 2016; schmidt et al., 2018) . other recent studies however indicate that the cd8 þ t cell response may not be the major determinant of severity of rsv-related pathology (collins and melero, 2011) . there are currently several recombinant rsv subunit vaccines in clinical trials, including the novavax rsv f vaccine representing the most promising candidate for licensing. this rsv f subunit targeting vaccine has been demonstrated to elicit the expression of circulating neutralizing antibodies against rsv (glenn et al., 2016) . toward the development of future adaptive immunity-inducing rsv vaccines, it has been demonstrated that the transfer of airway-resident t cells protect against rsv, where it has been suggested that, to lessen the burden of t cellemediated damage to airways, the induction of lung tissue t cells should be the focus of vaccine development (kinnear et al., 2018) . recently, the sev has been used as a component of a jennerian vaccine model for hpiv-1 and as a backbone for other viruses causing serious lower respiratory infections (lris), including other hpivs, rsv, and hmpv. sev-based vaccines have proven to be effective toward inducing b cell and t cell immune responses, and in the protection from hpiv-1, -2, and -3, and rsv, where they can also be used in combination with other vaccines to primeeboosts or to target one or more than one paramyxovirus pathogen (russell and hurwitz, 2016) . sev is attractive for use in human vaccination because it is a murine pathogen unable to infect humans (bousse et al., 2006) and therefore does not require attenuation as it can never revert to a human pathogenic phenotype (schickli et al., 2012) . another important feature supporting the use of sev as a pan-virus vaccination agent stems from its ability to grow transiently in mammalian cells, accommodating the endogenous expression of antigens with posttranslational modifications matching those of the target antigens and neutralizing epitopes (henrickson et al., 1991) , with endogenous expression of antigens ensuring robust activation of cd8 þ t cells able to destroy antigen-producing cells and terminate virus amplification (york and rock, 1996; russell and hurwitz, 2016) . in murine studies, sev could elicit rapid and durable respiratory mucosa and systemic hpiv-specific b cell and t cell responses (sealy et al., 2010; rudraraju et al., 2011) . clinical testing of human populations infected with rsv and hpivs is underway (adderson et al., 2015) . for a full review of current development of antiviral compounds and vaccine candidates tested against rsv, see costello et al., 2012. 2.4 adenovirus classification, epidemiology, immunology, and vaccinology human adenoviruses (hadvs) are classified in the mastadenovirus genus, containing seven known hadv species, from hadv-a to hadv-g, and with at least 57 unique known human serotypes (buckwalter et al., 2012) . adenoviruses are nonenveloped double-stranded dna viruses ranging from 65 to 80 nm in diameter and are composed of a protein capsid, a np core, and internal proteins. dna homology between hadv subgroups ranges from 48% to 99% (walls et al., 2003) . hadv infection rarely causes serious or fatal illness in immunocompetent individuals but may cause severe disease in immunocompromised, pediatric, and geriatric populations (lynch et al., 2011) . clinical disease symptoms associated with hadvs are dependent on hadv genotypes, with at least 69 recognized, and assigned to subgroups a through g. clinical symptoms include fever, rhinorrhea, pharyngitis, conjunctivitis, gastroenteritis, bronchitis, pneumonia, acute hemorrhagic cystitis, and meningoencephalitis (lynch et al., 2011) . recombination between hadvs are largely responsible for outbreaks of acute febrile respiratory disease in immunocompetent military recruits, where serotypes 4 and type 7 have been documented to account for approximately 60% of these respiratory illnesses (hilleman et al., 1957; dudding et al., 1973) , and are associated to other frequently occurring disorders including upper and lower respiratory illnesses, gastroenteritis, hepatitis, keratoconjunctivitis, meningoencephalitis, cystitis, and myocarditis in these immunocompetent populations, reviewed in lion (2014) . adenoviruses are endemic in pediatric populations (echavarria, 2008) . the incidence of adenovirus infection peaks in infants and children, where, globally, 5%e7% of respiratory tract infections in pediatric patients are ascribed to hadv (ghebremedhin, 2014) . recently, re-emergence of type 7d hadv has caused fatal outbreaks due to severe pneumonia syndromes in children from high-density populations . immune responses to adenovirus infection are dependent on primary sites of inoculation, methods of transmission, viral serotypes, and secretory ig antibody status of the infected host; igas are present in respiratory tract early following infection, and igg2 is present in serum and nasal secretions at later time points, reviewed in walls et al. (2003) . histopathological changes resulting from infection can be divided into two phases: the first phase of immune histopathology predominantly involves nonspecific, cytokine-mediated inflammatory recruitment of monocytes and macrophages, while the second phase involves t cell infiltration (prince et al., 1993) . t cellemediated immunity is believed to be required for hadv recovery from acute infections, and individuals lacking adaptive immunity are found to be at elevated risk of infection, with cd8 þ t cells as primary mediators of response to respiratory viruses, with relatively little contribution by cd4 þ cells (woodland et al., 2001) . however, following adenovirus exposure, cd4 þ t cells have been shown to be responsible for increasing proliferation status of peripheral blood mononuclear cells (pbmc), and cd4 þ t cells represent the major ctl subsets produced and recognize conserved antigens across adenovirus serotypes (flomenberg et al., 1995; regn et al., 2001) . adenoviruses have, however, evolved several hostevasion strategies, including inhibition of apoptosis, responses to ifn-g and tnf-a, and major histocompatibility complex (mhc) class i expression (mahr and gooding, 1999; wold et al., 1994 wold et al., , 1999 . the live, oral adenovirus vaccine was licensed in the 1970s for active immunization toward the prevention of febrile acute respiratory disease in military populations, where it initially reduced adenovirus-associated respiratory illnesses by over five-fold (dudding et al., 1973) . vaccine stock depletion and associated epidemics led to the manufacture of another vaccine in 2011, again denting adenovirus-associated disease burden by approximately 100-fold among recruits within the first 2 years of its introduction (radin et al., 2014) . these oral lyophilized vaccines replicate asymptomatically in the gut, inducing humoral and cell-mediated immunity, to confer longlasting protection from infection (berg et al., 2014) . due to their abilities to induce potent transgene product-specific t-and b-cell responses, adenovirus vectors are explored for use as vaccine carriers against a variety of many other pathogens (chen et al., 2010; harro et al., 2009; hill et al., 2010; radosevic et al., 2007) . alris by respiratory viruses are a major cause of morbidity and mortality, accounting for over 1.5 million deaths globally each year, and are predominantly resulting from human transmission of virus containing respiratory droplets. licensed vaccines are useful against several viruses causing these severe, often lethal, associated disorders. influenza has no borders and causes great economic burden, making it a prominent international concern. its rapid and unpredictable genetic drift causes human pandemics, where it has an annual potential of causing 5 million infections and 500,000 deaths worldwide. influenza vaccinology requiring constant yearly updates has stimulated interest in the development of universal t cell vaccines that can elicit both humoral and cellular immunity, whereby influenza-specific memory cd8 þ t cell responses against a range of influenza subtypes could be induced to clear infection in absence of neutralizing antibodies. rsv causes 34 million alris in children annually, resulting in 3 million hospitalizations and almost 200,000 deaths per year. rsv is cleared by balance of adaptive immune ctls and humoral neutralizing antibody responses, correlating most highly with cd4 þ helper and cd8 þ ctls during natural infections, and with memory cd8 þ and cd4 þ t cells following vaccination, with research endeavours targeting their strengthening by specific induction of lung tissue rsv targeting t cells. second, only to rsv, hpivs cause many ari-and lri-associated mortalities in children. hpivs can induce potent humoral, innate, and adaptive cd8 þ and cd4 þ t cell responses able to restrict their replication and where neutralizing antibodies can confer long-term protection against their associated disorders. hadvs infect both immunocompetent and immunocompromised humans and have been shown to cause up to 60% of respiratory disorders in hospitalized military personnel. despite their having evolved convoluted host-evasion strategies, adaptive t cell immunity against hadvs starts early in diseases phases and is key to recovery from acute natural infection, with its greatest contributions by cytotoxic cd8 þ t cells that require stimulating by cd4 þ t cells for their expansion. vaccines against hadvs induce both humoral and adaptive immunity, including potent transgene virus-specific t-and b-cell responses conferring long-term protection. success from hadv vaccinology has influenced explorations of adenovirus vectors as target carriers for vaccination against numerous other pathogens. diarrheal disorders remain a leading cause of morbidity and mortality worldwide, with these listed in the top five causes of death worldwide, and which are associated with global estimates at 4e6 million deaths per year; reviewed in clark and mckendrick (2004) . the majority of gastric infections are viral in origin, and viral gastroenteritis is one of the most common illnesses in all age groups and an important cause of morbidity in industrialized countries (chang et al., 2003) . the human risk of viral gastroenteritis in the united states alone is at least one per individual per year, with 450,000 adults and 160,000 children hospitalizations recorded and an associated 4000 mortalities per year (mead et al., 1999; mounts et al., 1999) . several viruses are responsible for viral gastroenteritis, where their transmission typically occurs from person-to-person by the oral-fecal route. viruses commonly causing gastroenteritis include rotavirus (rv; causing the most serious gastric disorders), norovirus, astrovirus, adenovirus, and coronavirus-like agents (table 1) . rvs are classified as a genus within the family reoviridae. these are nonenveloped viruses measuring 70 nm in diameter and have inner and outer capsids surrounding their cores containing double-stranded rna viral genomes encoding viral capsid (vp-1 to vp-6, and vp-7; vp4 outer capsid protein mediates virus attachment to cells) (bishop et al., 1973) and nonstructural (nsp-1 to nsp-6) proteins, reviewed in desselberger (2014) . rvs classify into seven serotypes (aeg), based on antigenic properties of the inner capsid vp6 protein, where subtypes aec represent human pathogens and are further subclassified into serotypes within these groups on the basis of differing outer capsid composition (anderson and weber, 2004; wilhelmi et al., 2003) . diarrhea is a major cause of death among children globally (liu et al., 2012) , and rv is the leading cause of severe diarrhea, globally causing an estimated 453,000 deaths in developing countries and 2.3 million pediatric patient hospitalizations parashar et al., 2003) . rv also represents a significant cause of disease in industrialized countries, with greater numbers of hospital admissions reported relative to developing countries (chang et al., 2003) . though group a rv causes the majority of endemic infections and can also lead to significant outbreaks in infant and geriatric populations (villena et al., 2003; marshall et al., 2003) , group b rvs are less common but can also lead to outbreaks and epidemics (sanekata et al., 2003; ahmed et al., 2004) , whereas group c rv is less often observed causing sporadic diseases. of the existing 10 g and eight p rv group a serotypes, g1 to 4, p (kostouros et al., 2003; clark and mckendrick, 2004) . studies of t cell responses to rv infection in humans have reported that most healthy adults and children have circulating rv-specific t cells, with approximately 50% of rv-cd4 þ t cells expressing the intestinal homing receptor a4b7, and with circulating rv-cd4 þ and rv-cd8 þ t cells secreting ifn-g or interleukin (il)-2 (makela et al., 2004; offit et al., 1992; yasukawa et al., 1990; rott et al., 1997; parra et al., 2014) . frequencies of circulating ifn-g þ rv t cells are comparable to those specific for other mucosal respiratory viruses (mesa et al., 2007) , but these often possess profiles of terminally differentiated effector cells that are usually associated to those unable to provide long-term immunity (parra et al., 2014) . (yen et al., 2014) . as in the case of natural neonatal rv infection, fair protection rates are achieved via humoral immunity using these vaccines. though these are unable to protect against rv reinfection, they do offer protection against severe associated clinical symptoms causing patient hospitalization. these vaccines offer both homotypic and heterotypic immunity, and protection often correlates with increases in rv typee specific igg or iga antibodies, reviewed in desselberger and huppertz (2011) . although rv vaccineeinduced humoral immunity substantially decreases disease burden, these vaccination strategies are less effective and difficult to implement in low-income countries requiring them most (patel et al., 2012) . as with natural rv infection, vaccines provide nonsterilizing immunity to children (angel et al., 2007) , where lack of establishment of long-term immunity against rv causes half of children's guardians to be at risk of becoming infected and presenting with severe associated disorders (rodriguez et al., 1987) . this further demonstrates that rv-specific t (rv-t) cells are crucial for the development of overall, long-term, protective immunity against rv (franco et al., 2006; offit et al., 1993) . indeed, in models of rv infection, vaccine-induced protective immune responses are dependent on antiviral cytokine production and by direct killing of rv-infected cells by t cell and b cell adaptive immune subsets (jiang et al., 2008; wen et al., 2016) . in addition, with observations that gut cd4 þ t cells may become tolerogenic or anergic in response to rv infection, stimulating t cells with rv antigen in the presence of il-2, il-12, or r59949, a pharmacological diacylglycerol kinase alpha inhibitor, causes increased pbmc frequencies of rv antigen-specific t effector cells, including rv-cd4 þ tnf-a þ , rv-cd4 þ ifn-g þ , and rv-cd8 þ ifn-g þ cells (parra et al., 2014) . diarrheal disorders cause an annual 4e6 million deaths worldwide. rv is the leading cause of severe diarrhea outbreaks in infant and geriatric populations, with global annual estimates of 453,000 deaths in developing countries and 2.3 million pediatric hospitalizations. most immunocompetent individuals have circulating rv-specific ctls at comparable frequencies to those elicited by other respiratory viruses, but which have terminally differentiated effector profiles rendering them incapable of conferring long-term protection against the reoccurrence of associated disorders. rv vaccineemediated protection from severe disorders is from humoral nonsterilizing immunity unable to protect against reinfection, yet vaccination programs are challenging to implement in countries requiring them the most. rv-specific t cells are key to long-term protection, and vaccineinduced protection is dependent on cytokine production and direct killing of infected cells by t cells. countermeasures against crucial helper cd4 þ t celledeveloping anergic states may assist the development of vaccines conferring long-term protection. an exanthem is a widespread eruptive skin rash that may be associated with fever or other systemic symptoms. more than 50 infectious agents causing exanthems have been identified (cherry, 1983) , where more than 70% of recorded cases of combined fever and widespread rash in pediatric populations were caused by viral infections, relative to the 20% resulting from bacterial infections (goodyear, laidler, price, kenny and harper, 1991) . correct diagnosis of these skin manifestations, resulting from direct inoculation of the infectious agent onto the cutaneous surface, or by dissemination from a distant site, is a main research theme on viral exanthems. this is because, while infections by many viral (i.e., paraviral) exanthems are benign and resolve spontaneously, others may rapidly lead to fatal conditions, reviewed in drago et al. (2017) . thus, special attention in diagnosing even vaccine-preventable viral exanthems must be applied to avoid the arising of serious complications in nonimmune pregnant women and their fetuses from the more harmful classes of viruses causing exanthems (white et al., 2012) . common exanthematous infections are typically caused by transmission of viruses from person-to-person (with exception of alphaviruses having a mosquito vector), and where a multitude of viruses are their causative agents, including rubeola virus, rubella virus, human parvovirus b19, human herpesvirus (hhv) type 6, varicella-zoster virus (vzv), variola, alphaviruses, and molluscum contagiosum virus (table 1) . numerous other exanthematous disorder causing viruses are not covered in this section, including ebola and zika, but which are becoming classified as emerging viral exanthems due to the increasing numbers of at-risk populations and the critical need to classify these diseases to minimize outbreaks and risk to pregnant women and fetuses (keighley et al., 2015) . rubeola, or measles virus (mev), belonging to the morbillivirus genus of the paramyxoviridae family, is a negative-sense rna virus having a nonsegmented genome and a lipid envelope, and measuring up to 250 nm in diameter, reviewed in griffin et al. (2012) . the 16 kb genome encodes eight proteins: the viral envelope is composed of hemagglutinin (h) and fusion (f) gps projecting from the matrix (m) protein lining its interior. the helical nucleocapsid is composed of the rna and nucleocapsid (n) protein packed within the envelope as a coil with the phosphoprotein (p) and large polymerase (l) proteins attached. the two ns proteins, c and v, regulate cellular response to infection and modulate ifn signaling (bellini et al., 1985) . humans are the only natural host of highly contagious mev virus spread by the respiratory route. despite the availability of a safe and efficacious vaccine, measles remains one of the most important viruses causing child morbidity and mortality worldwide (moss and griffin, 2006; wolfson et al., 2009) . infection by mev is associated with up to 10% of mortality rates in african children (grais et al., 2007; nandy et al., 2006) , and with 25% in unvaccinated refugee camp and virus-naive population mortalities (moss, 2007; shanks et al., 2011) . female mortality is a dominant feature disorders resulting from infection (garenne, 1994) , and many acute mortalities from secondary infections resulting from immune suppression induced by mev are also observed (beckford et al., 1985) . mev has a persistent and long latency infection period, often resulting in the development of subacute sclerosing panencephalitis (sspe) in males, causing fatal neurologic disease presenting itself many years following the original infection (bellini et al., 2005) . adaptive cellular immune responses are generally regarded as most important for clearance of mev. children with low plasma ig may recover from mev infection, while those with defects in cellular immunity develop progressive infections (albertyn et al., 2011; mcquaid et al., 1998) . mev-specific antibody and t cell responses coincide with the onset of the rash, whereby rash biopsies of mev-replicating, infected epithelial cells, have high levels of cd4 þ and cd8 þ t cell infiltrates (polack et al., 1999) . cd8 þ t cell subsets appear to be particularly important for control and clearance of infectious mev, where expanded circulating virus-specific ctls are found in the blood of patients suffering rash, and increases in cd8 þ t cells are also found in mev-induced pneumonias (jaye et al., 1998; mongkolsapaya et al., 1999; myou et al., 1993) . in addition, depending on the target tissue and cell type analyzed with regards to mev infection, though differentially rated, both cytotoxicity and ifn production have been implicated as key effector mechanisms for mev clearance (patterson et al., 2002; stubblefield park et al., 2011; finke et al., 1995) , with specific combinations of cd4 þ t cells, cd8 þ t cells, and b cells recorded as required for the control of primary mev infection (tishon et al., 2006) . protection against measles is based on mev-specific humoral, antibodybased, immunity. diagnostically, the current gold standard of protection is via quantification of neutralizing antibodies against the viral hemagglutinin (h) and fusion (f) surface gps (bouche et al., 2002; haralambieva et al., 2011; plotkin, 2010) . mev, however, triggers an aggressive immune response, involving both the humoral and cellular arms of the immune system (moss and griffin, 2012; de vries et al., 2012; buchanan and bonthius, 2012) . once measles has been cleared, it is memory t cells that can provide lifelong immunity against reinfection by mev (bester, 2016) . importantly, during mev infection, immune reactions to other pathogens are suppressed from weeks to years, leading to risk and susceptibility to secondary infections, and which is believed to be a driver of complications and mortality long after measles had been cleared. conversely, this measlesinduced immune "amnesia," sometimes disabling immune memory for up to 3 years, has been suggested to work toward herd protection against other infections and is supported by the association of measles vaccination with lowered mortality rates from other childhood infections (mina et al., 2015) . occasional spontaneous tumor regressions have also been observed to occur during natural measles infection, suggesting that mev infection may be adopted in the generation of safe and effective oncolytic viruses (russell and peng, 2009 ). rubella virus belongs to the togaviridae family and is the sole member of the rubivirus genus. rubella contains a single-stranded, positive-sense rna genome (frey, 1994) , and its viral particles measure between 50 and 85 nm in diameter (oshiro et al., 1969) and have a pleomorphic nucleocapsid surrounded by a host-derived lipid membrane (battisti et al., 2012) . the e1 and e2 rubella protein spikes are anchored to the external layer of the membrane, with membrane-bound e2 proteins bridging rows of e1 proteins, considered as the main immunodominant antigens responsible for controlling receptor-mediated endocytosis (petruzziello et al., 1996; katow and sugiura, 1985) . antibody levels against the neutralizing domain of e1 correlate with protection against rubella virus (mitchell et al., 1996; cordoba et al., 2000; wilson et al., 2006) . rubella virus is spread from person-to-person via the respiratory route and is the causative agent of rubella disease, commonly known as german measles (lambert et al., 2015) . although rarer in the united states, rubella infection remains a major health concern in developing countries (tosh et al., 2009) . although acquired rubella infection is not severe in adults, transplacental transfer of the virus to the developing fetus during maternal viremia can cause devastating consequences of congenital rubella syndrome (crs) (watson et al., 1998) , where more than 100,000 infants worldwide are born with crs each year . common crs symptoms include spontaneous abortion, premature delivery, fetal death, ocular abnormalities, neurological problems, abnormal cardiac development, and deafness (white et al., 2012) . congenital malformations due to crs may be present at birth, while other conditions such as diabetes mellitus, deafness, intellectual disability, and/or subacute encephalitis may develop months to years later (watson et al., 1998; white et al., 2012) . from mass immunization programs, the number of rubella cases has progressively declined and was no longer endemic in the united states as of 2004 but remains endemic in other countries, with a dramatic increase in reported cases the last decade (reef et al., 2011) . recently, africa and asian have seen 20-fold increases in rubella cases, representing a significant proportion of the over 121,000 global cases reported, but where neither of these regions has immunization policies in place to control rubella outbreaks (white et al., 2012) . a recent, 2013 rubella epidemic in japan reporting over 11,000 cases, with at least 13 crs cases (minakami et al., 2014) , has also served to demonstrate that partial vaccination strategies can lead to major outbreaks. in this case, vaccination was only provided to young women, while outbreaks affected the adult male populationsda phenomenon which has also been observed in other countries applying such vaccination strategies (paradowska-stankiewicz et al., 2013; janta et al., 2012) . once measles is cleared, memory t cells can provide lifelong immunity to mev (bester, 2016) , and distinct patterns of cellular immunity to rubella virus are observed and related to the time elapsed following vaccination (lambert et al., 2015) . predominant biomarkers of early cellular measles immunity are characterized by an immunosuppressive phenotype, with increases in il-10 and tnf-a and decreases in ifn-g and proliferative properties of circulating peripheral lymphocytes (pukhalsky et al., 2003) . late immunity is shifted to predominantly proinflammatory cytokine profiles via increased concentrations of il-6, granulocyte-macrophage colony-stimulating factor, and tnf-a, in combination with decreases in il-10 (dhiman et al., 2010) . human leukocyte antigens (hlas), known to play critical roles in immune response to viruses, contribute to the heterogeneity of the immune response to rubella virus as a result of their polymorphic nature, whereby hla class i and ii polymorphisms restrict the available repertoire of rubella antigens presented to t cells and therefore influence the subsequent immune response (mitchell et al., 1996; ou et al., 1994 ou et al., , 1998 . current efforts are placed on deciphering the immunogenetics of antirubella humoral and cell-mediated immune responses, with a focus on better understanding hla polymorphisms toward the development of vaccine candidates that utilize constructs comprised of hla-specific epitopes that can induce immunity across heterogenetic populations, reviewed in lambert et al. (2015) . both natural infection and vaccines induce humoral and cellular immune responses conferring protection against rubella (tosh et al., 2009) . while humoral responses have been conventionally used to measure and record protective immunity in human populations, cellular immune responses are intrinsic to humoral immunity (bautista-lopez et al., 2000; horstmann et al., 1985; ovsyannikova et al., 2004; nepom et al., 1997; vesikari et al., 1975; akaboshi et al., 2001; farzaneh et al., 2003) . since its induction into healthcare systems, immunization with live attenuated rubella virus vaccine has been demonstrated to be safe and effective at preventing infection, crs, and to interrupt endemic rubella transmission (lambert et al., 2015) . the live attenuated rubella vaccine strain ra27/3 has a proven track record for safety and immunogenicity efficacy (hilleman et al., 1968; plotkin, 1979) , where single doses have been demonstrated to potently induce humoral immunity and lifelong protection against infection, and where the vaccine has also been demonstrated to boost previously immunized persons (diaz-ortega et al., 2014) . from their safety and efficacy, use of recombinant rubella vectors has also been tested toward enhancing immune responses against siv and hiv epitopes, where increases in memory b cell repertoires have been observed upon re-exposure to rubella vectors (virnik et al., 2013) . durable hiv-specific cellular immunity has been observed from rubella vector boosting, with cytotoxic antigenspecific responses by central and effector memory cd4 þ and cd8 þ t cell subsets (rosati et al., 2015) . vzv, also known as hhv-3, is a virus of the varicellovirus genus from the herpesviridae family. humans are its only vector (hambleton and , where it specifically infects t cells, epithelial cells, and ganglia (gershon et al., 2015) . vzv viruses have diameters measuring up to 200 nm and are encoded by a linear double-stranded dna genome consisting of approximately 125 kb and encoding at least 70 unique genes, with all but the exception of 6, having homologs in herpes simplex virus (cohen, 2010) . vzv virions are composed of the viral dna, the capsid, the tegument surrounding the capsid, and the envelope surrounding the tegument and which incorporates the major viral gps (arvin, 1996) . during lytic infection phases, vzv produces at least 12 gps expressed on both virions and human cell surfaces. during this process, and which is common to other herpesviruses, gene expression is believed to proceed in an orderly cascade of immediate early genes, early genes, and late genes. during latent vzv infection, gene expression is restricted until reactivation for additional rounds of lytic infection (gershon and gershon, 2010) . vzv has extraordinarily high transmission rates and is highly communicable via the airborne transmission route, with concentrated virus coming from vesicles shedding from skin lesions, leading to cell-free contagious airborne viruses, and as evidenced by the fact that infected children without skin lesions are not contagious (tsolia et al., 1990; chen et al., 2004) . primary vzv infection causes varicella, also commonly known as chickenpox. as cellular immunity to vzv wanes in the elderly and immunocompromised populations, latent vzv becomes reactivated and causes zoster (i.e., shingles, herpes zoster), which is usually associated with chronic pain but also numerous other serious neurological and ocular disorders, as well as multiple visceral and gastrointestinal disorders, including ulcers, hepatitis, and pancreatitis (gershon et al., 2015; gilden et al., 2010) . available antiviral drugs and vaccines against varicella and zoster are safe and effective for treatment and prevention strategies (gershon and gershon, 2010) . varicella is globally endemic and is transmitted year-round, with frequent epidemics occurring every 2 to 3 years. outbreaks most commonly occur in nurseries and schools, in hospitals and other medical institutions, and in refugee camps and military and correctional facilities (izurieta et al., 1997; levy et al., 2003; longfield et al., 1990) . although it can often be a self-limiting disease, varicella can also result in death, where in developed countries, an estimated 5 of 1000 patients are hospitalized with serious complications, with up to three deaths per 100,000 patients (galil et al., 2002; rawson et al., 2001) . complications from varicella requiring hospitalization include bacterial superinfections of the skin, blood, bones, and lungs, as well as encephalitis and hemorrhagic manifestations in pediatric and immunocompromised populations (gershon et al., 2015) . importantly, acquiring vzv during early pregnancy often results in severe congenital defects in 1% of newborns (enders, 1984) . vzv is a great example of success through herd vaccination programs for children, dramatically influencing its epidemiology, and causing 95% declines of hospitalization cases in the united states (gershon et al., 2015) . following its transmission to the respiratory mucosa, vzv proliferates in the oral pharynx, where it infects human tonsillar activated memory cd4 þ t cells and induces their tissue-homing properties (sen et al., 2014) . vzv can be propagated to t cellerich regional lymph nodes for rapid proliferation and is then disseminated by the circulation to infect dermis, epidermis, and other organs (ku et al., 2002 (ku et al., , 2004 . lymphopenia is typically observed in patients during viral incubation, followed by an increase in leukocyte counts, correlating with the onset of rash until the resolution of viremia. during infection, vzv can be recovered from pbmcs in children exhibiting rash (ozaki et al., 1984; koropchak et al., 1992; sawyer et al., 1992) , is extensively observed in thymic lymphocytes (levin, 2014) , and observed in all t cell subsets examined (moffat et al., 1995) . though innate skin immunity can cause delays in multiplication of skin-bound vzv while the adaptive immune system mounts an attack, however, aggressive vzv replication in the skin results in characteristic varicella rash (ku et al., 2004) . high vzv titre-skin vesicles from rash provide cell-free virus for person-to-person transmission (chen et al., 2004) . vzv also latently infects neurons of cranial nerve ganglia, dorsal root ganglia, and enteric and autonomic ganglia (gershon and gershon, 2010) . vzv reactivation causes ganglia to become necrotic and hemorrhagic (head et al., 1997) , with vzv proteins found in neurons and non-neuronal cells, and where this vzv-induced ganglionitis is often also marked by the upregulation of mhc class i and ii proteins associated infiltration of cd4 þ and cd8 þ t cells (schmidbauer et al., 1992; steain et al., 2014; gowrishankar et al., 2010) . before vzv vaccines became available, approximately 30% of infected adults later developed shingles (yawn et al., 2007) . the single dose, lyophilized, live, attenuated vzv vaccine (i.e., zoster vaccine live (zvl), zostavax, merck) is indicated for prevention of latent vzv reactivation leading to shingles in individuals older than 50 years. zvl is licensed in over 55 countries, with 34 million distributed doses globally (willis et al., 2017) , and which has associated efficacy rates of over 50% in all ages tested (oxman et al., 2005; schmader et al., 2012) ; consistent with original clinical trial datasets (tseng et al., 2011; langan et al., 2013; marin et al., 2015) . however, increases in vzv susceptibility have arisen due to increasing aging populations, and in immune-suppressed organ transplant recipients, chemotherapy patients, hiv-infected individuals, and those suffering from chronic illnesses (forbes et al., 2014) . in these patients, earlier exposure to exogenous vzv protects against shingles by boosting cellular immunity (arvin et al., 1983; thomas et al., 2002) . the memory immune response following naturally acquired primary vzv infection is characterized by vzv igg and iga antibodies, as well as vzv-specific cd4 þ and cd8 þ t cells, where vzv-specific igg antibodies bind many vzv proteins and mediate virus neutralization and antibodydependent cytotoxicity, reviewed in arvin (2008). the frequency of vzv-specific memory proliferating t cells is estimated to be approximately one in 40,000 pbmc (hayward et al., 1986) . vsv-specific memory cytotoxic mhc class i-or class ii-restricted t cells producing ifn-g and tnf-a can recognize the vzv ge, gb, gc, gh, gi, ie62, and ie63 proteins and can be found to persist for over 20 years after varicella exposure (jenkins et al., 1998; huang et al., 1992; asanuma et al., 2000; diaz et al., 1989; hayward et al., 1986; sharp et al., 1992; sadzot-delvaux et al., 1997) . the ability of the live attenuated varicella vaccine to elicit vzv-specific igg and t cell immunity in naive hosts was established during its prelicensing clinical evaluations (gershon et al., 1992) , and where, as expected from its design, the magnitude of these vzv-specific immune responses correlated with infectious virus content and with antigen content of individual vaccine formulations (bergen et al., 1990; watson et al., 1993) . importantly, it was later discovered that providing two doses to children resulted in higher igg antibody titres and increased t cell proliferation and where experimental evidence suggesting that memory responses were sustained more effectively from such regimens (watson, 2008) . these observations led to the more recent recommendation of implementing of a two-dose regimen of varicella vaccine for all vaccine recipients (arvin, 2008) . studies of how regimens affect long-term protection by the adaptive t cell immune response to vaccination, as exemplified by vzv vaccination studies, have the potential to modify dosages and timelines to maximize overall and persisting beneficial long-term effects from vaccination against many other viruses. historically, smallpox was a severe human disease caused by the variola virus (varv), which was both highly lethal and highly contagious prior to its eradication from human populations in 1980 (moore et al., 2006) . varv belongs to the genus orthopoxvirus of the family poxviridae, which also includes zoonotic species: vaccinia virus (vacv), monkeypox virus, cowpox virus, and camelpox virus (shchelkunov, 2013) . orthopoxviruses are enveloped, brick-shaped viruses measuring 350 by 270 nm, and containing a double-stranded dna genomes encoding 150e200 genes, and measuring approximately 200 kb (garon et al., 1978) . unlike other dna viruses, these replicate as 'virus factories' in the cytoplasm of infected cells (pauli et al., 2010) . varv encodes approximately 200 proteins, where over 80 of these are found at terminal regions of the genome and are associated with host immune evasion. the origin of smallpox is unknown, but varv is considered to be one of the most deadly diseases of human history, decimating populations to such an extent that it significantly altered the course of human civilizations. smallpox is believed to have first appeared in 10,000 bc in africa, with the oldest credible confirmation found in sanskrit writings from 1500 bc and where smallpox lesions are believed to be observed on the mummified egyptian ruler ramses v (1100 bc) (ristanovic et al., 2016) . prior to its eradication in 1980, varv circulated in the human population for many centuries and repeatedly caused large-scale epidemics. in the 18th century for instance, smallpox caused the death of more than 400,000 europeans per year (babkin and babkina, 2015; smith and mcfadden, 2002) . despite varv eradication from the human population more than two decades ago, fears about its potential re-emergence or the threat of its use as a potential bioterrorism agent have not subsided. this has led to numerous debates concerning the destruction of existing viral stocks, currently maintained in the united states and russia. destruction of these stocks has been postponed for the benefit of further research elucidating varv mechanisms of pathogenesis toward the design of therapeutics as well as on efficacious vaccine strategies that may be required for potential future outbreak (smith and mcfadden, 2002; stone, 2002) . immune-evasion mechanisms by varv are the least understood among the orthopoxviruses due to difficulties of finding an appropriate host animal model (turner and moyer, 2002) , along with limited availability of authentic variola proteins since its eradication (massung et al., 1993) . thus only two variola proteins, namely smallpox inhibitor of complement enzymes (spice) and vaccinia virus complement control protein (vcp), have been characterized and are similar in structure (dunlop et al., 2003) . these viral antigens regulate the human complement system (yadav et al., 2008) , are important for stimulating innate immunity, and also have important features for adaptive immunity, shown to bolster antiviral t cell responses including ifn and cytokine expression (noris and remuzzi, 2013; moss and shisler, 2001) . vacv has been used more extensively for human immunization than any other vaccine and what was employed to provide cross-protection against varv toward smallpox eradication (jacobs et al., 2009 ). the first generation vacv/varv vaccines produced in the 1970s and 1980s (dryvax, apsv, lancyevaxina, l-ivp) contained live vacv , and induced robust humoral immunity is characterized by high antibody titers, neutralizing and opsonizing viral particles, fixing complement, hemagglutination, and antibody-dependent cell cytotoxicity (amanna et al., 2006; panchanathan et al., 2008) . these vaccines have since been observed to generate adaptive immune responses over many concentrations (frey et al., 2002; rock et al., 2006) , including the secretion of effector cytokines (e.g., ifn-g) and the lysing of infected cells (amanna et al., 2006; hammarlund et al., 2003) . most second-generation vaccines created for biodefense contain replication competent viruses (artenstein and grabenstein, 2008) and have comparable efficacies to dryvax. thirdgeneration vaccine formulations using attenuated vacv strains (lc16m8, mva, nyvac, dvvl) have increased safety profiles (artenstein, 2008; kennedy et al., 2009) . proof that adaptive cellular immunity is essential in preventing the spread of varv following immunization and in its generating overall protective immunity against smallpox comes from observations that individuals having t celledeficiency disorders suffered serious and sometimes fatal infections after vaccination, but that agammaglobulinemic children were not at risk of these adverse complications (rock et al., 2006) . varv vaccine induces strong cd4 þ and cd8 þ t cell responses, peaking after immunization and then contracting to provide stable memory t cell populations that remain detectable for decades (amanna et al., 2006; hammarlund et al., 2003) and with memory cd4 þ t cells persisting the longest (amara et al., 2004) . defects in cellular immunity lead to uncontrolled vaccinia infection (lane et al., 1970) , where cd4 þ and cd8 þ t cells are able to prevent mortality of b celledeficient animals infected with vacv (belyakov et al., 2003) and where cd4 þ t cells have the most protective overall effects (xu et al., 2004) , and are essential for optimal ctl function and memory formation (sun and bevan, 2003; kennedy et al., 2009) . vacv-specific cd4 þ and cd8 þ t cells recognize a diverse array of viral proteins, and cd8 þ t cell epitopes are predominantly found in early, non-structural genes and transcription factors (terajima et al., 2008) . cd4 þ t cell epitopes are from late viral products including membrane, structural proteins, and replicative enzymes (jing et al., 2008) , and linkage of b cell and cd4 þ t cell epitopes to varv proteins suggests t helper celleb cell interactions are those required for generation of robust vacv-specific antibody responses (sette et al., 2008) . in humans, varvspecific cd4 þ and cd8 þ t cells have been observed to persist for over 75 years following immunization (rock et al., 2006) . exanthem disorders by viruses represent more than 70% of cases of combined fever and widespread rash in pediatric populations, and their correct diagnosis is especially critical for the distinguishing of benign versus lethal viral strain variations that can cause lifelong morbidities in children born from infected mothers. mev is transmitted via human respiratory routes, and despite vaccine availability, still causes 10% of african children mortalities, and severe risk of sspe-derived fatalities years later in survivors. historically, in common with many other viruses, the gold standard diagnostic of protection is made by quantification of humoral neutralizing antibodies. adaptive cellular immune responses are, however, those most critical for mev clearance, where mev-specific t cell responses coincide with rashes densely infiltrated by cd4 þ and cd8 þ t cells. combinations of cd4 þ t cells, cd8 þ t cells, and b cells control primary infection, where cd8 þ t cells dominate for control and clearance, and memory t cells are able to provide lifelong protection. mev infection induces general longterm immunosuppression leading to vulnerability to other pathogens causing secondary infections, but this immunosuppression is believed, by some, to be simultaneously conferring herd protection and have been observed to induce spontaneous tumor regression. rubella virus infection has progressively declined from immunization programs but continues to be endemic in many countries, as a result of complete absence of or problematic or partial vaccination programs, still causing severe crs cases in 100,000 infants worldwide, per year. while humoral responses are conventionally used to measure protective immunity, it is adaptive immunity that confers protection. both natural infection and vaccination induce humoral and cellular immune responses, where memory t cells can provide lifelong immunity, with presence of cytolytic t cell biomarkers from vaccine-induced immunogenicity. vaccines in development can comprise hla-specific epitopes inducing immunity across heterogenetic populations. since rubella vaccines can boost the previously immunized, their vectors are being investigated for use toward immunization programs for unrelated viruses. vzv has extraordinarily high human transmission rates. primary vzv infection causes varicella, and before vaccination programs were initiated, would re-emerge from declines in adaptive immunity to cause zoster in 30% of in immunocompromised populations to cause the hospitalization of 1 of every 200 and the death of three per 100,000 patients. vzv represents a poster child of herd vaccination programs that led to 95% declines in hospitalization events. vzv infection rates are again on the rise in immunocompromised and immune-suppressed populations. it infects human tonsillar activated memory cd4 þ t cells that home to the lymph to then infect cd4 þ and cd8 þ t cells, followed by a lymphopenia resolved at rash onset. innate immunity controls vzv spread until adaptive immunity develops to fully counter the infection. vsv-specific memory ctls persist 20 years after varicella exposure, and observations that increased t cell proliferation with better-sustained memory responses result from multiple booster doses of vaccine have caused modifications in vaccination programs. smallpox by varv was one of the most deadly diseases in human history, causing more than 400,000 european casualties annually prior to its vaccine-mediated eradication. viral stocks are maintained from the necessity of developing new vaccines to counter potential future re-emergence of varv from natural-or bioterrorism-derived sources. characterized variola proteins amplify and strengthen t cell responses. first-generation vacv vaccine induced robust humoral immunity and adcc, in addition to generating adaptive immune responses marked by cytokines and cell lysis. varv vaccine induces strong initial effector cd4 þ and cd8 þ t cell responses having b cell linkage, then contacting to generate stable memory populations of varv-specific cd4 þ and cd8 þ t cells that can persist for over 75 years. accordingly, second-and third-generation vaccines created for biodefense are designed to stimulate adaptive cellular immunity. globally, liver cancer is the fifth most common of cancers, with an average of 374,000 cases per year, representing 7.2% of all cancers, and with mortality rates reflecting geographic incidence rates. almost 85% of liver cancers occur in developing countries, with over 20 of 100,000 individuals affected by these diseases. hepatocellular carcinoma (hcc) is the most common form of liver cancer, and approximately 80% of cases are associated with chronic infection by hepatitis b virus (hbv) or hepatitis c virus (hcv) (el-serag, 2012) . hepatitis viruses are so named because they display hepatotropism by preferentially infecting hepatocytes to cause liver inflammation, also known as viral hepatitis. infection by hbv and hcv promotes liver cirrhosis in most affected, leading to the development of hcc in up to 30% of patients (fattovich et al., 2004) . approximately 5% of the global population (350e400 million people) are chronically infected with hbv and strong correlations between hbv prevalence and hcc incidence and mortality. chronic hbv infection accounts for approximately 50% of hcc cases in adults and for all hcc cases in children (el-serag, 2012) . hepatitis transmission is from person-to-person contact with infected blood or body secretions or by the fecal-oral route and involving at least five specific viruses, namely hepatitis a, b, c, d, and e viruses (table 1) . infectious viral hepatitis is an important challenge to health worldwide: hepatitis a virus (hav) and hepatitis e virus (hev) are acute and endemic in many low-income countries, usually causing self-limiting hepatitis, whereas hbc and hcv also cause acute illness but usually lead to chronic and progressive liver fibrosis, cirrhosis, and an increased risk of hcc (stanaway et al., 2016) . hbv is controlled in adults but is chronically persistent from neonatal infection (shin et al., 2016) . hepatitis viruses differ in their virology. hbv is an enveloped dna virus that belongs to the hepadnaviridae family. it contains a 3200 bp, partially double-stranded relaxed-circular dna genome that is reverse transcribed via a pregenomic rna intermediate and encodes four overlapping open reading frames, which are translated to produce viral core protein, surface proteins, reverse transcriptase, and hbx (nguyen et al., 2008) . transmission of hbv results from exposure to infectious blood or body fluids containing blood, and hbv can integrate into the human genome, contributing to its genomic instability and ultimately to hcc (zhao et al., 2016) . hcv is also transmitted by infected blood; but unlike hbv, hcv does not integrate into the host genome . hcv is also a positivestranded rna virus but is classified in the hepacivirus genus within the flaviviridae family. its genome is 9.6 kb in length, includes an internal ribosome entry site, and encodes structural and ns proteins. the structural proteins form the viral particle and include the core protein and the envelope gps e1 and e2. the ns proteins include the p7 ion channel, the ns2-3 protease, the ns3 serine protease and rna helicase, the ns4a polypeptide, the ns4b and ns5a proteins, and the ns5b rnadependent rna polymerase (moradpour et al., 2007) . hepatitis d virus (hdv) is also transmitted by contact with infected blood or other body fluids. hdv is an enveloped, negative sense, singlestranded, closed circular rna virus, and requires hbv coinfection for its propagation, where infection with both viruses commonly results in severe liver pathologies. hdv genomic rna of hdv is composed of approximately 1700 bp, packaged with approximately 200 molecules of hepatitis delta antigen to form viral particles. hdv envelope surrounding its genome and hdag protein is composed of the three hbv small, medium, and large hbv hbsag envelope proteins. hdv also does not encode its own replicase or polymerase, and rather utilizes host cellular machineries for its replication (abbas and afzal, 2013) . hav and hev are positive-stranded nonenveloped rna viruses transmitted via the fecal-oral route, and unlike chronically persisting hbv and hcv, are typically cleared after acute infection of immunocompetent individuals (park and rehermann, 2014). hav is a hepatotropic virus belonging to the hepatovirus genus within the picornaviridae family. its genome consists of approximately 7500 bp and encompasses a single open reading frame coding for a single polyprotein, which is post-translationally processed into structural and ns proteins. the structural proteins of hav are divided into the polypeptides vp1, vp2, vp3, and vp4, forming the icosahedral capsid of the virus. ns proteins 2b, 2c, 3a, 3b, 3c, and 3d are involved in rna replication and viral polyprotein processing (martin and lemon, 2006) . hev of the family hepeviridae and genus orthohepevirus has a 7.2 kb genome having three opening reading frames encoding for the viral replicase, the capsid protein, and a small phosphoprotein required for the secretion of viral particles (debing et al., 2016) . hav and hev are waterborne viruses that usually cause acute hepatitis without progressing to chronic liver disease (joon et al., 2015) , where annually, over 100 million cases of hav and 28 million cases of hev infections have been recorded globally (makiala-mandanda et al., 2017) . hev outbreaks are reported in africa nearly every year, with some involving over 10,000 cases (kim et al., 2014) . hav is highly endemic in africa, infecting most children, conferring long-term immunity to reduce serious epidemics (jacobsen, 2014) . hbv, hcv, and hdv can be sexually, parenterally, or vertically transmitted and usually evolve into chronic hepatitis, liver cirrhosis, and hcc causing high morbidity and mortality rates, where globally, over 350 million people are chronically infected with hbv, 150 million with hcv, and 15 million with hdv (kramvis and kew, 2007; hughes et al., 2011; thursz and fontanet, 2014) . superinfection of hbv patients with hdv frequently accelerates the progression of hbv disease to liver cirrhosis, considerably increasing the burden of chronic liver disease (hughes et al., 2011) . hav, hbv and hcv are responsible for the majority of viral hepatitis cases, and there are similarities and differences in immune responses to infections by these three viruses, possibly explaining the distinct disease courses and outcomes of each hepatitis virus infection (shin et al., 2016) . type i and iii ifns, major components of the antiviral innate immune system, induce the expression of ifn-stimulated genes (isgs), observed to be much more highly induced by hcv than hav, and not at all by hbv, indicating that this virus is not recognized by the innate immune system (su et al., 2002; lanford et al., 2011; wieland et al., 2004) . another component of the innate immune system, nk cells, are also believed to be responsible for protection against hcv, where increased nk cells in protected individuals coincide with increased ifn-g and cytotoxicity (shin et al., 2016) . though virus-specific antibodies are produced by all viral hepatitis infections, these have differing roles according to the hepatitis virus infection. hav-specific antibodies with virus-neutralizing activity are induced by natural infection and vaccine immunization and confer lifelong protective immunity (walker et al., 2015; martin and lemon, 2006) . hbv surface antigen hbsag-specific antibodies are induced by infection and immunization with the recombinant protein and have virus-neutralizing activity conferring protective immunity (guidotti and chisari, 2006) . hcv-specific antibodies produced after infection do not offer long-term protection as these do not persist, are subject to loss of neutralizing activity from virus mutation, and are ineffective for cellto-cell hcv transmission (takaki et al., 2000; dowd et al., 2009; timpe et al., 2008) . t cells play critical roles during acute hcv and hbv infections, where robust and multiple epitope-specific cd8 þ t cell responses are assisted by cd4 þ t cells for spontaneous resolution of infection (shin et al., 2016) . this is supported by observations that the depletion of cd4 þ or cd8 þ t cells in chimpanzees delays rapid clearance and recovery from infection by these viruses (grakoui et al., 2003; thimme et al., 2003) . when hcv and hbv infections become chronically persistent, virus-specific t cells become exhausted and functionally impaired. in acute hcv infection, virus-specific t cells are only detected in the blood and liver after 8 weeks postinfection, and their appearance coincides with large declines of virus titres shin et al., 2006 shin et al., , 2011 . hbv virusespecific t cell responses are also important for spontaneous resolution of hbv infection, where their responses are observed to be vigorous, broad, and polyclonal in patients resolving primary infections and where their absences are associated with prolonged infection and delayed viral clearance (chisari et al., 2010; thimme et al., 2003) . cd8 þ t cells also play important roles in hav infection, where these have been observed to target multiple epitopes of hav, despite more recent results suggesting that hav is controlled by virus-specific cd4 þ t cells and not cd8 þ t cells (walker et al., 2015; shin et al., 2016) . finally, hepatitis virus infection results in liver injury, not directly caused by these viruses but rather by immunemediated mechanisms (guidotti and chisari, 2006) . liver injury biomarkers correlate with acute hav, hab, and hac infection (guidotti and chisari, 2006; park and rehermann, 2014; walker et al., 2015) and may result from cytotoxic activity of cd8 þ t cells, believed to induce apoptosis of hepatocytes in close proximity to their targeted cells (guidotti and chisari, 2006) , by il-22 producing th17-differentiated t cells, and by recruitment of nonspecific mononuclear cells by hbvspecific cytokine secreting cd8 þ t cells (iannacone et al., 2007; shin et al., 2016) . effective vaccines controlling hav and hbv have been available for over 2 decades, and an hev vaccine has also been licensed for use in china since 2011 (zhu et al., 2010; stanaway et al., 2016) . neonatal hbv vaccination has proven to be highly effective in inducing protective antibodies and preventing perinatal and horizontal transmission of hbv (lee et al., 1991) . however, observations that hbsag-specific ifn-g-or il-5-secreting pbmcs are absent in many adolescents suggest that booster vaccines should be administered to provide continued hbv immunization (lu et al., 2008) . hav vaccination provides long-term immunity in the general population and in immunocompromised patients infected with hiv (crum-cianflone et al., 2011). there is no existing vaccine for hcv, despite ongoing efforts toward their design and testing for their ability to generate prolonged cellular and humoral immune responses, reviewed in naderi et al. (2014) . in the absence of a vaccine, progress in hcv treatment includes oral treatments achieving cure in most patients, including those previously considered as difficult to treat cases (poordad et al., 2013; lawitz et al., 2013) . liver cancer is the fifth most common cancer, representing 7.2% of all cancers, with 374,000 annual cases from which 20 in 100,000 mortalities occur. hcc is the most common liver cancer, with 80% of cases resulting from chronic infection by hbv or hcv, with a significant 350 and 150 million chronically infected, respectively. in contrast, hav and hev cause acute hepatitis but do not progress to chronic liver diseases, and hdv infection depends on pre-existing hbv infection. hcv induces the expression of type i and iii isgs and nk cells, not at all present from hbv infection unrecognized by innate immunity. virus-specific antibodies are produced by all viral hepatitis infections but have differing roles across infections. robust and multiple epitope-specific cd8 þ t cell responses and dominant but depend on assistance from cd4 þ t cells for resolution of acute hav, hbv, and hcv infections. in chronic infections, cytolytic t cells either cause extensive liver injury to hepatocytes and/or become tolerant and functionally impaired. effective vaccines controlling hav and hbv provide protective antibodies. hav vaccination provides longterm immunity to the immunocompromised, but booster vaccination programs are required for persistence of hbv immunization. no vaccine is licensed for highly variable and rapidly mutating hcv, despite numerous ongoing efforts to generate those which will provide robust cellular and humoral immune responses. historically, the central nervous system (cns) has been considered to be an immunologically privileged site within the body (bailey et al., 2006; galea et al., 2007; engelhardt, 2008; prendergast and anderton, 2009 ). by definition, immunologically privileged sites, also including the brain, cornea, testis, and pregnant uterus, have a reduced or delayed ability to reject foreign tissue grafts compared with conventional sites within the body, such as skin (streilein, 2003; bailey et al., 2006; carson et al., 2006; mrass and weninger, 2006; kaplan and niederkorn, 2007) . though the cns is protected by a highly complex barrier system, a wide variety of viruses still manage to gain access to it and induce diseases. due to their sizes and tissue penetration strategies, the number of cns viral infections outweigh bacterial, fungal, and protozoa cns infections combined (romero and newland, 2003) . following cns infection, inflammatory events can arise in distinct anatomical regions such as the meninges (meningitis), brain (encephalitis), and spinal cord (myelitis) or can also simultaneously arise in multiple regions (meningoencephalitis, encephalomyelitis). for many neurotropic viruses, viral cytopathology plays a major role in cns dysfunction, reviewed in swanson and mcgavern (2015) . virus can breach the protective barriers of the cns in many ways, with the main route mechanism being via the blood, where inhaled or ingested viruses can move past the mucosa to establish infection in secondary lymphoid tissues and later be shed into circulating blood to cause broad systemic infections (swanson and mcgavern, 2015) . the cns parenchyma is protected from a plethora of agents carried in the circulation via an elaborate network called the blood-brain barrier (bbb) and the blood-cerebrospinal fluid barrier (ransohoff et al., 2003) . viruses have evolved and adapted to overcome these barriers (mcgavern and kang, 2011) , where some viruses can infect vascular endothelial cells, permitting direct passage across the bbb into the cns (verma et al., 2009; moses et al., 1993; coyne et al., 2007) . in addition, parts of the cns that are not completely protected by the bbb permit more rapid entry of several viruses (van den pol et al., 1999; wolinsky et al., 1974) . infected hematopoietic cells in circulating blood can also serve as "trojan horses" that can transport undetected virus into the cns (clay et al., 2007; tabor-godwin et al., 2010) . other mechanisms can include systemic viral infections leading to massive systemic inflammation and an ensuing bbb breakdown which opens the floodgates to cns infection by a variety of otherwise restricted infectious agents (arsenio-nunes et al., 1975; eugenin et al., 2006) . there are more than 30 recognized distinct virus strains that cause human neurological disease, and the majority of documented cases are caused by viruses that are transmitted to humans by blood-eating arthropod vectors, also known as arboviruses, that are mainly transmitted by mosquitoes and ticks and include polioviruses (pvs), alphaviruses, mosquito-borne flaviviruses, tick-borne orthobunyaviruses, mosquito-borne mammarenaviruses, and rabies virus (rabv; table 1 ). pv, the causative agent of poliomyelitis, more commonly referred to as polio, is a human enterovirus and member of the family of picornaviridae. typically spherical, nonenveloped picornaviruses range in diameter between 27 and 30 nm and have a positive-strand rna of 7000e9000 nucleotides, translated into a polyprotein (i.e., vp4-vp2-vp3-vp1-2a-2b poliovirus 2c-3a-3b-3c-3d), which yields 11 proteins upon its cleavage by viral proteases. picornavirus replication occurs in the cytoplasm of infected cells in association with intracellular membranes, where virions are released by cell lysis, ultimately killing cells and causing extensive damage to tissues. the host immune response against picornaviruses includes cytokine release, antibody production, and ctl activation, reviewed in dotzauer and kraemer (2012) . the viral genome of pv is a single-stranded rna of approximately 7500 nucleotides, enclosed in a nonenveloped capsid comprising 60 copies of four different polypeptides arranged with icosahedral symmetry (racaniello, 2006) . all three pv serotypes cause paralytic disease, and cd155 is the cellular receptor for all three serotypes, whereby pv interaction with cd155, expressed by many different cell types, leads to a conformational change of the virus particle and the following release of the rna genome into the cellular cytoplasm (mendelsohn et al., 1989; hogle, 2002) . once in the cytoplasm, the viral rna genome is translated, and the production of new infectious virions begins. pv infection results from ingested virus that replicates in the oropharyngeal and intestinal mucosa (sabin and ward, 1941) . from the primary sites of multiplication in the mucosa, pv virus drains into cervical and mesenteric lymph nodes and then into the blood, causing transient viremia symptoms. the person-to-person transmission of pv virus is through the fecal-oral route. once pv is shed in the feces, the majority of the natural human infection ends at this stage with a modest symptoms including sore throat, fever, and malaise (dotzauer and kraemer, 2012) . however, in 1%e2% of pv-infected individuals, the virus gains entry to the cns through neurons at neuromuscular junctions (nmjs) and replicates in motor neurons within the spinal cord, brain stem, or motor cortex and leads to the pv-characteristic flaccid muscle paralysis disorder poliomyelitis (racaniello, 2006; koyuncu et al., 2013) . approximately 10% of these paralytic cases result in death (roush et al., 2007) . following both pv infection and vaccination, neutralizing antibodies are generated to clear the virus, and these can be detected for many years, providing lifelong protection (libbey and fujinami, 2014) . vaccination with the injected inactivated pv vaccine prevents viral spread to the cns, whereas vaccination with the live-attenuated oral pv vaccine protects against infection of the intestinal tract and also prevents person-to-person spread of the virus (nathanson, 2008; griffin, 2010) . pv remains an important cause of neurologic disease as the three live-attenuated vaccine strains are at risk of recombining their genomes to revert to virulent form (griffin, 2010) . pv is endemic in afghanistan, pakistan, india, and nigeria, where political reasons, in part, are the most significant modality toward achieving pv eradication via vaccination. pv can usually be cleared by the adaptive immune response. under conditions of antibody deficiencies in humans, however, continuous fecal shedding of pv contributes to the establishment of persistent infection cycles (martin, 2006; nathanson, 2008; libbey and fujinami, 2014) . though less is known about the roles of adaptive t cell responses in controlling pv infections relative to that of neutralizing antibody responses, it is known that pv-specific cd4 þ t cells are induced in vaccinated individuals, where key epitopes have also been identified (graham et al., 1993; simons et al., 1993) . the induction of pv-specific cd4 þ t cells has been suggested to be the result of stimulating by pv-infected dcs and macrophages (wahid et al., 2005; dotzauer and kraemer, 2012) , where it has been demonstrated that hla class ii presentation remains intact in infected, antigen-presenting cells (apcs), and that cytolytic cd4 þ t cells produce ifn-g to lyse pv-infected cells for virus clearance. pv-specific cytotoxic, ifn-g-secreting cytotoxic cd8 þ t cell responses induced by infected macrophages have also been documented (wahid et al., 2005) , suggesting that both cd4 þ and cd8 þ cytolytic t cells partake in the adaptive immune reaction against pv (dotzauer and kraemer, 2012) . approximately 73 viruses are included in the flavivirus genus of the flaviviridae family of viruses, with 40 of its species associated with dengue, yellow fever (yf), japanese encephalitis (je), tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, 2003) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins assemble to generate the capsid, the envelope for receptor binding, membrane fusion and viral assembly, and the transmembrane proteins (prm) that assist in protein folding and function. the entry of flaviviruses into their target cells is mediated by the interaction of the e gp with host cell surface receptors (perera-lecoin et al., 2013) . flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, 1968) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism, where virus hides in inflammatory cells during their transit into the brain (solomon and vaughn, 2002) . ifn-dependent and complement system innate immune responses, along with humoral neutralizing antibodies, protect against virus dissemination and spread, reviewed in diamond (2003) . adaptive cellular immunity is also important toward the destruction of infected cells, whereby virus-specific ctls become activated, proliferate, and release inflammatory cytokines following exposure to flavivirus-infected cells (kesson et al., 1987; kurane et al., 1989a; liu et al., 1989; murali-krishna et al., 1996) . there is also evidence that flavivirus replication is enhanced by myeloid cells, as observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, 2003) . the je flavivirus is the leading cause of encephalitis and is amplified by waterfowl and only transmitted to humans by mosquito vectors, with no possibility of human-to-human transmission (erlanger et al., 2009) . pediatric and geriatric populations are at higher risk of infection by je (burchard et al., 2009) . while 99% of je infections remain asymptomatic, je can be devastating in symptomatic patients, causing mortality rates of 30% in these patient populations (batchelor and petersen, 2015) . after approximately 14 days of je incubation, these patients suffer from high fever, chills, headache, myalgia, and confusion, where pediatric patients also have symptoms of gastrointestinal pain, vomiting, and seizures. post-je infection, handicaps from persistent neurological deficits can last a lifetime in up to 50% of these survivors. as there is no treatment against je, the only method of prevention is avoidance of mosquitos and vaccination (batchelor and petersen, 2015) . the four available je vaccines are registered worldwide and used in national immunization programs for different age groups, including inactivated vero cell culture vaccine (je-vc) (ixiaro), inactivated mouse braine derived vaccine (je-mb), a cell cultureederived (primary hamster kidney) live-attenuated vaccine based on the sa 14-14-2 strain manufactured in china, and a live-attenuated chimeric vaccine based on the genes of yf 17d backbone combined with vero cellepropagated sa 14-14-2 strain (imojev) (chen et al., 2015) . t cell responses to je vaccination have been reported, where for sa14-14-2, t cell responses were detected in the majority following vaccination, and these cross-reacted with other flaviviruses (turtle et al., 2017) . je-specific t cell responses are observed in pbmcs isolated from je-infected patients and vaccinated individuals. cd4 þ and cd8 þ t cells directed against structural viral proteins were identified in vaccinated individuals, in contrast to specific cd4 þ and cd8 þ responses against ns or c proteins in infected patients (nathanson and cole, 1971) . these findings indicate that ns proteins, and especially ns3 have important roles in the initiation of t cell responses, as the main target of je-specific t cellemediated immune responses. in addition, cytolytic cd4 þ t cells clones that cross-reactive with other flaviviruses have been generated from individuals immunized with inactivated je vaccine (aihara et al., 1998) . finally, cd4 þ and cd8 þ and th1 t cells are believed to be primary determinants of protection from je infection (kumar et al., 2004a (kumar et al., , 2004b ). viruses from the alphavirus genus are members of the togaviridae family of viruses, a group of enveloped positive-sense rna viruses. these are mosquito-borne viruses causing two major types of human disease. the old world alphavirusesdsindbis, chikungunya, and ross river virusdcause arthritis and arthralgia, while the new world alphavirusesdeastern (eeev), western (weev), and venezuelan equine encephalitis virus (veev)dcause encephalitis (trobaugh and klimstra, 2017) . alphaviruses are small, icosahedral-shaped, enveloped viruses and are approximately 70 nm diameter in size (mancini et al., 2000; morgan et al., 1961; fuller, 1987) . alphavirus virions acquire host cell lipid membranes during viral assembly (fuller, 1987; acheson and tamm, 1967; vogel et al., 1986) , with 80 e1 and e2 viral gps spike protrusions, arranged in an icosahedral pattern embedded within their membranes and interacting with nucleocapsid (fuller, 1987; vogel et al., 1986; owen and kuhn, 1997) . alphavirus single-stranded, positive-sense, rna genomes are 12 kb long and consist of two large open reading frames encoding the ns and structural polyproteins that are subsequently cleaved by both viral and host proteases to create four ns proteins (nsp1 to 4) and five structural proteins (c, e3, e2, 6k, e1) (strauss et al., 1984; hardy and strauss, 1989) ; reviewed in leung et al. (2011) . of major concern are the new world eeev, weev, and veev alphaviruses, which are naturally transmitted by mosquitos, but where veev is also highly infectious via the aerosol route (zacks and paessler, 2010) . precise mechanisms of entry of alphaviruses into the cns remains elusive, however, once in, alphaviruses infect humans and equines neurons, causing neurologic symptoms from mild febrile illness to severe encephalitis resulting in death (ramakrishna et al., 2002; zacks and paessler, 2010) . development of severe encephalitis is believed to result from neuronal cell death from accelerated viral spread and host neuroinflammatory viral responses (paessler et al., 2006 (paessler et al., , 2007 . antibodies are protective against lethal meningoencephalitis when the virus is transmitted by insects, and virus-specific cd4 þ t cells are found to be important for protection from lethal meningoencephalitis from aerosol transmission routes (paessler et al., 2007; yun et al., 2009) ; reviewed in libbey and fujinami (2014) . veev remains an emerging disease threat by natural transmission as well as via its usage as a biological weapon. of the new world alphaviruses, veev is the most important human and equine pathogen, it having caused outbreaks of febrile and neurological disease primarily in latin america during the past century. past outbreaks have lasted several years and have involved up to 100,000 equine and human cases over large geographical regions, with the largest outbreaks on record were from the 1960s, where central colombia saw over 200,000 human cases and an estimated 100,000 equine deaths. more recent outbreaks in mexico and south america are behind the classification of veev as a re-emerging disease (weaver et al., 2004) . because veev can also be developed as a biological weapon amenable to use in warfare or terrorism, current global emphases on biological defenses have renewed interest in its virology (hawley and eitzen, 2001; weaver et al., 2004) . veev infection in humans typically causes nonlethal, incapacitating symptoms including fever, headache, malaise, myalgia, sore throat, and vomiting. up to 4% of rarer cases of cns involvement usually follow acute febrile phases, with associated severities of neurological disease ranging from somnolence and mild confusion, to seizures, ataxia, paralysis, and coma, with mortality rates ranging as high as 35% in infected children and 10% in infected adults (bowen et al., 1976) . veev has also been reported to cause long-term neurological deficits, abortions, and teratogenic effects (de la monte et al., 1985; rivas et al., 1997; weaver et al., 1996) . like veev, though the majority of human infections with eeev are asymptomatic, cns involvement results in severe neurological signs, lesions, and sequelae, with an estimated associated human mortality rate of 75%, and with its neurological manifestations including facial edema, paresis, paralysis, respiratory impairment, altered mental state, and seizures in children, many of these symptoms persisting long-term in surviving patients. in fatal cases of eeev, gross lesions in the brain include edema, meningeal congestion, hemorrhage, and malacia (deresiewicz et al., 1997) . as with veev and eeev, natural human cases of weev typically show an early, flu-like illness with associated fever, malaise, and headache. similar to eeev, weev results in cns involvement in a significant proportion of cases, including symptoms of somnolence, seizures, coma, and motor neuron dysfunction. ninety percent of infants infected with weev have severe cns symptoms (calisher, 1994) . human mortality rates from weev infection range from 3% to 15%, and neurological sequelae may become permanent features in survivors (steele and twenhafel, 2010) . alphavirus expression vectors based on sindbis, semliki forest, and veev have been demonstrated to induce strong cd8 þ t cell responses against their antigens (rayner et al., 2002; lundstrom, 2002 lundstrom, , 2003 riezebos-brilman et al., 2006; schlesinger and dubensky, 1999; polo et al., 2000) . both innate and adaptive immune responses can control viruses targeting cns neurons (griffin, 2003) . viral disruption of the type i ifn signaling pathways interferes with survival from veev, as well as of those infected with sindbis and west nile viruses (ryman et al., 2000; samuel and diamond, 2005; white et al., 2001) . virus-specific antibody responses are critical in limiting viral spread and facilitating clearance of infectious virus from neurons within the brain levine et al., 1991) . both alpha beta (ab) and gamma delta (gd) t cell responses have been demonstrated as being important for the control of veev (paessler et al., 2006) . t cell responses reduce mortality rates by direct killing of infected cells, producing antiviral cytokines and increasing production of virusspecific antibodies (bilzer and stitz, 1994; patterson et al., 2002; shrestha et al., 2006; sitati and diamond, 2006) . veev replicon particles delivered as an adjuvant have been demonstrated to induce activation of cd8 þ t cell responses (thompson et al., 2008) . more recently, t cells have been demonstrated to facilitate recovery from veev-induced encephalomyelitis in absence of antibodies, responsible for dramatic reduction in viral titres in cns, where cd4 þ t cells were the best t cell producers of ifn-g response and were more efficient at controlling veev in cns lesions than cd8 þ t cells, facilitating recovery from severe viral encephalomyelitis (brooke et al., 2010) . commercial equine vaccines marketed in the united states are generated with inactivated tc-83, which produces viremia, fever, and leukopenia in horses but generates robust neutralizing antibodies and veev protection from rechallenge (walton et al., 1972) . u.s. army special immunization programs provide inactivated c-84 to individuals failing to seroconvert in response to tc-83 boosters (pittman et al., 1996) ; however, neither of these vaccines can be shown to completely protect nonhuman primates against aerosol exposure (pratt et al., 1998) . a more stably attenuated veev vaccine candidate called v3526 has been produced, where preclinical testing has demonstrated it to be safe and immunogenic and possibly superior to tc-83 (pratt et al., 2003; hart et al., 2000; ludwig et al., 2001) . adaptive immune pbmc-derived biomarker signatures have been identified and able to efficiently stratify tc-83 vaccinated from naïve or nonresponding individuals (erwincohen et al., 2012) . rabv is the type species of the genus lyssavirus, within the rhabdoviridae family. rhabdoviruses are negative-sense, single-stranded rna viruses having a distinctive bullet-shaped structure. up to 10 viruses of lyssaviruses have the potential to cause rabies in humans. these have a 12,000 nucleotide genome encoding five proteins: nucleoprotein (n), phosphoprotein (p), matrix (m), glycoprotein (g), and rna-dependent-rna polymerase (l) (marston et al., 2007) . rabv causes acute encephalitis in mammals, causing fatality rates of almost 100%. rabv commonly infects many animals, including bats, skunks, foxes, and dogs and can also infect insects and plants. rabv in animal saliva spreads between hosts via bites or scratches. infected animals can survive for years, secreting infectious particles in their saliva, but untreated infection in humans generally results in rapidly fatal acute myeloencephalitis (koyuncu et al., 2013) . rabid dogs are the most important reservoirs for rabv, where dog bites account for more than 99% of human infections. rabv, like all members of lyssaviruses, is neurotropic and infects peripheral nerves close to the primary site of the bite. rabv then rapidly moves by retrograde axonal transport to the dorsal root ganglia where virus replication begins . rabv particles enter axons of motor neurons at the nmj via their binding to nicotinic acetylcholine receptors (e.g., nachr) and neural cell adhesion molecules (ugolini, 2011) . transneuronal rabv spread occurs between synaptically connected neurons, whereby viruses move from postsynaptic to presynaptic neurons. in humans, a relatively long asymptomatic incubation period after initial rabv infection can occur, sometimes lasting up to 1 year, and providing some time for cns infection intervention. however, death almost always ensues after rabv infection reaches the cns, with marked behavioral and neurological symptoms (koyuncu et al., 2013) . once rabv has entered the cns, it rapidly moves to the brain and is associated with an explosive increase in virus replication. initial symptoms include pain or paraesthesia close to the bite site and are often associated with fever, fatigue, and weakness in associated limbs. nonspecific neurological symptoms including headache and anxiety occur days prior to acute encephalitis (morrison and wenzel, 1985) . currently, there are no available therapies against disease symptoms once they develop, and death ensues within a number of days following cns-associated symptoms (jackson et al., 2003) and reviewed in johnson et al. (2010) . rabv replication begins following cns penetration, thereby limiting earlier possible detection of low-level primary antigens in the peripheral circulation. this delays antigen presentation, where antigens later but rapidly drain from the cns to local lymphoid tissues (knopf et al., 1998) . once b cells are stimulated, the next delaying obstacle is re-entry into the cns, but experimental models have demonstrated t and b cell infiltration of dorsal root ganglia, spinal cord, and brain (johnson et al., 2008) , with t cells as the major immune subsets, but where most of these cns-infiltrated t cells have fas-mediated apoptotic phenotypes (baloul and lafon, 2003) . further intrinsic complexities in immune responses are present in the cns, including tight mhc expression regulation (irwin et al., 1999) , and the expression of immunosuppressive factors by neuronal cells. additionally, the bbb remains intact during rabv infection (roy et al., 2007) . numerous studies have suggested that the virus suppresses the adaptive immune response, believed to be in part due to a deficit of adaptive immune effector cell accumulation within the cns due to a virally induced reduction in bbb permeability (libbey and fujinami, 2014; roy et al., 2007) . two rabv vaccines are licensed for human application, the human diploid cell vaccine manufactured by aventis pasteur and the purified chick embryo cell vaccine manufactured by chiron . pre-exposure vaccination given to healthcare personnel, laboratory workers, and travelers to endemic areas causes detectable igm and igg antibodies within a week following exposure, and long-term studies have provided evidence that igg antibodies provide the most effective protection against rabv due to its ability to penetrate tissues, in contrast to igm which cannot penetrate tissues (turner, 1978) . a multifaceted approach for human rabies eradication involving government support, disease awareness, and vaccination of at-risk humans and dogs will be required to achieve the goals of the world health organization in eradication of rabies by 2030 (fooks et al., 2017) . the cns is immunologically privileged and protected by a highly complex barrier system. viruses that have evolved to overcome these barriers can cause cns infections greatly outnumbering those from all bacterial, fungal, and protozoa infections combined. ingested pv multiplies in the oropharyngeal and intestinal mucosa and drains to cervical and mesenteric lymph nodes and then into the blood ahead of penetrating the cns to cause polio, with 10% of cases resulting in death. both neutralizing antibodies and the adaptive immune system can clear pv infection and may provide lifelong protection. vaccination combinations can induce pv-specific cytolytic cd4 þ and cd8 þ t cells for virus clearance, but their coadministration can pose the risk for reversion to virulence by recombination. the je flavivirus is amplified by waterfowl and transmitted to humans by mosquitoes, and while 99% of its infections are asymptomatic, mortality rates in 30% of infected individuals cause associated disorders that leave its survivors a lifetime of associated morbidities. as there is no existing je treatment, prevention involves either avoidance of mosquitoes or vaccination. flaviviruses evade the immune system to cross the bbb by an inflammatory celle mediated "trojan horse" mechanism. je dissemination is limited by innate immune responses, neutralizing antibodies produced by humoral immunity, and by virus-specific ctls. je vaccines are licensed worldwide, and the majority of vaccinated individuals have circulating je-specific cd4 þ and cd8 þ t cells that can cross-react with other flaviviruses. alphaviruses are transmitted by mosquito bites to infect neurons, causing mild to severe encephalitis resulting in death, with past outbreaks numbering in the hundreds of thousands. veev infection causes up to 35% mortality in children, 75% of which involve cns penetration, causing severe long-term neurological disorders. veev is not only a naturally emerging disease threat but is also a highly developed biological weapon amenable to warfare or terrorism due to its aerosol transmission route and associated lethal meningoencephalitis. ifn signaling pathways and ab and gd t cell response from innate and adaptive immunity can control veev targeting of cns, where virus-specific antibody responses are critical in limiting viral spread. in the absence of antibodies, veev replicon particles can induce t cell responses able to induce recovery from veev-induced encephalomyelitis, where cytotoxic cd4 þ t cells control veev in cns lesions. veev vaccines induce robust neutralizing antibodies for protection against rechallenge. tc-83 vaccine responders have circulating pbmc biomarkers, and military programs give boosters of c-84 to those failing to seroconvert. in contrast to these other viruses, rabv replication only begins after cns penetration, as facilitated by depth of bite by its canine vector, thereby limiting possible detection of primary viral antigen in the periphery and resulting in delayed and minimal innate and humoral responses. once rabv-related acute encephalitis symptoms begin, fatality is sure to follow due to absence of cns infiltration by adaptive immune effector cells as a result of virus-induced decreases in bbb permeability. other countermeasures against protection are tight mhc expression regulation and apoptotic phenotypes of bbb-infiltrated t cells. rabv vaccines cause increases in ig, but little is known concerning vaccine-associated adaptive immune responses. viral hemorrhagic fever (vhf) classification originates from the study of hantaviral hemorrhagic fever (hf) and was later extended to include crimeanecongo hf and omsk hf. vhf can results from infection by 23 enveloped rna viruses from four families: flaviviridae, filoviridae, arenaviridae, and bunyaviridae. vhf designation is given to severe febrile illnesses with abnormal vascular regulation and vascular damage (peters and zaki, 2002) . vascular dysregulation occurs early in the course of disease, visible as skin flushing, hypotension, and conjunctival vasodilation, whereby vascular damage with capillary leakage occurs as disease progresses, causing edema and serous effusions of pleural and peritoneal cavities. the terminal phase of vhf, or shock, arises from increased disease severity from combinations of vascular dysregulation and damage from capillary leakage (paessler and walker, 2013) . detailed mechanisms of hemorrhage and plasma leakage during vhf include endothelial injury, activation of the mononuclear phagocytic system, cytokine storm, platelet aggregation and consumption, activation of the coagulation cascade, and insufficiency of coagulation factors from severe hepatic damage (schnittler and feldmann, 2003; chen and cosgriff, 2000) . these mechanisms vary among diseases, cell and organ tropism of causative viruses, and host responses (paessler and walker, 2013) . flaviviruses, filoviruses, arenaviruses, and bunyaviruses are the main causes of hf (table 1) . these viruses continue to propagate as part of the life cycles of primates, bats, rodents, farm animals, mosquitoes, and ticks. infection by these viruses can cause mild vascular instability to fatal shock, with hemorrhage ranging from unnoticeable to life-threatening. pathogenic mechanisms of hfv are diverse and include hepatic necrosis leading to deregulation of coagulation factors, cytokine storm, increased permeability, and complement activation. overall disease severity by these viruses is varied, whereby ebola and marburg hf can cause high fatality rates, whereas yf and dengue infections can be asymptomatic. severe vhf is commonly correlated with ineffective immunity and high viral loads, and severe plasma leakage can occur from viral clearance and fever breaks in dengue hf (dhf). approximately 73 viruses are included in the flavivirus genus of the flaviviridae family of viruses, with 40 of these species associated with dengue, yf, je, tick-borne encephalitis, and west nile encephalitis as the most important arboviruses causing extensive global morbidity and mortality (diamond, 2003) . flaviviruses are enveloped viruses with single-stranded rna genomes that are translated in the cytoplasm to generate a single polyprotein that is then cleaved into structural and ns proteins by virus and host proteases. the various encoded viral proteins include capsid, envelope for receptor binding, membrane fusion and viral assembly, and transmembrane proteins (prm) that assist in protein folding and function. although the precise mechanism is unclear, flaviviruses are believed to evade the immune system to enter the brain and spinal cord via circulating blood (johnson and mims, 1968) , where these may cross the bbb by passive transport across the endothelium, by active replication in endothelial cells, or by a "trojan horse" mechanism utilizing inflammatory cells (solomon and vaughn, 2002) . ifn-dependent and complement system innate immune responses and humoral immunity, producing neutralizing antibodies limit dissemination of infection and protect against viral spread, reviewed in diamond (2003) . cellular immunity is also important toward eradication of infected cells, whereby infection induces the recognition of flavivirusinfected cells by virus-specific ctls, which then become activated, proliferate, and release inflammatory cytokines (kesson et al., 1987; kurane et al., 1989a; liu et al., 1989; murali-krishna et al., 1996) . however, there is also evidence that flavivirus replication is enhanced by myeloid cells and has been observed for dengue, yf, west nile, tick-borne, and je viruses (diamond, 2003) . yf virus (yfv) is important both historically and currently. it was once one of the most globally feared diseases terrorizing africa, europe, and the americas. hundreds of thousands were killed in the americas over a 250-year spandcrippling economies (watson and klimstra, 2017) . yfv is a member of the genus flavivirus of the flaviviridae family and contains a single-stranded rna genome of approximately 11 kb. yfv virions are icosahedral and are composed of nucleocapsid, composed of capsid (c) protein subunits and a surrounding lipid bilayer derived from host membranes. the viral envelope is studded with dimers of envelope (e) and membrane (m) proteins, for a total diameter of approximately 45 nm. as the major component of the virion surface, the e protein is responsible for cell-surface receptor binding, virion assembly, fusion, and immunogenicity. viral proteins are encoded in a single open reading frame and produced as a polyprotein later processed by proteolytic cleavage into structural (c, m, and e) and ns proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) , reviewed in gardner and ryman (2010) . during most yfv infections, the virus is transmitted by the bite of an infected aedes aegypti mosquito found in urban areas. infected patients often develop severe acute illness hemorrhagic yf disease, with associated symptoms of fever, nausea, vomiting, epigastric pain, hepatitis, jaundice, renal failure, hemorrhage, and shock, with 20%e60% of cases resulting in death (watson and klimstra, 2017) . yf is the prototypical vhf, sharing many pathophysiological features with other viral disorders only associated via similarities in syndromes, but with the exception that yf causes the most severe symptoms of hepatic dysfunction (monath and vasconcelos, 2015) . yfv remains endemic in south american and african countries, with monkeys as its reservoir, causing regular outbreaks of jungle yf, and resulting in as many as 200,000 infections per year causing 30,000 deaths. millions are at risk for infection in africa, where vaccination prevalence is low. the 2016 outbreak in angola serves as an example of yfv traveler-associated spreading to neighboring countries, where it reached as far as china, then naïve for virus (watson and klimstra, 2017) , and representing a prime population for a major outbreak of epic proportions (wasserman et al., 2016) . geographical shifting of mosquito populations to north america is also creating new risk for yfv, dengue, and zika infection of naïve populations (monaghan et al., 2016) . despite the availability of vaccination against yfv since the 1940s, large epidemics have still arisen, with dramatic surges of yfv in africa in the 1960s and the late 1980s, with each reporting over 100,000 cases. recent outbreaks have also affected brazil, paraguay and argentina, uganda, and sudan and ethiopia. immunity is the critical for reducing and eliminating viral infections, but other contributing factors to virus amplification are multifactorial and elusive, including the emergence of new viral strains and prolonged periods of hot and humid weather promoting insect propagation, reviewed in monath and vasconcelos (2015) . fifty-seven million people were vaccinated against yf across africa between 2007 and 2010. five hundred million doses of the live-attenuated yf 17d vaccine, representing the most effective vaccine ever created, have been distributed over the last 50 years (monath and vasconcelos, 2015) . both humoral and cellular immunity elicited by 17d are observed and well characterized, where neutralizing antibodies provide protection, but 17d also provides a robust, long-lived, and polyfunctional adaptive t cell immune response (watson and klimstra, 2017) . neutralizing antibodies remain the accepted correlate of protection against yfv, with 90% or greater of 17d immunized individuals developing neutralizing antibodies (gotuzzo et al., 2013) . 17d also elicits a complex modulation of innate immune cytokines, with elevated levels of plasma ifn-g 15 days postvaccination (neves et al., 2009) . restimulation of innate immune cell cultures of nk cells, neutrophils, and monocytes from 17d vaccinated humans with yf antigen results in the increased production of ifn-g, il-1beta, il-10, il-12, tnf-a, and il-10 (neves et al., 2009; gardner and ryman, 2010; luiza-silva et al., 2011; silva et al., 2011) . since its development, humoral immunity, as a gold standard of general vaccine development, was the most studied aspect of human immunity to 17d. however, recent studies of adaptive t cellemediated immunity to 17d have demonstrated that both cd4 þ and cd8 þ t cells strongly respond to 17d, with activated cd8 þ t cells detected as 3 days after vaccination , and cd4 þ t cells detected several days later kohler et al., 2012; blom et al., 2013) . increased cd8 þ t cell proliferation correlates directly with the levels of virus genomes in plasma, which peaks once virus is eliminated . cd8 þ t cell clones responding to 17d differentiate into central memory and effector memory subpopulations (dewitt et al., 2015) and are still detectable 25 years following vaccination (wieten et al., 2016) . 17d-specific cd8 þ t cells respond to epitopes contained from every protein product generated by the 17d polyprotein, and upon peptide restimulation, these 17d-specific cd8 þ t cells have activated cytotoxic profiles including increased expression of ifn-g, tnf-a, and mip1-b and il-2 granzyme b and cd107a (blom et al., 2013; akondy et al., 2009) but are not exhausted and retain long-lived memory and polyfunctional phenotypes for at least 2 years following 17d rechallenge (akondy et al., 2009) . dengue virus (denv), also a member of the single-stranded positivesense rna viruses from the flaviviridae family, causes visceral and cns disease in humans and is closely related to yfv, where denv fever has often been mistaken for yfv infection. far more serious is dhf, where additional symptoms develop, including hemorrhage and shock, and have mortality rates exceeding 30% if left untreated (rogers et al., 2006) . denv is a spherical, 50-nm virion, comprising of three structural proteins: capsid (c), premembrane and membrane (prm and m), and envelope (e). the e protein directs several critical steps of the viral replication cycle, including engagement with cellular attachment and entry factors, membrane fusion, and virion assembly. denv binds to target cells via glycosaminoglycans, c-type lectins such as dc-sign, the mannose receptor cd206, and immunomodulatory proteins (tim and tam receptors; diamond and pierson, 2015) . thus targets for denv infection include monocytes, macrophages, dcs, mast cells, and possibly hepatocytes and endothelial cells. following its entry into the cellular cytoplasm, the viral genomic 10.7 kb rna is translated into a single polyprotein, later cleaved into three structural and seven ns proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5 ) by viral ns3 and host cell proteases. twenty-five percent of denv infections cause both mild symptoms including dengue fever (df) to more severe and lethal dhf, causing shock via hemorrhagic and capillary leak syndrome. df can be characterized by abrupt onset febrile illness causing headache, severe muscle and joint pain, and rash, whereas dhf is characterized by rapid onset capillary leakage accompanied by significant thrombocytopenia and liver injury (halstead, 2007) . as with yf, denv origins are believed to be that of a sylvatic virus, with a natural life cycle involving multiple mosquito and vertebrate species from asia and africa (diallo et al., 2003) . denv adaptation to human demography is via mosquito vector aedes aegypti, breeding in urban areas (trpis and hausermann, 1986) . cases of denv infection have increased since the 1960s, with an estimated 50 million cases of df, and 500,000 cases of dhf occurring globally every year. there are no cures for denvassociated disorders, and vaccine development has been complicated by antibody-dependent enhancement of future heterotypic infection induced by vaccination (vaughn, 2000; halstead and deen, 2002) . thus avoidance and control of aedes aegypti is the best approach for limiting denv infection (rogers et al., 2006) . adaptive immune cd8 þ t cells vigorously and frequently recognize denv ns3, ns4b, and ns5 proteins, whereas the capsid, envelope, and ns3 proteins are the dominant targets for cd4 þ t cells (simmons et al., 2005; duangchinda et al., 2010; weiskopf et al., 2011 weiskopf et al., , 2013 rivino et al., 2013) . both cd4 þ and cd8 þ t cells are believed to contribute to protection against denv, as denv-specific cd4 þ t and cd8 þ t cells proliferate, produce ifn-g, and lyse target cells, from primary denv infection (kurane et al., 1989b; mathew et al., 1996; gagnon et al., 1999; livingston et al., 1995) . higher frequencies of denv-specific ifn-gproducing t cells are present in children with asymptomatic denv infection (hatch et al., 2011) . both cd4 þ and cd8 þ t cells contribute to protection against denv challenge (yauch et al., 2009 (yauch et al., , 2010 zompi et al., 2012; zellweger et al., 2013) , and hla alleles associated with increased risk of denv severity correlated with weak cd8 þ t cell responses and vice versa, implying a protective role for cd8 þ t cells against severe denv disease in humans (weiskopf et al., 2013) . in 2015, the first dengue vaccine (dengvaxia) was licensed in asian and south american countries for protection against all four denv serotypes, and while it demonstrated an good safety and efficacy in clinical trials, it has recently been withdrawn in the philippines due to its causing elevated disease severity if administered following infection (wichmann et al., 2017) . it has been suggested that failure of this and other live-attenuated tetravalent dengueeyf chimeric virus vaccines (guy et al., 2011) is the result of their lacking the ns proteins ns3, ns4b, and ns5, otherwise dominantly targeted by cd8 þ t cells (simmons et al., 2005; duangchinda et al., 2010; weiskopf et al., 2011 weiskopf et al., , 2013 rivino et al., 2013) , making it critical to accurately assess not only antibody responses but rather t cell responses in the context of denv vaccine development (weiskopf and sette, 2014) . lassa virus (lasv), causing lassa fever (lf), is an enveloped virus with two single-stranded rna segments and is another virus causing hf. lasv is an old world member of the arenavirida family of viruses. the single-stranded arenavirus genome consists of a small (s) and a large (l) rna segment, measuring 3.4 and 7 kb, respectively. the large segment encodes a small zinc-binding (z) protein which regulates transcription, replication, and viral budding, along with the rna polymerase (l). the small segment encodes the np and the two envelope glycoproteins (gp1 and gp2) responsible for cell entry, reviewed in russier et al. (2012) . lasv disorder is endemic in africa and its neighboring countries (safronetz et al., 2010; gunther et al., 2000) , and though infection rates are difficult to quantify due to limited survey infrastructure, classification of its clinical symptoms is common to other diseases. lasv is predicted to be responsible for approximately 300,000 infections and up to 6000 resultant deaths each year (ogbu et al., 2007; mccormick et al., 1987) . transmission to humans is via the rodent host mastomys natalensis (mccormick et al., 1987) . apcs, dcs, and macrophages are believed to be the first cells targeted by lasv infection (baize et al., 2004; mahanty et al., 2003) , which can rapidly speed up dissemination of lasv to multisystem organs due to their widespread physiological distributions in mucosal tissues and skin. due to their ease in motility across various organs and tissues, apcs are believed to the responsible for the spread of lasv for the establishment of systemic infection (hensley et al., 2011) . apc infection results in substantial virus release in the secondary lymphoid organs, the liver, hepatocytes, fibroblasts, and endothelial cells that are subsequently infected. lymphopenia of cd4 þ and cd8 þ t cells, nk cells, and b cells is observed early during disease onset, reviewed in russier et al. (2012) . lasv infection severities range from asymptomatic infection to fatal hf (fisher-hoch et al., 1995) and commonly resulting from other viral infections, nonspecific symptoms beginning several days after infection include fever, headache, arthralgia, myalgia, and severe asthenia. these early symptoms are typically followed by more severe symptoms of pharyngitis, conjunctivitis, cough, abdominal pain, diarrhea, and vomiting. in severely affected patients, cervical and facial edema, hemorrhages, renal and liver failures, and encephalopathy occur, and death follows systemic shock (edington and white, 1972) . survivors of lasv-related disorders have persisting lifelong morbidities and disabling conditions including deafness (cummins et al., 1990) . no vaccine has been licensed against lasv, and ribavirin is the only existing treatment, but is only effective if administered very early after infection and is not available for broad distribution in countries where lasv is endemic (mccormick et al., 1986) . t cells play a crucial role in the outcome of severe lasv infection, which has been associated with defective t cell responses since the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, t cell responses have been demonstrated to play critical roles in the control of lasv, where strong memory cd4 þ t cell responses directed against lasv np and gp proteins are observed in lasvseropositive healthy individuals from endemic regions (ter meulen et al., 2004) . high serum concentrations of il-8 and cxcl10 chemokines that attract and activate t cells are associated with nonfatal lasv infections (christensen et al., 2006; dufour et al., 2002) and vice versa in fatality cases (mahanty et al., 2001) . the control of acute lf has been correlated with increases in circulating activated cd4 þ and cd8 þ t cells in response to lasv infection or antigen (baize et al., 2009) . in vaccine studies, protection against a lethal lasv rechallenge is associated with the induction of t cell immunity (fisher-hoch et al., 2000; geisbert et al., 2005) . however, in comparison to other viruses, lasv-infected dcs are unable to mount effector t cell responses (pannetier et al., 2011) . human and nonhuman primate studies have demonstrated that lasv np and gp proteins are the main viral antigens recognized by activated t cells (meulen et al., 2004; ter meulen et al., 2000; fisher-hoch et al., 2000; fisher-hoch and mccormick, 2001, 2004; geisbert et al., 2005) , suggesting that vaccines using these proteins to induce long-term memory t cell expansion will best control the spread of lf. ebola virus (ebov) causes a rapidly fatal hf for which there is currently no treatment (muyembe-tamfum et al., 2012; team et al., 2014) . ebov is a member of the filoviridae family, which are filamentous, negative-stranded rna viruses that cause severe human disease. filoviruses viruses are variable, with long filaments measuring 80 nm in diameter and which can reach lengths of up to 1000 nm, with many turns and branches and which have tendency to curve to resemble the number 6. viruses are composed of nucleocapsid, matrix, and envelope proteins, whereby seven genes encode np, the viral proteins vp24-vp30-vp35-vp40, l (polymerase), and the gp (hoenen et al., 2006) , expressed as gp1 and gp2, and regulating virus production and release (mohan et al., 2015) . nps embed the genome in complex with vp30 and vp35 for rna synthesis. vp40 and vp24 proteins are localized in virus matrix space (watanabe et al., 2007; hoenen et al., 2010) . ebov is transmitted to humans via mucosal surfaces, skin injury, and vertical transmission (feldmann and geisbert, 2011) , and with the exception of t cells, can infect almost all human cells using various different attachment mechanisms, reviewed in falasca et al. (2015) . both innate and adaptive immune responses are involved in ebov pathogenesis, where innate immune deregulation involves inhibition of type-i ifn response and perturbation of cytokine signaling, along with impairment of dc and nk cells, and adaptive immune deregulation involves both humoral and cell-mediated immunity (falasca et al., 2015) . because high levels of ebov replication are associated to multiple cell types, its associated systemic dissemination results in a highly complex pathogenesis model, including detrimental immune suppression and hyperactivation, and leading to disordered coagulation and tissue damage, that, in the absence of treatment, results in rapid multiple organ failure and death within days of symptomatic infection (baseler et al., 2017) . for 50 years, ebov and related filoviruses have been repeatedly re-emerging to cause large epidemics of highly fatal hf. ongoing ebov outbreaks in africa has brought this virus to the forefront of research, with over 20,000 reported cases of infection and an associated 8000 deaths (mcelroy et al., 2015) . natural serologic response to ebov infection involves virus-specific igm and igg antibody responses sometimes detected early, but usually later, once symptoms begin rowe et al., 1999) . ebovinfected dcs are impaired in cytokine production required for t cell activation (mahanty et al., 2003) , whereas infected macrophages are unable to mature (bosio et al., 2003) . ebov is classified as an immunosuppressive virus since numerous of its proteins interfere with immune responses by inducing t cell apoptosis, lymphopenia, and absence of antibody responses in fatal cases (basler and amarasinghe, 2009 ). classification of ebov-targeting mechanisms has been compromised by lack of infrastructure for adequate biosafety containment level facilities required to analyze this deadly virus. however, observations of the adaptive t cell immune response have shown that ebov correlates with fatal outcomes by causing aberrant cytokine responses (baize et al., 1999 (baize et al., , 2002 wauquier et al., 2010; villinger et al., 1999; ansari, 2014) , decreased cd4 þ and cd8 þ t cells, and increased apoptotic t cell phenotypes (baize et al., 1999; wauquier et al., 2010; geisbert et al., 2000; bradfute et al., 2010; gupta et al., 2007) . in recent work, ebov induced increased cd4 þ and cd8 þ t cell activation against the viral np, with cd8 þ t cells demonstrating the largest increases in expression of activation and proliferation biomarkers, with sustained activation following ebov clearance and following patient discharge, suggesting continued antigen stimulation after resolution of the disease (mcelroy et al., 2015) . recently, an rvsv-zebov recombinant, replicationecompetent vesicular stomatitis virusebased vaccine expressing a surface gp of zaire ebolavirus, demonstrate a 100% efficacy in preventing ebov disease in contacts and contacts of contacts of recently confirmed cases in guinea, west africa (henao-restrepo, 2017 #550). this vaccine produced rapid innate immune responses after a single dose, suggested to lead to longerterm full protection by providing an essential period of restricted virus replication during the development of specific adaptive responses (marzi, 2015 #551) . crimean-congo hemorrhagic fever virus (cchfv) is a tick-borne virus causing hf resulting in human fatalities. cchfv is a member of the nairoviridae family of viruses from the genus of orthonairovirus and the order of the bunyaviridae viruses. it has a single-stranded, negative-sense rna genome possessing three segments: the large (l), medium (m), and small (s) segments (casals, 1969; clerx et al., 1981) . the l segment encodes the viral rna-dependent rna polymerase responsible for mrna synthesis and rna replication (honig et al., 2004) . the m-segment encodes numerous ns and two structural gps (gn and gc) responsible for cell tropism and attachment and are targets for neutralizing antibodies. the s-segment encodes the viral np binding the rna segments toward formation of ribonucleoprotein complexes (altamura et al., 2007; sanchez et al., 2006) . though hf by cchfv infection in humans is not among the most common viral disorders reported, it remains important because it is fatal in up to 30% of cases (bente et al., 2013; goedhals et al., 2017) . transmission of cchfv to humans occurs through contact with infected animal blood, or ticks, belonging to the genus hyalomma, as its primary vectors and providing transit from one infected human to another (mousavi-jazi et al., 2012) . cchfv human infection involves sudden onset of acute symptoms, including high fever, headache, myalgia, and petechial rash, followed by hemorrhage progressing to multiorgan failure, with leukopenia, thrombocytopenia, and elevated liver enzymes as hallmarks of the overall disorder (begum et al., 1970; sanchez et al., 2006) . outbreak-associated fatality rates are varied but can reach 70% (mousavi-jazi et al., 2012) . there is currently no licensed vaccine, and use of ribavirin as treatment has been investigated but remains controversial (begum et al., 1970; bente et al., 2010) . distribution of cchfv infection and associated disease follows geographical spread of the principal vectors (bente et al., 2013; whitehouse, 2004) . clinical cchfv disorders are described in africa, asia, the middle and east eastern europe, and have recently emerged in other countries including turkey, india, spain, and greece, and with almost 10,000 cases reported in turkey between 2002 and 2015 (maltezou et al., 2010; papa et al., 2008; leblebicioglu et al., 2016) . typically, transient igm and igg antibody responses develop within days following primary cchfv infection and can persist long-term (shepherd et al., 1989; burt et al., 2013) , but where lack thereof usually results in fatality (shepherd et al., 1989) . igm and igg antibodies have however not been correlated with clearance, viral load, or outcomes (duh et al., 2007) , implying that innate and t cell immunity must be critical for viral clearance. neutralizing antibodies also do not cause protection, and nonneutralizing antibodies may assist in antibody-dependent cell-mediated cytotoxicity (bertolotti-ciarlet et al., 2005) . thus, as immune correlates of protection for cchfv are not well documented, vaccine design has aimed at targeting the cchfv np or gps. only an inactivated vaccine is available, and even though the attaining of immunogenicity has required its administration in multiple doses, it was demonstrated to reduce infections and induce both neutralizing antibody responses and t cell responses to np peptides (mousavi-jazi et al., 2012) . another promising approach is the use of modified vacv ankara recombinant vaccine expressing the viral gps, which induce cellular and humoral responses and which are observed to provide protection from lethal disease in mice (buttigieg et al., 2014; dowall et al., 2016) . although t cell responses are known to play a role in protection from and clearance of viral infections, it was only recently that specific cd8 þ t cell epitopes against gn and gc were shown to stimulate ifn-g production, whereby responses were detectable several years after the acute cchfv infection, even in the absence of continued antigenic stimulation, and where ifn-g-producing cd8 þ t cells were confirmed as responsible for providing long-term protection (goedhals et al., 2017) . vhfs causes high fatality rates and are commonly correlated with ineffective immunity and high viral loads. yfv is important for both historical and current reasons. historically, it crippled economies and families by killing hundreds of thousands over 250 years. yfv is transmitted by mosquitos in urban areas, causing as many as 200,000 infections and 30,000 deaths annually. like other mosquito vector viruses, yfv is an existing and re-emerging threat due to increased geological spread by both world travelers and shifting mosquito breeding geographies. flaviviruses evade immunity to enter the brain and spinal cord via circulating blood, crossing the bbb via "trojan horse" mechanism. dissemination of infection and protection against yfv is elicited by ifn-signaling, complement system, and other innate, humoral, and adaptive immune responses. neutralizing antibodies are still the gold standard correlates of protection, but evidence that a robust, long-lived, and polyfunctional adaptive cd4 þ and cd8 þ t cell immune response is what is provided, early after vaccination by the 500 million doses of effective yf 17d vaccine distributed globally. importantly, these are nonexhausted, polyfunctional central memory and effector memory t cell subsets that are detected for over 25 years following vaccination. far more serious is dhf by infection by denv, contributing to either mild or severe hf, with mortality rates exceeding 30% in the untreated. as with yfv, denv uses mosquitos adapted to urban areas as their vectors. fifty million cases of df and 500,000 cases of dhf occur globally each year, with no available cures for its associated disorders and where vaccine development has been complicated by antibody-dependent vaccineinduced enhancement of future heterotypic infections. cytotoxic cd8 þ and cd4 þ t cells, however, vigorously and frequently recognize denv proteins and are believed to contribute protection against primary infection. the denv vaccine dengvaxia has been recently withdrawn, with its failure posited to result from its lack of denv proteins specifically targeted by cd8 þ t cells, and demonstrates that is essential to accurately assess t cell responses in the context of denv development. lasv also causes mild and severe vhf and may cause 300,000 infections and up to 6000 deaths each year. lasv is transmitted to humans by a rodent vector and first targets apc, dc, and macrophages, contributing to rapid multisystem and organ lasv dissemination due to their widespread physiological distributions. lasv also causes lymphopenia of cd4 þ and cd8 þ t cells, nk cells, and b cells, and survivors of lasv-related disorders can be expected to maintain lifelong morbidities. there is no licensed lasv vaccine as of yet, complicated by the fact that the very cells responsible for stimulating t cell antigen responses are those infected by the virus. however, control of acute lf is correlated with increased circulating strong memory cd4 þ and cd8 þ t cells in response to lasv infection or rechallenge and lasv antigen. ebov also causes mass hf. it can infect almost all human cells with exception of lymphocytes and has no known treatment despite documented involvement of innate and adaptive immune responses. classification of ebov targeting mechanisms are compromised by lack of infrastructure for adequate biosafety containment level facilities. ebov causes dcs to be impaired in cytokine production for t cells activation and inhibits macrophages maturation, thus classifying it as an immunosuppressive virus, with fatalities correlating with its induction of lymphopenia the absence of antibody responses. ebov, however, causes increased cd4 þ and cd8 þ t cell activation, with recorded persistence of activated cd8 þ t cells in survivors. the knowledge that cd4 þ and cd8 þ t cell responses are against the ebov np will be useful for the design of efficient targeting vaccines. cchfv also causes hf yielding fatalities in up to 30% of cases. there is no current vaccine, and cchfv has recently reemerged in naïve countries in response to geographical relocation of its primary tick vector. infection by cchfv causes transient ig antibody responses, inversely correlating with sure fatality, but not correlating with clearance, viral load, or outcomes. no protection is granted by neutralizing antibodies either, implying that innate and t cell arms of immunity are critical for viral clearance. with little to no immune correlates of protection for cchfv, vaccine design has aimed at targeting the cchfv np or gps, where immunogenicity requires the administration of multiple doses to induce both neutralizing antibody responses and t cell responses. only recently, specific cd8 þ t cell epitopes against these cchfv proteins have been confirmed to stimulate cytotoxic cd8 þ t cells providing protection in survivors. despite many documented instances of virus eradication from populations by earlier vaccination strategies, there is a continuous imminent risk of outbreaks of pandemic proportions by existing and emerging viruses, as a result of anti-vaccination campaigns, unforeseen viral strain recombination, exposure of naïve populations to viruses by infected world travellers, a rapidly growing and aging and immunocompromised world population, acts of bioterrorism, and use of viruses as biological weapons in war. past strategies in the development of certain vaccines have often been lucky in their efficacies due to their eliciting both humoral and long-lasting adaptive t cell immunity, whereas others have failed and have only induced shorter lived humoral responses that do not always confer long-lasting protection. the precise and likely overlapping mechanisms dictating lifelong immunity conferred by successful vaccines against smallpox, measles, mumps, rubella, polio, and yf are not completely understood. what has become clear however is that innate immunity usually primes adaptive immunity to confer this long-term protection. it is not a lack of existing immune-monitoring methodologies but rather perhaps a lack of their correct implementation in the continued analysis of vaccinated individuals that are hampering the gaining of this important knowledge. therefore it seems there is an urgent need for the standardization of vaccine methodologies adequately measuring effector memory t cell responses and host-immune evasion mechanisms in appropriate animal models and humans and through the use of multiparametric immune-monitoring platforms able to simultaneously document numerous immune cell phenotypes across time postexposure, vaccination, or rechallenge. the importance of global development of standardized procedures of biospecimen banking from vaccinated individuals cannot be understated and will provide unprecedented statistical power in the analysis of the balance between the innate and adaptive immune arms conferring lifelong resistance to infection. the standardization of these methodologies should better assist future rational design of vaccines and boost confidence toward the mass production and stockpiling of these critical prophylactic measures against otherwise crippling and lethal viral disorders. finke, d., brinckmann, u.g., ter meulen, v., liebert, u.g., 1995. gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis. j. virol. 69, 5469e5474. fisher-hoch, s.p., mccormick, j.b., 2001 . towards a human lassa fever vaccine. rev. med. virol. 11, 331e341. fisher-hoch, s.p., mccormick, j.b., 2004 . lassa fever vaccine. expert rev. vaccin. 3, 189e197. fisher-hoch, s.p., tomori, o., nasidi, a., perez-oronoz, g.i., fakile, y., hutwagner, l., mccormick, j.b., 1995. review of cases of nosocomial lassa fever in nigeria: the high price of poor medical practice. bmj 311, 857e859. fisher-hoch, s.p., hutwagner, l., brown, b., mccormick, j.b., 2000 . effective vaccine for lassa fever. j. virol. 74, 6777e6783. life cycle and pathogenesis of hepatitis d virus: a review replication of semliki forest virus: an electron microscopic study safety and immunogenicity of an intranasal sendai virus-based human parainfluenza virus type 1 vaccine in 3-to 6-year-old children genetic analysis of group b human rotaviruses detected in bangladesh in 2000 and establishment and characterization of japanese encephalitis virus-specific, human cd4(þ) t-cell clones: flavivirus cross-reactivity, protein recognition, and cytotoxic activity the yellow fever virus vaccine induces a broad and polyfunctional human memory cd8 þ t cell response initial viral load determines the magnitude of the human cd8 t cell response to yellow fever vaccination silent casualties from the measles outbreak in south africa identification of a novel c-terminal cleavage of crimean-congo hemorrhagic fever virus pregn that leads to generation of an nsm protein immunity and immunological memory following smallpox vaccination long-lived poxvirus immunity, robust cd4 help, and better persistence of cd4 than cd8 t cells evasion of interferon responses by ebola and marburg viruses japanese encephalitis: a review of clinical guidelines and vaccine availability in asia cryo-electron tomography of rubella virus development and durability of measles antigen-specific lymphoproliferative response after mmr vaccination factors associated with fatal cases of measles. a retrospective autopsy study tick-borne viruses of west pakistan. ii. hazara virus, a new agent isolated from ixodes redikorzevi ticks from the kaghan valley, w. pakistan measles virus p gene codes for two proteins subacute sclerosing panencephalitis: more cases of this fatal disease are prevented by measles immunisation than was previously recognized shared modes of protection against poxvirus infection by attenuated and conventional smallpox vaccine viruses pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity immune responses in macaques to a prototype recombinant adenovirus live oral human papillomavirus 16 vaccine the immunogenicity of the oka/merck varicella vaccine in relation to infectious varicella-zoster virus and relative viral antigen content universal influenza vaccines: shifting to better vaccines potent cd8 þ t-cell immunogenicity in humans of a novel heterosubtypic influenza a vaccine, mva-npþm1 cellular localization and antigenic characterization of crimean-congo hemorrhagic fever virus glycoproteins measles and measles vaccination: a review immune-mediated brain atrophy. cd8 þ t cells contribute to tissue destruction during borna disease virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis global, regional, and national causes of child mortality in 2008: a systematic analysis temporal dynamics of the primary human t cell response to yellow fever virus 17d as it matures from an effectorto a memory-type response ebola and marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation neutralizing b cell response in measles human parainfluenza virus type 1 but not sendai virus replicates in human respiratory cells despite ifn treatment clinical aspects of human venezuelan equine encephalitis in texas mechanisms and consequences of ebolavirus-induced lymphocyte apoptosis t cells facilitate recovery from venezuelan equine encephalitis virus-induced encephalomyelitis in the absence of antibody measles virus and associated central nervous system sequelae real-time qualitative pcr for 57 human adenovirus types from multiple specimen sources expert opinion on vaccination of travelers against japanese encephalitis human defined antigenic region on the nucleoprotein of crimean-congo hemorrhagic fever virus identified using truncated proteins and a bioinformatics approach a novel vaccine against crimean-congo haemorrhagic fever protects 100% of animals against lethal challenge in a mouse model medically important arboviruses of the united states and canada disproportionate recruitment of cd+ t cells into the central nervous system by professional antigen-presenting cells antigenic similarity between the virus causing crimean hemorrhagic fever and congo virus disease burden and risk factors for hospitalizations associated with rotavirus infection among children in new york state hemorrhagic fever virus-induced changes in hemostasis and vascular biology mannose 6-phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster adenovirus-based vaccines: comparison of vectors from three species of adenoviridae current recommendations for the japanese encephalitis vaccine viral exanthems pathogenesis of hepatitis b virus infection cxcl10 is the key ligand for cxcr3 on cd8 þ effector t cells involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system a review of viral gastroenteritis neuroinvasion of fluorescein-positive monocytes in acute simian immunodeficiency virus infection structural characteristics of nairoviruses (genus nairovirus, bunyaviridae) the varicella-zoster virus genome progress in understanding and controlling respiratory syncytial virus: still crazy after all these years the seattle virus watch. vi. observations of infections with and illness due to parainfluenza, mumps and respiratory syncytial viruses and mycoplasma pneumoniae evaluation of antibodies against a rubella virus neutralizing domain for determination of immune status targeting rsv with vaccines and small molecule drugs poliovirus entry into human brain microvascular cells requires receptor-induced activation of shp-2 acute sensorineural deafness in lassa fever estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study the systemic pathology of venezuelan equine encephalitis virus infection in humans the pathogenesis of measles update on hepatitis e virology: implications for clinical practice clinical and neuroradiographic manifestations of eastern equine encephalitis immune responses to rotavirus infection and vaccination and associated correlates of protection dynamics of the cytotoxic t cell response to a model of acute viral infection predominant inflammatory cytokine secretion pattern in response to two doses of live rubella vaccine in healthy vaccinees amplification of the sylvatic cycle of dengue virus type 2, senegal, 1999-2000: entomologic findings and epidemiologic considerations evasion of innate and adaptive immunity by flaviviruses molecular insight into dengue virus pathogenesis and its implications for disease control a critical role for induced igm in the protection against west nile virus infection t lymphocyte cytotoxicity with natural varicella-zoster virus infection and after immunisation with live attenuated varicella vaccine booster immune response in children 6-7 years of age, randomly assigned to four groups with two mmr vaccines applied by aerosol or by injection innate and adaptive immune responses against picornaviruses and their counteractions: an overview protective effects of a modified vaccinia ankarabased vaccine candidate against crimean-congo haemorrhagic fever virus require both cellular and humoral responses selection pressure from neutralizing antibodies drives sequence evolution during acute infection with hepatitis c virus contemporary infectious exanthems: an update immunodominant t-cell responses to dengue virus ns3 are associated with dhf acute respiratory disease in military trainees: the adenovirus surveillance program ifngamma-inducible protein 10 (ip-10; cxcl10)-deficient mice reveal a role for ip-10 in effector t cell generation and trafficking viral load as predictor of crimean-congo hemorrhagic fever outcome variola virus immune evasion proteins adenoviruses in immunocompromised hosts the pathology of lassa fever universal vaccine based on ectodomain of matrix protein 2 of influenza a: fc receptors and alveolar macrophages mediate protection epidemiology of viral hepatitis and hepatocellular carcinoma the blood-central nervous system barriers actively control immune cell entry into the central nervous system past, present, and future of japanese encephalitis host responses to live-attenuated venezuelan equine encephalitis virus (tc-83): comparison of naive, vaccine responder and nonresponder to tc-83 challenge in human peripheral blood mononuclear cells ccl2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (hiv)-infected leukocytes across the blood-brain barrier: a potential mechanism of hiv-cns invasion and neuroaids analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection molecular mechanisms of ebola virus pathogenesis: focus on cell death hepatocellular carcinoma in cirrhosis: incidence and risk factors characterization of human proliferative t cell responses to adenovirus quantification of risk factors for herpes zoster: population based case-control study immunity and correlates of protection for rotavirus vaccines national institute of, a., infectious diseases smallpox vaccine study the t¼4 envelope of sindbis virus is organized by interactions with a complementary t¼3 capsid bystander target cell lysis and cytokine production by dengue virus-specific human cd4(þ) cytotoxic t-lymphocyte clones what is immune privilege (not)? trends immunol 28 hospitalizations for varicella in the united states yellow fever: a reemerging threat sex differences in measles mortality: a world review visualization of an inverted terminal repetition in vaccinia virus dna apoptosis induced in vitro and in vivo during infection by ebola and marburg viruses development of a new vaccine for the prevention of lassa fever perspectives on vaccines against varicella-zoster virus infections varicella vaccine: the american experience varicella zoster virus infection human adenovirus: viral pathogen with increasing importance neurological disease produced by varicella zoster virus reactivation without rash melanoma differentiation-associated gene 5 (mda5) is involved in the innate immune response to paramyxoviridae infection in vivo a randomized, blinded, controlled, dose-ranging study of a respiratory syncytial virus recombinant fusion (f) nanoparticle vaccine in healthy women of childbearing age risk of primary infection and reinfection with respiratory syncytial virus long-lived cd8 þ t cell responses following crimean-congo haemorrhagic fever virus infection efficacy and duration of immunity after yellow fever vaccination: systematic review on the need for a booster every 10 years rhoa signaling is required for respiratory syncytial virus-induced syncytium formation and filamentous virion morphology analysis of the human t-cell response to picornaviruses: identification of t-cell epitopes close to b-cell epitopes in poliovirus immune responses to rna-virus infections of the cns emergence and re-emergence of viral diseases of the central nervous system unacceptably high mortality related to measles epidemics in niger hcv persistence and immune evasion in the absence of memory t cell help human influenza viruses and cd8(þ) t cell responses measles virus, immune control, and persistence respiratory syncytial virus: infection, detection, and new options for prevention and treatment imported lassa fever in germany: molecular characterization of a new lassa virus strain ebola virus infection of human pbmcs causes massive death of macrophages, cd4 and cd8 t cell sub-populations in vitro from research to phase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine respiratory syncytial viral infection in children with compromised immune function the burden of respiratory syncytial virus infection in young children preventing varicella-zoster disease duration of antiviral immunity after smallpox vaccination a large observational study to concurrently assess persistence of measles specific b-cell and t-cell immunity in individuals following two doses of mmr vaccine processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the c-terminal half of nsp2 and functions both in cis and in trans safety and immunogenicity of adenovirus-vectored near-consensus hiv type 1 clade b gag vaccines in healthy adults improved mucosal protection against venezuelan equine encephalitis virus is induced by the molecularly defined, liveattenuated v3526 vaccine candidate intracellular cytokine production by dengue virus-specific t cells correlates with subclinical secondary infection biological weapons-a primer for microbiologists specific lysis of varicella zoster virus-infected b lymphoblasts by human t cells efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ca suffit!) lower respiratory viral infections in immunocompetent children neutralizing epitopes of human parainfluenza virus type 3 are conformational and cannot be imitated by synthetic peptides national disease burden of respiratory viruses detected in children by polymerase chain reaction pathogenesis of lassa fever in cynomolgus macaques prime-boost vectored malaria vaccines: progress and prospects appraisal of occurrence of adenovirus-caused respiratory illness in military populations live, attenuated rubellavirus vaccine ebola virus: unravelling pathogenesis to combat a deadly disease oligomerization of ebola virus vp40 is essential for particle morphogenesis and regulation of viral transcription poliovirus cell entry: common structural themes in viral cell entry pathways crimean-congo hemorrhagic fever virus genome l rna segment and encoded protein persistence of vaccine-induced immune responses to rubella: comparison with natural infection delayed clearance of sendai virus in mice lacking class i mhc-restricted cd8 þ t cells cytokine-dependent induction of cd4 þ t cells with cytotoxic potential during influenza virus infection specific lysis of targets expressing varicella-zoster virus gpi or gpiv by cd4 þ human t-cell clones hepatitis delta virus intracellular ifn-gamma expression in natural killer cells precedes lung cd8 þ t cell recruitment during respiratory syncytial virus infection hbv pathogenesis in animal models: recent advances on the role of platelets basis of rabies virus neurovirulence in mice: expression of major histocompatibility complex class i and class ii mrnas postlicensure effectiveness of varicella vaccine during an outbreak in a child care center management of rabies in humans vaccinia virus vaccines: past, present and future hepatitis a virus in west africa: is an epidemiological transition beginning? ongoing rubella outbreak among adolescents in salaj ex vivo analysis of cytotoxic t lymphocytes to measles antigens during infection and after vaccination in gambian children fc or not fc; that is the question: antibody fc-receptor interactions are key to universal influenza vaccine design interleukin (il)-10, il-12, and interferon-gamma production in primary and memory immune responses to varicella-zoster virus qualitative and quantitative characteristics of rotavirus-specific cd8 t cells vary depending on the route of infection an extremely diverse cd4 response to vaccinia virus in humans is revealed by proteome-wide t-cell profiling pathogenesis for viral infections of the nervous system inflammatory responses in the nervous system of mice infected with a street isolate of rabies virus the immune response to rabies virus infection and vaccination prevalence of hepatitis a virus (hav) and hepatitis e virus (hev) in the patients presenting with acute viral hepatitis regional immunity and immune privilege evaluation of a live-attenuated human parainfluenza type 1 vaccine in adults and children antibody response to individual rubella virus proteins in congenital and other rubella virus infections viral exanthems the immunology of smallpox vaccines the primary in vivo murine cytotoxic t cell response to the flavivirus, west nile community respiratory virus infections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immunodeficiency virus infection a systematic review of the epidemiology of hepatitis e virus in africa airway t cells protect against rsv infection in the absence of antibody antigen-dependent intrathecal antibody synthesis in the normal rat brain: tissue entry and local retention of antigen-specific b cells the early cellular signatures of protective immunity induced by live viral vaccination investigation of varicella-zoster virus infection by polymerase chain reaction in the immunocompetent host with acute varicella molecular characterization of rotavirus strains from children in toronto virus infections in the nervous system epidemiology of hepatitis b virus in africa, its genotypes and clinical associations of genotypes clinical virology of ebola hemorrhagic fever (ehf): virus, virus antigen, and igg and igm antibody findings among ehf patients in kikwit, democratic republic of the congo tropism of varicellazoster virus for human tonsillar cd4(þ) t lymphocytes that express activation, memory, and skin homing markers varicella-zoster virus transfer to skin by t cells and modulation of viral replication by epidermal cell interferon-alpha impaired t helper 1 function of nonstructural protein 3-specific t cells in japanese patients with encephalitis with neurological sequelae conserved amino acids 193-324 of non-structural protein 3 are a dominant source of peptide determinants for cd4 þ and cd8 þ t cells in a healthy japanese encephalitis virus-endemic cohort human t cell responses to dengue virus antigens. proliferative responses and interferon gamma production dengue virus-specific human t cell clones. serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity immunity against heterosubtypic influenza virus induced by adenovirus and mva expressing nucleoprotein and matrix protein-1 complications of smallpox vaccination, 1968: results of ten statewide surveys acute hepatitis a virus infection is associated with a limited type i interferon response and persistence of intrahepatic viral rna herpes zoster vaccine effectiveness against incident herpes zoster and post-herpetic neuralgia in an older us population: a cohort study sofosbuvir for previously untreated chronic hepatitis c infection crimean-congo hemorrhagic fever in turkey: current status and future challenges the protective efficacy of recombinant hepatitis b vaccine in newborn infants of hepatitis b e antigen-positive-hepatitis b surface antigen carrier mothers vaccine-elicited cd8 þ t cells protect against respiratory syncytial virus strain a2-line19f-induced pathogenesis in balb/c mice replication of alphaviruses: a review on the entry process of alphaviruses into cells varicella-zoster virus and virus dna in the blood and oropharynx of people with latent or active varicella-zoster virus infections antibody-mediated clearance of alphavirus infection from neurons pox in the docks: varicella outbreak in an australian prison system adaptive immune response to viral infections in the central nervous system hepatitis c virus-associated cancer adenovirus infections in immunocompetent and immunocompromised patients identification of cytolytic lymphocytes in west nile virus-infected murine central nervous system unicef, 2012. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since dengue virus-specific, hla-b35-restricted, human cd8 þ cytotoxic t lymphocyte (ctl) clones. recognition of ns3 amino acids 500 to 508 by ctl clones of two different serotype specificities varicella outbreaks in army recruits from puerto rico. varicella susceptibility in a population from the tropics humoral and cellular immune responses to a hepatitis b vaccine booster 15-18 years after neonatal immunisation comparative neurovirulence of attenuated and non-attenuated strains of venezuelan equine encephalitis virus in mice cytokine signatures of innate and adaptive immunity in 17dd yellow fever vaccinated children and its association with the level of neutralizing antibody alphavirus vectors for vaccine production and gene therapy. expert rev. vaccin. 2, 447e459. lynch 3rd low levels of interleukin-8 and interferon-inducible protein-10 in serum are associated with fatal infections in acute lassa fever cutting edge: impairment of dendritic cells and adaptive immunity by ebola and lassa viruses detection of respiratory viruses by molecular methods immune evasion by adenoviruses rotavirus-specific t-cell responses in young prospectively followed-up children high prevalence and diversity of hepatitis viruses in suspected cases of yellow fever in the democratic republic of congo crimean-congo hemorrhagic fever in europe: current situation calls for preparedness cryo-electron microscopy reveals the functional organization of an enveloped virus, semliki forest virus herpes zoster vaccine effectiveness and manifestations of herpes zoster and associated pain by vaccination status rotavirus detection and characterisation in outbreaks of gastroenteritis in aged-care facilities comparative analysis of the full genome sequence of european bat lyssavirus type 1 and type 2 with other lyssaviruses and evidence for a conserved transcription termination and polyadenylation motif in the g-l 3' non-translated region vaccine-derived poliovirus from long term excretors and the end game of polio eradication hepatitis a virus: from discovery to vaccines ebola vaccine. vsv-ebov rapidly protects macaques against infection with the 2014/15 ebola virus outbreak strain potential virulence determinants in terminal regions of variola smallpox virus genome dominant recognition by human cd8 þ cytotoxic t lymphocytes of dengue virus nonstructural proteins ns3 and ns1.2a lassa fever. effective therapy with ribavirin a prospective study of the epidemiology and ecology of lassa fever human ebola virus infection results in substantial immune activation illuminating viral infections in the nervous system the immunologic response to infection with respiratory syncytial virus in infants distribution of measles virus in the central nervous system of hiv-seropositive children food-related illness and death in the united states cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily interaction of rotavirus with human peripheral blood mononuclear cells: plasmacytoid dendritic cells play a role in stimulating memory rotavirus specific t cells in vitro old and new world arenaviruses share a highly conserved epitope in the fusion domain of the glycoprotein 2, which is recognized by lassa virus-specific human cd4 þ t-cell clones long-term measles-induced immunomodulation increases overall childhood infectious disease mortality causes of a nationwide rubella outbreak in japan rubella reimmunisation: comparative analysis of the immunoglobulin g response to rubella virus vaccine in previously seronegative and seropositive individuals tropism of varicella-zoster virus for human cd4 þ and cd8 þ t lymphocytes and epidermal cells in scid-hu mice less is more: ebola virus surface glycoprotein expression levels regulate virus production and infectivity yellow fever antigen-specific expansion of cytotoxic t lymphocytes in acute measles virus infection replication of hepatitis c virus structure and development of viruses as observed in the electron microscope. v. western equine encephalomyelitis virus rabies: a review and current approach for the clinician human immunodeficiency virus infection of human brain capillary endothelial cells occurs via a cd4/galactosylceramideindependent mechanism measles still has a devastating impact in unvaccinated populations global measles elimination immunology 101 at poxvirus u: immune evasion genes trends in hospitalizations associated with gastroenteritis among adults in the united states healthy individuals' immune response to the bulgarian crimean-congo hemorrhagic fever virus vaccine. vaccine immune cell migration as a means to control immune privilege: lessons from the cns and tumors protection of adult but not newborn mice against lethal intracerebral challenge with japanese encephalitis virus by adoptively transferred virus-specific cytotoxic t lymphocytes: requirement for l3t4 þ t cells current approaches to the development of vaccines effective against parainfluenza viruses. bull. world health organ. 66, 391e397. muyembe-tamfum bronchoalveolar lavage cell analysis in measles viral pneumonia hepatitis c virus and vaccine development global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis case-fatality rate during a measles outbreak in eastern niger in 2003 the pathogenesis of poliomyelitis: what we don't know immunosuppression: a means to assess the role of the immune response in acute virus infections recognition of contiguous allele-specific peptide elements in the rubella virus e1 envelope protein tlr expression and nk cell activation after human yellow fever vaccination hepatitis b virus-cell interactions and pathogenesis respiratory viruses other than influenza virus: impact and therapeutic advances overview of complement activation and regulation rotavirus-specific helper t cell responses in newborns, infants, children, and adults rotavirus-specific humoral and cellular immune response after primary, symptomatic infection lassa fever in west african sub-region: an overview electron microscopic studies of rubella virus analysis of overlapping t-and b-cell antigenic sites on rubella virus e1 envelope protein. influence of hla-dr4 polymorphism on t-cell clonal recognition promiscuous t-cell recognition of a rubella capsid protein epitope restricted by drb1*0403 and drb1*0901 molecules sharing an hla dr supertype the contribution of hla class i antigens in immune status following two doses of rubella vaccination alphavirus budding is dependent on the interaction between the nucleocapsid and hydrophobic amino acids on the cytoplasmic domain of the e2 envelope glycoprotein shingles prevention study viremic phase in nonimmunocompromised children with varicella pathogenesis of the viral hemorrhagic fevers replication and clearance of venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric sin/vee viruses alpha-beta t cells provide protection against lethal encephalitis in the murine model of veev infection correlates of protective immunity in poxvirus infection: where does antibody stand? human dendritic cells infected with the nonpathogenic mopeia virus induce stronger t-cell responses than those infected with lassa virus ongoing outbreak of rubella among young male adults in poland: increased risk of congenital rubella infections global illness and deaths caused by rotavirus disease in children circulating rotavirus-specific t cells have a poor functional profile fulfilling the promise of rotavirus vaccines: how far have we come since licensure? immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent orthopox viruses: infections in humans viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis flavivirus entry receptors: an update role of the endothelium in viral hemorrhagic fevers pathway of rubella virus infectious entry into vero cells long-term duration of detectable neutralizing antibodies after administration of live-attenuated vee vaccine and following booster vaccination with inactivated vee vaccine rubella vaccination correlates of protection induced by vaccination immune cell entry to central nervous systemcuirent understanding and prospective therapeutic targets production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody alphavirus dna and particle replicons for vaccines and gene therapy exploratory study of oral combination antiviral therapy for hepatitis c examination of influenza specific t cell responses after influenza virus challenge in individuals vaccinated with mva-npþm1 vaccine use of telemetry to assess vaccine-induced protection against parenteral and aerosol infections of venezuelan equine encephalitis virus in non-human primates genetically engineered, live attenuated vaccines for venezuelan equine encephalitis: testing in animal models pathogenesis of adenovirus type 5 pneumonia in cotton rats (sigmodon hispidus) cytokine profile after rubella vaccine inoculation: evidence of the immunosuppressive effect of vaccination dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: cd4 and cd8 t-cell epitope mapping and role of gamma interferon mechanisms of central nervous system viral persistence: the critical role of antibody and b cells three or more routes for leukocyte migration into the central nervous system deaths from chickenpox in england and wales 1995-7: analysis of routine mortality data alphavirus vectors and vaccination progress toward control of rubella and prevention of congenital rubella syndrome-worldwide ex vivo generation of cytotoxic t lymphocytes specific for one or two distinct viruses for the prophylaxis of patients receiving an allogeneic bone marrow transplant recombinant alphaviruses as vectors for anti-tumour and anti-microbial immunotherapy smallpox as an actual biothreat: lessons learned from its outbreak in ex-yugoslavia in 1972 epidemic venezuelan equine encephalitis in la guajira, colombia differential targeting of viral components by cd4 þ versus cd8 þ t lymphocytes in dengue virus infection rubella and congenital rubella syndrome: global update cellular immune responses to diluted and undiluted aventis pasteur smallpox vaccine longitudinal study of rotavirus infection and gastroenteritis in families served by a pediatric medical practice: clinical and epidemiologic observations the global distribution of yellow fever and dengue viral meningitis and encephalitis: traditional and emerging viral agents recombinant rubella vectors elicit siv gag-specific t cell responses with cytotoxic potential in rhesus macaques expression of mucosal homing receptor alpha4beta7 by circulating cd4 þ cells with memory for intestinal rotavirus historical comparisons of morbidity and mortality for vaccine-preventable diseases in the united states clinical, virologic, and immunologic follow-up of convalescent ebola hemorrhagic fever patients and their household contacts, kikwit, democratic republic of the congo. commission de lutte contre les epidemies a kikwit failure to open the blood-brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection sendai virus as a backbone for vaccines against rsv and other human paramyxoviruses measles virus for cancer therapy immune responses and lassa virus infection alpha/ beta interferon protects adult mice from fatal sindbis virus infection and is an important determinant of cell and tissue tropism the natural history of human poliomyelitis: ii. elimination of the virus recognition of the latency-associated immediate early protein ie63 of varicella-zoster virus by human memory t lymphocytes detection of lassa virus alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival crimean-congo hemorrhagic fever virus glycoprotein precursor is cleaved by furin-like and ski-1 proteases to generate a novel 38-kilodalton glycoprotein human group b rotavirus infections cause severe diarrhea in children and adults in bangladesh detection of varicella-zoster virus dna in the oropharynx and blood of patients with varicella nonclinical phenotypic and genotypic analyses of a phase 1 pediatric respiratory syncytial virus vaccine candidate medi-559 (ra2cp248/ 404/1030deltash) at permissive and non-permissive temperatures alphavirus vectors for gene expression and vaccines efficacy, safety, and tolerability of herpes zoster vaccine in persons aged 50-59 years presence, distribution and spread of productive varicella zoster virus infection in nervous tissues progress in the development of human parainfluenza virus vaccines memory cd8 t cells mediate severe immunopathology following respiratory syncytial virus infection viral hemorrhagic fever-a vascular disease pathogenesis of acute respiratory illness caused by human parainfluenza viruses robust iga and igg-producing antibody forming cells in the diffuse-nalt and lungs of sendai virus-vaccinated cotton rats associate with rapid protection against human parainfluenza virus-type 1 single-cell mass cytometry analysis of human tonsil t cell remodeling by varicella zoster virus selective cd4 þ t cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities extreme mortality after first introduction of measles virus to the polynesian island of rotuma kinetics and viral protein specificity of the cytotoxic t lymphocyte response in healthy adults immunised with live attenuated varicella vaccine an increasing danger of zoonotic orthopoxvirus infections antibody response in crimean-congo hemorrhagic fever virus-induced type i ifn stimulates generation of immunoproteasomes at the site of infection immune responses and immunopathology in acute and chronic viral hepatitis cd8 þ t cells require perforin to clear west nile virus from infected neurons characterization of main cytokine sources from the innate and adaptive immune responses following primary 17dd yellow fever vaccination in adults early t-cell responses to dengue virus epitopes in vietnamese adults with secondary dengue virus infections characterization of poliovirus-specific t lymphocytes in the peripheral blood of sabin-vaccinated humans cd4 þ t-cell responses are required for clearance of west nile virus from the central nervous system pathogenesis and clinical features of japanese encephalitis and west nile virus infections influenza and memory t cells: how to awake the force. vaccines (basel) expression of the f and hn glycoproteins of human parainfluenza virus type 3 by recombinant vaccinia viruses: contributions of the individual proteins to host immunity the global burden of viral hepatitis from analysis of t cell responses during active varicella-zoster virus reactivation in human ganglia review paper: pathology of animal models of alphavirus encephalitis interview about immune privilege with memory cd8 t cells in rsv infection: friend or foe? smallpox. who puts off destruction of u.s., russian caches complete nucleotide sequence of the genomic rna of sindbis virus t cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue defective cd8 t cell memory following acute infection without cd4 t cell help receptor specificities of human respiroviruses viral diseases of the central nervous system a novel population of myeloid cells responding to coxsackievirus infection assists in the dissemination of virus within the neonatal cns cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis c estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis ebola virus disease in west africa-the first 9 months of the epidemic and forward projections characterization of human cd4(þ) t-cell clones recognizing conserved and variable epitopes of the lassa virus nucleoprotein vaccinia virus-specific cd8(þ) t-cell responses target a group of epitopes without a strong immunodominance hierarchy in humans viral and immunological determinants of hepatitis c virus clearance, persistence, and disease cd8(þ) t cells mediate viral clearance and disease pathogenesis during acute hepatitis b virus infection contacts with varicella or with children and protection against herpes zoster in adults: a case-control study alphavirus replicon particles acting as adjuvants promote cd8 þ t cell responses to co-delivered antigen hcv transmission in industrialized countries and resourceconstrained areas hepatitis c virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies cd4 t cell control primary measles virus infection of the cns: regulation is dependent on combined activity with either cd8 t cells or with b cells: cd4, cd8 or b cells alone are ineffective clearance of an influenza a virus by cd4 þ t cells is inefficient in the absence of b cells correlation between rubella antibody levels and cytokine measures of cell-mediated immunity alphaviruses suppress host immunity by preventing myeloid cell replication and antagonizing innate immune responses dispersal and other population parameters of aedes aegypti in an african village and their possible significance in epidemiology of vector-borne diseases herpes zoster vaccine in older adults and the risk of subsequent herpes zoster disease live attenuated varicella vaccine: evidence that the virus is attenuated and the importance of skin lesions in transmission of varicella-zoster virus. national institute of allergy and infectious diseases varicella vaccine collaborative study group immunoglobulin (igg) and (igm) antibody responses to rabies vaccine cellular immune responses to live attenuated japanese encephalitis (je) vaccine sa14-14-2 in adults in a je/dengue co-endemic area rabies virus as a transneuronal tracer of neuronal connections cytomegalovirus cell tropism, replication, and gene transfer in brain invited commentary: dengue lessons from cuba west nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: transmigration across the in vitro blood-brain barrier cell-mediated immunity in rubella assayed by cytotoxicity of supernatants from rubella virus-stimulated human lymphocyte cultures a large infantile gastroenteritis outbreak in albania caused by multiple emerging rotavirus genotypes markedly elevated levels of interferon (ifn)-gamma, ifn-alpha, interleukin (il)-2, il-10, and tumor necrosis factor-alpha associated with fatal ebola virus infection enhanced expression of hiv and siv vaccine antigens in the structural gene region of live attenuated rubella viral vectors and their incorporation into virions envelope structure of semliki forest virus reconstructed from cryo-electron micrographs virus-specific cd4 þ and cd8 þ cytotoxic t-cell responses and long-term t-cell memory in individuals vaccinated against polio reassessing immune control of hepatitis a virus adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients experimental infection of horses with an attenuated venezuelan equine encephalomyelitis vaccine (strain tc-83) molecular basis for broad neuraminidase immunity: conserved epitopes in seasonal and pandemic h1n1 as well as h5n1 influenza viruses yellow fever cases in asia: primed for an epidemic ebola virus (ebov) vp24 inhibits transcription and replication of the ebov genome humoral and cell-mediated immune responses in children and adults after 1 and 2 doses of varicella vaccine t cell-mediated immunity towards yellow fever virus and useful animal models the effect of decreasing amounts of live virus, while antigen content remains constant, on immunogenicity of oka/merck varicella vaccine measles, mumps, and rubella-vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunisation practices (acip) human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis re-emergence of epidemic venezuelan equine encephalomyelitis in south america. vee study group venezuelan equine encephalitis parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization t-cell immunity to infection with dengue virus in humans insights into hla-restricted t cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design comprehensive analysis of dengue virus-specific responses supports an hla-linked protective role for cd8 þ t cells respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases b-cell-deficient and cd8 t-cell-depleted gnotobiotic pigs for the study of human rotavirus vaccineinduced protective immune responses role of alpha/beta interferon in venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region measles, mumps, and rubella who influenza (seasonal) 2018. available online live-attenuated tetravalent dengue vaccines: the needs and challenges of post-licensure evaluation of vaccine safety and effectiveness genomic analysis of the host response to hepatitis b virus infection a single 17d yellow fever vaccination provides lifelong immunity; characterization of yellow-fever-specific neutralizing antibody and t-cell responses after vaccination viruses causing gastroenteritis herpes zoster vaccine live: a 10year review of post-marketing safety experience humoral immune response to primary rubella virus infection adenovirus proteins that subvert host defenses immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic estimates of measles case fatality ratios: a comprehensive review of community-based studies ultrastructure of mumps virus replication in newborn hamster central nervous system cellular and humoral immunity against vaccinia virus infection of mice identification of hot spots in the variola virus complement inhibitor (spice) for human complement regulation rotavirus induces proliferative response and augments non-specific cytotoxic activity of lymphocytes in humans a protective role for dengue virus-specific cd8 þ t cells cd4 þ t cells are not required for the induction of dengue virus-specific cd8 þ t cell or antibody responses but contribute to protection after vaccination a population-based study of the incidence and complication rates of herpes zoster before zoster vaccine introduction rotavirus vaccines: current status and future considerations antigen processing and presentation by the class i major histocompatibility complex fatal community-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate cd4 þ t cells provide protection against acute lethal encephalitis caused by venezuelan equine encephalitis virus role of humoral versus cellular responses induced by a protective dengue vaccine candidate infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium genomic and oncogenic preference of hbv integration in hepatocellular carcinoma efficacy and safety of a recombinant hepatitis e vaccine in healthy adults: a large-scale protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive b and t cells key: cord-032183-yqqqe325 authors: ning, qin; wu, ting; su, hai-bin; ma, ke; qi, jun-ying; ni, ming; wu, di title: antiviral therapy for aechb and severe hepatitis b (liver failure) date: 2019-05-21 journal: acute exacerbation of chronic hepatitis b doi: 10.1007/978-94-024-1603-9_5 sha: doc_id: 32183 cord_uid: yqqqe325 this chapter describes the principles of antiviral therapy, treatment strategies, medications and recommendations for aechb, hbv-aclf, hbv-related liver cirrhosis, hbv-related hcc, and liver transplantation. 1. severe exacerbation of chronic hepatitis b is closely related to continuous hbv replication. therefore, inhibiting hbv replication to reduce viral load may block disease progression and improve the quality of life of these patients. etv or tdf has been recommend first-line drug for the treatment of aechb. 2. a hyperactive immune response due to continuous hbv replication is the main mechanism for development of severe hepatitis b. in addition to comprehensive treatment, early administration of potent nucleoside analogs can rapidly reduce hbv dna concentration, relieve immune injury induced by hbv, and reduce liver inflammation and patient mortality. antiviral agents have become important in the treatment of severe exacerbation of chronic hepatitis b. 3. long-term antiviral treatment with nucleoside analogs can delay or reverse the progress of liver cirrhosis. virologic response, viral resistance and adverse drug reactions should be closely monitored during treatment. the treatment should be optimized for maximum effect based on each patient’s responses. 4. effective antiviral therapy can suppress hbv replication and reduce the incidence of hbv-related hcc. patients with hbv-related hcc should receive individualized and optimal multidisciplinary comprehensive treatment. anti-viral drugs with high efficacy, low resistance and low adverse drug reactions should be selected to improve the patient’s quality of life and prolong survival time. 5. methods to prevent hbv reinfection after liver transplantation include passive immunization (hbig), antiviral treatment (nucleoside analogs) and active immunization (hepatitis b vaccine). 6. clinical trials involving sequential combination therapy with nuc and peg-ifn have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response. combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. dence of hbv-related hcc. patients with hbv-related hcc should receive individualized and optimal multidisciplinary comprehensive treatment. anti-viral drugs with high efficacy, low resistance and low adverse drug reactions should be selected to improve the patient's quality of life and prolong survival time. 5. methods to prevent hbv reinfection after liver transplantation include passive immunization (hbig), antiviral treatment (nucleoside analogs) and active immunization (hepatitis b vaccine). 6 . clinical trials involving sequential combination therapy with nuc and peg-ifn have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response. combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. ting wu and qin ning chb is a progressive disease, and its development is the interaction between hbv and the host's immune response. host's immune system eliminates hbv through cytolysis and non-cytolysis mechanism, which leads to hepatic inflammation, hepatocyte necrosis and apoptosis. continuous hepatic inflammation can lead to liver fibrosis, liver cirrhosis, liver failure, and even liver cancer gradually. hbv replication plays a key role in the disease evolution. therefore, suppressing hbv replication is crucial in treatment of chb. the development of continuous liver inflammation, cirrhosis and liver cancer is closely related to the sustainable hbv replication in chb patients. hence, inhibiting hbv replication and reducing hbv dna load is the key to block the disease progression and improve the survival quality in the treatment of chb [1] . the fundamental goal of chb treatment is to eliminate hbv or suppress hbv permanently, thus to reduce viral pathogenicity and infectivity, relieve or inhibit hepatic necroinflammation. however, the existing antiviral drugs cannot remove the intercellular covalently closed circular dna (cccdna). therefore, the goal of antiviral treatment is to sustainably suppress the virus, reduce or prevent hepatic injury and disease progression. the short-term goal of clinical practice is sustainable hbv suppression, alt normalization and prevention from decompensated liver cirrhosis (initial response), releasing the hepatic necroinflammation and liver fibrosis during and after treatment. the long-term and ultimate goal is prevention from liver decompensation, relief or halt of the progression to liver cirrhosis or hcc, and prolonged survival period (sustained response) [2] [3] [4] [5] [6] [7] . hbv is difficultly to be eliminated, and there is no drug can absolutely eradicate hbv infection so far. many drugs can function against hbv, but only two classes are internationally recognized as antiviral drugs: interferon and nucleoside analogues (fig. 5.1 ). common guidelines for management of chb (table 5 the recommendations for the treatment of hbeag positive patients with chb in guidelines (table 5. 2) [3] [4] [5] [6] 8] . the recommendations for the treatment of hbeag negative patients with chb in guidelines (table 5. 3) [3] [4] [5] [6] 8] . the recommendations for the treatment of cirrhotic patients with chb in guidelines (table 5 .4) [3] [4] [5] [6] . the recommendations for liver biopsy in guidelines (table 5 .5) [3] [4] [5] [6] . the recommendations for chb initial treatment options in guidelines (table 5 .6) [3] [4] [5] [6] 8] . the recommendations for chb cirrhosis initial treatment options in guidelines (table 5 .7) [3] [4] [5] [6] . the recommendations for chb treatment duration in guidelines (table 5 .8) [3] [4] [5] [6] 8] . 1. antiviral treatment strategies for hbeag positive patients ( fig. 5.2 ). 2. antiviral treatment strategies for hbeag negative patients (fig. 5.3 ). the recommendations for management of lamivudine drug-resistance in guidelines (table 5 .9) [3] [4] [5] [6] 8] . start antiviral treatment to avoid liver transplantation entecavir and tenofovir as the first-line choice, or pegylated interferons aasld 2018 pegylated interferon-α, entecavir or tenofovir are preferred; interferon-α/pegylated interferon-α, lamivudine, adefovir, entecavir, tenofovir or telbivudine can be used; adefovir or entecavir for interferon nonresponders or patients with interferon contraindication. csld/csid 2015 pegylated interferon-α, entecavir who 2015 tenofovir and entecavir as the first-line choice ifn may be considered when hbv dna viral load and genotyping are available, or co-infection with hdv nas with low resistance first; use interferon with caution, start from small doses decompensation nas with low resistance first; interferon is forbidden the recommendations for management of adefovir drug-resistance in guidelines (table 5 .10) [3] [4] [5] [6] 8] . consensus of antiviral treatment in special populations with chronic hepatitis b was put forward by china expert committee of antiviral treatment in special population with chronic hepatitis b in 2010. in china, hbv infection is one of the main causes of liver failure. hbv related liver failure can be further divided into acute liver failure, subacute liver failure, acuteon-chronic liver failure and chronic liver failure. nucleoside analogues can be safely used in the treatment of hbv related liver failure, and improve the prognosis of patients. nucleoside analogues treatment can improve the survival rate and reduce the incidence of complications in hbv related acute and subacute liver failure patients. therefore, nucleoside analogues treatment should be early applied in hbsag positive and hbv dna detectable patients with acute and subacute liver failure, and nucleoside analogues can quickly inhibit virus, including lamivudine, entecavir and tenofovir, are recommended for these patients. drug resistance should be monitored in long-term nucleoside analogues treatment. remaining hbv cannot be completely excluded even when hbsag and hbv dna are undetectable during the treatment, therefore the antiviral treatment should continue to hbsag seroconversion. antiviral treatment is unnecessary for anti-hbs positive patients at first visit [9] . for patients with hbv related primary liver cancer, liver cancer resection, radiofrequency ablation and interventional therapy all can lead to hbv reactivation and liver function damage aggravation, antiviral treatment is decided depending d on liver compensatory situation. ifn-α can exhibit both anti-virus and anti-cancer effect, delay the tumor recurrence and prolong the median survival period. ifn-α should be the preference if the patients can tolerate ifn-α. if the patients have contraindications to ifn-α, lamivudine, adefovir or entecavir can be chosen depending on hbv dna loads, cirrhosis compensatory situation and kidney function. for patients undergoing hepatic arterial chemotherapy, prophylactic nucleoside analogues treatment should be given before chemotherapy. for patients with advanced liver cancer, portal vein branch thrombosis, but without contraindications to ifn-α, ifn-α treatment can extend survival period [9] [10] [11] [12] [13] [14] [15] . patients awaiting liver transplantation because of hbv-related end-stage liver disease or liver cancer should be given nucleoside analogues with strong hbv inhibition and low drug-resistance, or nucleotides analogues combination treatment, in order to reduce viral load and prevent graft re-infection. lamivudine and (or) adefovir combination with hbig can be safely and effectively prevent graft re-infection, and reduce the re-infection rate to below 10%. hbv-associated liver transplant patients require lifelong treatment of antiviral drugs for the prevention of hepatitis b recurrence. hbsag-negative patients receiving anti-hbs positive donor liver should also receive long-term treatment of lamivudine or preventive treatment of hbig [1, 9, [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . according to who, elderly chb patients refers to chb patients aged ≥60 years old. generally speaking, according to current guidelines, ≥60 years of age is not a contraindication to antiviral therapy, so their treatment can refer to the relevant guidelines, but their desire, risks and benefits of treatment should be comprehensive evaluated. especially for the patients using ifn-α, the expected survival and liver function compensatory situation, possible side effects, underlying hypertension, diabetes, coronary heart disease, and the improvement of liver function should be comprehensive evaluated. additionally, the treatment response, side effects, blood sugar, kidney function and occurrence of liver cancer should be closely monitored during and after treatment [9] . children chb patients are usually in the immune tolerance phase of hbv infection, hence they could not receive antiviral treatment, but should be closely followed up. fda has approved of ifn-α (2-17 years of age), lamivudine (2-17 years of age), and adefovir (12-17 years of age) for use in children. recommended ifn-α dose is 6 miu/m 2 of body surface area three times per week, and the maximum dose is 10 miu/m 2 total body surface area. it is showed that ifn-α effects the same in children as in adult. recommended lamivudine dose is 3 mg/(kg day) with a maximum dose of 100 mg/day. recommended adefovir dosage and usage are the same with adult patients [9, [28] [29] [30] [31] [32] [33] . mother to child transmission is the main route of transmission of hbv infection in china, in order to block the transmission of hbv, antiviral therapy in pregnant chb patients is very important. firstly, antiviral treatment should be completed before pregnancy as possible. it is recommended to consider pregnant 6 months later after interferon or nucleoside analogs treatment. secondly, unwanted pregnancies should be terminated during ifn-α treatment because of its pregnancy toxicity. pregnancy safety of nucleosides analogs has not been proved by any clinical trials, but a large number of studies have shown that lamivudine and tenofovir were safe, so the treatment with lamivudine could continue under the premise of full communication with patients. telbivudine, adefovir or entecavir treatment may switch to lamivudine treatment. pregnant chb patients with slight alt elevation, should be monitored closely or given liver protection and symptomatic treatment, and given antiviral treatment after delivery. pregnant chb patients with poor liver function could be given lamivudine treatment after full consultations with patients and signed informed consent forms. serum hbv dna load in pregnant chb patients is the key factors of mother to child transmission, and effective antiviral treatment can significantly reduce the transmission incidence. according to the findings, lamivudine or telbivudine treatment could start in [28] [29] [30] [31] [32] [33] [34] week of pregnancy to block transmission, and the withdrawal can refer to the patients with immunosuppressive agents or chemotherapy. in addition, women with husbands receiving ifn-α antiviral treatment, should only consider pregnancy 6 months later after withdrawal. there is no evidence at present that nucleosides analogs have a negative impact on sperm and embryo, pregnancy could be taken into account under the premise of full communication with patient [9, [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] . hbv and hcv co-infection increases the incidence of severe hepatitis, liver cirrhosis and liver cancer. when co-infection, hcv may inhibit hbv generally, and different treatment should be given depending on hbv and hcv viral load and alt level (table 5 .11) [9, [46] [47] [48] [49] [50] [51] [52] [53] . hbv and hiv co-infection increases hbv dna load, reduces spontaneous hbeag seroconversion rate, aggravates liver damage and increases mortality in patients with liver disease. anti-hbv regimens should combine with highly active antiretroviral therapy (haart). if anti-hbv and hiv treatment is needed, anti-hbv drugs such as tenofovir + lamivudine or tenofovir + truvada could be used in haart. if haart only contains lamivudine as anti-hbv drugs, hbv drug resistance should be closely monitored and treatment should be adjusted in time. if haart is unnecessary, adefovir, telbivudine and ifn-α can be used for anti-hbv treatment. because lamivudine, tenofovir and entecavir monotherapy induced risk of hiv drug-resistance, lamivudine, tenofovir and entecavir treatment are not recommend to these patients [9, [54] [55] [56] [57] [58] [59] [60] . anti-hbv treatment is critical for hepatitis b virus associated glomerulonephritis (hbv-ag). patient diagnosed with hbv-ag must start anti-viral therapy as long as hbv dna is detectable. a number of studies show that kidney disease alleviated significantly after lamivudine treatment, along with hbv dna decline and hbeag clearance. adefovir has been shown to increase serum creatinine level in some patients in clinical trials, therefore should be carefully chosen. there is not enough clinical evidence of telbivudine and entecavir treatment for hbv-ag. there is no consensus of nucleoside analogue treatment for hbv-ag currently. safety and efficacy of ifn-α and pegylated ifn-α treatment for hbv-ag have not been proved [9, [61] [62] [63] [64] [65] [66] [67] . elevation of hbv dna can be observed in 20-50% of the hbsag-positive patients receiving immunosuppressive agents or cytotoxic therapy, including corticosteroids, anti-cd20 and anti-tnf. some patients suffer from transaminase elevation and jaundice, and severe patients develop to fulminant liver failure even death. nucleoside analogues prophylactic treatment can decrease hbv reactivation. regardless of the hbv dna level, hbsag carriers should receive nucleoside analogues antiviral treatment 2-3 weeks before immunosuppressive or cytotoxic therapy. antiviral drugs inhibit hbv dna rapid, such as lamivudine, telbivudine and entecavir, are preferred for prophylaxis. most patients cannot tolerate the recurrent aggravations induced by drug-resistance. prophylaxis decision should be made depending on baseline hbv dna load and duration of immunosuppressive therapy or cytotoxic therapy. antiviral drugs with low resistance are recommended if prophylaxis will last more than 12 months. treatment duration: if baseline hbv dna ≤10 5 copies/ml, treatment should be continued to 6 months after immunosuppressive therapy or cytotoxic therapy completed; if baseline hbv dna >10 5 copies/ ml, treatment should be continued to the referring withdrawal standard of ordinary chb patients. however, ifn-α is not recommended because of the bone marrow suppression. there is no consensus of prophylactic antiviral treatment for hbsag-negative and anti-hbc-positive patients during the immunosuppressive or cytotoxic therapy. serum hbv marker and hbv dna level should be monitored [9, [68] [69] [70] [71] [72] [73] . hbv infection itself is not correlated with thyroid dysfunction. ifn-α can aggravate underlying autoimmune thyroid disease or induce emerging thyroid disease in some patients because of its immunomodulatory activity and direct thyroid toxicity. uncontrolled thyroid dysfunction contraindicates ifn-α antiviral therapy. patients with previous thyroid dysfunction or high titer of thyroid autoantibody (tpo-ab >18 iu/ml) before treatment should be monitored for thyroid function during ifn-α antiviral treatment. patients with emerging thyroid dysfunction during treatment should terminate antiviral treatment. majority of thyroid dysfunction emerged during treatment in patients without history of thyroid dysfunction is reversible, and can restore after ifn-α withdrawal [74] [75] [76] [77] . resistance to nucleoside analogues is a serious problem in chb treatment, which does make the long-term treatment strategies become a difficulty. a variety of factors may be associated with resistance to nucleoside analogues, including nucleoside analogue type, hbv dna load at initial therapy, liver fibrosis/ cirrhosis, and previous nucleoside analogues treatment. in addition, male gender, high body mass index and alcohol abuse are also the risk factors of resistance mutations in antiviral therapy. however, a growing number of studies suggest that early virological response is an important indicator to predict drug resistance [78, 79] . select nucleoside analogues treatment indications reasonably. nucleoside analogues treatment is not recommended for the hbv infected patients in immune tolerant phase or non-active phase, especially those who are younger, if they do not receive immunosuppressive therapy or chemotherapy. for the chb patients with first flare, especially those who are younger, nucleoside analogues should be given with caution after fully analysis of inductions. select nucleoside analogues treatment strategies reasonably. treatment should be consulted the guideline on prevention and treatment of chronic hepatitis b in china. for patients with antiviral therapy indications, drugs with strong antiviral activity and low resistance are recommended if nucleoside analogues are chosen. the previous antiretroviral therapy, including drugs, treatment response and resistance mutations, should be understood in order to avoid nucleoside analogues with cross-resistance. furthermore, sequential monotherapy treatment should be avoided for multi-drug resistance. improve patients' compliance. prescribed time and adequate medication should be repeatedly emphasized to the patients during nucleoside analogues treatment. according to the clinical trial, more than 30% of cases with virologic breakthrough are resulting from poor compliance. gradual dose reduction will significantly increase the risk of resistance and is forbidden. regularly detect hbv dna load and genotypic resistance and timely adjust treatment. hbv dna load is the most important indicators of drug resistance in nucleoside analogue antiviral therapy. hbv dna levels should be monitored regularly during treatment. numerous clinical trial data show that early virological response is an important predictor of drug-resistance. therefore, aasld and easl guidelines both recommend adjusting treatment plan based on evr to improve the efficacy and reduce the incidence of drug resistance [6, [78] [79] [80] [81] . patients with normal alt and mild inflammation or fibrosis (<g1s1) before treatment can stop the anti-viral treatment, and need to be monitored closely for antiretroviral therapy again promptly if aura symptoms happened. most patients with nucleoside analogues resistant, especially the decompensated cirrhotic patients, need rescue therapy timely. virologic breakthrough usually precede biochemical breakthrough, and rescue therapy before biochemical breakthrough can avoid sudden hepatitis flare and aggravation. management of lamivudine and adefovir resistance refers to guide recommendations above. add adefovir or tenofovir (the latter has not been approved by sfda, and the safety of combination of entecavir and tenofovir needs further study), or switch to ifn-α or pegylated ifn-α for patients with entecavir resistance. add adefovir or tenofovir for patients with telbivudine resistance. tenofovir resistance has not detected, if happened, entecavir, telbivudine, lamivudine or emtricitabine can be added theoretically. for lamivudine and adefovir multidrug resistance, switch to emtricitabine or combination of entecavir and tenofovir (but not been approved by sfda), or switch to ifn-α or pegylated ifn-α treatment [6, 80, 81] . definition of refractory chb: chb patients with treatment failure or poor efficacy and chb patients have been confirmed poor efficacy by evidence-based medicine, using anti-hbv drugs, including nucleoside analogues and (or) interferon, under the existing guidelines or recommendations for various reasons and (or) factors. refractory chb patients include: (1) patients with primary non-response, partial virological response and (or) virologic breakthrough, drug-resistant hbv mutations and clinical drug-resistant. (2) patients with serological (hbeag) no response or partial response, namely hbeag-positive patients without hbeag loss or hbeag seroconversion after initial treatment of more than 1 year. (3) high baseline hbv viral load: hbeag-positive patients with hbv dna >10 9 copies/ml, hbeagnegative patients with hbv dna >10 7 copies/ml. (4) cirrhotic (compensated and decompensated) patients and aechb patients. (5) other patients with hbv reactivation after transplantation, immunosuppressive therapy, combination of other viral infections such as hcv and hiv, combination of metabolic/autoimmune diseases such as insulin resistance, hyperlipidemia and (or) non-alcoholic steatohepatitis and fiber cholestatic hepatitis. the present guidelines for the treatment of refractory chb involve virus strains resistant mutations and virologic breakthrough, partial virological response, cirrhosis, virus reactivation after liver transplantation, receiving immunosuppressive therapy, and combination of other viral infections (hcv and hiv). majority of the treatment recommendations are based on expert consensus, clinical experience, or clinical studies of small sample. however, for patients with serological no-response or partial response, high baseline hbv viral load, aechb, insulin resistance and non-alcoholic steatohepatitis, and fiber cholestatic hepatitis, there is no clear guidance. currently ongoing hepatitis b related clinical researches more concentrated in drug selection and optimization of treatment-naïve patients, and only a small portion and small-scale studies involve refractory chb. anti-hbv treatment was not taken into account in aecdhb in the past. it is thought that immune pathological damage is the key in the development of aechb, and hbv is just a promoter. the role of hepatitis virus in development of aechb has not been paid enough attention. with the study of aechb mechanism deep going, more and more scholars have realized that constant hbv replication induced hyperactive immune response is a major factor in exacerbation. when hbv induces hypersensitivity, a large amount of immune complexes generate and activate the immune network, leading to serious hepatocyte damage through the following mechanisms: (1) th1 cells activate, release interleukin-2 (il-2), and mediate cytotoxicity of cytotoxic t cell (ctl), macrophages and natural killer (nk) cells; (2) macrophages activated by hbv and endotoxin release cytokines (including tumor necrosis factor tnf-α and fgl2), inducing direct hepatocyte damage or secondary damage by microcirculation disturbance; (3) fas ligands (fas-l) express increasingly on the surface of infected hepatocytes, conjunct to fas expressed by ctl, and induced apoptosis. antiviral therapy can quickly suppress hbv replication, reduce intercellular viral spread, and inhibit the membrane target antigen expression, so as to inhibit ctl attack and relieve hepatocyte injury and necrosis. antiviral treatment at early stage in disease is the pivot to terminate intense cellular and humoral immunity. therefore, it is advocated to start antiviral treatment for severe hepatitis patients with hbv replication. some scholars believe that viral load is an important indicator for aechb, although it does not directly related to liver damage. hbeag and hbv dna seroclearance is important for remission and positive for improving cure rate of severe hepatitis. therefore, early effective anti-viral therapy can reduce the viral load, suppress virus generation by infected hepatocytes, decrease infection of newborn hepatocytes, alleviate liver inflammation and is beneficial to liver recovery. antiviral therapy has become an effective treatment for aechb [82] . whether antiviral therapy increase or alleviate immune response in the advanced stage of severe hepatitis had been controversial. however, recent studies reach a consensus that antiviral treatment can slow disease progression and improve recent survival rate. in most early studies, lamivudine failed to improve liver function and survival in aechb compared to placebo [83] [84] [85] [86] [87] . however, a study from taiwan showed that lamivudine could improve survival in patients with baseline total bilirubin less than 342 mmol/l (20 mg/dl) [88] . wong and chan reported that antiviral therapy in aechb cannot improve resent survival, but could prevent further deterioration [89] . in early stage, there is intense immune response, high viral load, severe inflammation, and ongoing immune liver damage. viral load can affect the progression and prognosis. patients with hbv dna positive have a relative poor prognosis because of more virus antigen on hepatocyte surface activating immune injury. in middle-advanced stage, the impact of hbv dna on progression and prognosis would weaken because immune response has alleviated after self-regulation. therefore, hbv dna level in early severe chb is a significant indicator for prognosis, and antiviral therapy is essential. in middle-advanced stage, hbv dna level has little influence on the prognosis, and antiviral therapy is meaningful to prevent recurrence. in addition, lamivudine can obtain sustained virologic response, but long-term treatment may induce viral resistance and virologic breakthrough, which reduce the efficacy of antiviral therapy [90] . most recent studies suggest that antiviral therapy for hbv dna positive patients in early severe hepatitis can postpone disease progression and improve recent survival rate [91] . ma et al. analyzed 248 cases of hbv-aclf retrospectively. 124 patients added entecavir on the basis of standard medical treatment, another 124 patients only received standard medical treatment without nucleoside analogues. the 1-month and 3-month survival rate of entecavir-treated patients were 72.58% (90/124) and 61.59% (76/124) respectively, and significantly higher than those of control with 53.23% (66/124) and 61.29% (57/124). entecavir-treated patients get a significant improved meld scores compared to control post treatment, suggesting that entecavir can postpone progression of hbv-aclf and improve recent survival [92] . lin et al. investigated 120 hbv-aclf cases with entecavir treatment, and concluded that entecavir can significantly increase the survival rate [93] . hu et al. investigated the efficacy of lamivudine and entecavir on hbv-aclf. after 1-month treatment, survival rates are similar, but clinical improvement rate in lamivudine and entecavir group were significantly higher than basic treatment group. after 6-month treatment, the cumulative survival rates of lamivudine and entecavir group were 65.8%, 60.1% respectively and significantly higher than the basic treatment group (42%). in patients with baseline hbv dna >10 7 iu/ml, cumulative survival rate in antiviral treatment group were higher than basic treatment group. patients with pretreatment meld scores >30 had a lower cumulative survival rate than patients with meld scores ≤2, but obtained better response to antiviral therapy. it also showed that there was no significant difference in efficacy using entecavir and lamivudine treatment for hbv-aclf [94] . tillmann et al. summarized 14 cases of aechb with lamivudine treatment. it suggested that patients with lamivudine treatment had an overall survival rate without transplant of 78.2%, but patients without lamivudine only 45.7% [95] . garg et al. found that tenofovir can significantly reduce hbv dna levels in hbv-aclf, improve ctp and meld score, and decrease mortality. hbv dna decrease of more than 2lg copies/ml after 2-week treatment is a predictor of good prognosis. liver failure is a common syndrome, and its incidence is increasing with the use of alcohol and growing epidemic of obesity and diabetes. liver failure is defined as inability of the liver to perform its normal, metabolic, excretory and biotransformation functions by chinese medical association [96] . its manifestation includes coagulopathy, jaundice, hepatic encephalopathy (he) and ascites. liver failure can be divided into four classes: acute liver failure (alf), sub-acute liver failure (salf), acute on chronic liver failure (aclf), and chronic liver failure (clf) according to histopathological characteristics and the progression of disease. alf is a syndrome with liver function deterioration rapidly accompanied with a grade ii or higher he within 2 weeks illness duration. salf onset is slowly than alf that symptoms occur within 2-26 weeks. liver failure occurred with known or unknown chronic liver disease refer to aclf. clf is defined as progressive deterioration and decompensation of liver function in patients with liver cirrhosis, mainly manifested with complications of portal hypertension. based on the severity of clinical manifestations, sub-acute and acute-on-chronic liver failure can be divided into early, middle, and end stages. early stage has severe fatigue and gastrointestinal symptoms, total bilirubin level is more than 171 μmol/l or daily increase of total bilirubin is more than 17 μmol/l and prothrombin activity (pta) is less than 40%. middle stage has stage ii he and/or ascites and pta ≤30%. end stage has refractory complications such as stage iii or higher he, hepatorenal syndrome, massive hemorrhage of the upper alimentary tract, severe infection and refractory fluid and electrolyte imbalance, pta ≤20%. otherwise, asian pacific association for the study of the liver (apasl) define aclf as a severe liver injury, leading to coagulation abnormality usually with an inr ≥1.5, and any degree of mental alteration (he) in a patient with pre-existing liver disease and with an illness duration less than 4 weeks [97] . etiology, epidemic and precipitating factors of liver failure are different between western countries and china. non-infection factors such as alcohol and drug induced liver failure are predominant in western countries [98] . however, hepatitis b virus (hbv) infection is the main reason to induce liver failure [99] . we retrospective analysis the etiology of 1977 cases of liver failure came from 13 provinces in china northern area from 2002 to 2006 and found hbv infection induced liver failure was about 82.8% [99] . so far, liver transplantation is the most effective way to treat hbv induced liver failure. but due to the cost and shortage of organ donor, liver transplantation can't be used widely. comprehensive treatment including supportive therapy, antiviral therapy and immunoregulation therapy is the main way to treat hbv related liver failure in china. but mortality of liver failure is high and total curative rate is only about 35.56% because of the complicate pathophysiology in liver failure [100] . the precise mechanism underlying the liver injury caused by hbv-aclf and the factors contributing to the progression of liver failure remain unknown. generally, virus factors, host factors, and their interaction determine the outcome of aclf. hbv dna replication is one of the key factors causing the progression from liver damage to liver failure. the hbv dna level is closely associated with the risk of hepatocellular carcinoma development, and hbv dna suppression significantly improves the prognosis of cirrhosis. current clinical guidelines advocate oral antiviral treatment in decompensated cirrhosis and sustained hbv dna suppression to reduce sequelae [4, 96, 97, 101] . (liver failure) eight different hbv genotypes (a-h) have been described based on their genomic heterogeneity. many studies showed that the severity of hbv infection correlated with hbv genetype. in the asia-pacific countries, genotype b and c hbv are predominant with genotype c hbv associated with delayed hbeag seroconversion and more aggressive disease activity as compared to other hbv genotypes [102] . patients infected with hbv genetype c have high hbv dna level and high hbeag positive rate than people infected with other hbv genetypes. these patients have low response to anti-viral therapy and progress to liver failure, particular in patients infected with genetype c2. zhang et al. analyses 2922 hepatitis b patients and found that the most common hbv genetype was b and c in chronic hepatitis b patients [103] . patients infected with genetype c was more predisposed to chronic and cirrhosis and hepatic carcinoma. genetype b and c had no influence on illness progression in acute and mild patients, but hbeag positive rate and hbv dna level were high in patients infected with genetype c. further studies showed that hbv bcp/pre c mutation was associated with hbv genetype. a1846t and c1913 mutation probably associated with aclf. c1913 was an independent prognostic factor in aclf patients [104] . hbv dna polymerase lack proof function which can lead to viral mutation during replication. in addition, hbv exist in many quasispecies. under select press, quasispecies variance can cause the change of hbv replication, pathogenicity, antigen epitope which lead to influence immune response and viral resistance. over immune response can result in severe liver injures. hbv pro core/core, pre s, p gene and multi gene mutation were found to be associated with liver failure [105] . one of the functions of hbeag is to induce immune tolerance. in the absence of hbeag, patients harboring pre-core mutant hbv may have a more rigorous immunological response. chronic infection with pre-core mutants has been often associated with multiple flares with interspersed asymptomatic periods. mutations at the basal core promoter (bcp) regions lead to decreased hbeag synthesis, active liver histology, and increased viral replication. these exacerbations are seen to lead to fulminant hepatic failure [89, 106, 107] . we investigated hbv bcp a1762t/g1764a double mutation in liver failure patients [104] . longitudinal study showed that nucleotide mutation sites were occurred more in hbv-aclf than cirrhosis patients among which nt53, nt1846, nt1896 and nt1913 were associated with hbv-aclf. t1846 mutation was found exist more in genetype b than genetype c (57.1% vs 30.4%), a/g1913 mutation is found frequently in hbeag negative patient than positive patients (28% vs 13.2%). these indicated that pre core/bcp mutation associated with the occurrence of liver failure and influence patients outcome. hbx is a multifunction regulatory protein which can influence gene transcription, activate signal transduction, enhance viral replication, accelerate protein degradation and regulate cell apoptosis. hbx can participate in the process of hbv pre s and bcp/pre core mutation liver failure caused by chronic hbv infection (i.e., chronic severe hepatitis b) is a common life-threatening disease in china. the pathogenesis of chronic severe hepatitis b is complex and is currently not completely understood. however, one widely accepted mechanism is the induction of cellular immune responses mediated primarily by cytotoxic t lymphocytes (ctls) and delayed-type hypersensitivity t cells. these immune responses are induced by viral protein antigens expressed in the target cell surface due to the active replication of hbv and eventually result in large areas of liver cell necrosis [108] . the specific mechanisms may involve several factors [109] . using a hybridization assay for hbv dna and a conventional enzyme immunoassay to measure the hbeag level, an earlier study showed significant parallel increases in serum hbeag and hbv dna levels and accumulation of intracellular viral proteins several weeks before the hepatitis flare. in addition, there was a subsequent increase in anti-hbe production and hbeag/anti-hbe immune complex formation, implicating the important role of the immune response to hbv in initiating the hepatitis flare [110] . immunohistologic studies during the hepatitis flares have shown cd8+ t cells in the mononuclear cell infiltrates, strong membranous expression of human leukocyte antigen class i (hla-i), and cytoplasmic or membranous/submembranous hepatitis b core antigen (hbcag) expression [111, 112] . some findings suggest that hepatitis b flares are the results of dynamic changes of the innate and adaptive immune responses with hla-i restricted, ctl mediated immune cytolysis of hbv antigen(s) expressing hepatocytes and its downstream apoptotic mechanisms [113, 114] . the activated th1 cells release interleukin-2 and excite cytotoxic effects of ctls, macrophages, natural killer cells, and lymphokines [102] . macrophages, activated by hbv and endotoxins, release various cytokines mainly with tumor necrosis factor (tnf)-a, which directly damage liver cells and also result in secondary injury of liver cells through disturbances in microcirculation. fas ligands (fas l) are highly expressed in the surface of hbv-infected liver cells and combine with fas expressed by ctls, together inducing hepatocellular apoptosis. therefore, antiviral therapy early in chronic severe hepatitis b is beneficial for suppressing intense cellular immune responses induced by hbv replication. if hbv replication is suppressed rapidly, the immune pathological responses of chronic severe hepatitis b may be reduced, thus effectively blocking the disease progression. the administration of anti-hbv therapy in chronic severe hepatitis b (acute-or subacute-on-chronic liver failure) is still undergoing research, and limited data are presently available. so far, anti hbv treatment drugs include interferon and oral antiviral drugs. interferon application is contraindicated in the treatment of liver failure due to its limited anti-viral efficacy, significant adverse drug effects, and induction of immune enhancement, which can further result in aggravation of liver damage. oral anti hbv drugs including adefovir dipivoxil (adv), lamivudine (lam), telbivudine (tbv), entecavir (etv) and tenofovir (tdf) have few adverse effects. antiviral therapy may have the advantage of shortening the replication and thereby reduce disease duration without the side effects of interferon. therefore, many studies have been carried out to find the efficacy of oral antiviral drugs on patients with liver failure. the effect of antiviral therapy in hbv related acute liver failure is controversial. some studies reported that antiviral therapy can't improve outcome because hbvdna can be eradicated spontaneously due to enhanced immune response in liver failure patients. but hbv infection is the initial factor to induce over immune response that can lead to liver damage. early treatment with antiviral therapy can inhibit hbv dna replication and attenuate immune reaction which can reduce liver damage, hepatocyte apoptosis and necrosis. tillmann et al. [115] reported that 17 acute hbv related liver failure patients were treated with lam and 20 patients were treated with placebo. encephalopathy occurred in 3 (17.6%) and 11 (68.6%) patients, respectively (p = 0.005). it demonstrated that early use of antiviral drugs can reduce the rate of encephalopathy and mortality. in addition, a prospective study about etv on 6 acute liver failure patients demonstrated that etv can reduce hbv dna load significantly and 5 patients achieved anti hbsag conversion [116] . therefore, monitor closely and early use antiviral drugs to reduce hepatocyte apoptosis and necrosis on patients with hbv related acute liver failure are very important. the objective of antiviral treatment for hbv-aclf is to reduce viral load at an appreciably high rate, thereby promoting reduction in hepatocyte cell death and improved survival outcomes by prevention of decompensation related multiorgan complications in this group of severely ill patients. several studies have delineated the fact that low pretreatment hbv dna load and a rapid decrement in viral load improves outcomes in hbv-aclf [117] , whereas a study from india reported that a 2 log decrease in hbv dna at week 2 improved survival benefit in patients with hbv-aclf [118] . antiviral therapy also promotes chances of stabilization to liver transplant time and improves transplant outcomes. studies have debated on the issue of antiviral therapy related improvement in the long term [119] . lam decreased viral load significantly, but did not result in significant biochemical or clinical improvement compared with those patients given placebo. mortality of patients receiving nucleoside analog therapy was significantly lower than the placebo group, which indicated that antiviral therapy improved prognosis of patients with hbv-aclf if implemented as soon as possible [117] . even in the age of effective antiviral therapy, early transfer to a transplantation facility should be considered before managing conservatively by medical means. the apasl consensus guidelines on aclf describe the value of early and prompt institution of antiviral therapy in hbv-aclf. hbv dna levels are now not an indication for commencement of antivirals in hbv-aclf reactivation, as earlier starting of such therapy, even prophylactic, has been found to have great survival benefit in the long run. from 2006 to 2009, early and middle stage hbv-aclf patients in our hospital were recruited in a prospective study to evaluate the efficacy and safety of lam and etv [120] . no antiviral therapy was used in control group. this study showed that lam or etv can reduce 3 and 6 months mortality, improve survival rate on patients with aclf-hbv. three and 6 months cumulative survival rate of lam and etv therapy were 69.2% and 65.8%, 67% and 60.1%, respectively, which was higher than control group (42% and 42%, p = 0.045 and 0.04). no significant different on survival rate between lam and etv (p = 0.723). this study also showed that meld score was an effective prognostic predict factor on patients with aclf-hbv. patients with meld score less than 30 had a good outcome. another study also demonstrated that for hbeag-negative patients with hbv-aclf, when entecavir was added to comprehensive therapy, a meld score ≥30 predicted very poor prognosis due to fatal liver failure [121] . hu et al. made a survival analysis on 190 hbv-aclf patients and results indicated that nucleoside analog application in early and middle stage hbv-aclf can improve survival rate and prolong patients' life. median survival time was 5.7 and 1.79 months in patients treated with and without antiviral therapy. another study indicated that patients with meld score 30-40 and hbv dna load decrease 2 log had a better outcome than patients with hbv dna load decrease less than 2 log within 4 weeks treatment [122] . in addition, mortality had no relationship with hbv dna load if meld score >40. xiao et al. also demonstrated that nucleoside analog treatment is an independent prognosis predict factor in 219 hbv induced liver failure. lam and etv had no difference. although antiviral therapy can improve patients survival rate, treatment with and without nucleoside analog had no difference in late stage liver failure patients. currently, liver transplantation is the ultimate therapeutic option for decompensated cirrhosis patients. however, liver transplantation can't be used in all patients because of the shortage of donor organs. therefore, the aim to treat decompensated cirrhosis is to decrease the occurrence of disease associated complications and the liver associated mortality rate [123] . the natural history of decompensated hbv-related cirrhosis is affected by high hbv replication which may exist in some decompensated hbv-related cirrhosis patients [123] . hepatic necroinflammation and fibrosis progression are improved after sustained viral suppression is achieved which can prevent decompensation in cirrhosis [124] . oral nas treatment are strongly recommended in most clinical guidelines for decompensated hbv-related cirrhosis patients no matter what hbv dna replication level is [109, 110] . yao et al. [125] found that ctp scores was reduced more than three points in 69% lam treated patients with severely decompensated cirrhosis. furthermore, ctp scores was decreased to less than seven point in 38% of these patients, and their statuses on the united network of organ sharing waiting list changed to inactive. a randomized controlled trial in asia demonstrated less liver-related morbidity in the lam-treated patients with hbv associated advanced compensated cirrhosis compared to the untreated controls because of the reduced incidence of hepatic decompensation and lower risk of hcc. increased ctp scores were noted in 3.4% of the patients in the lam group compared to 8.8% of the patients in the placebo group (p = 0.02) [126] . fontana et al. showed most deaths caused by liver related complications occurred within the first 6 months in patients with decompensated hbv-related cirrhosis treated with lam. pretreatment high hbv dna replication level, serum bilirubin and creatinine were associated with 6-months mortality rates significantly [127] . this finding indicates early antiviral therapy might be important. lamivudine lam is a nucleoside analogue that inhibits hbv dna synthesis which was the first oral drug to treat chronic hbv infection in 1998. its mechanism is to compete with nature cytidine to inhibit hbv polymerase, then terminate hbv replication. chan et al. [128] studied the effect of lamivudine in treatment of severe hepatitis-b-related acute exacerbations leading to aclf in 28 patients as against 18 controls. it was concluded that lamivudine had no survival benefit compared with conventional treatment in severe aggravations of chronic hepatitis b and that liver transplantation should be considered in these patients with thrombocytopenia and high bilirubin. however, another meta study [129] analysis 242 studies to evaluate the short-term effect of lamivudine (lmv) treatment for severe chronic hepatitis b. they found that the survival rates and pta of the test group were distinctively higher than those of the control group at weeks 4, 8, and 12 of the treatment course. the hbv-dna negative change rate was distinctively higher throughout the 12 weeks of lmv treatment. for patients who started lmv treatment in the middle stage, the mortality rate of the test group was lower. they concluded that lmv decreased hbv-dna levels in the serum, improved liver function in patients, and enhanced survival rate during the early and medium stages of severe chronic hepatitis b. tsubota et al. [130] studied 25 patients with spontaneous severe acute exacerbation treated with lamivudine, and found that lamivudine monotherapy did not prevent hepatic failure deterioration significantly, but it resulted in long-term benefits. baseline serum bilirubin, pre-existing cirrhosis and baseline pt were independent determinants of prognosis. adv is an acyclic nucleotide analogue of adenosine monophosphate. use of adefovir for hbv-aclf has been rare. in two case reports, adefovir dipivoxil failed to salvage cases of lamivudine resistance after jaundice and liver failure developed. a lower antiviral potency and the potential risk of nephrotoxicity of adv remain a problem for routine use as a first-line treatment. it is hence not advisable to use adefovir as a first-line drug in the treatment of acute severe exacerbation. but considered to the high viral resistant to lam, adv plus lam can reduce the incidence of lam resistance. the combination of adv and lam can be used in liver failure patients. 128 patients with decompensated cirrhosis caused by lam-resistant hbv were treated with adv and hbv dna level become undetectable occurred in 81% of patients and ctp scores were improved [131] . but another study focused on long time outcome found that resistance to adv was 20% and renal toxicity was confirmed in 3% of patients [132] . etv is a cyclopentyl guanosine analogue that can inhibit hbv polymerase's function. compared with lam and adv, etv has a more potent activity against wild type hbv [133, 134] . etv has been studied in in cirrhotic patients. one korea study showed ctp and meld scores were improved in 55 patients treated with etv for 12 month. the 2-year cumulative incidence of hcc was 6.9%, and the cumulative death rate was 17% [90] . many studies have been carried out to compared the efficacy of etv and lam to treat hbv related aclf. the short-term efficacy of entecavir vs lamivudine was similar and the degree of pretreatment liver failure significantly affected the outcome of treatment [135, 136] . in summary, the pros and cons of lam vs etv in decompensated or severe acute exacerbation of chronic hepatitis b were etv being more effective in promoting faster viral load decrement. also the available clinical evidence suggests that clinicians treating chronic hepatitis b patients with acute hbv exacerbation or decompensated liver disease should use the most potent nucleoside analogs available [137] . tbv is a synthetic thymidine nucleoside analogue that has potent antiviral activity against hbv. one study investigated the short-term efficacy and safety of tbv therapy in liver failure patients caused by chronic hepatitis b virus (hbv) infection [138] . in this study, 20 patients were treated with tbv, and the other 18 patients were treated with lam. hbv dna levels in the tbv group fell to the lower limit of detection after the fifth week, which was more rapid than in the lam group. in addition, the total bilirubin and prothrombin time activity of the patients with tbv treatment showed a more significant improvement as compared to the patients treated with lam from the start of the fifth week. they concluded that tbv treatment is superior to lam treatment in improving the condition of patients with liver failure as a result of chronic hbv infection in the short term. but viral resistance is also a major concern. a study in decompensated cirrhosis patients with hbv infection showed genotypic resistance was developed in 27% of the tbv patients during the 2-year period [139] . therefore, tbv used as a first-line treatment has limitations in patients with hbv-related decompensated cirrhosis. tdf is an acyclic nucleotide analogue with potent inhibition of hbv polymerase/ reverse transcriptase. in a seminal study by garg et al. [90] , consecutive patients with aclf due to spontaneous reactivation of chronic hepatitis b were randomized to receive either tdf or placebo. the primary endpoint was survival at 3 month. more than 2 log reduction in hbv dna levels at 2 weeks was associated with survival rate. the authors concluded that tdf had potent activity to reduce hbv-dna levels, improve ctp and meld scores, and reduce mortality in patients with severe spontaneous reactivation of chronic hepatitis b presenting as aclf, and that reduction in hbv-dna levels at 2 week should be considered a desirable goal and a good predictor of survival. until now, no studies have reported viral resistance to tdf. therefore, tdf and etv can be considered to be the first-line therapy because their potent activity against hbv replication and high resistance barrier. in addition, the data about tdf to treat hbv-aclf is limited. thus, larger prospective and multicenter studies are encouraged to evaluate further the effect of tdf on shortterm mortality of patients with hbv associated aclf. there still lack multicenter, larger samples, prospective and randomized clinical trial to test the efficacy of antiviral therapy. but it is likely that antiviral therapy with nucleos(t)ide can improve patients survival rate in patients with hbv-related liver failure [140] . therefore, antiviral therapy is reasonable to try in patients with high hbv dna replication. in recent year, lam and other nucleoside analogue have been used in severe hepatitis b wildly. the efficacy of antiviral therapy is correlated with the time to start. many clinical trials demonstrated that early antiviral treatment is important for patients with liver failure [141] . antiviral therapy used in early and middle stage liver failure can improve patients survival rate. in patients with late stage, such as serum total bilirubin is more than 342 μmol/l, it's rare that patients can get benefit from antiviral therapy. therefore, early use antiviral therapy can reduce viral load, inhibit viral replication, reduce new hepatocyte be infected by hbv again and alleviate liver inflammation and all of that are benefit to hepatocyte regeneration. the criteria for antiviral therapy dependent on hbv dna level in serum. some people suggested antiviral therapy should be used in patients with hbeag positive and hbv dna >10 4 copies/ml or hbeag negative and hbv dna >10 3 copies/ ml. however, take into account over immune response in liver failure patients that associated with viral eradication, even patients with hbsag positive and hbv dna undetectable should be considered for antiviral therapy. in our opinion, antiviral therapy should be used in all liver failure patients with hbv replication immediately. the feature of oral antiviral drugs should be considered when used in liver failure patients. side effects of nucleos(t)ide, including elevate ck, myopathies and lactate acidosis, can occurred during treatment. we observed the change and effect of elevated ck during the treatment of etv and lam in liver failure patients. we found that no different of ck elevation in both drugs. ck elevation was consistence with infection and hepatic renal syndrome. but long term safety of nas has not been confirmed in liver failure patients. antiviral therapy should be used for life because the nas treatment can't eradicate cccdna in the hepatocyte. some cirrhotic patients developed viral resistance to lam during long-term lam therapy which cause virologic response loss [142, 143] . in antiviral treatment naïve patient, the lam resistance rate is up to 70% after 5 years of continuous therapy and the annual resistance rate is up to 20% [6] , compared with that etv resistance rate is less than 0.5% in patients treated with etv at 4 years [144] . therefore, lam should be used with careful monitoring for the development of resistance. and etv or tdf rather than lam is recommended to be used as the first-line therapy in patients with hbv infection because of its high genetic barrier and potent activity against hbv replication [145] . multiorgan failure occurred rapidly frequently in liver failure patients. although some patients can recover treated by comprehensive and antiviral therapy, all patients should be evaluated for liver transplantation. it's challenge to study the effect of na, how and when to use na and how to treat viral resistance to na in liver failure patients. further studies are needed to evaluate patient outcomes after antiviral therapy with na in liver failure patients. for patients with chronic hepatitis b (chb), hbv continue replication caused progression of liver disease, eventually lead to cirrhosis or hepatocellular carcinoma. the principle of treatment for hbv-related cirrhosis is a comprehensive treatment, including antiviral, anti-inflammatory, hepatoprotective treatment and anti-fibrosis, which antiviral treatment is the key point. since 1999, listed lamivudine, there were many new ideas and concepts on treatment for hbv-related cirrhosis, liver failure. a large number of evidence-based medicine evidence suggests that sustained suppression of hbv by anti-viral treatment can reduce liver inflammation and fibrosis, reduce or delay disease progression, and ultimately improve survival rates and quality of life. internationally there are many chb antiviral therapy management guidelines, but antiviral therapy for hbv-related cirrhosis was still a hot and difficult issue in clinic. this article reviews some of the new progress and new perspectives of antiviral therapy liver cirrhosis based on the recent research. management is guided by recommendations from the american association for the society of liver disease (aasld) [6] , european association for the study of liver (easl) [4] , asian pacific association for the study of liver (apasl) [146] , and society of hepatology and infectious diseases of chinese medical association (cma) have formed a consensus [3] . guideline 2015 edition issued by the chinese medical association clearly stated that the overall goal of treatment is to chb was to suppress hbv as long as possible, relieve inflammation or necrosis of liver cells and liver fibrosis, prevent the progression of cirrhosis, reduce and prevent the occurrence of hepatic decompensation, hcc and its complications, thereby improving the quality of life and survival rate. for patients with cirrhosis, whether compensated or non-compensated, antiviral treatment can delay or reduce hepatic decompensation and hcc occurs, does not change the final outcome of end-stage liver cirrhosis. in the guideline 2010 edition issued by the chinese medical association, the goal of antiviral therapy in hbv compensated cirrhotic patients is to prevent progression of the disease to decompensated cirrhosis, end-stage liver disease, hepatocellular carcinoma. the goal of antiviral therapy in hbv-related decompensated cirrhosis is to improve the hepatic disease severity, improve the clinical symptoms and quality of life, and prolong patient's survival. for the endpoint of the antiviral treatment, the definition of each guide is slightly different. guideline 2018 edition issued by aasld explicitly mentioned these patients with compensated cirrhosis should receive long-term treatment. however, treatment maybe stopped in hbeag positive patients if they have confirmed hbeag seroconversion and have completed at least 6 months of consolidation therapy and in hbeag negative patients if they have confirmed hbsag clearance. for these patients with decompensated cirrhosis life-long treatment is recommended. guidelines 2008 issued by american society of digestive disease [147] recommend long-term treatment until negative hbv dna and hbsag disappears. in the guideline 2017 edition issued by easl, endpoint of antiviral treatment has even been further subdivided into "ideal endpoint", "satisfactory endpoint" and "basic endpoint", where "ideal endpoint" means to achieve hbsag clearance with/without hbsag seroconversion. patients with liver cirrhosis generally have characteristics of longer course of disease, most of mother to child transmission, the large proportion of treated patients, high complexity of quasispecies and higher risk of resistance. the clinical treatment decisions in these patients need to consider a variety of factors, including long-term treatment, delay disease progression, improve histology, low resistance rates, good safety and patient tolerance, etc. there are differences in current guidelines of countries in antiviral therapy indications for liver cirrhosis, mainly cutoff viral load. for compensated cirrhosis, 2012 apasl guideline pointed hbv dna >2000 iu/ml, 2009 aasld guideline that the hbv dna >2000 iu/ml or hbv dna <2000 iu/ml but elevated alt should be treated with antiviral treatment, and 2012 easl guidelines as long as detectable hbv dna should be treated. 2010 chinese guideline point that whether normal or elevated alt, hbeag(+) and hbv dna ≥10 4 copies/ml, hbeag negative and hbv dna ≥10 3 copies/ml should be antiviral treated. for decompensated cirrhosis, the recommendations of the guidelines is basically the same: as long as hbv dna can be detected, even if the viral load is low, it should be treated. currently there are two types of anti-hbv drugs, including interferons and nucleos(t)ide analogues. among them, decompensated cirrhosis is a contraindication to interferon. even compensated cirrhosis, due to the risk of hepatic decompensation, the use of interferon should be very careful. patients need to be closely monitored in the clinical application process, dose adjustments, the injection interval prolonged, and assistant drug to reduce adverse reactions and other measures so that the patient can safely complete drug treatment. on the contrary, the nucleos(t) ide analogues with strong antiviral effect, ease of use, safety, and well tolerance were recommended as the preferred treatment of liver cirrhosis by the major guidelines. currently, lamivudine (lam), adefovir dipivoxil (adv), telbivudine (ldt), entecavir (etv), and tenofovir disoproxil fumarate (tdf) have been approved for chb therapy. different nucleoside drug efficacy and safety varies. cirrhotic patients should be treated with potent, fast, direct inhibition of viral replication drugs. in 2018 aasld 2017 easl and 2015 apasl guidelines, for decompensated cirrhosis nucleoside (acid) analogues with potent effect and low resistance, such as entecavir or tenofovir should be selected. the reveal-hbv study of chen [148] has established an hbv viral load paradigm in the natural history of chb. serum hbv dna level has been shown to be significantly and independently associated with incidence of hepatocellular carcinoma (hcc) and cirrhosis. liaw et al. [126] evaluated the effectiveness of antiviral therapy in preventing disease progression in patients with chb and advanced fibrosis or cirrhosis. this is a large-scale, multi-center, randomized, double-blind, placebo-controlled prospective study. the study has changed the concept of the world, especially the chinese doctors to treat chb and liver cirrhosis, and promote the development of antiviral treatment for chb and liver cirrhosis. results showed that 3-year follow-up hepatocellular carcinoma occurred in 17 patients (3.9%) who received lam and 16 patients (7.4%) who received placebo (p = 0.047). this study also found that lam therapy reduced the risk of hcc by 51%, the risk of disease progression to fibrosis/cirrhosis by 55%. it first confirmed that antiviral therapy with lam could delay disease progression, reduce the incidence of hcc. in the study of patients with decompensated cirrhosis has also been confirmed lam was well tolerated, could effectively stabilize or improve liver function, and delay progression of liver disease, reduce liver transplantation. hann et al. [149] conducted a prospective, multicenter study evaluated lam in 75 decompensated cirrhosis patients, 93% of whom were not waiting for liver transplantation. all 75 patients tested hbsag(+) and 62% tested positive for hbeag(+) at baseline. in 64% of patients hbv dna levels were detectable by the branched chain dna assay. the virus in 69% of these patients after 6 months treatment and in 64% overall became undetectable by the bdna assay. alt, bilirubin and albumin level improved throughout treatment. from 10 at baseline to 8 at last visit the median ctp score also improved. after a median of 13.1 months of treatment, a virologic breakthrough occurred in only 18% patients. treatment of lam can improve liver function in nontransplantation candidates with decompensated cirrhosis. a meta-analysis from huang et al. [150] indicated that lam and ldt significantly decrease the mortality rate and disease severity in decompensated cirrhosis patients. eight studies (total 511 patients) were included. data showed that lam and ldt significantly decreased the mortality rate (rr 0.36, 95% ci 0.25-0.54), improved the ctp scores (mean difference −3.23, 95% ci −3.98 to −2.48). in the study of patients with liver cirrhosis showed clinical improvement after treatment with lam 3-6 months. and even in patients with clinical improvement, they may develop to hcc, therefore such patients still need early treatment, and close monitoring of hcc. it is showed good efficacy and safety in the lam treatment of hbv related compensated or decompensated liver cirrhosis. however, in the long course of lam treatment viral resistance could not be ignored. more importantly, if these patients with chronic liver disease for a long time, poor liver reserve function, not promptly be treated, condition will deteriorate or even lead to death due to viral resistance. in clinical use of lam, the proportion of viral resistance increased year by year, 14%, 38%, 49%, 66%, the first year, second year, third year, fourth year, respectively. after the occurrence of viral resistance, some patients will be worsening, and even hepatic decompensation. therefore it is emphasized that patients with compensated or decompensated liver cirrhosis by lam therapy need to be improved compliance, closely monitoring and follow-up, to be adjusted treatment based on serum hbv dna response situation. some patients with decompensated cirrhosis or high viral load should be considered an initial combined with adefovir dipivoxil. the rates of adv resistance in lam-resistant subjects with hbv chronic hepatitis and cirrhosis are reported to be 6.4% and 25.4% after 1 and 2 years, respectively [151] . nevertheless, adv use as a rescue therapy is affected by a primary nonresponse in 8-15% of patients. in a study of adv add-on lam rescue therapy in lamivudine-resistant patients, kim et al. [152] reviewed 167 patients with adv add-on rescue treatment for 5 years. after 5 years treatment 86.9% patients had complete virological response. alt in 92.5% patients normalized, hbeag seroconversion occurred in 16.7% patients. a study from woo et al. [153] was to determine the long-term clinical outcomes after adv rescue therapy in decompensated patients infected with lamivudine-resistant hbv. in total, 128 patients with a decompensated state and lamivudine-resistant hbv were treated with adv at a dosage of 10 mg/day for a median of 33 months in this multicenter cohort study. following adv treatment, 86 (72.3%) of 119 patients experienced a decrease in child-pugh score of at least 2 points, and the overall end-stage liver disease score decreased from 16 ± 5 to 14 ± 10 (mean ± sd, p < 0.001) during the follow-up period. with adv treatment, 67 patients (56.3%) had undetectable serum hbv dna (detection limit, 0.5 pg/ml). virologic breakthrough occurred in 38 patients (36.1%) and 9 patients had a suboptimal adv response. the overall survival rate was 89.9% (107/119), and a suboptimal response to adv treatment was associated with both no improvement in child-pugh score (≥2 points; p = 0.001) and high mortality following adv rescue therapy (p = 0.012). three years of adv treatment was effective and safe in decompensated patients with lamivudine-resistant hbv. the globe [154] trial was one of the largest international multi-center clinical trials of ldt treatment of chb. the safety and efficacy of ldt versus lam monotherapy has been compared for 2 years in chb patients. hbeag(+) and hbeag(−) patients were treated 104 weeks with ldt or lam once daily. the patients treated by ldt achieved superior therapeutic response versus these treated by lam in hbeag positive (63% vs 48%; p < 0.001) and hbeag negative (78% vs 66%; p = 0.007) patients. in both the hbeag positive and the hbeag negative groups, greater hbv dna suppression and less resistance was observed in patients treated by ldt than lam. after 52 weeks of therapy in the phase iii globe study, hbv resistance (breakthrough and resistance mutations) to ldt occurred in 3% of patients with hbeag(+) and 2% of patients with hbeag(−). after 104 weeks of therapy, hbv dna rebound in 17.8-21.6% of hbeag(+) and 7.3-8.6% of hbeag(−) ldt-treated patients associated with breakthrough and resistance mutations [155] . ldt is not active against lamivudine-resistant hbv. in the globe study, patients who failed lam therapy showed cross-resistance to ldt. ldt was relegated to second-line status in the management of chronic hbv infection due to increasing resistance over time. in aasld and easl guidelines ldt is not recommended as first-line drug for monotherapy. when necessary the combined adv or tdf is recommended. the "roadmap concept" was derived primarily from a phase iii global registration study of ldt. an optimized strategy based on the roadmap concept is supposed to improve the clinical outcomes of patients with suboptimal antiviral response. sun et al. [156] conducted the efficacy optimization of response to telbivudine (effort) study to investigate the efficacy and safety of the roadmap strategy by adding adv to ldt for suboptimal responders. in all, 606 hbeag(+), na naive patients with chb were randomized to the mono or optimize group. patients in the optimize group were treated with ldt for 24 weeks. subsequently, patients with hbv dna ≥300 copies/ml at 24 weeks were added adv to 104 weeks, while those with hbv dna <300 copies/ml continued monotherapy. mono group received ldt monotherapy and added adv if development of viral breakthrough. due to suboptimal response 68% patients in the optimize group had been added adv. in the optimize group, more patients at 104 weeks achieved hbv dna <300 copies/ml versus the mono group (76.7% vs 61.2%, p < 0.001), and with less genotypic resistance (2.7% vs 25.8%, p < 0.001). combination therapy showed an additive antiviral and low resistance potency. in two groups all patients were well tolerated. patients with ldt which are suboptimal virological responders at 24 weeks are recommended to add adv. these patients with ldt monotherapy can be benefited from combination therapy without increased side effects. chan et al. [139] studied the safety and efficacy of ldt and lam in hbv-related decompensated cirrhosis patients. in this double-blind trial, 232 treatment-naive patients with decompensated cirrhosis in 80 academic hospitals were randomized (1:1) to receive ldt or lam for 104 weeks. a modified endpoint was used hbv dna <300 copies/ml and alt normalization. in intent-to-treat analysis (missing = failure) response rates were 56.3% vs 38.0% after 76 weeks (p = 0.018) and 45.6% vs 32.9% after 104 weeks (p = 0.093) for ldt vs lam. cumulative death and hcc rates were 16% and 15% in patients with ldt, and 22% and 16% compared to lam, respectively. cumulative genotypic resistance rate were 27% in patients with ldt, and 36% compared to lam during a 2-year period. comparable to lam, ldt can effectively stabilize liver function and is well tolerated. however, these two drugs are not recommended as first-line drugs due to high virological breakthrough rates. international clinical trials [133, 157] and independent registrational studies in china have shown that etv achieved statistically superior virological and biochemical responses compared with lam. shim et al. [158] evaluated etv as first-line monotherapy in 70 patients with hbv-related decompensated cirrhosis primarily treated with 0.5 mg/day etv. there was no significant differences between groups in mean hbv dna changes, rates of hbeag seroconversion or hbeag loss, the proportion of patients with alt normalization after treatment. after 12 months of treatment, the meld score and the ctp score in decompensated patients improved significantly. in these patients, 66% achieved ctp class a status and 49% showing a decrease of ctp score p2 points. the cumulative death rates and hcc rates in 2 years were 17% and 6.9%, respectively. zoutendijk et al. [159] conducted a study to investigate the effect of etv on disease progression in patients treated with etv. all 372 patients were divided into three groups, chronic hepatitis b without cirrhosis group (n = 274), decompensated cirrhosis group (n = 9) and cirrhosis group (n = 89). the virological response (vr) was not influenced by the severity of liver disease (p = 0.62). in cirrhosis group, the probability of developing clinical events was higher (hr 15.41 (95% ci 3.42-69.54), p < 0.001) compared to two other groups during a median follow-up of 20 months. vr was associated with a lower probability of disease progression (hr 0.29 (95% ci 0.08-1.00), p = 0.05). patients with compensated and decompensated cirrhosis who have achieved vr can achieve significant clinical benefits (hr 0.22 (95% ci 0.05-0.99), p = 0.04). in patients with cirrhosis, virological response to etv treatment lead to a lower probability of disease progression. the association between disease progression and viral replication was reduced with a threshold of 2000 iu/ml. it suggested that complete viral suppression was essential for patients with na treatment, especially in cirrhosis patients. tenofovir disoproxil fumarate (tdf) is an acyclic adenine nucleotide analogue. tdf as a potent, oral antiviral with low resistance rates, has been recommend firstline drug for the treatment of chb in a number of international guidelines. a study [160] evaluated the effects on chb patients with fibrosis and cirrhosis treated with tdf of at least 5 years. 489 (76%) patients of 641 completed 240 weeks treatment. 54% patients (348/641) had biopsy results at both baseline and 240 weeks. 87% patients (304/348) had histological improvement, and 51% (176/348) had regression of fibrosis at 240 weeks (p < 0.0001). 28% (96/348) cirrhosis patients (ishak score 5 or 6) at baseline, 74% (71/96) no longer had cirrhosis (≥1 unit decrease in score). the histological response and regression of fibrosis seen in this study are probably due to the potent viral suppression achieved with long-term use of tdf. liaw et al. [161] conducted a clinical trial to observe 112 chb patients with decompensated liver disease received either etv (n = 22), emtricitabine (ftc)/ tdf (n = 45), or tdf (n = 45). after 48 weeks treatment, hbv dna was <69 iu/ ml (400 copies/ml) occurred in 72.7% (etv), 87.8% (ftc/tdf), and 70.5% (tdf) of patients. alt normalization occurred in 55% (etv), 76% (ftc/tdf), and 57% (tdf). hbeag loss/seroconversion rate were: 0/0% (etv), 27/13% (ftc/ tdf), and 21/21% (tdf). in three groups meld scores and ctp scores both improved. this study demonstrated that all nas were well tolerated in chb patients with decompensated liver disease and can effectively improve virologic and biochemical parameters. response-guided therapy 4006 study [126] suggested continuous treatment with lam (10 years) delayed clinical progression in patients with chronic hepatitis and advanced fibrosis by significantly reducing the incidence of the risk of hepatocellular carcinoma and hepatic decompensation. the biggest risk is the occurrence of viral resistance in the longterm antiviral therapy. although rescue therapy can save some patients regain virologic response, but improper treatment or less often sicker or even canceling the original clinical benefit. although treatment can save some patients regain virologic response, but improper or untimely treatment often leads to deterioration or even canceling the existing clinical benefit. therefore, in the long-term antiviral therapy in patients with cirrhosis, how to overcome and prevent resistance, maximize the clinical benefit of antiviral therapy (including histological improvement and prevent and delay disease progression) is required by clinicians to consider. response-guided therapy refers to select appropriate medication according to the baseline characteristics of patients, and based on the patient's response to treatment, especially for those who did not achieve an early virological response, timely to adjust treatment to achieve a better long-term results. response-guided therapy is a hot point in the current study of antiviral treatment for chb, it is also an important strategy and important measure for the prevention of viral resistance. some experts even believe that all of antiviral therapy will need to be optimized. response-guided therapy means optimization based on baseline characteristics and early virologic response. numerous studies have demonstrated that baseline parameters such as low viral load, high serum alt level, high inflammation activity score prompted by liver biopsy and early virological response predict better longterm effect. in 2007 keeffe's [80] "road map concept", response-guided therapy according to virologic response at 24 weeks has been recommended. combination therapy is an important part of response-guided therapy. combination therapy includes an initial stage combination, the combination in the course of treatment (poor response or resistance), the combination treatment for treated patients with relapse. in 2009 easl guideline it was referred that adv or tdf with lam combination treatment need to consider in patients with liver cirrhosis. in 2009 aasld guideline lam or adv was recommend initial treatment for patients with decompensated cirrhosis, but their combination is recommended to reduce the risk of resistance and rapid inhibition of virus. the combined treatment for lam+adv is the most clinically used and studied. in first-generation na lam, ymdd mutation (rtm204v/i) developed in up to 65% of patients in 5 years [162] . the combination of adv dipivoxil and lam was found to lower the rates of resistance to lam and serum hbv dna levels, and fasten the rates of alt normalization in hbeag(+) patients, with similar rates of hbeag seroconversion [163] . in china a prospective cohort study [164] from eight medical centers was conducted to observe the effect of response-guided combination therapy with lam and adv in patients with chb. according to hbv dna level at 24 weeks, a total of 100 patients with chb and cirrhosis treated lam were divided into three groups: complete response group (arm a, n = 49, hbv dna ≤60 iu/ ml), partial response group (arm b, n = 31, hbv dna: 60-2000 iu/ml) and inadequate response group (arm c, n = 20, hbv dna >2000 iu/ml). patients was added adv at week 24 in arm c, but at week 48 in arms a and b. at 144 weeks undetectable rate of hbv dna and ymdd mutation rate in three arms was 95.96%, 66.67%, 35.29% (p = 0.000) and 0%, 3.23%, 15% (p = 0.015), respectively. the data showed that early complete virologic response at 24 weeks was associated with maintained viral suppression. hbv dna level of these patients without complete virological response at week 24, adding adv therapy further decreased by 1 log 10 iu/ml. all patients achieved biochemical improvement including alt/ast decline and alb elevation. in patients with hbv dna breakthrough due to ymdd mutations, adv and lam combination therapy did not lead to further multiple drug resistance. in chb patients with compensated liver cirrhosis, continuous hbv suppression for longterm and liver function improvement could be obtained by optimized responseguided add-on therapy of lam and adv. in recent years, some new antiviral drugs have gradually entered people's field of vision. truvada is a fixed-dose combination of two antiretroviral drugs (emtricitabine and tdf) approved by the fda for anti hiv therapy. the molecular structure of ftc was similar to that of lam, and the drug resistance was also similar to lam. in 2005 a double-blind study [165] evaluated 48 weeks treatment of 25, 100 or 200 mg once daily doses of emtricitabine in patients with chronic hepatitis b. then these patients were followed 200 mg emtricitabine treatment for an additional 48 weeks. after 2 years, 85% of the patients had normal alt, 33% seroconverted to anti-hbe and 53% had serum hbv dna ≤4700 copies/ml. eighteen percent of the patients treated with 200 mg emtricitabine developed resistance mutations after 2 years. emtricitabine 200 mg once daily was chosen as the optimal dose for chb based on these data. emtricitabine was well tolerated and confirmed a potent antiviral response for up to 2 years. in a randomized double-blind, 96-week trial [166] , patients were divided (1:1) to groups given a combination of emtricitabine (ftc, 200 mg; n = 139) and tdf (300 mg, ftc/tdf) or monotherapy of tdf (300 mg, n = 141). patients were hbeag(+) or hbeag(−), with levels of hbv dna ≥3 log 10 iu/ml and lam resistance mutations (rtm204i/v ± rtl180m). after 96 weeks of treatment, 86.3% in the ftc/tdf group and 89.4% of patients in the tdf group had levels of hbv dna <69 iu/ml (p = 0.43). hbeag loss and seroconversion was not significant difference between groups; only 1 patient (0.7%) in the ftc/tdf group lost hbsag. no additional benefit was observed with the combination therapy of emtricitabine and tdf vs tdf monotherapy. prolonged therapy with an oral nucleoside or nucleotide can lead to the development of antiviral resistance. loss of initial response and hbv dna rebound induce the development of resistance. these patients with resistance may develop biochemical breakthrough and histologic deterioration. sometimes severe exacerbations occur due to virus resistance in patients with cirrhosis. there are many risk factors for resistance development, such as potency of the antiviral agent, pretreatment hbv dna titer, period of treatment, nucleotide antiviral therapy or oral nucleoside history, and the degree of genetic barrier to resistance to the individual drug. thus, either etv or tdf, which possess the lowest genotypic resistance, should be used as the initial therapy. patients should be evaluated in the course of treatment, and these patients with poor response should be treated combination therapy early. managing resistance recommendations vary but generally involve either adding a drug in a separate class or switching to a more potent drug within the same class. in clinical practice, most members of the panel generally avoid monotherapy in patients with resistance and either use add-on therapy with tdf or etv or switch to tenofovir/emtricitabine. however, in patients with lam resistance, there are data providing compelling reassurance that tdf monotherapy is sufficient [166] . data suggest that tdf may also be sufficient for patients with adefovir resistance [167] was limited. however, with newer anti-hbv agents such as etv and tdf, viral resistance in previously treatment-naïve patients is very rare and the vast majority of cases of virologic breakthrough in clinical practice are due to nonadherence [168, 169] . to see treatment measures in sect. 5.1. clinical trials and cohorts from clinical practice have shown that nas are generally well-tolerated and safe [170] . rare serious adverse reactions includes renal insufficiency, myositis, rhabdomyolysis, lactic acidosis, etc. some of these drugs can induce impairment of mitochondrial replication with mitochondrial dysfunction or loss due to a low level of activity against the human mitochondrial dna polymerase gamma. lam has been well-tolerated and effective in patients with hbv related decompensated cirrhosis [127, 171] . adefovir dipivoxil and tdf are associated with a dose-dependent renal toxicity, except for ldt, a drug that may even improve creatinine clearance [172] . 1.7% patients had elevation of serum creatinine ≥0.5 mg/dl above baseline have been reported after 7 years of tdf therapy in chb patients [173] . hadziyannis et al. [132] conducted a cohort study to investigate the efficacy, safety, and resistance profile of adefovir dipivoxil treatment for up to 240 weeks in patients with hbeag(−) chb that was lost when 48 weeks adv treatment was discontinued. a total of 125 hbeag(−) chb patients treated with adv for 5 years. serum creatinine elevations (0.5 mg/dl above baseline) occurs in 3% of these patients. similarly, 8% of the 65 hbeag(+) chb patients treated adv for 5 years had reversible creatinine elevations, 3% developed hypophosphatemia, and 5% had albuminuria [174] . there were a growing number of studies of lam and adv combination therapy in both treatment-experienced and treatment-naïve patients with lamivudine-resistant hbv. lampertico et al. [175] conducted a study to investigate the risk of resistance in the long term of lam and adv combination therapy in lamivudine-experienced chb patients. the results showed that 8% of 145 patients with lamivudine resistance developed mild nephrotoxicity. after increasing the adv-dosing interval, all these patients were able to continue combination therapy. before treatment estimated creatinine clearance and serum creatinine levels should be tested in all chb patients treated with nas. in patients with creatinine clearance <50 ml dosing adjustments are needed, regardless of the type of nas. etv preclinical research data showed that a higher incidence of solid tumors in animals was associated with prolonged administration of high-dose compared to placebo. however, in clinical trials prolonged administration of etv was not associated with an increased incidence of malignancy. in a clinical trials conducted by lai et al. [134] the severity and frequency of laboratory and clinical adverse events were similar among lam-treated and etv-treated patients. furthermore no evidence of serious adverse events and mitochondrial were observed in patients with etv treatment for up to 5 years [176] . in recent years, in patients with liver cirrhosis in high meld score (>20) taking etv, lactic acidosis, and even death cases were reported. although rare happening, it still has to be pay more attention by the clinician, and needs for future research. ldt may cause myopathy and peripheral neuropathy. in treatment of ldt combined with peg-ifn-2a, moderate-severe peripheral neuropathy may occur in patients. therefore ldt was forbidden in combination with peg-ifn. in these studies [155, 177] , patients treated with ldt at 2 years had a significantly higher incidence of severe elevations of serum creatine phosphokinase (i.e., seven times upper limit of normal) compared to patients treated with lam, 12.9% and 4.1%, respectively. although most of them are asymptomatic, there are still two cases of patients with ldt-induced symptomatic myopathy had to be terminated treatment. in 2010 chinese guideline, it has been stated that careful medical history investigation before treatment was needed to reduce the risk. in the course of treatment, patients with serum creatinine, ck or lactate dehydrogenase significantly increased, and accompanied by myalgia or weakness, should be immediately tested. once diagnosed with uremia, myositis, rhabdomyolysis or lactic acidosis, patients should be promptly discontinued treatment or switched to other drugs, and given the appropriate treatment. antiviral therapy with nucleos(t)ide analogue is an important means to delay or reverse progression of liver fibrosis and cirrhosis. cirrhotic patients need long-term or even lifelong antiviral treatment. all of the five antiviral agents could effectively inhibit virus replication, improve biochemical and pathological parameters in chb cirrhotic patients with good tolerance. patients should be fully assessed baseline characteristics before treatment, and closely monitored therapeutic response and adverse reactions during treatment, then to optimized treatment. according to both drug resistance and efficacy profile, etv and tdf are superior to ldt, adv, and lam, and can be recommended as the first-line drug for nuc-naïve patients with hbv related decompensated cirrhosis. finally, hbv related cirrhosis patients treated oral nuc(s) must be frequently laboratory and clinical assessed to insure medication compliance and surveillance for clinical and virological response as well as drug resistance, drug side effects, and hepatocellular carcinoma. in the world hepatocellular carcinoma is one of the most frequent malignancies: estimated 782,000 new cancer cases worldwide occurred in 2012 (50% in china alone). it is the fifth most common cancer in men (554,000 cases, 7.5% of the total) and the ninth in women (228,000 cases, 3.4%) [178] . hepatocellular carcinoma is the second most common cause of cancer death worldwide, estimated to be responsible for nearly 745,000 deaths in 2012 (9.1% of the total). given the very poor prognosis for liver cancer (the ratio of mortality to incidence is 0.95), the geographical patterns in incidence and mortality are quite similar [179] . chronic infections with hepatitis c virus (hcv) and hepatitis b virus (hbv) are the major recognized risk factors for hcc worldwide [180] , hbv being most common in eastern asia and hcv in mediterranean countries [181] . current hcc treatment is the comprehensive treatment based on resection, liver transplantation, or percutaneous local ablative treatment. more and more studies indicated that after resection antiviral therapy effectively inhibit hbv replication and sequentially decrease the rate recurrence of hcc. chronic hepatitis b is the most frequent etiology of hcc. chen [182] conducted a prospective cohort study to evaluate the relationship between mortality and past hbv dna level for 11 years of follow-up. hbv dna level had been measured on stored samples in 2763 hbsag(+) adults from cohort entry (1992-1993). there was a significant increase in mortality in patients with hcc across viral load categories (p < 0.001). compared to the hbv undetected category, the relative risk for hcc mortality in the high viral load group was 11.2 (95% ci 3.6-35.0) and 1.7 (95% ci 0.5-5.7) in the low viral load group. the relative risk for chronic liver disease mortality were 15.2 (95% ci 2.1-109.8) and 1.5 (95% ci 0.2-12.1), respectively (p < 0.001). with increased follow-up time, the rr associated with high viral load did not change. in surviving cohort patients evaluated for liver disease in 2003, the disease significantly associated with viral load. the data showed that increased mortality from hcc and cld was associated with viral load in patients infected hbv. hbv dna level may be a useful prognostic indicator in chb patients, and treatment interventions to inhibition of virus replication should be explored. the reveal-hbv study of chen [148] assessed the relationship between risk of serum hbv dna level and hcc. from 1991 to 1992, this prospective cohort study in taiwan enrolled 3653 participants who were hbsag(+) and 30-65 years from a community-based cancer screening program. during 41,779 person-years of follow-up and a mean follow-up of 11.4 years, there were 164 incident cases of liver cancer and 346 deaths. the incidence of liver cancer grew in patients with hbv dna level at baseline in a dose-response relationship ranging from 0.108% personyears for an hbv dna <300 copies/ml to 1.152% person-years for an hbv dna 1 × 10 6 copies/ml or greater. the cumulative incidence rates of liver cancer in these patients were 1.3% and 14.9%, respectively. after adjustment for age, sex, alcohol consumption, cigarette smoking, serum alt level, hbeag, and liver cirrhosis at baseline, the biological gradient of liver cancer by hbv dna levels were significant different (p < 0.001). the dose-response relationship was most prominent for hbeag(−) patients without liver cirrhosis and with normal serum alt levels at baseline. chb patients with persistent elevation of viral load had the highest liver cancer risk during follow-up. these data proved that high level of hbv dna (≥10,000 copies/ml) was a strong risk factor of liver cancer independent of liver cirrhosis, hbeag, and serum alt level. these data showed that the correlation between hcc and hbv dna level was more closely than alt. the current guidelines [183] [184] [185] for management of chb are of the view that: for patients with chronic hepatitis b the primary goal of treatment is to permanently suppress or eliminate hepatitis b virus replication. thus hepatic infectivity and pathogenicity could be decreased, and thereby necroinflammation could be stopped or reduced. the short-term goal in clinical terms is to reduce hepatic inflammation, to prevent the development of hepatic fibrosis and decompensation, to ensure a sustained loss of hbv-dna and alt normalization. the long-term goal is to prevent progression to cirrhosis and hcc, to prevent alt flares that may lead to hepatic decompensation, and finally prolong the survival time. therefore, the ideal treatment for patients with chb should be able to effectively reduce the hbv dna level, thereby inhibit or stop the deterioration of liver disease, reduce the incidence of severe exacerbation and hcc. hbv chronic infection is a major risk predictor for the development of hepatocellular carcinoma. the hepatocarcinogenesis in chb patients has been extensively investigated, and a number of predictors relate to occurrence of hepatocellular carcinoma. the most significant predictors associated with hepatocellular carcinoma include chronic hcv and hbv infection, aflatoxin b1, chronic alcohol consumption and virtually all cirrhosis-inducing conditions [186] . for hepatocellular carcinoma in human, chronic infection of hbv was considered as the major environmental etiological factor [187] . hbv-induced hepatocarcinogenesis can involve an array of processes, including host-viral interactions, sustained cycles of necrosis-inflammation-regeneration, viral-endoplasmic-reticulum interactions (induction of oxidative stress), viral integration into the host genome (and associated host dna deletions) and the targeted activation of oncogenic pathways by various viral proteins. a predominant risk factor for hcc is chronic active hepatitis. the mechanisms of chronic active hepatitis consist of a combination of complementary, effects, several involved in liver cell inflammation, and necrosis thus fibrosis and cytokine synthesis. the underlying chronic active hepatitis inflammatory is a major risk factor for the higher hcc occurrence in patients with progressive cirrhosis. fundamentally important information on these issues have been provided in animal models for hepadna virus infection [188] . integration of hbv dna results continuous replication of the virus, which induces occurrence of genetic alterations. the hbv-dna sequences are integrated into cellular dna in most (approximately 90%) liver-tumor samples from hbsagpositive patients [189] . hbv genome integration should be viewed as a "driver" of liver carcinogenesis. in the impact of genes, some of the other genes involved, and play an important role in the carcinogenesis process. in addition, hbv dna integration effects on host cells including cell gene deletion, chromosomal rearrangements, genomic dna copy number variation, loss of chromosomal heterozygosity, etc. [190, 191] . in the host cells, the integration of hbv damage mechanisms to protect the integrity of the chromosome. hepatitis b virus x protein (hbx) exhibits pleiotropic effects on different pathways involved in intracellular signaling and transcriptional activation that modulate cell responses to protein degradation, genotoxic stress, apoptosis and cell division; these biological effects might contribute to the potential transforming activities of hbx. hbx has been confirmed to interact with p53, accordingly inactivating several essential p53-dependent activities, including p53-mediated apoptosis transactivation properties of p53, regulation of cell cycle dna repair genes and tumor suppressor genes. hbx may also play a role in tumorigenesis of hepatocellular carcinoma through modulation of angiogenesis pathway. indeed, hbx expression induces upregulation of the vascular endothelial growth factor (vegf) transcription and stabilizes hypoxia inducible factor (hif)-1 [192, 193] . moreover, hbx causes multipolar spindle formation, chromosome segregation defects, and appearance of multinucleate cells by inducing aberrant centrosome duplication; these biological actions might be due to sequestration of the nuclear transport receptor crm1 in the cytoplasm [194] . it has been frequently reported that hbx with mutations in amino acids 130 and 131 may be associated with the severity of chb. studies indicated that these mutations had also been detected in hcc tissue and arose before the development of hcc [195] . other studies [196] have pointed out that many signaling pathways have been outlined as common targets deregulated during hepatocarcinogenesis, including the wnt/β-catenin, tgf-β pathway, ras/mapk pathway, pi3k/akt/mtor pathway, jak/stat pathway, pkc pathway, etc. in addition, fibrinogen-like protein 2 (fgl2), hgf, igfs and other coagulation factors, growth factors and other angiogenesis gene may also be involved in the occurrence and development of hcc [197] . 1. wnt/β-catenin pathway: the wnt signaling pathway is an evolutionary highly conserved pathway and involved in the regulation of proliferation, motility, cell/ cell interaction, organogenesis and axis formation. the accumulation and expression of β-catenin in the nucleus were decreased, and cell proliferation was suppressed followed by up-regulated gsk-3β activity due to hbx induction [198] . hbx mutants may participate in the development and progression of hcc, at least in part through the wnt-5a pathway [199] . 2. tgf-β pathway: tgf-β is a central regulatory factor in control of hepatocyte proliferation and death. paradoxically, either under-or overexpression seem to have deleterious consequences resulting in an increased turnover of liver cells and thereby predisposing to cancer progression [200] . in both cases the escape from the antiproliferative, proapoptotic action of tgf-β would be a prerequisite for tumor progression. at the stage of tumor occurrence, tgf-β can promote tumor cell invasion, metastasis, but suppresses tumor growth in liver damage stage. tgf-β receptor i kinase inhibitors, blocking the tgf-β signaling pathway, show anti-tumor effect [201] . 3. ras/mapk pathway: ras/mapk signaling pathway is a signal cascade waterfall reaction caused by external signal activated receptor in the cytoplasm, involving a variety of connectors, nucleotide exchange factor, small gtp binding protein. hbx retains the ability to overcome ras-induced senescence in human cells immortalized by htert, although hbx alone could neither immortalize nor transform human cells. the ability of hbx to collaborate with active ras in cell transformation may explain its role in hcc [202] . 4. pi3k/akt/mtor pathway: the pi3k/akt/mtor protein cascade is one of major signaling pathways associated with receptor tyrosine kinases (rtks) [203] . in nontransformed cells, a tumor suppressor pten (phosphatase and tensin homolog deleted from chromosome 10), which inhibits this pathway by blocking akt activation and reversing the pi3k reaction, control the pi3k/akt/ mtor pathway. in almost half of the studied hccs, pten expression was reduced or absent, and hepatocyte-specific abrogation of pten expression in mice results in the development of hcc [204] . 5. jak/stat pathway: the jak/stat pathway is activated by more than 40 cytokines and growth factors and involves in multiple cell functions such as differentiation, proliferation, and apoptosis [205] . in this pathway, the cytokines induce phosphorylation of the janus tyrosine kinases (jak1, 2 and 3, tyk2), followed by activation of stat1-6 [206] . both hbv and hcv are able to induce jak/stat pathways [207] . in hcc, phosphorylation of jak1, jak2, and tyk2 tyrosine kinases was not detected in normal livers but increased significantly from surrounding non-neoplastic livers to hccs. activation of stat1, stat3, and stat5 was statistically higher in tumors than in respective surrounding livers, with pstat3 being higher in hcc with poor prognosis than hcc with better prognosis. the levels of jak/stat targets, including bcl-xl, mcl-1, cyclin d1, and c-myc were markedly increased in the majority of hccs [208] . 6. pkc pathway: pkc isozymes have a central role in cellular signaling transduction involved in cell proliferation, differentiation, apoptosis and angiogenesis [209] . pkc-α, pkc-δ, and pkc-ι have been found to be over-expressed in human hcc cell lines. the focus of pkc research in hcc has predominantly been on pkc-α. its expression is significantly increased in cancerous tissue and is correlated with tumor size and tnm stage. in addition, over-expression of the mrna of this isozyme has been associated with a shorter survival rate, and thus may be a marker for disease prognosis in cancer patients [210] . 7. fgl2: fgl2 could directly generate thrombin from prothrombin without activation of the conventional coagulation cascade. it was confirmed to be overexpressed in various human malignant tumors [211] . the hfg12 was associated to the hypercoagulability in cancer and may induce tumor metastasis and angiogenesis via cytokine induction [212] . fgl2 was overexpressed in hcc tissues and co-localized with fibrin deposition. fgl2 contributed to hcc tumor angiogenesis and growth in a thrombin-dependent manner, and down regulation of its expression might be of therapeutic significance in hcc [211] . to investigate whether prevention of hcc by the hbv vaccine and to identify the predictors of hcc for vaccinated birth cohorts, a population-based study [213] in taiwan the study shows strong evidence that the hbv vaccine reduce the incidence of hcc. those who received incomplete hbv vaccination (i.e., less than three administrations of the vaccine) during infancy and infants of hbeag-and hbsagseropositive mothers without hbig injection at birth had higher risk of developing hcc. approximately 30% of children with hcc born to hbeag and hbsag carrier mothers did not receive hbig at birth. improvements of the hbig injection rate within 24 h after birth in infants of high-risk mothers should be implemented. this study has limitations in that the role of host factors, such as genetic polymorphisms, hbv genotype, virus mutation, were not studied, which could influence the interpretation of the data. a number of studies on the long-term treatment of interferon (ifn) or na for patients with hbv showed the prevention of hcc. a meta-analysis [214] compared risk of hcc in chb patients who received ifn or na. a total of 17 studies were included in this review. ifn treatment (12 studies; n = 2742) showed a significantly reduced risk of hcc for patients treated by ifn compared to controls (rr 0.66, 95% ci 0.48-0.89; 12 studies) and for compensated cirrhotic patients (rr 0.53, 95% ci 0.36-0.78; 6 studies). there was no statistical heterogeneity for these comparisons. na treatment (5 studies; n = 2289) showed a significantly reduced risk of hcc for patients treated by nas compared with controls (rr 0.22, 95% ci 0.10-0.50; 5 studies). na treatment demonstrated a more profound reduction in hcc risk of 78% compared to ifn which produced only a modest effect of 34%. this is perhaps not a surprising finding, as the viral load is found to be the most important factor leading to cirrhosis and cancer development in the liver. the more effective reduction in hcc risk may be related to the more profound effects of viral suppression of oral anti-viral agents than ifn [215] . across subgroups there was a significantly reduced risk of hcc: hbeag(+) patients (rr 0.21, 95% ci 0.10-0.44; five studies); compensated cirrhotic patients (rr 0.17, 95% ci 0.04-0.79; three studies); non-cirrhotic patients (rr 0.21, 95% ci 0.10-0.47; two studies); patients with drug resistance (rr 0.52, 95% ci 0.28-0.97; three studies); and those without drug resistance (rr 0.37, 95% ci 0.17-0.77; three studies). in subgroup analysis of ifn studies, a more significant reduction in hcc risk among those with early cirrhosis was found. the effects of ifn could be beyond its viral suppressive activities. previous studies have shown that at least ifn-α2b has inhibitory activities on hepatic stellate cells (hscs) activation by suppressing the effects of il-1β, tnf-α and probably inducing apoptosis of hsc [216, 217] . as hscs play a central role in fibrogenesis, the effects of ifn-α on hscs are worthy of further investigation. on the other hand, the anti-cancer effect of nas, and probably ifn, was more prominent among hbeag-positive than among hbeag-negative patients. this discrepant results based on hbeag status is consistent with the fact that while hbeag(+) patients usually have a higher hbv dna level, treatment of hbeag(−) patients is more difficult and sustained virological responses are uncommon [216, 217] . high risk population hbv-hcc refers to patients who are the middle-aged men with high hbv load, with hbv and hcv co-infection, with family history of liver cancer, alcoholics, and with diabetes mellitus. the long-term effect of antiviral therapy for patients with hbv showed the prevention of hcc. however, according to current national management guidelines for chb, there are still some patients without antiviral treatment. thus, some of the high risk population of hbv-hcc may lost the opportunity of early interventional treatment. current guidelines recommend liver biopsy to assess the degree of necroinflammation and liver fibrosis prior to treatment initiation in patients with increased hbv dna and/or mild elevated alt levels (1-2 times the uln). for patients older than 40 years, liver biopsy should be considered. in those with "high normal" alt levels liver biopsy is strongly recommended [183] . although liver biopsy remains the gold standard for assessing hepatic fibrosis, its use has several limitations including sampling error and intra-or inter-observer sampling variability [4] . inadequate liver biopsy may further pose misleading histological information that precludes cirrhotic patients from antiviral treatment [218] . in the report by tong et al. [219] , 5 of 9 patients who developed hcc were diagnosed as having chronic hepatitis by histology and therefore did not fulfill the recommended treatment criteria. these patients probably had normal alt and/or intermediate hbv dna levels (between 10,000 and 100,000 copies/ml). in this report, 7 of 15 patients with cirrhosis who developed hcc could not be identified by the treatment recommendations. in other words, patients with cirrhosis have a significant risk of developing hcc even when their hbv dna levels are not high. patients with elevated alt between 0.5 and 1 times the uln also was a strong risk predictor of hcc or complications [220, 221] , a claim supported by a korean population study. the reveal study suggested that high hbv dna level significantly increased risk of hcc independent of liver cirrhosis, hbeag, and serum alt level. hbv dna consistently replicates and is integrated into the host genome, adding to the coexistence of metabolic disorders, inflammatory responses and oxidative injuries, which induce genetic instability and an imbalance of cell growth and apoptotic tolerance signals. these are all biologic driving forces for hcc development in chb patients. therefore, we must pay more attention to the effect of continuous hbv replication on the prognosis of patients. any antiviral drugs, if not completely clear the virus but can reduce the viral load, it may reduce the risk of patients with hcc. the risk factors for hcc include progression to cirrhosis, longer duration of hbv infection, higher serum viral load (≥10 5 copies/ml), males, age >40 years, alcohol, ethnic groups native to regions of east asia and sub-saharan africa, the virus genotype (genotype a in african population or genotype c in asian population), co-infection with human immunodeficiency viruses (hiv) or hepatitis c, d, decompensated liver cirrhosis, persistent inflammation of the liver, continuous hbeag positive, and a family history of liver cancer [222] . cirrhosis is the most important independent risk factor for hcc. up to 70-90% of hcc occur in patients with cirrhosis. effective antiviral therapy inhibits hbv replication, reduces serum viral load and accelerates serum conversion of hbeag, which may therefore alleviate liver damage and reduce the development of cirrhosis. all the patients with the above risk factors should be received antiviral therapy. ifn-α is an immune modulator inducing antiviral, immune regulation, anti-tumor and anti-fibrosis effects. its antiviral mechanism is a complex mode of action including the destabilization of viral nucleocapsid, inhibition of viral genome transcription, activation of natural killer (nk)/nkt cells. however, disadvantages of ifn shortcomings are prominent, such as the high cost of peg-ifn, intolerance to ifn therapy in patients with cirrhosis. compared with ifn, na is safer, better tolerance for these patients. current guidelines from apasl, easl and aasld, do not provide treatment recommendations for patients with hbv-hcc. the chinese expert consensus [223] on antiviral therapy to treat hbv/hcv-related hcc has been published in 2014. this expert consensus indicated that promptly initiation of antiviral therapy is not only important for preventing the incidence of hcc in patients chb, but also essential for reducing hbv reactivation, improving liver function, delaying or reducing recurrence of hcc, and prolonging survival of patients with hbv-hcc after palliative and curative therapies. it puts forward the overall principle and target of antiviral therapy of hbv-hcc, and emphasizes the antiviral therapy is a part of comprehensive treatment. at present, suitable treatment for hbv-hcc is multidisciplinary comprehensive treatment. a large number of evidence-based medical evidence suggested, standard anti-hbv treatment for these patients help to improve the overall curative effect, prevent the recurrence of the tumor, and improve the os. therefore, anti-hbv therapy should be taken as a very important part of comprehensive treatment of hbv-hcc (fig. 5.4) . following curative liver resection for hcc, 50-90% of postoperative death is caused by recurrent disease [224] . high serum hbv dna levels were a strong predictor of hcc. effective control of hbv replication with antiviral therapy may lower its recurrence after liver resection. in 2000, kubo et al. [225] first reported the relationship between recurrence of hbv-hcc after resection and hbv dna level. later in another study [226] he pointed out that patients with high hbv dna levels (more than 5 meq/ml) have high risk with recurrence and poor prognosis. on the contrary, kim et al. [227] included 230 consecutive patients undergoing curative resection and found that, sustained hbv viremia (>5 log copies/ml) increased recurrence, but did not have a marked effect on survival. an et al. [228] investigated the hbv dna changing patterns and their effects on outcome in hbv-hcc patients with resection. all 188 patients were followed up. data from 115 alive patients without recurrence at 12 months were collected. the mean period of follow-up was 48.5 months and the mean age was 53 years. for the entire population with multivariate analysis, tumor size >5 cm (p = 0.047), hbv dna >10 4 copies/ml, child-pugh class b (p = 0.017) at the time of resection (p = 0.003), and vascular invasion (p = 0.028) were independently risk factors of hcc recurrence. on multivariate analysis for 115 patients, sustained hbv dna level <10 4 copies/ml was the only risk predictor for both longer survival (or 3.76; 95% ci 1.61-8.78; p = 0.002) and low recurrence (or 3.13; 95% ci 1.55-6.35; p = 0.002). that clarified that a sustained high hbv dna is among the most important risk factors of adverse outcome after liver resection of hbv related hcc. the sustained suppression of hbv dna <10 4 copies/ml strongly benefit to long-term survival and recurrence-free. kim et al. failed to show the difference in survival between the sustained viremia (>5 log copies/ml) and non-viremia groups despite the high recurrence rate in the sustained viremia group. the reason may be that researchers have used a higher hbv dna cut-off value (>10 5 copies/ml) to differentiate between patients with high and low viremia. in an's results, they found that a lower hbv dna level cutoff value of 10 4 copies/ml is superior to >10 5 copies/ml in predicting outcomes after resection. it is therefore needed to suppress further hbv dna to a lesser level in order to obtain better clinical outcomes after surgery. studies have shown that positive hbeag was a risk predictor for recurrence of patients after resection of hcc. sun et al. [229] evaluated the impact of hbeag on patients' survival and tumor recurrence after curative resection of hbv-hcc. all 203 patients undergone curative resection with small hcc (⩽3 cm) were divided into hbeag(+) and hbeag(−) groups. postoperative outcomes and clinicopathological factors of two groups were compared, and risk predictors for recurrence and survival were investigated. hbeag(−) patients had higher 5-year disease free survival rates (52.9% vs 37.4%, p = 0.046) and 5-year overall survival rates (76% vs 53.9%, p = 0.002). there was no significant difference in tumor factors and operative morbidity between hbeag(+) and hbeag(−) groups, but more macronodular cirrhosis, higher serum alanine aminotransferase levels, and younger age were found in the hbeag(+) group. in patients for multivariate analysis, macronodular cirrhosis, hbeag(+) and age >50 years were independent risk predictors for overall survival, and multiple tumor nodules and hbeag(+) were independent predictors for disease free survival. in patients with small hcc after curative resection, hbeag(+) was a significant risk factor of early recurrence (within 1 year). because of the adverse effects, the impact of ifn-α therapy after curative resection on recurrence of hcc and the os among patients with hbv infection are controversial. theoretically, the effect of postoperative ifn-α therapy on recurrence might be related with the direct suppression of tumor growth and metastasis, the inhibition of hbv replication, down-regulating expression of vascular endovascular growth factor (vegf), antiangiogenesis effect, and modulating some factors in tumor microenvironment. however the results of clinical trials are not the same. in recent years, more and more studies show that reasonable application of ifn-α can prevent the recurrence of the tumor and prolong the survival time of the patients. qu et al. [230] conducted a retrospective study to investigate the impact of ifn-α therapy on survival and recurrence after curative resection in patients with hbv-hcc. of 568 hbv-hcc patients with curative resection, 101 patients were treated postoperative by ifn-α therapy (5 million units three times every week for 18 months). patients with postoperative ifn-α therapy had higher os rates (p = 0.010, hr: 0.612, 95% ci: 0.422-0.889). there was no significant difference in dfs rates between the two groups (p = 0.086, hr: 0.786, 95% ci: 0.597-1.035). on multivariate analysis, postoperative ifn-α therapy was an independent factor for os (p = 0.010, hr: 0.611, 95% ci: 0.421-0.887) and significantly reduced early recurrence (p = 0.005, hr: 0.562, 95% ci: 0.375-0.840). however, patients with or without postoperative ifn-α therapy had similar cumulative late recurrence rates (hr: 1.205, 95% ci: 0.781-1.858, p = 0.399). sun et al. [231] evaluated the effects of postoperative ifn-α treatment on survival and recurrence in patients with hbv related hcc. all 236 patients were randomized after curative resection into ifn-α treatment (n = 118, 5 μg i.m. tiw for 18 months) and control groups (n = 118). if recurrence was diagnosed, treatment was terminated, these recurrence patients was managed in the same way in both groups. all clinicopathological parameters in two groups were analyzed. the median os was 63.8 months in the treatment group and 38.8 months in the control group (p = 0.0003); the median dfs period was 17.7 versus 31.2 months (p = 0.142). that concluded that ifn-α therapy improved the os of hbv-hcc patients after curative resection, probably by postponing recurrence. someya et al. [232] investigated 80 consecutive patients with hbv cirrhosis and hcc who underwent potentially curative ablation for hcc. eleven patients received long-term ifn therapy. initial dna was high (>6.0 log copies/ml) in 41 patients and low (<6.0 log copies/m) in 39. hcc recurrence rates in the high dna group and low dna group were 82.6% and 46.9% at the fifth year, and 91.3% and 73.5% at the tenth year, respectively (p = 0.0103). similarly, recurrence rates after treatment of hcc in the abnormal ast group (n = 38) and normal ast group (<38 iu/l, n = 42) were 84.0% and 50.6% at the fifth year, and 100% and 71.3% at the tenth year, respectively (p = 0.0003). five of 42 patients with normal ast, and 6 of the 38 patients with abnormal ast, received ifn-α after confirmation of tumor ablation. in the subgroup of abnormal ast, tumor recurrence rates in the untreated and ifn-α groups were 37.9% and 16.7% at the end of the first year, 60.1% and 16.7% at the second year, and 83.4% and 16.7% at the third year, respectively (p = 0.0139). on multivariate analysis, ifn-α significantly reduced the recurrence rate (p = 0.037, hr = 0.21) even after adjusting for background characteristics. pathogenic mechanism of hbv-hcc mainly related with the integration of hbv dna into host hepatocytes. therefore, inhibition of inflammation and viral replication can reduce the hbv dna level and the risk of hcc. after the resection the residual liver is still cirrhosis, still have a high risk of new cancer. hbv-hcc occurrence seems to have the relationship with the hbv greater than the liver repeatedly inflammation [233] . tang et al. [234] reported that high hbv dna levels is associated with increased risk for development of hcc. ifn has a dual role of antiviral and immune regulation. ifn as immune regulator can not only activate or mediated macrophages, nk cells and cytotoxic t lymphocyte, but also adjust the antibody. its antiviral activity includes induction of 2,5 oligonucleotide adenosine monophosphate synthetase and protein kinase. moreover, ifn also has the anti-fibrosis, anti-proliferation and anti-tumor effects. the experimental study [235] confirmed that ifn exerts potent growth inhibitory effects on the hcc cell line plc/prf/5 both in vitro and in vivo and its mode of action in this animal model system appears to be predominantly mediated by a direct antiproliferative effect on tumor cells. breitenstein et al. [11] searched 7 cochrane central register of controlled trials between january 1998 to october 2007 and evaluated the effects of ifn-α or -β in patients after surgical resection or ablation of hbv-hcc. seven rcts (n = 620 patients) were included in a meta-analysis review. patients treated with ifn had a significantly decreased mortality rate than control group (rr 0.65, 95% ci 0.52-0.80, p < 0.001) and significantly reduced risk of tumor recurrence (rr 0.86, 95% ci 0.76-0.97, p = 0.013). as 6 of the 7 trials used ifn-α, it is interesting that the only study [236] on ifn-β showed the largest beneficial effect on tumor recurrence. due to the small number of cases in this study, further clinical evaluation of ifn-β in the adjuvant setting of hcc treatment seems to be indicated. the rate of treatment discontinuation ranged from 8% to 20% because of the side-effects of ifn which were dose dependent and often serious. severe adverse effects of the adjuvant ifn treatment leading either to treatment disruption or dose reduction occurred in up to a quarter of the patients. in particular, work is needed to optimize the type and dosage of ifn to minimize side-effects, and to study the combination of ifn treatment with other (neo)adjuvant agents. reasonable application of nas can prevent the recurrence of hcc and prolong the survival time of the patients. a comparative nonrandomized study [224] of postoperative antiviral treatment was conducted on 71 hcc patients who underwent curative hepatectomy. the authors assessed the impact of antiviral therapy for patients who underwent partial hepatectomy for hbv-hcc in the immuneactive phase of hbv infection. all 43 patients in the treatment group treated by lamivudine (lam) with or without adefovir dipivoxil (adv), while 36 patients in control group received no antiviral treatment. at 6-month postoperation, the treatment group also had a significantly greater increase in residual liver volume per unit surface area following hepatectomy (78.0 ± 40.1 cm 3 /m 2 vs. 35.8 ± 56.0 cm 3 / m 2 ). the os rate was a significant difference between two groups. the os rates of 1-and 2-year were 33.3% and 0%, respectively, for the control group, and 41.9% and 7.0%, respectively, for the treatment group. these results revealed that antiviral therapy with nas effectively relieved hbv-induced liver damage, improved liver function, promoted hepatocyte regeneration, and increased volume of residual liver, thus enhancing tolerance to subsequent therapy. there were no serious adverse effects to lam therapy in this study. however, the most significant problem associated with long-term therapy with lam is emergence of resistance. in this study, the emergence of ymdd mutants was observed in 6 of 43 patients in the lam group. administration of adv successfully controlled the virus. in a meta-analysis, wong et al. [237] assessed whether anti-viral therapy with nas could prevent hbv-hcc patients from tumor recurrence after curative treatment. a total number of 551 patients from 9 cohort studies were included: 347 patients without antiviral treatment (control group) and 204 patients with antiviral treatment group. lam was the primary antiviral therapy in the majority of patients. patients with lam resistance was treated by either switching to entecavir (etv) or adding adv therapy. thirteen patients received etv as primary antiviral therapy. most of the antiviral therapies were started after the curative treatment of hcc. the recurrence rate of hcc in the antiviral treatment group was significant lower compared to control group (55% and 58%; p = 0.04). in the antiviral treatment group the risk of hcc was reduced by 41%. there were also significant differences in favour of antiviral treatment group in terms of overall mortality (38% vs. 42%; p < 0.001) and liver-related mortality (0% vs. 8%; p = 0.02). hcc patients with anti-viral therapy after curative treatment may be reduced the risk of hcc recurrence for 41%. besides, antiviral therapy significantly improved os, as overall mortality was reduced by 78%. after curative treatment of hcc, patients should be monitored regularly concerning their viral status for consideration of antiviral therapy. antiviral therapy was beneficial as it not only might reduce hcc recurrence and liver failure secondary to reactivation of hbv due to viral suppression (90% reduction in the mortality secondary to liver failure in the antiviral therapy group). after ablation, the use of ifn or nas can reduce the recurrence of hcc, improve liver function, thus enhancing tolerance to subsequent therapy and prolong the survival time of the patients. recurrence in patients with hbv-hcc after ablation was common. chung et al. [238] assessed the correlation between viral load and intrahepatic recurrence and predictors of intrahepatic recurrence after percutaneous ablation. hbv-hcc patients undergoing percutaneous ethanol injection (pei) or radiofrequency ablation (rfa), between 2004.10 and 2008.12 were prospectively enrolled. a total of 145 patients (mean age, 55.3 years; male, 81.4%) were included. ninety patients (62.1%) had serum hbv dna ≥2000 iu/ml. the median followup duration was 28.9 months (range, 12.0-57.0) and 43.4% patients (n = 63) experienced intrahepatic tumor recurrence. on multivariate analysis, hbeag(+) was an independent risk predictor of late recurrence (≥1 year) (p = 0.012; hr 0.288) and intrahepatic recurrence (p = 0.026; hr 0.473). the afp level also significantly predicted late recurrence (p = 0.005; hr, 1.001). however, neither serum hbv dna titers nor the ablation method were correlated with intrahepatic recurrence. xia et al. [239] conducted a study to investigate the risk factors of recurrence in patients with hbv-hcc after rfa. all 152 patients with small hcc enrolled in this study. in 67 patients the intrahepatic recurrence rate was 44.1% by median follow-up of 35 months. on univariate analysis, meld score, afp, hbv dna, precollagen iii, and hyaluronic acid were independent risk factors for recurrence. on multivariate analysis, hyaluronic acid and hbv dna were independent risk factors for recurrence. the cumulative 1-, 3-, and 5-year dfs rates were 86.8%, 41.2%, and 22.8% in the high hbv dna group (>1 × 10 5 copies/ml) and 96.4%, 65.8%, and 36.7% in the low hbv dna group (≤1 × 10 5 copies/ml), respectively. that showed significant difference between the two groups (p = 0.003). goto et al. [240] reported the similar results that serum hbv dna load (>4.0 log 10 copies/ml) were associated with the recurrence. thus reasonable antiviral therapy can improve liver function and prevent the recurrence of the tumor. lin et al. [241] assessed the impact of ifn-α in preventing hcc recurrence. thirty eligible patients were randomized into three groups: ifn-α-continuous group (n = 11, ifn-α 3 mu tiw for 24 months), ifn-α-intermittent group (n = 9, ifn-α 3 mu daily for 10 days every month for 6 months followed by 3 mu daily for 10 days every 3 months for a further 18 months), and control group (n = 10, no ifn-α therapy). the three groups were comparable in terms of demographics, laboratory data, and etiology at entry and hcc characteristics. after a median follow-up of 27 months, 90% patients (n = 9) in the control group and 45% patients (n = 9) in 2 treatment groups (3 patients in the ifn-α-intermittent group and 6 patients in the ifn-α-continuous group) developed an hcc recurrence (p = 0.021). cumulative hcc recurrence rates in the control groups ifn-α-intermittent, ifn-α-continuous, and were 40%, 22.2%, and 27.3% at the end of 1 year and 90%, 33.3%, and 54.6% at the end of 4 years (p = 0.0375), respectively (control vs. ifn-α-continuous group, p = 0.0822; vs. ifn-α-intermittent group, p = 0.0123). the cumulative hcc recurrence rate of the patients treated with ifn-α and the control group was 25% and 40% at the end of 1 year and 47% and 90% at the end of 4 years, respectively (p = 0.0135) if both ifn-α groups were combined. the conclusion was that hcc recurrence may be reduced by ifn-α therapy after medical ablation therapy for primary tumors. antiviral, anti-tumor and anti-angiogenesis effect of inf can effectively resist the risk factors of recurrence after ablation. some patients do not tolerate the adverse reaction of ifn, still should be treated with nas to inhibit viral replication, relieve the liver inflammation, improve liver function, enhancing tolerance to subsequent repeated ablation. yoshida et al. [242] evaluated the efficacy of lam in hbv-hcc patients who were treated with rfa. complete ablation of hcc was achieved in 104 patients in this study. after rfa, 33 patients was treated by lam. there were 24 (73%) patients with serum hbv-dna negative conversion. four patients showed alt elevation and redetection of hbv-dna. there was no difference in recurrence-free survival and overall survival between the two groups. in the lam group no specific adverse effect was observed. the conclusion was that lam for patients with hbv-hcc after rfa was safe and liver function was improved. kuzuya et al. [13] evaluated the impact of antiviral therapy with lam on patients after initial treatment for hbv-hcc. comparison was made between 33 patients who did not received lam therapy after treatment for hcc (control group) and 16 patients who received at a dose of 100 mg/day (lam group) in terms of changes in survival, hcc recurrence, and remnant liver function. there was no significant difference in cumulative recurrence rates of hcc between the two groups (p = 0.622). however, median ctp score at the time of hcc recurrence was significantly different; 7 (range 5-12) in the control group versus 5 (range 5-6) in the lam group (p = 0.005). in the lam group, all patients were able to receive curative treatment for recurrent hcc. in contrast in the control group, 10 of 15 patients were unable to receive curative optimal therapy for recurrent hcc due to deterioration of remnant liver function. in the lam group, the cumulative survival rates of patients tended to be higher than those of patients in the control group (p = 0.063). the conclusion is that lam therapy is beneficial for hbv-hcc patients after initial treatment because it contributes to improving remnant liver function, accordingly decreasing the probability of liver failure and increasing the possibility to receive available treatment for recurrent hcc. clinical evidence showed hbv reactivation may occur in chronic hbv carriers with tumor during chemotherapy, then followed by hepatic decompensation and various complications. hbv reactivation occurs in nearly between 19% and 44% [243] [244] [245] . similarly, hbv reactivation may occur in patients with hbv-hcc after tace. some of these patients even treated with lam still occur hepatic decompensation or liver failure and eventually death, because of the delay of lam antiviral therapy, suggesting that these patients need to be treated by antiviral drugs before tace. moreover, more studies [246] [247] [248] also suggested that before chemotherapy early antiviral therapy can significantly reduce the chemotherapy-induced reactivation of hbv. tace is local treatment, different from systemic chemotherapy. therefore, early antiviral treatment before tace in patients with hbv-hcc can reduce the occurrence of postoperative virus reactivation, reduce the hepatitis flare caused by hbv, and reduce the mortality of acute exacerbation of chb. in 2006, a study about reactivation of hbv in patients with hbv-hcc undergoing tace of jang et al. [249] was published in hepatology. in a prospective and randomized study, 73 consecutive patients with hbv-hcc undergoing tace (cisplatin 60 mg/m 2 and epirubicin 50 mg/m 2 ) at monthly intervals were assigned to receive lam 100 mg daily from the start of tace (preemptive group) or not (control group). during the study, 2.8% patients (1/36) in the preemptive group and 29.7% patients (11/37) in the control group developed hepatitis due to hbv reactivation (p = 0.002). in addition, there were significantly more incidences of severe grade of hepatitis (p = 0.035) and overall hepatitis (p = 0.021) in the control group. on multivariate analysis, hbv dna level >10 4 copies/ml in baseline was the only independent predictor of hepatitis due to hbv reactivation during chemo-lipiodolization (p = 0.046). these data demonstrated preemptive lam therapy effectively reduced hepatitis due to hbv reactivation and hepatic morbidity during tace. preemptive therapy should be considered in hcc patients with an hbv dna level >10 4 copies/ml. preemptive antiviral therapy would effectively reduce liver-related morbidity attributable to hbv reactivation and would allow more prolonged chemotherapy. this study also suggested that preemptive lam therapy decreases the severity of clinical hepatitis if it develops during tace. zhu et al. [250] investigated the efficacy of adjuvant tace with or without antiviral therapy for hbv-hcc patients after radical hepatectomy. this study enrolled 176 patients, 58 of whom were treated using tace combined with antiviral therapy and 118 using tace alone. analysis of all patients suggested that overall survival of the combination therapy group was better compared to the tace-only group (p = 0.048), while disease free survival was similar between the two groups (p = 0.322). analysis of only propensity score-matched pairs proved 5-year overall survival in the combination therapy group was significantly better (64.6% vs. 37.5%, p = 0.033) and also suggested better 5-year disease free survival (37.9% vs. 14.6%, p = 0.048). for patients after hcc recurrence, radical hepatectomy was the treatment choice for a significantly larger proportion of patients from the combination therapy group than from the tace-only group (p = 0.018). these data suggested that combining tace with antiviral therapy significantly improved overall survival and potentially disease free survival relative to tace alone in hbv-hcc patients. combination tace with antiviral therapy also improves remnant liver function, increasing the chance of curative resection in case of tumor recurrence. combination tace with antiviral therapy may benefit to prevent recurrence of hcc after radical hepatectomy. it has been observed that hbv reactivation occurs after the end of radiotherapy in a way similar to that after cytotoxic chemotherapy [251] . radiotherapy to hcc can damage immune system, and cause leukocytes decreased, following by hbv reactivation. so antiviral therapy before radiotherapy for hbv dna positive patients is necessary. kim et al. [251] evaluated the impact of three-dimensional conformal radiotherapy (3d-crt) on hbv reactivation and hepatitis exacerbation in hbv-hcc patients. this study enrolled 48 hbv-hcc patients who underwent 3d-crt to the liver. group 1 (n = 16) treated lam before and during 3d-crt and group 2 (n = 32) did not treat with nas before 3d-crt. to investigate spontaneous hbv reactivation, 43 hcc patients received no specific treatment for chb or hcc were included as a control group. the cumulative rate of radiation-induced liver disease in groups 2 was higher than group 1 (12.5% vs. 21.8%, p > 0.05). the cumulative rate of hbv reactivation was significantly higher in group 2 (21.8%) than in group 1 (0%) or the control group (2.3%) (p < 0.05 each). there was no significant difference in cumulative rate of chb exacerbation between groups 1 (0%) and 2 (12.5%) or the control group (2.3%) (p > 0.05 each). that demonstrated that hbv reactivation and consequent chb exacerbation should be considered in hbv-hcc patients after 3d-crt and antiviral therapy should be recommended to prevent liver function after rt. in study of huang et al. [252] , the rate of hbv reactivation and chb exacerbation was 24.6% (17/69) and 21.7% (15/69), respectively. there was a relatively high rate of hbv reactivation in those patients and whose prognosis was unfavorable. the serum hbv dna level and some dosimetric parameters (normal liver volume, v20, and dmean) were the prognosis factors for hbv reactivation and should been considered carefully before crt. the goal of anti-hbv therapy is to effectively reduce the hbv dna level, thereby reduce the incidence of cirrhosis and hcc. although antiviral therapy is recommended in guidelines from apasl, easl and aasld, the specific procedures are not the same. and these guidelines do not give treatment recommendations for patients with hbv-hcc. current clinical studies have confirmed that early antiviral therapy is necessary for the prevention of liver function and reduce the integration of the viral dna. although antiviral therapy inhibits viral replication, the integration of viral dna continued. there are two distinct types of hcc recurrence: tumors grown from dissemination of the primary tumor and de novo tumors arising from the "field effect" in diseased liver [253, 254] . this may argue for an earlier antiviral intervention, as adjuvant therapy after the resection for the hcc patients with a high hbv dna level to prevent recurrence. anti-hbv therapy were performed in the light of the recently updated hbv treatment guidelines, on the recurrence and prognosis of hcc. to substantiate the beneficial effects of antiviral therapies, future randomized clinical trials (rcts) with longer follow-up, larger sample size, and regular hbv dna level monitoring will be needed to conduct. the molecular mechanisms of preventing tumor recurrence also need to be further studied. jun-ying qi and ming ni liver transplant is an effective treatment for hbv-related end-stage liver disease. the risk of hbv reinfection after liver transplant is the main limiting factor for long-term survival rate. combination therapy of lamivudine and hepatitis b immunoglobulin (hbig) reduced the rate of recurrence. however, considering the disadvantages of high dose hbig and high rate of lamivudine resistance, other therapies that composed by entecavir, tenofovir, or lamivudine plus adefovir, with or without hbig have been used in several liver transplant centers. other researchers have used posttransplant hbv vaccination for achieving a lasting endogenous anti-hbs antibody, yet the efficacy is still controversial. the combination hbig/nucleotide (acid) prophylaxis should be converted to oral prophylaxis within 1 or 2 years after liver transplantation. recently, the discovery of sodium taurocholate co-transporting polypeptide (ntcp) as the cellular receptor for hbv entry has opened up many channels of investigation, which indicate the possibility of using ntcp inhibitor in the prophylaxis of hepatitis b recurrence post lt. liver transplant (lt) is an effective treatment for hepatitis b virus (hbv)-related end-stage liver disease (such as acute or chronic liver failure, cirrhosis, hepatocellular carcinoma and so on). china has become the world's second-largest lt country as there are nearly 3000 operations annually. until the end of 2010, there were 19,330 cases of liver transplant had been completed in china, 80% of which were due to hepatitis b-related liver disease, and nearly 40% recipients with detectable hbv dna. the risk of hbv reinfection after lt is the main limiting factor for long-term survival rate. the rate of hbv reinfection is as high as 80% without antiviral prophylaxis [255] . lt recipients with recurrent hepatitis b develop an aggressive form of fibrosing cholestatic hepatitis, cirrhosis or graft failure within 2 years [256, 257] , which lead to death or re-lt. combined treatment of hepatitis b immunoglobulin (hbig) and nucleos(t)ide analogues (nas) reduced the hbv recurrence rate to 5-10% after 2 years of liver transplantation [258] [259] [260] [261] [262] . [263] . another problem in patients with hbv disease post lt is the presence of extrahepatic reservoirs of the virus that are continuous latent source of hbv recurrence [264] . on the other hand, late recurrence is related to low anti-hbs titer or the development of hbs viral escape mutations or ymdd mutations [265] . the strategies to prevent hbv reinfection after lt involve three stages: pre-, at and post-transplant. currently, the strategies include passive immunization (hbig), antiviral therapy (nas) and active immunization (hepatitis b vaccine). hbig was the first drug to effectively prevent hbv recurrence. limited duration of hbig therapy (<12 months) [10,000 iu iv at lt, 10,000 iu iv daily for 8 days after lt, then iv at different intervals to maintain anti-hbs titers >100 iu/l] delayed but did not prevent hbv reinfection [266] . the efficacy of this treatment seemed to be dependent on the viral load pretransplant. there was 96% developed recurrent hepatitis b 2 years after transplant in patients with detectable hbv dna in serum. the recurrent rate were 29% in patients who were hbv dna negative pretransplant [267] . this problem was partially resolved by using higher doses of hbig. monthly fixed doses of 10,000 iu of hbig (to keep anti-hbs levels >500 iu/l) or different hbig doses adjusted to maintain anti-hbs >500 iu/l for the first 6 months after liver transplant significantly reduced the rate of recurrence in patients with detectable hbv dna pretransplant [268] [269] [270] . however, using high doses of hbig was very expensive. lamivudine had apparent effect on hbv dna replication, decreasing the positive rate of hbv dna in patients undergoing or waiting for liver transplantation. data from 20 north american transplant centers showed that treatment with lamivudine improved pre-liver transplantation and liver transplantation-free survival of patients awaiting liver transplantation for hbv-related cirrhosis [271] . the early results of monotherapy of using lamivudine to prevent hbv recurrence post-lt were promising. in nine of ten survivors, hbsag and hbv dna were negative, and liver biopsy showed no evidence of recurrent by week 24 [272] . however, 50% patients re-infected with lamivudine-resistant hbv by 8-15 months post-transplant [273] . hbig and lamivudine are different in action mechanisms. hbig is a specific passive immune agents prepared from individual plasma who has been infected by hbv or injected hepatitis b vaccine. high concentration of hbig can neutralize hbv and block its infection of hepatocytes. lamivudine is a potent inhibitor of hbv-associated dna polymerase to block hbv replication. therefore hbig and lamivudine play a complementary role to each other. combined treatment of high dose iv hbig and lamivudine had been the accepted standard prophylaxis for post-lt hbv recurrence. lamivudine was used pre-lt for reducing the viral load in the peri-lt period in most centers. hbig was used at a dose of 10,000 iu daily for the first week post-operative and then either at a fixed dose of 10,000 iu/month or with different doses to keep anti-hbs titers >100 iu/l [260, 261, [274] [275] [276] . some centers had targeted anti-hbs titers >500 iu/l in hbv dna positive patients for 3-6 months post-lt. compared to the monotherapy of hbig or lamivudine, these combined treatments are highly effective [261, 277] . however, the long-term use of hbig has many disadvantages, such as high cost, the need for injection, headache, flushing, and chest pain [270, 278] . moreover, the long-term use of lamivudine induces viral resistance, which leads to a high rate of recurrence post-lt [162] . a number of studies have shown that im hbig has similar kinetics and produces roughly equivalent trough concentrations of anti-hbs compared to iv equivalent doses of hbig but less expensive [279] . the largest reported data of prophylaxis with using of im hbig comes from investigators in australia and new zealand [280] . im 400 or 800 iu hbig daily for 1 week from transplantation and monthly thereafter. lamivudine, 100 mg/day, was administered to candidates waiting for transplantation with hepatitis b surface antigen (hbsag)-positive and continued posttransplantation. thirty-seven patients with positive hbsag (34 patients had hepatitis b, 2 patients had hepatitis b and c, and 1 patient had hepatitis b, c, and d) underwent liver transplantation using this protocol. thirty-six patients were hbv dna positive. the therapy had no significant adverse events and was well tolerated. all patients were hbv dna negative and 31 patients were hbsag negative at latest follow-up. this investigation suggested that low-dose hbig combined with lamivudine prevented recurrence of hepatitis b posttransplantation is more cost-effective. long-term results of this protocol showed that the actual rate of hbv recurrence at 5 years was 4% in 147 hbsag-positive patients underwent liver transplantation [281] . recently, entecavir and tenofovir have been approved as the first-line regimen for the treatment of chronic hepatitis b. these new nas have replaced lamivudine as the prophylaxis of hbv recurrence post lt. according to easl clinical practice guidelines, to achieve the lowest level of hbv dna pre-lt, nas with high barrier to resistance is recommended as pre-transplant antiviral therapy for all hbsag positive patients undergoing liver transplantation [4] . hu et al [282] reported a lower hepatitis b recurrence rate in patients received entecavir than those received lamivudine. a total of 145 patients were administered entecavir combined with lowdose hbig, and 171 patients received lamivudine plus low-dose hbig in the control group. two patients in the entecavir group developed hbv recurrence with no evidence of viral resistance in the median 36 months follow-up time. eleven patients in the lamivudine group developed hbv recurrence, three of whom were proved hbv resistance in the median 77 months follow-up period. further analysis demonstrated that hcc at the time of liver transplantation and low anti-hbs titer post-liver transplantation were independent risk factors for hbv recurrence. perrillo et al. [20] investigated the efficacy of entecavir combined with various hbig regimens after liver transplantation. sixty-one patients with hbv-related liver disease took 1.0 mg/day of entecavir plus various hbig regimens. in the median 72 weeks follow-up period, only 2 patients showed positivity hbsag but hbv dna remained undetected. na et al. [283] showed that 4 of 262 recipients who received entecavir plus hbig experienced hbv recurrence after liver transplantation in the median 49 months follow-up time. among these 4 patients, 3 had received lamivudine followed by entecavir. studies concerning the efficacy of tenofovir in the prophylaxis of hbv reinfection post-lt are limited. jiménez-pérez et al. [284] reported that four patients received tenofovir plus hbig with or without entecavir for the prophylaxis of hepatitis b recurrence. no hepatitis b recurrence was observed in these four patients at 12 months. several researchers have investigated if it was possible to stop hbig after an initial successful prophylaxis with combined hbig/lamivudine. in one largest prospective study, 29 patients who were hbvdna negative before liver transplantation received prophylaxis with hbig/lamivudine for 1 month after transplantation, then they were randomized to continue combination prophylaxis or lamivudine monotherapy [285] . the early results showed that there was no recurrence case in either group by 18 months. however, 15-20% of the patients who were converted to lamivudine monotherapy had viral breakthrough because of lamivudine resistance in longer follow-up [286] . an alternative choice was to change from hbig/lamivudine to a combination of antiviral drugs had a higher barrier of resistance than lamivudine. several studies indicated that this method may be more effective [287, 288] . in a prospective study, 16 of 34 patients receiving prophylaxis with low-dose im hbig/ lamivudine 12 months post-lt were changed to adefovir/lamivudine combination therapy, whereas the other patients continued previous prophylaxis [288] . at a median of 21 months post-switch, patients in both group had no recurrence. one a low titer of hbsag in serum was detected in 1 patient in the adefovir/lamivudine group but hbv dna was negative. this change in therapy improved the life quality of patients and significantly saved the cost. more recently, a multicenter, prospective study demonstrated the results of hbig-sparing regimen combined with lamivudine plus adefovir dipivoxil initiated at the time of waiting for liver transplantation and continued post-transplantation [289] . twenty-six patients were recruited into this study at the time of listing for transplantation. twelve patients had lam exposure before the study, but none had lamivudine resistance. to the 20 patients who underwent transplantation, 800 iu/day of intramuscular hbig was given immediately after transplantation for 7 days. all transplanted patients remained alive without hbv recurrence at a median of 57 months after transplantation. after the completion of this prospective study, the regimen was modified that no perioperative hbig was administered if the pretransplant hbv dna level <3 log(10) iu/ml. another 28 patients with hbv-related liver disease underwent transplantation (18 without hbig). all the patients remained alive without hbv recurrence at a median of 22 months post-transplantation. this study indicated that combination of lamivudine and adefovir initiated at the time of listing for transplantation was safe and effective prevention of hbv recurrent without high costs and long-term hbig therapy. other researchers used posttransplant hbv vaccination for achieving a durable endogenous anti-hbs antibody. two studies reported that 60-80% of patients achieved an anti-hbs titer >10 iu/l following cessation of hbig and immunization with 1-3 courses of recombinant im hbv vaccine [290, 291] . however, other investigations using the same protocol have failed to replicate these results [292, 293] . moreover, the low response (16-62%) was reported in cirrhotic patients awaiting for lt [294] . more recently, di paolo et al investigated the efficacy of 1 year, monthly vaccination together with hbig plus lamivudine in lt patients. one year after vaccination, 44.4% patients maintained anti-hbs titers more than 100 iu/l [295] . these results suggested that hbig can be considered as an additional strategy in the prophylaxis against hbv recurrence post lt: (1) vaccine administration should be long-lasting (e.g. 1 year); (2) passive prophylaxis with hbig should preferably be maintained during the initial phase of vaccination and nas should be maintained during the entire vaccination period. lamivudine is the most widely used na to prevent hepatitis b recurrence. however, lamivudine resistance can result in hepatic decompensation and increases the rate of post-transplant recurrence. newer nas with lower resistance rates should therefore replace lamivudine in hepatitis b prophylaxis. schiff et al [296] investigated the effect of adefovir dipivoxil as the rescue therapy in listing or post-lt patients with chronic hepatitis b and lamivudine-resistance. among listing patients, the percentage of hbv dna levels undetectable at weeks 48 and 96 was 59% and 65%, respectively. after 48 weeks, liver function normalized in 77% and 76% of these patients respectively. and coagulation function normalized in 60% and 84% of these patients respectively. among post-transplantation patients, the percentage of serum hbv dna levels undetectable at weeks 48 and 96 was 40% and 65%, respectively. after 48 weeks, liver function and coagulation function normalized in 51%, 81%, 76%, and 56% of these patients, respectively. among listing patients who underwent liver transplantation, prevention of graft reinfection over a median of 35 weeks was similar among patients who did or did not receive hbig. hbsag was detected on the first test only in 6% and 9% of patients who did or did not receive hbig, respectively. serum hbv dna was detected on follow-up in 6% and 0% of patients who did or did not receive hbig, respectively. adefovir dipivoxilrelated adverse events occurred in 4% of patients and led to drug withdrawal. cumulative resistance rate were 0%, 2%, and 2% at 48, 96, and 144 weeks, respectively. in conclusion, adefovir dipivoxil is safe and effective in prevention of graft reinfection with or without hbig for listing or post-transplant chb patients with lamivudine-resistance. more recently, one study indicated that late hbig replaced by adefovir dipivoxil (at least 12 months post-transplant) can prevent late hbv recurrence at less cost [288] . in a prospective open-labeled study, lamivudine plus adefovir dipivoxil given from the time of listing was well tolerated, prevented lamivudine resistance pre-transplantation and post-transplantation, regardless of the baseline hbv-dna level [289] . the rescue therapy for patients with lamivudine or telbivudine resistance is to add adefovir or tenofovir, or change to tenofovir + emtricitabine. for patients with adefovir resistance, the approach is to add lamivudine or entecavir, or switch to tenofovir + emtricitabine. for patients with entecavir resistance, the approach is to add adefovir or tenofovir. combination therapy is still effective for some patients with multi-antiviral drugs-resistance according to evidence based medicine and clinical practice [297] . regular monitoring and follow-up for patients post lt is also very important. items include liver function, hepatitis b markers, hbv dna quantitative, mutant, blood concentration of immunosuppressive drug and ultrasound examination. for hepatitis b recurrence patients, therapy include: support treatment, hepatocyte protection, anti hbv therapy, immunosuppressant regimen adjustment (withdrawal, reduction or change immunosuppressive agents) and liver retransplant. hepatitis b is a major cause of liver failure in asia, although the use of hbig plus lamivudine can effectively prevent hbv reinfection in liver transplantation, but the cost is high. active immunization approach is still controversial. combined hbig/ nucleos(t)ide prophylaxis should be considered to switch to oral prophylaxis at 1 or 2 years post-lt, particularly in patients with low hbv dna loads before antiviral therapy or hbv dna negative at lt, and in patients with liver failure due to hbv or hdv coinfection, since these patients are at lower risk of recurrence once hbig is ceased. recently, the seminal discovery of sodium taurocholate co-transporting polypeptide (ntcp) as the cellular receptor for hbv entry has opened up many channels of investigation, making hbv entry amenable to therapeutic intervention. several fda approved drugs with ntcp inhibiting activity were tested for their ability to inhibit hbv infection of the cell line [298] [299] [300] . these investigations indicate the possibility of using ntcp inhibitor in the prophylaxis of hepatitis b recurrence post lt. di wu and qin ning both host and viral factors are associated with the chronicity of hbv infection. hbv has a capability of escaping the host immune responses. more importantly, the hbv genome forms a stable minichromosome, namely covalently closed circular dna (cccdna), in the nuclei of infected hepatocytes, enabling hbv to persist its infection [301] . the goal of anti-hbv therapy is to prevent the progression of hbv-related liver disease, which may be achieved initially through sustained immunologic control over hbv, and ultimately, by completely eliminating the virus [4, 302, 303] . however, due to the fact that hbv cccdna persists stably at a very low level even after the loss of hbsag in chronic infected patients, elimination of hbv (complete cure) is rarely achieved. it is suggested that serum hbsag could represent a surrogate marker of intrahepatic cccdna and a marker of host immune control of hbv infection. seroclearance of hbsag is found to be associated with functional remission and improved long-term clinical outcomes in patients with chronic hepatitis b, under this circumstance, even though hbv genome cannot be cleared, the host immune system is in general sufficient to control the few persisting infected hepatocytes [304] [305] [306] . therefore, hbsag seroclearance with or without the appearance of hbsab (functional cure) is considered the ideal endpoint for anti-hbv therapy, representing durable immunologic control over the virus and complete suppression of hbv replication, which is the critical step towards complete cure for hepatitis b [4, 302, 303] . nuc and ifn or its pegylated form, peg-ifn, are the only two types of approved antiviral therapeutics. as the ideal endpoint for anti-hbv treatment, hbsag loss is achieved in very few patients after long-term nuc or 48-week courses of peg-ifn therapy [307] [308] [309] . these current standard antiviral therapies can only suppress the hbv replication and viral protein production, but cannot eliminate hbv cccdna and cure chronic hbv infection. therefore, new treatment approaches such as optimal combination therapy with the approved antivirals or emerging novel therapeutics are needed to improve rates of hbsag loss and, ideally, hbsag seroconversion. different characteristics, mechanisms of action and antiviral activities of nuc and ifn provide the possibility of combining these two types of agents for improving chances of sustained post-treatment response, thereby allowing the discontinuation of nuc without virus relapse, through harnessing both immunomodulatory and direct antiviral mechanisms [310, 311] . according to the updated chinese guidelines, asian-pacific guidelines, as well as european guidelines for the treatment of chronic hepatitis b, sequential therapy with additional peg-ifn or switching to peg-ifn can be considered in chb patients who have achieved virological remission by long-term nuc treatment [312] , though clinical trials evaluating either simultaneous or sequential combination therapy with nuc and ifn for chb patients drew different conclusions. several previous studies exploring the efficacy of simultaneous combination with peg-ifn and lam or adv have demonstrated that the therapeutic strategy led to higher rates of virological response during treatment, but did not improve durable post-treatment responses [308, 309, 313, 314 ]. an exploratory study showed that combination treatment with peg-ifn plus adv for 48 weeks led to remarkable decline in serum hbv dna level and intrahepatic cccdna, which was significantly correlated with reduced serum hbsag [315] . a recent study evaluating the efficacy of combination therapy with ldt and peg-ifn in hbeag-positive chb patients have demonstrated that the combination therapy led to greater reductions in hbv dna and hbsag levels, however, it may contribute to an increased risk of unexpected severe peripheral neuropathy, combination therapy with ldt and peg-ifn should not be used [316] . in a prospective, active-controlled randomized trial evaluated loss of hbsag in patients receiving the combination of tdf and peg-ifn for a finite duration, chb patients were randomly assigned to receive combination therapy for 48 weeks, combination therapy for 16 weeks followed by tdf for 32 weeks, tdf for 120 weeks, or peg-ifn for 48 weeks. the study demonstrated that, 9.1% of patients receiving 48-week course of combination therapy with tdf and peg-ifn had hbsag loss, which was significantly higher than those receiving tdf or peg-ifn given alone [317] . however, it is worth noting that a prolonged followup of these subgroups of patients is required to determine the durability of treatment response and long-term benefits. although simultaneous combination of peg-ifn and nuc other than tdf may not improve sustained response rate, the optimal approach for combination treatment remains to be determine and should take into consideration the time schedule of drug administration. late breaking clinical trials have demonstrated that sequential combination therapy with nuc and ifn, either "switch" or "add-on", showed more promising results, with higher probabilities of hbeag seroconversion and hbsag loss than nuc monotherapy. an observation study has shown that the add-on of peg-ifn to a stable nuc therapy in chb patients with suppression of hbv dna, induced hbsag seroconversion in 2 out of 12 patients [318] . a prospective study demonstrated that additional of peg-ifn to a long-term nuc treatment in hbeag-negative patients with undetectable hbv dna, led to a durable hbsag loss in 6 out of 10 patients [319] . a global multicentered, randomized controlled trial (ares study) assessed the effectiveness of add-on peg-ifn to etv therapy in hbeag positive patients, compared to etv monotherapy, 24 weeks of peg-ifn add-on therapy did not improve response rates (defined as hbeag loss with hbv dna <200 iu/ml at week 48), but led to greater viral decline and appeared to prevent relapse after stopping etv, which may facilitate the discontinuation of nuc treatment [320] . another randomized controlled trial has shown that neither etv pretreatment nor etv add-on to peg-ifn demonstrated superiority in sustained posttreatment response compared with 48 weeks of peg-ifn alfa-2a monotherapy in treatmentnaive hbeag-positive patients [321] . a prospective, randomized controlled trial (osst study) reported that switching to 48-week course of peg-ifn in hbeag positive chb patients who achieved virologic remission after long-term etv treatment led to significantly increased rates of hbeag seroconversion and hbsag loss (8.5%) [322] . data from 1-year follow-up of these patients who received sequential therapy showed that rates of hbeag seroconversion increased from 17.7% at the end of treatment to 38.7% at 1-year post-treatment, besides, hbsag loss was durable in 6 of 7 patients [323] . these results are in consistent with findings from earlier studies exploring sequential combination therapy with nuc and ifn but only tested in a small number of patients [324, 325] . an exploratory study assessed the efficacy of sequential therapy in genotypes a, b, c and e chb patients with high hbv viremia, patients receiving etv alone for 12 weeks, followed by etv plus peg-ifn for 12 weeks, lastly only peg-ifn for 36 weeks achieved significantly higher rates of hbeag and hbsag seroconversion than those receiving peg-ifn monotherapy for 48 weeks [326] . an interim analysis from new switch study demonstrated that sequential combination therapy with etv and peg-ifn for 48 weeks in nuc-experienced hbeag positive chb patients who achieved partial responses, with hbv dna suppression and hbeag loss, led to a high rate of hbsag loss (17.3%) [327] . accumulating evidences suggest that quantitative hbsag (qhbsag) is a useful marker for guiding treatment decision, e.g. individualizing the treatment, implementing stopping rules for ending or extending ifn treatment [328] . recently, several studies identified that hbsag loss occurred in patients with a low baseline qhbsag and high on-treatment reduction, therefore, a baseline or response-guided approach based on hbsag kinetics may help identify chb patients with the greatest chance of benefit. the osst study has demonstrated that patients undergoing long-term etv treatment with low hbsag titers (<1500 iu/ml) and hbeag loss were suitable for sequential therapy with peg-ifn as they had a good chance of both hbsag loss (22.2%) and hbeag seroconversion (33.3%). patients whose hbsag levels declined to 200 iu/ml at week 12 of sequential therapy have the greatest chance of achieving hbsag loss. while, patients whose hbsag levels were >1500 iu/ml at week 12 might consider stopping peg-ifn treatment as they had a minimal chance of achieving hbeag seroconversion and hbsag loss [322] . these findings are consistent with results from previous studies and interim analyses from the new switch study, suggesting that qhbsag identifies nuc-treated patients who are the best candidates for sequential therapy, and allows response-guided treatment [327] [328] [329] . however, given the small number of patients included in the exploratory analyses, these results need to be interpreted cautiously and warrant further investigation and validation. differences in study designs and characteristics of patients make it difficult to determine the optimal combination therapy with nuc and peg-ifn for chb patients at this stage. nevertheless, we could speculate that once suppression of hbv viremia has been achieved by pretreatment with nuc, the additional use of peg-ifn would be more beneficial. these new therapeutic strategies require further investigation before being introduced into routine clinical practice. complete cure of hbv infection depends not only on the deep suppression of hbv replication, but also on the induction of durable antiviral immune response [330] . besides the approved therapeutics, several novel therapeutic approaches including direct acting antivirals (daa) targeting different stages of the life cycle of hbv (including hbv entry, hbv genome processing, virus protein assembly, etc.) as well as immunological approaches are currently under early stage of preclinical or clinical investigation, these promising therapeutics may act in a synergistic way with currently available antiviral agents and have the potential to achieve a cure of hbv infection. specific inhibition of hbv entry may be a promising therapeutic concept to control hbv infection. a currently identified cellular receptor for hbv entry, the sodium taurocholate co-transporting polypeptide (ntcp), is an emerging target providing new research possibilities and allowing the development of hbv entry inhibitors [331] . cyclosporine a (csa) can interfere with the binding between large envelope protein and ntcp, and thus prevent hbv entry into cultured hepatocytes [298, 332] . myrcludex-b also can inhibit the binding of the hbv envelope proteins to ntcp, blocking the hbv/hepatitis d virus (hdv)'s entry, which is now under clinical development [333] . however, these entry inhibitors can prevent new hbv infection [334, 335] , but do not directly target on cccdna or eliminate the preexisting hbv infection. therefore, antiviral strategies combining entry inhibitors with anti-hbv agents might be superior to their use as monotherapy by taking advantage of synergy. therapeutic approaches targeting cccdna for hbv cure aim to directly degrade or alternatively block cccdna formation, or silence cccdna transcription. rnaguided nucleases, such as the clustered regularly interspaced short palindromic repeats (crispr)/cas9 [336, 337] , might be the most promising strategy to target cccdna. however, the risk of undesired off-target mutagenesis and delivery constitute the major limits. histone modifications, e.g. inhibitors of histone acetyltransferase, offer great potential as therapeutic candidates for chb patients through transcriptional silencing of cccdna [301, 338] . activation of ifn-a and lymphotoxin beta receptor (ltβr) has been shown to induce cytidine deaminases of the apobec3 family, triggering degradation of cccdna in hbv cell culture model systems. these novel strategies will make elimination of hbv a real possibility [339] . several attempts have been made to develop capsid assembly modulators/core inhibitors, which can be divided into two main classes. the first class, including phenylpropenamides (ppas) and sulfamoylbenzamide derivatives, e.g. at-61 and at-130, can inhibit the entry of pregenomic rna (pgrna) into the immature nucleocapsid [340, 341] resulting in nucleocapsid with normal size and geometry but empty of nucleic acid. the other class, including heteroaryldihydropyrimidines (haps) antiviral compounds, e.g. bay41-4109 [342] and nvr 3-778, can directly inhibit the nucleocapsid formation, resulting in virus particles with abnormal size and structure. ppas and haps show synergy in vitro with nucleoside reverse transcriptase inhibitors (nrtis) and overcome resistance to nrti [340, 343] , highlighting the value for developing combination therapy. post-transcriptional gene silencing by rna interference (rnai), is a new therapeutic approach to hepatitis b [344, 345] . inhibiting protein production by targeting hbv messenger rna (mrna) for translational repression or degradation can impair virus replication and augment the hbv-specific immune response. several rnai-based drug candidates have currently entered early-phase clinical development for the treatment of chb, including aln-hbv and tkm-hbv [346, 347] , showing clinical efficacy significant declines in hbv dna, hbsag, and cccdna levels. despite lingering concerns about delivery, the risk of resistance and safety, the rnai-based therapeutics might be promising when combined with other antiviral agents. future trials with rnai-based therapeutics in combination with ifn or other antivirals are required to determine whether these agents would act synergistically to reduce viral antigen production, activate and restore the host immune responses, and subsequently eliminate hbv infection. hbsag production and secretion is capable of altering the host immune response by inducing t cell exhaustion and tolerance to hbv, which partially mediate hbv persistence. control of hbsag secretion may help restore t cell function, suggesting the possibility of developing anti-hbv treatments targeting hbsag production and release. nucleic acid polymer (nap) could prevent hbsag release from infected hepatocytes, leading to a restoration of the immune response. newly developed hbsag release inhibitors, e.g., rep 2139 and rep 2165 [348] [349] [350] , appear potent in preventing the release of hbsag in humans and thereby reducing serum hbsag levels and also potentially promoting surface antibody seroconversion. however, it remains to be seen whether these compounds may cause detrimental intrahepatocyte accumulation of hbsag. emerging exciting advances have also led to new promising approaches to attenuate hbv-induced immune impairment, such as toll like receptor (tlr) agonists [351, 352] , pleiotropic cytokines [353] [354] [355] , programmed cell death-1 (pd-1) and its ligand pd-l1 blockages [356] , therapeutic vaccines [357, 358] , etc. tlr agonists can activate intracellular innate pathways and stimulate both innate and adaptive immune responses. a recently developed oral active agonist of tlr-7, gs-9620, has been shown to enhance ifn-a and isg expression and activate nk cells, t cells and b cells in animal studies [351, 352] , however, early human studies have shown limited efficacy of the tlr-7 agonist at tolerated doses, and further research into this tlr7 agonist was subsequently discontinued. therapeutic cytokines play critical roles in the control of hbv infection and mediate a non-cytolytic clearance of the virus [353] [354] [355] . several studies investigated the antiviral activities and therapeutic potential of cytokines including granulocyte-macrophage colony-stimulating factor (gmcsf), il-2, il-7, etc. a previous study has shown that hbsag vaccine in combination with lam or il-2 could induce antiviral immune response and consequently elimination of hbv may be achieved in chb patients [353] . a prospective study investigated whether additional gmcsf could enhanced the immunomodulatory effect of ifn, demonstrating that the combination treatment with gmcsf and ifn was effective in patients who had previously failed ifn monotherapy [354] . a recent prospective, randomized controlled trial (anchor study) evaluated whether sequential combination therapy with nuc, peg-ifn and gmcsf could induce hbsag loss in chb patients treated with long-term nuc and demonstrated that for patients who achieved virological suppression with nuc, this sequential combination therapy significantly increases rates of hbsag loss and hbsab appearance [359] . the difficulties in eliminating cccdna and breaking the immune tolerance constitute the major obstacles for a cure of hbv infection. combination of potent daa with immunotherapeutic approach may help overcome both persistence of cccdna and immune escape, creating synergies, reducing resistance and creating the potential for durable and sustained post-treatment virological and serologic responses. theoretically, nuc treatment may suppress viral replication, partially restore function in exhausted t cells and allow hbv-specific t cells to be more receptive to peg-ifn treatment. although at this stage which agents or which combination may be preferable remains to be determined, clinical trials involving sequential combination therapy with nuc and peg-ifn, either "switch" or "add-on", have shown statistically significant decline in hbsag levels on treatment and high rates of sustained post-treatment serologic response [360, 361] . combination therapy with novel daa and immunotherapeutic approach may hold promise to overcome both cccdna persistence and immune escape, representing a critical step towards hbv cure. several large prospective trials (e.g. cost and ocean study) investigating the effectiveness and long-term benefit of sequential combination treatment with nuc and peg-ifn are currently being carried out. chinese society of infectious diseases, chinese medical association, severe liver diseases and artificial liver group, chinese society of hepatology, chinese medical association. guideline for diagnosis and treatment of liver failure treatment of chronic hepatitis b: update of the recommendations from the european association for the study of the liver. easl 2017 clinical practice guidelines on the management of hepatitis b virus infection asian-pacific clinical practice guidelines on the management of hepatitis b: a 2015 update update on prevention, diagnosis, and treatment of chronic hepatitis b: aasld 2018 hepatitis b guidance treatment of chronic hepatitis b: evolution over two decades world health organization. guidelines for the prevention, care and treatment of persons with chronic hepatitis b infection committee of experts on antiviral therapy for special patients with chronic hepatitis b. [expert consensus on antiviral therapy for special patients with chronic hepatitis b: an update in clinical and virological factors associated with hepatitis b virus reactivation in hbsagnegative and anti-hbc antibodies-positive patients undergoing chemotherapy and/or autologous stem cell transplantation for cancer systematic review and meta-analysis of interferon after curative treatment of hepatocellular carcinoma in patients with viral hepatitis a systematic review and metaanalysis of adjuvant interferon therapy after curative treatment for patients with viral hepatitis-related hepatocellular carcinoma efficacy of antiviral therapy with lamivudine after initial treatment for hepatitis b virus-related hepatocellular carcinoma prophylactic lamivudine administration prevents exacerbation of liver damage in hbe antigen positive patients with hepatocellular carcinoma undergoing transhepatic arterial infusion chemotherapy fait (fu arterial infusion and interferon therapy) for hepatocellular carcinoma hepatitis b immunoglobulin and/or nucleos(t)ide analogues for prophylaxis against hepatitis b virus recurrence after liver transplantation: a systematic review high genetic barrier nucleos(t)ide analogue(s) for prophylaxis from hepatitis b virus recurrence after liver transplantation: a systematic review oral nucleoside/nucleotide analogs without hepatitis b immune globulin after liver transplantation for hepatitis b entecavir monotherapy is effective in suppressing hepatitis b virus after liver transplantation entecavir and hepatitis b immune globulin in patients undergoing liver transplantation for chronic hepatitis b antibodies to hepatitis b surface antigen prevent viral reactivation in recipients of liver grafts from anti-hbc positive donors comparison of different immunoprophylaxis regimens after liver transplantation with hepatitis b core antibody-positive donors: a systematic review preoperative antiviral treatment and postoperative prophylaxis in hbv-dna positive patients undergoing liver transplantation randomized trial of emtricitabine/tenofovir disoproxil fumarate after hepatitis b immunoglobulin withdrawal after liver transplantation management of the hepatitis b virus in the liver transplantation setting: a european and an american perspective is hepatitis b immunoglobulin necessary in prophylaxis of hepatitis b recurrence after liver transplantation? a meta-analysis prophylaxis against recurrence of hepatitis b virus after liver transplantation: a retrospective analysis spanning 20 years treatment of children with hbeag-positive chronic hepatitis b: a systematic review and meta-analysis treatment of children with chronic hepatitis b virus infection in the united states: patient selection and therapeutic options clinical trial of lamivudine in children with chronic hepatitis b chronic hepatitis b virus infection in children and adolescents in japan chronic hepatitis b in children and adolescents long-term lamivudine therapy for children with hbeag-positive chronic hepatitis b evaluation and management of hepatitis b in pregnancy: a survey of current practices hepatitis b virus and human immunodeficiency virus drugs in pregnancy: findings from the antiretroviral pregnancy registry management of chronic hepatitis b in pregnancy virologic responses to add-on adefovir dipivoxil treatment versus entecavir monotherapy in children with lamivudine-resistant chronic hepatitis b management of chronic hepatitis b in children: an unresolved issue the effects of telbivudine in late pregnancy to prevent intrauterine transmission of the hepatitis b virus: a systematic review and meta-analysis antiviral treatment among pregnant women with chronic hepatitis b a prospective and open-label study for the efficacy and safety of telbivudine in pregnancy for the prevention of perinatal transmission of hepatitis b virus infection clinical course and management of acute and chronic viral hepatitis during pregnancy hbv treatment and pregnancy management of hepatitis b in pregnancy: weighing the options motherto-infant transmission of hepatitis b virus infection: significance of maternal viral load and strategies for intervention hepatitis b and c virus coinfection: a novel model system reveals the absence of direct viral interference hepatitis b virus/hepatitis c virus coinfection: epidemiology, clinical features, viral interactions and treatment hepatitis b reactivation during successful treatment of hepatitis c with sofosbuvir and simeprevir clinical outcomes of hepatitis b virus coinfection in a united states cohort of hepatitis c virus-infected patients treatment of patients with dual hepatitis c virus and hepatitis b virus infection: resolved and unresolved issues updates on the treatment and outcomes of dual chronic hepatitis c and b virus infection dual chronic hepatitis b virus and hepatitis c virus infection clinical characteristics and current management of hepatitis b and c in china resistance patterns and response to entecavir intensification among hiv-hbv-coinfected adults with persistent hbv viremia patterns and causes of suboptimal response to tenofovir-based therapy in individuals coinfected with hiv and hepatitis b virus management and treatment of chronic hepatitis b virus infection in hiv positive and negative patients: the epib 2008 study strategies for managing coinfection with hepatitis b virus and hiv management and treatment of chronic hepatitis b in hiv-positive patients hepatitis b and human immunodeficiency virus coinfection hepatitis b virus (hbv) coinfection accelerates immunologic progression in patients with primary hiv infection in an area of hyperendemicity for hbv infection complete remission of nephrotic syndrome of hepatitis b virus-associated membranous glomerulopathy after lamivudine monotherapy hepatitis b virus-related membranous nephropathy treated with entecavir successful treatment of hepatitis b virus-associated membranous nephropathy with entecavir and immunosuppressive agents successful treatment of hepatitis b virus-associated membranous nephropathy with lamivudine complete remission of hepatitis b virus-associated nephrotic syndrome from iga nephropathy following peginterferon therapy treatment of hepatitis b virus-associated membranous nephritis patients in chinese: an open parallel controlled trial the efficacy of anti-viral therapy on hepatitis b virus-associated glomerulonephritis: a systematic review and meta-analysis preemptive adefovir versus lamivudine for prevention of hepatitis b reactivation in chronic hepatitis b patients undergoing chemotherapy prevention of acute exacerbation of chronic hepatitis b infection in cancer patients receiving chemotherapy in a hepatitis b virus endemic area entecavir vs lamivudine for prevention of hepatitis b virus reactivation among patients with untreated diffuse large b-cell lymphoma receiving r-chop chemotherapy: a randomized clinical trial systematic review: the effect of preventive lamivudine on hepatitis b reactivation during chemotherapy management of the hbv reactivation in isolated hbcab positive patients affected with non hodgkin lymphoma hepatitis b virus reactivation associated with anti-neoplastic therapy thyroid abnormalities in chronic viral hepatitis and their relationship to interferon alfa therapy can pegylated interferon alpha 2a cause development of thyroid disorders in patients with chronic hepatitis b? cases of interferon-alpha and interferon-beta-induced thyroiditis prevalence of thyroid autoantibodies in hepatitis c and hepatitis b infection in china chronic hepatitis b: preventing, detecting, and managing viral resistance antiviral drug-resistant hbv: standardization of nomenclature and assays and recommendations for management report of an international workshop: roadmap for management of patients receiving oral therapy for chronic hepatitis b adefovir rapidly suppresses hepatitis b in hbeag-negative patients developing genotypic resistance to lamivudine apoptosis: a mechanism of acute and chronic liver injury the role of lamivudine and predictors of mortality in severe flare-up of chronic hepatitis b with jaundice lamivudine treatment for fulminant hepatic failure due to acute exacerbation of chronic hepatitis b infection lamivudine monotherapy for spontaneous severe acute exacerbation of chronic hepatitis b lamivudine treatment for decompensated cirrhosis resulting from chronic hepatitis b the effect of lamivudine therapy in hepatic decompensation during acute exacerbation of chronic hepatitis b severe acute exacerbation of chronic hepatitis b: a unique presentation of a common disease tenofovir improves the outcome in patients with spontaneous reactivation of hepatitis b presenting as acute-on-chronic liver failure entecavir treatment prevents disease progression in hepatitis b virus-related acute-on-chronic liver failure: establishment of a novel logistical regression model management of severe acute to fulminant hepatitis b: to treat or not to treat or when to treat? organization committee of 13th asia-pacific congress of clinical microbiology and infection. microbiology and infection consensus guidelines for diagnosis and treatment of liver failure acute-on-chronic liver failure: consensus recommendations of the asian pacific association for the study of the liver (apasl) acute liver failure related to hepatitis b virus etiological analysis of 1977 patients with acute liver failure, subacute liver failure and acute-on-chronic liver failure analysis of prognostic factors for patients with acute-onchronic liver failure aasld position paper: the management of acute liver failure: update potent antiviral therapy improves survival in acute on chronic liver failure due to hepatitis b virus reactivation association between hbv genotype and chronic/ severe liver disease with hbv infection in chinese patients t1846 and a/g1913 are associated with acute on chronic liver failure in patients infected with hepatitis b virus genotypes b and c hepatitis b virus genotype and basal core promoter/precore mutations are associated with hepatitis b-related acute-on-chronic liver failure without pre-existing liver cirrhosis distribution of hepatitis b viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from chiba hbeag negative variants and their role in the natural history of chronic hepatitis b virus infection acute-on chronic liver failure: current concepts on definition, pathogenesis, pathogenesis, clinical manifestations and potential therapeutic interventions hepatitis b flares in chronic hepatitis b: pathogenesis, natural course, and management serology of acute exacerbation in chronic hepatitis b virus infection intrahepatic distribution of hbsag and hbcag in chronic hepatitis b virus infection: hepatocyte with cytoplasmic/membranous hbcag as a possible target for immune hepatocytolysis hla class i antigen display on hepatocyte membrane in chronic hepatitis b virus infection: its role in the pathogenesis of chronic type b hepatitis hepatitis flares and hepatitis b e antigen seroconversion: implication in anti-hepatitis b virus therapy pathogenesis and clinical significance of spontaneous exacerbation and remissions in chronic hbv infection safety and efficacy of lamivudine in patients with severe acute or fulminant hepatitis b, a multicenter experience hepatitis b-associated acute liver failure: immediate treatment with entecavir inhibits hepatitis b virus replication and potentially its sequelae survival and prognostic factors in hepatitis b virus-related acute-on-chronic liver failure clinical profile and predictors of mortality in patients of acute-on-chronic liver failure hepatitis e virus as an etiology of acute exacerbation of previously unrecognized asymptomatic patients with hepatitis b virus-related chronic liver disease lamivudine and entecavir significantly improved the prognosis of early-to-mid stage hepatitis b related acute on chronic liver failure what meld score mandates use of entecavir for aclf-hbv hbeag-negative patients? survival analysis of 190 patients with hbv-related acuteon-chronic liver failure does antiviral therapy reduce complications of cirrhosis? hepatitis b-associated fibrosis and fibrosis/cirrhosis regression with nucleoside and nucleotide analogs lamivudine treatment in patients with severely decompensated cirrhosis due to replicating hepatitis b infection lamivudine for patients with chronic hepatitis b and advanced liver disease determinants of early mortality in patients with decompensated chronic hepatitis b treated with antiviral therapy the role of lamivudine and predictors of mortality in severe flare-up of chronic hepatitis b with jaundice meta-analysis of the short-term effects of lamivudine treatment for severe chronic hepatitis b lamivudine monotherapy for spontaneous severe acute exacerbation of chronic hepatitis b adefovir dipivoxil therapy for lamivudine resistant hepatitis b in pre-and post-liver transplantation patients long-term therapy with adefovir dipivoxil for hbeag negative chronic hepatitis b for up to 5 years a comparison of entecavir and lamivudine for hbeagpositive chronic hepatitis b entecavir versus lamivudine for patients with hbeagnegative chronic hepatitis b entecavir versus lamivudine therapy for patients with chronic hepatitis b-associated liver failure: a meta-analysis entecavir vs. lamivudine in chronic hepatitis b patients with severe acute exacerbation and hepatic decompensation the pros and cons of lamivudine vs. entecavir in decompensated or severe acute exacerbation of chronic hepatitis b efficacy and safety of telbivudine therapy in liver failure patients with chronic hepatitis b virus infection randomized clinical trial: efficacy and safety of telbivudine and lamivudine in treatment-naïve patients with hbv-related decompensated cirrhosis nucleoside analogues improve the short-term and long-term prognosis of patients with hepatitis b virus-related acute-on-chronic liver failure the study of efficacy of lamivudine in patients with severe acute hepatitis b hbv-dna suppression and disease course in hbv cirrhosis patients on long-term lamivudine therapy clinical outcome of hbeag-negative chronic hepatitis b in relation to virological response to lamivudine long-term continuous entecavir therapy in nucleos(t) ide-naïve chronic hepatitis b patients update in the management of chronic hepatitis b asian-pacific consensus statement on the management of chronic hepatitis b: a 2012 update a treatment algorithm for the management of chronic hepatitis b virus infection in the united states: 2008 update risk of hepatocellular carcinoma across a biological gradient of serum hepatitis b virus dna level united states lamivudine compassionate use study group. a united states compassionate use study of lamivudine treatment in nontransplantation candidates with decompensated hepatitis b virus-related cirrhosis a meta-analysis of nucleos(t)ide analogues in patients with decompensated cirrhosis due to hepatitis b resistance to adefovir dipivoxil in lamivudine resistant chronic hepatitis b patients treated with adefovir dipivoxil outcome of adefovir add-on lamivudine rescue therapy of up to 5 years in patients with lamivudine-resistant chronic hepatitis b rescue therapy with adefovir in decompensated liver cirrhosis patients with lamivudine-resistant hepatitis b virus globe study group. 2-year globe trial results: telbivudine is superior to lamivudine in patients with chronic hepatitis b telbivudine for the management of chronic hepatitis b virus infection the 104-week efficacy and safety of telbivudine-based optimization strategy in chronic hepatitis b patients: a randomized, controlled study efficacy and safety of entecavir compared to lamivudine in nucleoside-naïve patients with chronic hepatitis b: a randomized double-blind trial in china efficacy of entecavir in treatment-naïve patients with hepatitis b virus-related decompensated cirrhosis virgil surveillance study group. virological response to entecavir is associated with a better clinical outcome in chronic hepatitis b patients with cirrhosis regression of cirrhosis during treatment with tenofovir disoproxil fumarate for chronic hepatitis b: a 5-year open-label follow-up study tenofovir disoproxil fumarate (tdf), emtricitabine/tdf, and entecavir in patients with decompensated chronic hepatitis b liver disease long-term safety of lamivudine treatment in patients with chronic hepatitis b lamivudine compared with lamivudine and adefovir dipivoxil for the treatment of hbeag-positive chronic hepatitis b response-guided treatment of cirrhotic chronic hepatitis b patients: multicenter prospective study safety and antiviral activity of emtricitabine (ftc) for the treatment of chronic hepatitis b infection: a two-year study randomized comparison of tenofovir disoproxil fumarate vs emtricitabine and tenofovir disoproxil fumarate in patients with lamivudine-resistant chronic hepatitis b long-term efficacy and safety of emtricitabine plus tenofovir df vs. tenofovir df monotherapy in adefovir-experienced chronic hepatitis b patients medication nonadherence with long-term management of patients with hepatitis b e antigen-negative chronic hepatitis b virological breakthrough and resistance in patients with chronic hepatitis b receiving nucleos(t)ide analogues in clinical practice kinetics of hepatitis b surface antigen decline during 3 years of telbivudine treatment in hepatitis b e antigen-positive patients extended lamivudine treatment in patients with chronic hepatitis b enhances hepatitis b e antigen seroconversion rates: results after 3 years of therapy telbivudine prophylaxis for hepatitis b virus recurrence after liver transplantation improves renal function seven-year efficacy and safety of treatment with tenofovir disoproxil fumarate for chronic hepatitis b virus infection long-term efficacy and safety of adefovir dipivoxil for the treatment of hepatitis b e antigen-positive chronic hepatitis b low resistance to adefovir combined with lamivudine: a 3-year study of 145 lamivudine-resistant hepatitis b patients entecavir therapy for up to 96 weeks in patients with hbeag-positive chronic hepatitis b globe study group. telbivudine versus lamivudine in patients with chronic hepatitis b cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012 hepatocellular carcinoma epidemiology the global health burden of infection-associated cancers in the year 2002 epidemiology and prevention of hepatocellular carcinoma past hbv viral load as predictor of mortality and morbidity from hcc and chronic liver disease in a prospective study chronic hepatitis b guideline working party of the asian-pacific association for the study of the liver. asian-pacific consensus statement on the management of chronic hepatitis b: a 2008 update european association for the study of the liver. easl clinical practice guidelines: management of chronic hepatitis b chronic hepatitis b: update 2009 hepatitis b virus-related hepatocellular carcinoma: epidemiology and pathogenic role of viral factors hepatocellular carcinoma in the woodchuck model of hepatitis b virus infection molecular bases for the development of hepatitis b virus (hbv)-related hepatocellular carcinoma (hcc) evidence for increased in vitro recombination with insertion of human hepatitis b virus dna characterization of integration patterns and flanking cellular sequences of hepatitis b virus in childhood hepatocellular carcinomas hepatitis b virus x protein induces the expression of mta1 and hdac1, which enhances hypoxia signaling in hepatocellular carcinoma cells role of hypoxia-inducible factor-alpha in hepatitis-b-virus x protein-mediated mdr1 activation hbxip, cellular target of hepatitis b virus oncoprotein, is a regulator of centrosome dynamics and cytokinesis specific mutations of hepatitis b virus in plasma predict liver cancer development genetics of hepatocellular tumors dysregulation of growth factor signaling in human hepatocellular carcinoma hbx inhibits the growth of ccl13-hbx-stable cells via the gsk-3beta/beta-catenin cascade mutations in the c-terminus of the x protein of hepatitis b virus regulate wnt-5a expression in hepatoma huh7 cells: cdna microarray and proteomic analyses biology of transforming growth factor beta in hepatocarcinogenesis targeting transforming growth factor (tgf)-betari inhibits activation of beta1 integrin and blocks vascular invasion in hepatocellular carcinoma hepatitis b virus x protein overcomes oncogenic ras-induced senescence in human immortalized cells the phosphatidylinositol 3-kinase akt pathway in human cancer hepatocytespecific pten deficiency results in steatohepatitis and hepatocellular carcinomas signaling through the jak/stat pathway, recent advances and future challenges activation of stat proteins and growth control mitotic aberration coupled with centrosome amplification is induced by hepatitis b virus x oncoprotein via the ras-mitogenactivated protein/extracellular signal-regulated kinase-mitogen-activated protein pathway ubiquitous activation of ras and jak/stat pathways in human hcc protein kinase c and other diacylglycerol effectors in cancer overexpression of protein kinase c alpha mrna in human hepatocellular carcinoma: a potential marker of disease prognosis downregulation of fgl2/prothrombinase delays hcclm6 xenograft tumour growth and decreases tumour angiogenesis fibrinogen-like protein 2/fibroleukin prothrombinase contributes to tumor hypercoagulability via il-2 and ifn-gamma taiwan hepatoma study group. decreased incidence of hepatocellular carcinoma in hepatitis b vaccinees: a 20-year follow-up study meta-analysis: treatment of hepatitis b infection reduces risk of hepatocellular carcinoma hepatitis b virus genotype and dna levels and hepatocellular carcinoma: a prospective study in men antifibrogenic effect of ifn-alpha2b on hepatic stellate cell activation by human hepatocytes rat liver fibrosis regresses better with pegylated interferon alpha-2b and ursodeoxycholic acid treatment than spontaneously recovery revisiting the treatment recommendations for chronic hepatitis b treatment recommendations for chronic hepatitis b: an evaluation of current guidelines based on a natural history study in the united states prognostic determinants for chronic hepatitis b in asians: therapeutic implications normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective cohort study epidemiology of viral hepatitis and hepatocellular carcinoma expert consensus on antiviral therapy to treat hepatitis b/c virus-related hepatocellular carcinoma a comparative study of antiviral therapy after resection of hepatocellular carcinoma in the immune-active phase of hepatitis b virus infection effect of viral status on recurrence after liver resection for patients with hepatitis b virus-related hepatocellular carcinoma virologic and biochemical changes and prognosis after liver resection for hepatitis b virus-related hepatocellular carcinoma persistent hepatitis b viral replication affects recurrence of hepatocellular carcinoma after curative resection sustained low hepatitis b viral load predicts good outcome after curative resection in patients with hepatocellular carcinoma positive serum hepatitis b e antigen is associated with higher risk of early recurrence and poorer survival in patients after curative resection of hepatitis b-related hepatocellular carcinoma interferon-α therapy after curative resection prevents early recurrence and improves survival in patients with hepatitis b virus-related hepatocellular carcinoma postoperative interferon alpha treatment postponed recurrence and improved os in patients after curative resection of hbv-related hepatocellular carcinoma: a randomized clinical trial interferon lowers tumor recurrence rate after surgical resection or ablation of hepatocellular carcinoma: a pilot study of patients with hepatitis b virus-related cirrhosis consistently low hepatitis b virus dna saves patients from hepatocellular carcinogenesis in hbv-related cirrhosis. a nested case-control study using 96 untreated patients hepatitis b viremia is associated with increased risk of hepatocellular carcinoma in chronic carriers human lymphoblastoid interferon. in vitro and in vivo studies in hepatocellular carcinoma interferon beta prevents recurrence of hepatocellular carcinoma after complete resection or ablation of the primary tumor-a prospective randomized study of hepatitis c virus-related liver cancer meta-analysis: the efficacy of anti-viral therapy in prevention of recurrence after curative treatment of chronic hepatitis b-related hepatocellular carcinoma negative hepatitis b envelope antigen predicts intrahepatic recurrence in hepatitis b virus-related hepatocellular carcinoma after ablation therapy high serum hyaluronic acid and hbv viral load are main prognostic factors of local recurrence after complete radiofrequency ablation of hepatitis b-related small hepatocellular carcinoma influence of serum hbv dna load on recurrence of hepatocellular carcinoma after treatment with percutaneous radiofrequency ablation prospective randomized controlled study of interferon-alpha in preventing hepatocellular carcinoma recurrence after medical ablation therapy for primary tumors safety and efficacy of lamivudine after radiofrequency ablation in patients with hepatitis b virus-related hepatocellular carcinoma reactivation of hepatitis b virus replication in patients receiving cytotoxic therapy frequency of hepatitis b virus reactivation in cancer patients undergoing cytotoxic chemotherapy: a prospective study of 626 patients with identification of risk factors lamivudine for the treatment of hepatitis b virus reactivation following chemotherapy for non-hodgkin's lymphoma prophylactic lamivudine prevents hepatitis b reactivation in chemotherapy patients preemptive use of interferon or lamivudine for hepatitis b reactivation in patients with aggressive lymphoma receiving chemotherapy hepatitis b virus reactivation in breast cancer patients undergoing cytotoxic chemotherapy and the role of preemptive lamivudine administration a randomized controlled study of preemptive lamivudine in patients receiving transarterial chemolipiodolization comparative efficacy of postoperative transarterial chemoembolization with or without antiviral therapy for hepatitis b virus-related hepatocellular carcinoma hepatitis b virus reactivation after three-dimensional conformal radiotherapy in patients with hepatitis b virus-related hepatocellular carcinoma risk factors for hepatitis b virus reactivation after conformal radiotherapy in patients with hepatocellular carcinoma recurrence of hepatocellular carcinoma practice guidelines committee hepatitis viruses and liver transplantation hepatitis b: progress in the last 15 years impact of virologic breakthrough and hbig regimen on hepatitis b recurrence after liver transplantation prevention of hepatitis b virus recurrence after liver transplantation in cirrhotic patients treated with lamivudine and passive immunoprophylaxis prophylaxis against hepatitis b recurrence following liver transplantation using combination lamivudine and hepatitis b immune globulin lamivudine and low-dose hepatitis b immune globulin for prophylaxis of hepatitis b reinfection after liver transplantation possible role of mutations in the ymdd motif prior to transplantation as a risk factor for reinfection an efficacy and cost-effectiveness analysis of combination hepatitis b immune globulin and lamivudine to prevent recurrent hepatitis b after orthotopic liver transplantation compared with hepatitis b immune globulin monotherapy increasing applicability of liver transplantation for patients with hepatitis b-related liver disease liver transplantation in european patients with the hepatitis b surface antigen prevention of hepatitis b recurrence after liver transplantation recurrent viral liver disease (hepatitis b and c) after liver transplantation liver transplantation in hbs antigen (hbsag) carriers. prevention of hepatitis b virus (hbv) recurrence by passive immunization passive immunoprophylaxis after liver transplantation in hbsag-positive patients improved outcome of orthotopic liver transplantation for chronic hepatitis b cirrhosis with aggressive passive immunization improved clinical outcomes with liver transplantation for hepatitis b-induced chronic liver failure using passive immunization prophylaxis in liver transplant recipients using a fixed dosing schedule of hepatitis b immunoglobulin national institutes of health hepatitis b virus orthotopic liver transplantation study group. effect of lamivudine treatment on survival of 309 north american patients awaiting liver transplantation for chronic hepatitis b lamivudine prophylaxis against reinfection in liver transplantation for hepatitis b cirrhosis high pre-treatment serum hepatitis b virus titre predicts failure of lamivudine prophylaxis and graft re-infection after liver transplantation prevention of hepatitis b virus recurrence after liver transplantation in cirrhotic patients treated with lamivudine and passive immunoprophylaxis prophylaxis against hepatitis b recurrence following liver transplantation using combination lamivudine and hepatitis b immune globulin hbv dna persistence 10 years after liver transplantation despite successful anti-hbs passive immunoprophylaxis successful hepatitis b reinfection prophylaxis with lamivudine and hepatitis b immune globulin in patients with positive hbv-dna at time of liver transplantation hepatitis b immune globulin preparations and use in liver transplantation antibody to hepatitis b surface antigen trough levels and half-lives do not differ after intravenous and intramuscular hepatitis b immunoglobulin administration after liver transplantation combination low-dose hepatitis b immune globulin and lamivudine therapy provides effective prophylaxis against posttransplantation hepatitis b australasian liver transplant study group. lamivudine plus low-dose hepatitis b immunoglobulin to prevent recurrent hepatitis b following liver transplantation section 14. combination of entecavir plus low-dose on-demand hepatitis b immunoglobulin is effective with very low hepatitis b recurrence after liver transplantation prevention and risk factors of hepatitis b recurrence after living donor liver transplantation efficacy and safety of entecavir and/or tenofovir for prophylaxis and treatment of hepatitis b recurrence post-liver transplant adherence to lamivudine after an early withdrawal of hepatitis b immune globulin plays an important role in the long-term prevention of hepatitis b virus recurrence a randomized study comparing lamivudine monotherapy after a short course of hepatitis b immune globulin (hbig) and lamivudine with long-term lamivudine plus hbig in the prevention of hepatitis b virus recurrence after liver transplantation combination therapy in liver transplant recipients with hepatitis b virus without hepatitis b immune globulin a randomized study of adefovir dipivoxil in place of hbig in combination with lamivudine as post-liver transplantation hepatitis b prophylaxis combination of lamivudine and adefovir without hepatitis b immune globulin is safe and effective prophylaxis against hepatitis b virus recurrence in hepatitis b surface antigen-positive liver transplant candidates hepatitis b immunoglobulin discontinuation followed by hepatitis b virus vaccination: a new strategy in the prophylaxis of hepatitis b virus recurrence after liver transplantation prophylaxis of recurrent hepatitis b virus by vaccination after liver transplant: preliminary results failure of a reinforced triple course of hepatitis b vaccination in patients transplanted for hbv-related cirrhosis failure of hepatitis b vaccination in patients receiving lamivudine prophylaxis after liver transplantation for chronic hepatitis b hepatitis b vaccination in liver transplant candidates one-year vaccination against hepatitis b virus with a mpl-vaccine in liver transplant patients for hbv-related cirrhosis adefovir dipivoxil study 45 international investigators group. adefovir dipivoxil for wait-listed and post-liver transplantation patients with lamivudine-resistant hepatitis b: final long-term results how to diagnose and treat hepatitis b virus antiviral drug resistance in the liver transplant setting cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilin-independent interference with the ntcp receptor strategies to inhibit entry of hbv and hdv into hepatocytes the fda approved drug irbesartan inhibits hbv-infection in hepg2 cells stably expressing sodium taurocholate co-transporting polypeptide control of cccdna function in hepatitis b virus infection new perspectives in the therapy of chronic hepatitis b effectiveness of hepatitis b treatment in clinical practice serum hepatitis b surface antigen quantitation can reflect hepatitis b virus in the liver and predict treatment response early serum hbsag drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in hbeag-negative patients hbsag quantification: useful for monitoring natural history and treatment outcome tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis b peginterferon alfa-2a hbeag-negative chronic hepatitis b study group. peginterferon alfa-2a alone, lamivudine alone, and the two in combination in patients with hbeag-negative chronic hepatitis b peginterferon alfa-2a hbeag-positive chronic hepatitis b study group. peginterferon alfa-2a, lamivudine, and the combination for hbeag-positive chronic hepatitis b dissecting the divergent effects of interferon-alpha on immune cells: time to rethink combination therapy in chronic hepatitis b? an integration of deep viral suppression with sequential immune modulation (cocktail therapy) to restore antiviral capacity: the future of chronic hepatitis b? the guideline of prevention and treatment for chronic hepatitis b: a 2015 update a randomized controlled trial of pegylated interferon-alpha2a plus adefovir dipivoxil for hepatitis b e antigen-negative chronic hepatitis b pegylated interferon alfa-2b alone or in combination with lamivudine for hbeag-positive chronic hepatitis b: a randomised trial peginterferon alpha-2b plus adefovir induce strong cccdna decline and hbsag reduction in patients with chronic hepatitis b telbivudine plus pegylated interferon alfa-2a in a randomized study in chronic hepatitis b is associated with an unexpected high rate of peripheral neuropathy study 149 investigators. combination of tenofovir disoproxil fumarate and peginterferon alfa-2a increases loss of hepatitis b surface antigen in patients with chronic hepatitis b adding pegylated interferon to a current nucleos(t)ide therapy leads to hbsag seroconversion in a subgroup of patients with chronic hepatitis b add-on peg-interferon leads to loss of hbsag in patients with hbeag-negative chronic hepatitis and hbv dna fully suppressed by long-term nucleotide analogs adding pegylated interferon to entecavir for hepatitis b e antigen-positive chronic hepatitis b: a multicenter randomized trial (ares study) a randomized, open-label clinical study of combined pegylated interferon alfa-2a (40kd) and entecavir treatment for hepatitis b "e" antigen-positive chronic hepatitis b switching from entecavir to pegifn alfa-2a in patients with hbeag-positive chronic hepatitis b: a randomised open-label trial (osst trial) sustained immune control in hbeagpositive patients who switched from entecavir therapy to pegylated interferon-α2a: 1 year follow-up of the osst study sequential therapy with adefovir dipivoxil and pegylated interferon alfa-2a for hbeag-negative patients higher efficacy of sequential therapy with interferon-alpha and lamivudine combination compared to lamivudine monotherapy in hbeag positive chronic hepatitis b patients sequential therapy with entecavir and peg-inf in patients affected by chronic hepatitis b and high levels of hbv-dna with non-d genotypes predictive value of baseline and on-treatment qhbsag level in hbeag positive chb patients who switched from nucs to pegylated interferon a-2a: a further analysis from new switch study the role of hbsag levels in the current management of chronic hbv infection hepatitis b surface antigen: association with sustained response to peginterferon alfa-2a in hepatitis b e antigenpositive patients review article: novel therapies for hepatitis b virus cure-advances and perspectives sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus cyclosporin a and its analogs inhibit hepatitis b virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (ntcp) the entry inhibitor myrcludex-b efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis b virus a novel tricyclic polyketide, vanitaracin a, specifically inhibits the entry of hepatitis b and d viruses by targeting sodium taurocholate cotransporting polypeptide ntcp opens the door for hepatitis b virus infection targeting hepatitis b virus cccdna using crispr/ cas9 crispr/cas9 cleavage of viral dna efficiently suppresses hepatitis b virus hepatitis b virus replication is regulated by the acetylation status of hepatitis b virus cccdna-bound h3 and h4 histones specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna phenylpropenamide derivatives at-61 and at-130 inhibit replication of wild-type and lamivudine-resistant strains of hepatitis b virus in vitro the phenylpropenamide derivative at-130 blocks hbv replication at the level of viral rna packaging inhibition of hepatitis b virus replication by bay 41-4109 and its association with nucleocapsid disassembly the main hepatitis b virus (hbv) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of hbv replication role for a bidentate ribonuclease in the initiation step of rna interference an rna-directed nuclease mediates posttranscriptional gene silencing in drosophila cells hepatocyte-targeted rnai therapeutics for the treatment of chronic hepatitis b virus infection aiming for cure in hbv and hdv infection rep 9 ac: a potent hbsag release inhibitor that elicits durable immunological control of chronic hbv infection nucleic acid polymers rep 9 ac/rep 9 ac′ elicit sustained immunologic control of chronic hbv infection update on the safety and efficacy of rep 2139 monotherapy and subsequent combination therapy with pegylated interferon alpha-2a in chronic hbv/hdv co-infection in caucasian patients gs-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees functional activation of nk and cd8+ t cells in vitro by the toll-like receptor 7 agonist gs-9620 clinical and immunological efficacy of intradermal vaccine plus lamivudine with or without interleukin-2 in patients with chronic hepatitis b efficacy of granulocyte-macrophage colonystimulating factor or lamivudine combination with recombinant interferon in non-responders to interferon in hepatitis b virus-related chronic liver disease patients control and eradication strategies of hepatitis b virus enhancing virus-specific immunity in vivo by combining therapeutic vaccination and pd-l1 blockade in chronic hepadnaviral infection safety, tolerability and immunogenicity of gs-4774, a hepatitis b virus-specific therapeutic vaccine, in healthy subjects: a randomized study tg1050, an immunotherapeutic to treat chronic hepatitis b, induces robust t cells and exerts an antiviral effect in hbv-persistent mice combination/ sequential therapy with etv, peg-ifn alpha-2b and gmcsf enhanced hbsag loss and appearance of hbsab in na suppressed chb patients (the anchor a study): an interim analysis toward a cure for hepatitis b virus infection: combination therapy involving viral suppression and immune modulation and long-term outcome viral hepatitis. hbv cure-can we pin our hopes on immunotherapy? key: cord-030369-4dn02a35 authors: peng, liang; gao, zhi-liang; wang, yu-ming; he, deng-ming; zhao, jing-ming; bai, xue-fan; wang, xiao-jing title: clinical manifestations and laboratory tests of aechb and severe hepatitis (liver failure) date: 2019-05-21 journal: acute exacerbation of chronic hepatitis b doi: 10.1007/978-94-024-1603-9_1 sha: doc_id: 30369 cord_uid: 4dn02a35 this chapter describes the clinical symptoms and signs of aechb and hbv aclf, classification, grading of hbv aclf and their features, diagnostic principles and standards in liver pathology, biochemistry, and virology of hbv aclf. 1. liver failure is defined as serious damage to the liver cause by a variety of etiologies, leading to liver function disorder or even decompensation, and clinical syndromes with coagulopathy, jaundice, hepatic encephalopathy, and ascites. 2. severe hepatitis b can be indicated pathologically by apparent hepatocellular necrosis, including extensive multifocal, confluent, bridging, sub-massive or massive necrosis. 3. laboratory tests during the course of severe exacerbation of chronic hepatitis b can reflect pathological changes and liver function in a timely manner, providing objective and informative reference data for evaluation of disease severity and treatment efficacy. among the most important laboratory tests are those for prothrombin activity, international normalized ratio, and increases in total bilirubin concentration. 4. severe hepatitis b is associated with interactions between the virus and host factors. detection of hbv dna, hbv genotype, quasispecies and hbv mutation can provide important theoretical bases for the prevention, control or mitigation of the progress of severe hepatitis b. 5. noninvasive imaging modalities can be used to visualize the entire liver and parts of it. measuring liver volume to evaluate liver size and liver reserve capacity is regarded as important in diagnosis, surgical approach and prognostic evaluation of patients with severe exacerbation of chronic hepatitis b and liver failure. 6. model for end-stage liver disease (meld) is the first quantitative method developed to assess whether a patient with liver failure requires a liver transplant. the predictive value of the meld model has been improved by the meld-na, imeld, and meso models. several other valuable prognostic models have been developed. for example, for patients with hbv-aclf, the established tppm scoring system was found to be more predictive than meld score. can reflect pathological changes and liver function in a timely manner, providing objective and informative reference data for evaluation of disease severity and treatment efficacy. among the most important laboratory tests are those for prothrombin activity, international normalized ratio, and increases in total bilirubin concentration. 4. severe hepatitis b is associated with interactions between the virus and host factors. detection of hbv dna, hbv genotype, quasispecies and hbv mutation can provide important theoretical bases for the prevention, control or mitigation of the progress of severe hepatitis b. 5. noninvasive imaging modalities can be used to visualize the entire liver and parts of it. measuring liver volume to evaluate liver size and liver reserve capacity is regarded as important in diagnosis, surgical approach and prognostic evaluation of patients with severe exacerbation of chronic hepatitis b and liver failure. 6. model for end-stage liver disease (meld) is the first quantitative method developed to assess whether a patient with liver failure requires a liver transplant. the predictive value of the meld model has been improved by the meld-na, imeld, and meso models. several other valuable prognostic models have been developed. for example, for patients with hbv-aclf, the established tppm scoring system was found to be more predictive than meld score. liang peng, zl huang, yy mei and zhi-liang gao currently, both clinical and pathophysiological diagnoses are made of severe hepatitis (liver failure) in china. according to the guideline for the prevention and treatment of viral hepatitis (2000), severe hepatitis is classified as acute severe hepatitis, subacute severe hepatitis, and chronic severe hepatitis. acute severe hepatitis is initially diagnosed due to acute jaundice that rapidly progresses to liver failure within 2 weeks. subacute severe hepatitis can be identified in patients with acute jaundice hepatitis that progresses to liver failure anywhere from 15 days to 24 weeks. chronic severe hepatitis often develops with pre-existing chronic liver diseases. the clinical manifestations of chronic severe hepatitis are similar to those of subacute severe hepatitis in some patients, or, in some patients, appear similar to decompensated cirrhosis at disease onset. the diagnostic criteria for severe hepatitis in china remain to be fully developed and hence have not been introduced internationally. to meet the clinical requirements and standardize the diagnosis and therapy of liver failure, the branch of infectious diseases and the branch of hepatology of the chinese medical association invited experts in china to develop the first guidelines for the diagnosis and therapy of liver failure in 2006. in those guidelines, liver failure refers to severe liver damage caused by multiple factors. that damage to the liver results in either the severe impairment or decompensation of synthesis, detoxication, excretion, and biotransformation in the liver and subsequent clinical manifestations characterized by coagulation disorder, jaundice, hepatic encephalopathy, and ascites. on the basis of pathological features and disease progression, liver failure is classified as acute liver failure (alf), subacute liver failure (salf), acute-on-chronic liver failure (aclf), and chronic liver failure (clf). alf is characterized by the rapid appearance of clinical manifestations. patients with alf usually develop a clinical syndrome of liver failure characterized by highgrade hepatic encephalopathy (he, >grade 2) within 2 weeks. patients with salf typically present with a clinical syndrome of liver failure anywhere from 15 days to 26 weeks. finally, aclf refers to the acute decompensation of the liver function in the presence of pre-existing chronic liver diseases, and clf refers to chronic decompensation of the liver function characterized by ascites or portal hypertension, coagulation disorder, and he due to progressive liver dysfunction in the presence of hepatic cirrhosis. the published guidelines systemically and extensively reflect the current status of the diagnosis and therapy of liver failure. in addition, the guidelines, for the first time, focus on liver failure rather than severe hepatitis, which broadens our horizons and highlights practicability. in china, acute severe hepatitis, subacute severe hepatitis, and chronic severe hepatitis correspond closely to alf, slf, and aclf, respectively, as illustrated in table 1 .1. in some patients, chronic severe hepatitis is similar to clf in other countries. on the basis of available guidelines for liver failure, we define severe hepatitis b as liver failure due to hepatitis b virus infection. clf is the most common, and alf and slf are rare. acute exacerbation of chronic hepatitis b (aechb) is a dynamic process, including mild, moderate, severe chronic hepatitis b and chronic aclf defined in above guidelines. the reference index of abnormality in laboratory examination is shown in table 1 .2. in addition to viral replication, other factors also contribute to the pathogenesis of hepatitis b-induced liver failure, such as concomitant infection of other hepatitis viruses (especially the hepatitis e virus), immune status, pregnancy, drug and/or alcohol consumption, concomitant bacterial infection, mental stress, and concomitant disease processes (e.g., hyperthyroidism). the liver is the largest solid organ in humans and has complex functions. hepatic parenchymal cells are responsible for metabolism, secretion, synthesis and bioconversion. factors that can cause severe damage to hepatocytes (i.e., parenchymal cells, kupffer cells) may result in disorders of metabolism, secretion, synthesis, detoxication and immunity. in turn, that damage can lead to jaundice, liver shrinkage, coagulation dysfunction, hemorrhage, secondary infection, hepatorenal syndrome, he, and other clinical entities described in detail here. the physical condition of patients deteriorates, and affected individuals usually develop weakness, extreme fatigue, and a severely diminished quality of life. they frequently require assistance to perform basic personal needs, such washing their face, brushing their teeth, and using the toilet. in the early stage of jaundice, in addition to developing extreme fatigue, gastrointestinal symptoms become evident, including extremely poor appetite, anorexia, intolerance of oily foods, nausea, vomiting, abdominal discomfort, and hiccups. in the jaundice stage, the gastrointestinal symptoms deteriorate further. patients can develop refractory vomiting, hiccups, evident abdominal distension, and reduced/ lack of borborygmus. clinically, patients initially note their urine color darkens, becoming a strong-tea like color. next, a yellowish pigmentation of the ski and conjunctival membranes develops. that jaundice progressively becomes deeper, characterized by hepatocellular jaundice. in this stage, serum bilirubin increases rapidly. in fact, the daily increment in serum bilirubin may be >17 μmol/l (>1 mg/dl). the sulfur-containing amino acids in the intestine are degraded into mercaptans that have the odor of rotting fruit. mercaptans cannot be metabolized in the liver and are therefore excreted from the respiratory tract. this distinctive odor is specifically noted in patients with he. the severity of hepatic foetor may, in some cases, reflect the severity of liver injury. the occurrence of coagulation dysfunction is primarily ascribed to the reduced synthesis of coagulation factors by the liver. a majority of the both coagulation and anticoagulant factors are synthesized in the liver. in addition, some coagulationrelated factors and their inhibitors are also metabolized in the liver. the outcome of coagulation dysfunction is dependent on the severity of damage to the hepatocytes. thus, even patients in an early stage of liver failure may present with coagulation dysfunction. prothrombin (pt) activity is often abnormal in the early stages of liver failure and may therefore serve as a sensitive indicator for the prognosis of liver failure. common clinical manifestations of coagulation dysfunction are mucocutaneous bleeding (i.e., spontaneous bruising, gingival bleeding, subconjunctival hemorrhage), ecchymosis at the site of injection/puncture, and purpura in more severe cases. gastrointestinal bleeding is also common in affected individuals, whereas bleeding into/from the genitourinary tract, lung, kidney, and retroperitoneum is rare but occasionally observed in some patients. if intracranial hemorrhage develops, it is frequently life threatening. in ahf, the incidence of bleeding and severe bleeding is as high as 73 and >30%, respectively. another cause of coagulation dysfunction is thrombocytopenia and platelet dysfunction. of the two, thrombocytopenia is more common. because platelets are derived from megakaryocytes in bone marrow, bone marrow fibrosis and either reduced bone marrow regeneration or invasion of lymphoma cells in the bone marrow can reduce the number of platelets. platelets perform multiple activities, including adhesion, aggregation, release, and shrinking blood clots. additionally, they play an important role in coagulation. platelet dysfunction may also increase capillary permeability and fragility, which may cause either spontaneous bleeding of the skin and mucous membranes or difficult hemostasis following vascular injury. in patients with slf, thrombocytopenia is mainly diagnosed in the latter stage of disease in which massive hepatocyte necrosis leads to posthepatic cirrhosis, portal hypertension, and hypersplenism. in clf patients, thrombocytopenia might be present, and hepatocyte necrosis may aggravate portal hypertension and hypersplenism, resulting in worsening thrombocytopenia. splenomegaly and splenic sinus hyperplasia increase the phagocytosis and destruction of platelets. further, splenomegaly can cause enlargement of the platelet pool within the spleen. as a result, the platelets in the spleen may account for >90% of platelets in the body. the above pathological changes may finally cause a reduction in the circulating platelets. the reason for thrombocytopenia in liver disease patients without hypersplenism is still poorly understood and might be ascribed to following factors (1) the hepatitis b virus may significantly inhibit the megakaryocyte system of the bone marrow, resulting in reduced production of platelets; (2) the thrombopoietin (tpo) level is reduced. the division of megakaryocytes into platelets in the bone marrow is controlled by both megakaryocyte colony stimulating factor (meg-csf) and tpo. meg-csf primarily regulates the proliferation of megakaryocyte progenitor cells, whereas tpo stimulates the maturation of megakaryocytes and production of platelets. tpo is almost exclusively produced by hepatocytes, and only a minority of tpo is produced in the kidney and other organs. tpo is a key factor affecting the production of platelets, and the synthesis of tpo is reduced significantly in patients with either severe hepatitis or hepatic cirrhosis, which affects the production of platelets. in patients with parenchymal liver diseases, abnormalities of platelets are present in both quality and quantity. for example, when the platelet membrane glycoprotein gpi6-ix is reduced, the aggregation of platelets following ristocetin treatment and the shrinkage of blood clots are markedly compromised; and (3) patients with liver diseases usually develop immune dysfunction and are therefore susceptible to infection. bacterial toxins and systemic inflammatory response syndrome may also cause thrombocytopenia. one published study of icu patients found that infection was an independent risk factor of thrombocytopenia. he he is both a neuropsychiatric syndrome, a type of central nervous system dysfunction, and metabolic disturbance due to hepatocellular dysfunction and portosystemic shunting. he is clinically characterized by mental and neurological abnormalities, such as abnormal personality and behaviors, irritability, sleep perversion, drowsiness, and complete loss of consciousness or coma. he is one of the major causes of severe complications and death in patients with liver failure and is typically classified into one of the four following stages: stage 1: the prodromal stage. this stage usually manifests with mild abnormal personality changes and behaviors, such as euphoric excitement, indifference, taciturnity, being sloppily dressed, and inappropriate defecation/urination. the affected individual can usually provide correct responses to questions but they are inarticulate and have slow speech. flapping tremor/hepatic tremor might also be present. to test for flapping tremor, patients are asked to close their eyes with their arms stretching straight, elbows flexed, palms in dorsal extension, with separated fingers. a positive response is determined when the metacarpophalangeal joint, wrist, elbow, and shoulder show irregular movements (jitter) when held in that position within 30 s. physicians may also ask the patients to hold the their hand for 1 min. if the physician feels the hand tremor, the test suggests a positive diagnosis of flapping tremor. the condition is caused by afferent dysfunction of joint-reticular formation of the brainstem and a characteristic neurological manifestation. that said, flapping tremor has no specificity fro he and can also be found in patients with either uremia or hypoxemia due to chronic respiratory disease/heart failure. the presence of flapping tremor in a patient with severe liver disease, however, is helpful for early diagnosis of he. patients with he usually have a normal electroencephalogram. stage 1 of he lasts anywhere from several days to several weeks. several patients with he in the prodromal stage may have no evidence of clinical symptoms; therefore, misdiagnosis is possible. stage 2: the precoma stage. patients with he in this stage usually presents with confusion, sleep and behavioral disorders, and symptoms as described in the prodromal stage further deteriorate. patients suffer from disorientation and understanding disorders as well as conceptual confusion over time, place, and person. patients are unable to perform simple intellectual composition (e.g., building blocks, arranging matchstick into pentagon), and have decreased computing capacity (e.g., 100-7 and continuing). slurred speech, writing disorders, and abnormal behaviors are also common. sleep perversion and daytime sleep and night awaking may be present. further, hallucinations, fear, and mania are also observed, and some patients can be misdiagnosed with mental diseases. patients with liver failure in this stage usually have evident neurological signs such as tendon hyperreflexia, increased muscle tone, ankle clonus, and presence of the babinski sign. flapping tremor and an abnormal electroencephalogram can also be observed. patients may also suffer from uncontrolled muscular activities and ataxia. stage 3: the lethargic stage. patients with he in the lethargic stage mainly manifest lethargy and insanity, and neurological signs continue and deteriorate. in the majority of time, patients are in a lethargic state, but can be waken up. patients respond to questioning, but may present confusion and hallucination. flapping tremor is also present. muscular tension increases, and there is resistance in the passive limb movements. pyramidal signs and abnormal waves in eeg can also be noted. stage 4: the coma stage. patients have complete loss of consciousness and are unable to be awakened. in a light coma, patients are responsive to painful stimuli and uncomfortable postures, have tendon hyperreflexia, and increased muscular tension. patients in this stage are usually unable to co-operate during an examination, and a flapping tremor may not be inducible. in a deep coma, various reflexes disappear; muscular tension reduces; pupils become dilated; and there are paroxysmal convulsions, ankle clonus, hyperventilation, and abnormalities on an electroencephalogram. stage of he is an important indicator of severity of disease. it may reflect not only the severity of brain damage but also the severity of liver disease. it is important to recognize that there is no clear boundary between two neighboring stages and that there might be some overlap between two neighboring stages (therefore missing the middle stage of he). when the disease condition either deteriorates or improves after therapy, the severity of he may be reduced by one or two stages. as mentioned above, the initial symptoms of he are personality changes. patients with extrovert personalities (i.e., lively, cheerful) may become depressed, whereas patients with introverted personalities (i.e., withdrawn, reticent) may become euphoric and garrulous. the second most common symptom is a change in behaviors. initially, patients have sloppy behaviors, such as meaningless behaviors like scattering garbage all over the place and defecating/urinating anywhere, looking at clothes, and touching the bed. those changes are usually only identified by close observation and careful experience. there are also changes in sleep habits. patients are often drowsy during the daytime but have difficult sleeping at night or show sleep perversion, which predicts imminent he. hepatic foetor is also an important feature of he. he patients usually have brain edema and present with nausea, vomiting, dizziness, headache, and either irregular breathing or even apnea. as blood pressure increases there might be a paroxysmal or sustained increase in systolic blood pressure. bradycardia may be also observed. muscular tension can increase or the patient can develop a decerebrate posture or even opisthotonus with severe he. the pupillary light reflex can become blunt/absent, the pupils can become mydriatic, and anisocoria can occur. achilles and knee tendon hyperreflexia may be observed. it is important to note that some signs might not be obvious in a patient with late-stage he. in clinical practice, clinicians may indirectly evaluate the severity of brain edema according to chemosis. accurate evaluation of brain edema is dependent on the subdural, epidural, or cerebral parenchymal measurement of intracranial pressure. the normal intracranial pressure is <2.7 kpa (20 mmhg), and brain edema is diagnosed once intracranial pressure is >20 mmhg. the most important sign of he is flapping tremor, which means the presence of he in stage ii. in addition, thinking and intelligence tests (such as number connection test, signature test, mapping test, and computing capability test) are abnormal in he patients. in some he patients (especially those with hyperammonemia due to he), slow waves with high amplitude may be observed on electroencephalogram, and positive-evoked potential is also a characteristic change. brain edema is a complication of alf. typical clinical manifestations of brain edema are sustained increase in blood pressure, abnormal pupils, irregular respiration, and papilledema. more than 80% of patients with he in stage 3 or 4 are likely to develop brain edema, and severe brain edema may result in cerebral hernia. brain edema has the clinical presentations of increased intracranial pressure and cerebral dysfunction, which can sometimes overlap with the manifestations of he. it is therefore sometimes difficult to differentiate the two, potentially resulting in misdiagnosis. he patients with brain edema may present dysphoria, irascibility, and increased muscular tension, which are more common than in patients with he without brain edema. if there are concomitant changes in pupils and respiration together with convulsions and/or seizures, cerebral hernia is suspected. in the late stages of liver failure, patients may develop intracranial hemorrhage, causing respiratory and circulatory arrest and even sudden death. thus, once cardiopulmonary arrest of unknown cause is present, intracranial hemorrhage should be considered. concomitant gastrointestinal bleeding in patients with severe hepatitis can be caused by multiple factors, including (1) decreased coagulation factor synthesis by hepatocytes and/or significant inactivation of active coagulation factors in the liver; (2) endotoxemia and disseminated intravascular coagulation consuming a large amount of coagulation factors; (3) hypersplenism causing abnormalities in the quality and quantity of the platelets; (4) portal hypertension causing the rupture of esophageal and gastric varices; and (5) stress response in severe hepatitis leading to diffuse gastric corrosive erosion. of the possible complications occurring in liver failure patients, bleeding is the most common and severe. in clinical practice, gastrointestinal bleeding with severe hepatitis seems to make the primary disease worse. it may worsen liver ischemia and hypoxia and aggravate liver dysfunction and ascites. blood in the gastrointestinal tract can be degraded into ammonia and increase the production of sulfur-like substance, resulting in he. in addition, bleeding may reduce immune function, which make infections difficult to control. the reduction in effective circulating blood volume may also induce hepatorenal syndrome. taken together, bleeding may cause multiple organ dysfunction, thereby complicating treatment and reducing the success rate of therapy. the causes of upper gastrointestinal bleeding are different among patients with different types of liver failure. in alf and slf, bleeding is related to reduced synthesis of coagulation-related factors and stress-induced gastric mucosal lesions. in clf, however, rupture of esophageal and gastric varices and gastric mucosal lesions secondary to portal hypertension are the main causes of gastrointestinal bleeding. in some cases, there is more than one cause of bleeding. in liver failure, the ability of the monocyte-macrophage system to clear intestinerelated endotoxins is reduced significantly, which may lead to intestine-related endotoxemia and deterioration of liver function. this clearly forms a vicious cycle and may cause multiple organ failure if it is severe enough. in addition, patients usually have compromised immune function and are susceptible to infection. invasive manipulations and use of broad-spectrum antibiotics and immunosuppressants further increase the possibility of secondary infection. concomitant infection in liver failure patients has the following characteristics: (1) a high incidence; (2) infection may occur at different sites either simultaneously or sequentially, and abdominal and biliary tract infection is the most common. once pulmonary infection is present, the disease condition will likely deteriorate, directly causing death; (3) a majority of infections are nosocomial infection, and pathogens are usually resistant to common antibiotics, making therapy challenging; (4) the pathogens causing infection are diverse but mainly gram-negative bacteria, although the incidence of gram-positive and fungal infections is increasing; (5) infection is closely related to the prognosis for liver failure patients. in sum, the more severe the disease, the higher the incidence of infection is and secondary infection may worsen the condition or cause death. the early diagnosis of secondary infections is based on clinical findings such as signs of infections (i.e., fever, increase in peripheral white blood cells, deterioration of primary disease, specific symptoms of infection of a particular organ). some patients may not present with an obvious fever and instead only show focal signs of infection. for example, in spontaneous bacterial peritonitis, examination could reveal abdominal tenderness and rebound tenderness and a slight increase in peripheral white blood cells and polymorphonuclear proportion (although they are in normal ranges). in contrast, pulmonary infections can present only with fever while the respiratory symptoms are not obvious/absent and thoracic radiographs fail to show abnormalities in affected patients. in such cases, computed tomography is required to identify the pulmonary lesions. in addition, liver failure patients are vulnerable to fungal infection, especially for those receiving long-term therapy with broad-spectrum antibiotics. gastrointestinal candidiasis is the most common fungal infection. oral candida albicans infection is characterized by thickening and a bean residue-like coating on the tongue, gastrointestinal fungal infection is characterized by increased stool frequency and stool with mucus, and pulmonary fungal infection (especially aspergillus infection) is a severe complication of liver failure that can progress rapidly and has a high mortality rate. once a pulmonary fungal infection is suspected, computed tomography of the thorax should be performed to confirm the diagnosis, and effective antifungal therapy should be initiated as early as possible. hepatorenal syndrome (hrs) refers to progressive functional renal failure in the absence of primary kidney disease in patients with severe liver diseases. hrs is most often diagnosed in the late stages of severe hepatitis and hepatic cirrhosis. the main clinical manifestations of hrs include: 1. late stages of liver failure; 2. renal failure after a reduction in effective circulating blood volume (e.g., water and electrolyte disorder, following paracentesis for ascites, excessive urination due to diuresis, gastrointestinal bleeding, secondary infection, vomiting, and diarrhea). however, hrs may present abruptly with no evident/discoverable causes; 3. hrs is often found in patients with moderate to severe ascites; 4. hrs has no significant relationship with jaundice and he; and 5. blood pressure reduces during hrs. thus, when patients are treated with propranolol for portal hypertension, physicians should pay attention to the baseline blood pressure because reduction in blood pressure after pharmacotherapy may reduce the blood supply to the kidney and decrease glomerular filtration rate, inducing hrs; 6. the abrupt decrease in urine output suggests the presence of hrs. diuretics usually fail to increase the urine output. patients often have a reduction in urine sodium and concomitant hyponatremia; 7. urinalysis shows similarities to prerenal azotemia but displays opposite features to acute tubular necrosis; 8. the symptoms of uremia may overlap with those of liver failure and cause the deterioration of original symptoms. in patients with progressive liver diseases, secondary renal dysfunction is closely related to the deterioration of their general condition, suggesting the aggravation of liver failure. in addition, the presence of uremia may contribute to metabolic complications. coagulation dysfunction in liver disease patients may be deteriorated due to compromised aggregation of platelets during uremia. uremia may also aggravate immune dysfunction. on the basis of clinical characteristics, hrs can be classified into two types. type i hrs is rare, has an acute onset, and is characterized by progressive renal dysfunction. serum creatinine may be either 2× that at baseline (i.e., >221 μmol/l or 2.5 mg/dl) within 2 weeks or creatinine clearance decreases by 50% within 24 h (i.e., creatinine clearance of <20 ml/min). patients with type i hrs have a poor prognosis, with 80% of patients dying within 2 weeks of diagnosis and only 10% of patients can survive for >3 months. the course of disease is short, and symptoms of uremia are not obvious. in type ii hrs, which is often found in clf patients with pre-existing hepatic cirrhosis, has a chronic onset. ascites patients with type ii hrs are usually nonresponsive to diuretics. in type ii hrs, renal failure shows a slow progression (lasting for several weeks to months), but the survival rate of patients is lower than that of hepatic cirrhosis patients with ascites. the main clinical consequence is refractory ascites nonresponsive to diuretics in patients with type ii hrs. a follow-up study of 234 hepatic cirrhosis patients with ascites showed the accumulative incidence of hrs was 18% within 1 year and 39% within 5 years. a retrospective study showed about 17% of patients with ascites on admission had hrs and hrs patients accounted for 50% of hepatic cirrhosis patients died. however, for hepatic cirrhosis patients, the 2-year and 5-year incidence of hrs is 32% and 41% after development of ascites. a majority of patients (80-95%) die within 3 weeks after development of azotemia. hps refers to a series of pathophysiological changes and clinical manifestations (including hypoxemia) due to abnormal pulmonary vascular dilation, gas exchange disorder, and abnormal arterial oxygenation. abnormal arterial oxygenation due to a gas exchange disorder may increase the alveolar-arterial oxygen pressure difference. hypoxemia is an important pathophysiological basis of hps, and hps is a severe pulmonary complication of end-stage liver disease that is clinically characterized by dyspnea and cyanosis. hps was first reported by rydell hoffbauer in 1956, but it wasn't until 1977 that kenned and knudson proposed the full concept of hps. hps per se refers to pulmonary vascular dilation and the shunting of venous blood with low oxygenation to arteries in the presence of severe liver disease. hps is mainly identified in patients with clf (child c hepatic cirrhosis). in addition, patients with either acute or chronic liver disease may present with a pulmonary vascular abnormality and arterial hypoxemia. hps occurs most commonly in patients with hepatic cirrhosis secondary to chronic liver disease, including hepatitis-induced cirrhosis, cryptogenic cirrhosis, alcoholic cirrhosis, and primary biliary cirrhosis, all of which have similar pathophysiological processes as hps. in hps, severe ascites, portal hypertension, and arterial hypoxemia (pao 2 < 10 kpa) may be related to the intrapulmonary vascular shunt, excessive production of nitric oxide, lung ventilation-perfusion imbalance, and interstitial fibrosis. the incidence of hps varies among studies. the incidence of hps is about 5-29% in chronic liver disease patients but higher in patients with hepatic cirrhosis. the most common clinical manifestations of hps are dyspnea, hypoxemia, and cyanosis caused by intrapulmonary vascular dilation and poor arterial oxygenation in the presence of primary liver disease: patients usually progressively develop respiratory manifestations (e.g., cyanosis, dyspnea, clubbed-fingers/toes, orthostatic hypoxia, supine breathing). progressive dyspnea is the most common pulmonary symptom of hps, and cyanosis is a unique and reliable clinical sign. supine breathing and orthostatic hypoxia are characteristic manifestations of hps. pulmonary examination often fails to identify clinically important signs, and hps is not associated with the either the cause or severity of liver disease. in a fraction of patients with stable liver disease, there is progressive lung dysfunction. research shows that hps is associated with esophageal varices and spider angiomas. intrapulmonary vascular dilation (i.e., pulmonary spider angiomas) is frequently found in liver disease patients with subcutaneous spider angiomas susceptible to hypoxemia. spider angiomas have been regarded as a marker of extrahepatic involvement. if patients have no primary heart and lung disease, concomitant lung disease (such as chronic bronchitis, emphysema, pneumonia, and pleural effusion) may coexist with hps. affected patients usually have obvious respiratory symptoms; therefore, physicians should differentiate between the conditions. hps is an independent risk factor for prognosis. specifically, studies have reported that the median survival time is 10.6 months after the diagnosis of hps. to date, no effective strategies have been developed for the therapy of hps. orthotopic liver transplantation should be performed as early as possible for hps patients. in liver failure, there is massive hepatocyte necrosis that may cause a reduction in glycogenolysis and abnormal gluconeogenesis. thus, patients are vulnerable to hypoglycemia, shock, coma, and impaired glucose tolerance. the synthetic function of the liver is impaired in such patients, and the serum level of cholesterol and triglycerides decreases. serum cholesterol has been used as an indicator for the prediction of prognosis of liver failure patients. the frequent use of diuretics can cause water and electrolyte imbalance, of which hypokalemia and hyponatremia are the most common. such imbalances may also induce he and brain edema. ap is a rare, but severe, complication of liver failure. the inciting cause(s) and pathogenesis of ap in patients with viral hepatitis remain unclear but might be associated with viral infection, biliary tract lesions, drugs (steroids and diuretics), and other factors. the reported incidence is 0.2-3%, but one autopsy study shows that the incidence of ap is as high as 33% in patients with severe hepatitis and hepatic cirrhosis. evidence shows that the incidence of ap is relatively high in patients with advanced liver failure. further, high serum bilirubin, low albumin, and a significant reduction in prothrombin activity may predict a poor prognosis for ap patients with severe hepatitis and a high mortality. two of the following three criteria are required for the diagnosis of ap: (1) patients have abdominal pain characteristic of ap; (2) serum amylase and/or lipase is ≥3× the upper limit of normal; and (3) there are characteristics of ap on medical imaging. that said, in cases of severe hepatitis with concomitant ap, the symptoms of ap are usually atypical, diverse, and easy to be masked by symptoms of severe hepatitis. thus, severe hepatitis is often considered the cause of abdominal distension, nausea, and vomiting, even in the presence of ap. in some patients, ap may be misdiagnosed as spontaneous bacterial peritonitis, cholecystitis, or gastritis, which may delay treatment and therefore worsen the patient's condition. the clinical manifestations of ap are usually atypical in patients with preexisting liver failure, therefore, physicians should highlight the diagnosis of ap in affected patients. when the following findings are observed, ap should be suspected and laboratory and imaging examinations should be performed as soon as possible for the confirmed diagnosis: 1. patients with severe hepatitis develop abrupt and persistent upper abdominal pain/peritoneal irritation that is nonresponsive to general antispasmodics; 2. patients present with severe vomiting, severe sialorrhea of unknown cause, and refractory hiccups; 3. patients manifest repeated and transient episodes of conscious disturbance, which are refractory and not caused by hepatic coma and hypoglycemia-like reaction; 4. patients have prior chronic cholecystitis or gallstones, receive treatment with diuretics or steroids, and have symptoms and signs described in (1) after exclusion of spontaneous peritonitis. for patients with severe hepatitis, routine blood testing and urine amylase detection should be performed dynamically. imaging examinations can be performed simultaneously. abdominal ultrasonography may be performed within 24-48 h after the onset of abdominal pain, which is helpful for the morphological change in the pancreas and the exclusion of biliary tract disease. however, gas in the gastrointestinal tract during ap may affect the performance of ultrasonography and make accurate diagnosis of ap impossible. thus, computed tomography is recommended as a standard imaging examination for the diagnosis of ap. computed tomography is helpful for the early diagnosis and subsequently timely therapy, which may improve the prognosis. to facilitate the determination of therapeutic efficacy and the evaluation of prognosis, the branch of infectious and parasitic diseases and branch of hepatology of chinese medical association published the guideline for the prevention and therapy of viral hepatitis in 2000 (xi'an conference). on the basis of those guidelines, severe hepatitis can be classified as early, intermediate, and advanced severe hepatitis. specifically, early severe hepatitis meets the diagnostic criteria for severe hepatitis (i.e., severe fatigue, gastrointestinal symptoms, deepening jaundice, serum bilirubin >10 × the upper limit of normal, prothrombin activation of ≤30-40%, or pathological characteristics), but patients have no evidence of he and no ascites. intermediate severe hepatitis patients have grade 2 he or obvious ascites, bleeding tendency (i.e., bleeding point, ecchymosis, and a prothrombin activation of ≤20-30%). advanced severe hepatitis patients develop refractory complications and hrs, gastrointestinal bleeding, severe bleeding tendency (i.e., ecchymosis at the injection site), severe infection, refractory electrolyte imbalance, he >grade 2 brain edema, or a prothrombin activation of ≤20%. currently, some investigators classify the natural history of liver failure into the following: prejaundice stage, bilirubin increase stage, bilirubin plateau stage, and bilirubin reduction stage. those stages are based on disease progression, serum bilirubin level, and recovery of liver failure patients. in the prejaundice stage, patients have fatigue, anorexia, and an intolerance of oil. they deteriorate gradually, the urine becomes yellow, liver function detection usually shows a significant increase in aspartate aminotransferase and alanine aminotransferase (higher than several thousand), and the prothrombin activity increases. serum bilirubin increases progressively (i.e., a daily increment of >17.1 μmol/l), and symptoms (fatigue, anorexia) deteriorate after the appearance of jaundice (which is different than manifestations of acute jaundice hepatitis). when the serum bilirubin peaks and remains relatively stable, the disease may be in the bilirubin plateau stage in some patients with no severe complications but present improved mental status and appetite. with the regeneration of hepatocytes, the disease progresses into the bilirubin reduction stage, in which the coagulation, mental status, and appetite improve. when the disease recommences its deterioration, it may progress from the so-called bilirubin increase stage directly to the end stage. in patients with alf, the bilirubin plateau stage is not obvious, and patients might die shortly after disease onset. if patients survive alf, the disease may be pathologically classified as a hepatocyte edema type, and liver function will improve in a short period. not all types of liver failure (including severe hepatitis b) have clear stages based on their natural history and characteristics, and the respective features are discussed in detail as described in the following sections. there is still no consensus on the definition of alf. in 2005, the us acute liver failure study group published guidelines for the management of acute liver failure. in those guidelines, they emphasized that liver failure within 26 weeks after onset can be diagnosed with alf in mother to child transmission of hepatitis b infection (or autoimmune hepatitis), although it has the possibility of progressing into hepatic hepatitis. in addition, some physicians propose that liver failure with an abrupt attack either secondary to chronic hepatitis b or in the presence of other hepatitis virus infection can also be classified as alf. the pathological basis of alf may be classified as necrosis-and degeneration-dominant (acute edema) type. in alf of the necrosis-dominant type, hepatocytes become diffuse and massive necrosis occurs soon after disease onset. in alf of the degeneration-dominant type, hepatocytes show diffuse and severe swelling. alf secondary to acute hepatitis b virus (hbv) infection is rare in clinical practice. patients with alf secondary to acute hbv infection usually have no history of hbv infection, are relatively young, and often have predisposing factors (e.g., stress, absence of rest after disease onset, malnutrition, alcoholism, use of liver damaging drugs, pregnancy, concomitant infection). moreover, it usually progresses rapidly, and patients may develop coagulation dysfunction before the jaundice becomes evident. such patients present with symptoms of liver failure characterized by he >grade 2 within 2 weeks, a prothrombin activation ≤40%, an obvious bleeding tendency (i.e., massive petechiae at an injection site), patients have no ascites, disease progresses rapidly and has a poor prognosis, and patients frequently die of complications such as brain edema or cerebral hernia within 3 weeks. some patients may recover rapidly after appropriate therapy and are usually diagnosed with liver failure of extensive hepatocyte swelling. after recovery, the risk for hepatic cirrhosis is relatively low. another situation is the presence of a history of hbv infection in which patients have a good liver condition and no evidence of/mild liver lesions. for hbv patients with alf, the liver condition is good (as in alf patients without prior hbv infection) and both alf patients with and without prior hbv infection share pathological basis, pattern of disease onset, and clinical course. alf usually progresses rapidly, and the four stages of alf (i.e., prejaundice stage, bilirubin increase stage, bilirubin plateau stage, and bilirubin reduction stage) are difficult to identify. alf may result in high mortality, and a majority of patients directly develop alf of the bilirubin increase stage or even terminal stage. pathologically, slf not only has extensive hepatocyte necrosis but also an obvious inflammatory reaction and formation of regenerative nodules in residual hepatocytes. slf usually has an origin of alf. when slf occurs in patients with or without mild liver lesions, it often shows an abrupt onset. in the early stages, slf is similar to acute icteric hepatitis and patients progressively deteriorate. affected individuals may also develop clinical symptoms of liver failure from 15 days to 26 weeks, including severe fatigue, loss of appetite, frequent vomiting, and deepening jaundice (i.e., a daily increment of >17.1 μmol/l or > 1 mg/dl and an increase in serum bilirubin of >171 μmol/l or 10 mg/dl). patients usually have hepatic foetor, refractory abdominal distension, ascites (susceptible to concomitant peritonitis), evident bleeding tendencies, and mental and neurological symptoms. in the late stages, hepatorenal syndrome may be present and patients often develop complications (such as gastrointestinal bleeding and hepatic coma) before death. the liver either shrinks or remains normal in size. the course of slf lasts for several weeks to several months. patients surviving slf following therapy usually develop postnecrotic hepatic cirrhosis. clinically, slf can be divided into two types. first, the ascites type results in profound jaundice (serum bilirubin of ≥171 μmol/l or > 10 × the upper limit of normal), ascites, and evident bleeding tendencies (i.e., a pta ≤40%). he might be absent or present in the late stages. patients often die of hrs, upper gastrointestinal bleeding, severe secondary infection, and intracranial hemorrhage. slf of the ascites type accounts for a majority of slf. second is the encephalopathy type. such patients have he as an initial symptom and present manifestations as in ash except for course of disease lasting for >14 days. patients usually die from either brain edema or cerebral hernia. slf of the encephalopathy type is also not rare. slf often has an abrupt onset, and the four stages (i.e., the prejaundice, bilirubin increase, bilirubin plateau, and bilirubin reduction stage) of liver failure are difficult to identify. it is usually associated with a high mortality rate. the pathological basis of aclf is similar to that of slf; therefore, they both share clinical characteristics. a majority of patients with aclf have ascites, spontaneous peritonitis, and biliary tract infection. in the late stages, patients may develop portal hypertension and other complications, repetitive he and hrs, and most die of gastrointestinal bleeding and hrs. according to the guideline for the prevention and therapy of viral hepatitis (2000), a fraction of patients with csh meeting the diagnostic criteria can be grouped with aclf. that is, patients have either chronic hepatitis or compensated hepatitis cirrhosis that remain stable, but some predisposing factors cause the deterioration of liver function, which, thereafter, progresses to liver failure. aclf refers to acute decompensated liver function in the presence of chronic liver disease. the previously mentioned guidelines emphasize pre-existing chronic liver disease and liver failure due to acute liver dysfunction. it is important to note that controversy regarding the basis of chronic liver disease persists. in 2002, an english physician proposed that aclf was diagnosed in chronic liver disease patients with compensated liver function presenting with acute aggravation of liver function within 2-4 weeks due to accidents characterized by jaundice, he, and/or hrs. german physicians subsequently proposed that the diagnostic criteria for aclf included (1) the liver has the histological, laboratory, or ultrasound evidence of hepatic cirrhosis; and (2) patients develop jaundice, ascites, coagulation dysfunction, and/or grade 2-4 he, meeting the definition of decompensated liver function. the guideline for the diagnosis and therapy of liver failure (2006) does not detail chronic liver diseases as a basis of liver failure. however, in general, hbv carrier status may not serve as a baseline liver disease for patients with either chronic hepatitis or hepatic cirrhosis. the term aclf also highlights that acute or subacute deterioration of liver function occurs, which rapidly progresses to liver failure. patients often display an abrupt onset and develop severe fatigue and evident gastrointestinal symptoms. in the early stages, there is acute liver damage; therefore, patients usually present with a significant increase in transaminase levels. thereafter, the disease condition becomes aggravated, and patients may manifest symptoms of liver failure. aclf can also be divided into the brain type and ascites type, of which aclf of the brain type has a higher incidence. further, the four stages (prejaundice, bilirubin increase, bilirubin plateau, and bilirubin reduction) of liver failure are very clear in patients with aclf. one goal for physicians and researchers is to determine individualized therapy for aclf patients according to the specific stage of aclf. patients with clf usually have decompensated hepatic cirrhosis that progressively evolves into chronic liver failure, resulting in clinical manifestations of chronic decompensated liver dysfunction characterized by ascites, portal hypertension, coagulation dysfunction, and he. the pathological basis of chronic liver failure is hepatic cirrhosis, chronic and progressive aggravation of hepatocyte injury, and reduction in hepatocytes that are unable to maintain normal liver function. physical examination usually shows signs of chronic liver diseases (such as liver palms and spider angiomas), imaging examination shows characteristics of chronic liver diseases (such as spleen thickening), and laboratory examination also supports the diagnosis of chronic liver diseases (increased gamma-globulin and reduced/inverted albumin/globulin ratio). of note, a majority of patients have no clear history of liver disease and may initially be misdiagnosed with alf. further examinations may provide evidence of hepatic cirrhosis. when patients with hepatic cirrhosis become decompensated, the liver dysfunction usually presents with acute deterioration due to complications or gradually aggravates in a small fraction of patients. on the basis of the above findings, liver failure secondary to decompensated hepatic cirrhosis can be divided into slowly progressive liver failure and acutely deteriorating liver failure. the former shows a chronic status of liver failure and is characterized by repetitive ascites and he. the latter shows an acute deterioration of liver function in the presence of chronic liver dysfunction, which is similar to aclf in the disease onset and clinical course. hepatopulmonary syndrome is often noted in acutely deteriorated liver failure, and patients usually die of heavy gastrointestinal bleeding, hrs, and severe infection. clf is generally characterized by slow progression of liver failure, and the course of clf is relatively long. the four stages (prejaundice, bilirubin increase, bilirubin plateau, and bilirubin reduction) of liver failure are difficult to identify. as such, finding ways to best preserve residual hepatic function reserve is one of the important therapeutic goals in affected individuals. yu-ming wang, deng-ming he liver failure is a clinical syndrome with high mortality by severe liver damage. it is caused by a variety of causes, results in serious obstacles or decompensation of liver synthesis, detoxication, excretion and biotransformation and appears with coagulation disorders, jaundice, hepatic encephalopathy and ascites as main manifestation. hepatic failure can be divided into acute liver failure (alf), subacute liver failure (salf), acute-on-chronic liver failure (aclf), and chronic liver failure (clf). although the incidence of liver failure is not high in western countries, the relevant papers, reviews, conferences and other exchanges have increased markedly in recent years. aasld, easl and apasl had established a thematic seminar and the definition diagnosis and classification of liver failure has been consistent. at the same time, there are different understandings. therefore, it is necessary to discuss the main differences of liver failure diagnosis and classification, so as to develop a more rational diagnosis and classification scheme. the classification of liver failure involves classification of hepatic injury. a variety of factors (drugs, virus, alcohol, etc.) can cause liver cell damage. although course and prognosis of liver cell damage are different, the most common mechanism is inflammation. wieland et al. found that there were two mechanisms of liver cell injury in the immune clearance of hbv; non-soluble cell damage occurring early and soluble cell damage, early mainly non-soluble cell injury, by the study of gorillas with hbv infected. in 1994, bonino et al. proposed the theory of nonsoluble liver cell damage in the study of fibrosing cholestatic hepatitis (fch) study. however, this theory was ignored because many scholars believed that it ignored the background of immunosuppression. at that time, fch was still considered as the injury of endoplasmic reticulum and golgi apparatus by the excess replication of hbv as well as overexpression of hbv antigen during immune inhibition. however, in 2008, masayoshi et al. reported that most effective antibodies had been detected in children after living donor liver transplantation who received chickenpox vaccine, attenuated vaccines in children, such as measles, rubella, and mumps. according to this, immune suppression cannot stop antibody production and in fch, non-cellular immune injury may be present. recently, we found that there were two types of hbv reactivation in immune-suppressed; high alt type (>10 × uln) and low alt type (<5 × uln) and both with bad prognosis. we proposed that there may be different injury mechanisms, both immune and non-immune damage. in 2000, rolando et al. found that 56.8% of acute liver failure patients with systemic inflammatory response syndrome (sirs), and there was significant correlation between the progress of hepatic encephalopathy and infection and between the degree of hepatic encephalopathy and the occurred rate of infection. infection aggravates degree of illness and fatality rate in liver failure patients. accordingly, we speculate that serious primary liver injury can cause injury to other organs by cytokines, while injury to other organs can aggravate liver injury. corresponding with this, we summarized relevant literature and referred that alf can occur secondary in the development process of multiple organ failure induced by non-primary liver damage. this suggests that this type of liver failure is a special type of liver failure, which is a result of rapid changes in internal environment and inflammatory factors induce liver damage. therefore, liver failure can be divided into one with primary injury and with secondary injury. liver failure mainly caused by non-soluble cell injury is extremely rare; as "paralyzed type", "stunned type" or "edematous type", and with the good prognosis. in 2005, small-for-size syndrome after partial liver transplantation was defined by dahm et al., which is a hepatic failure due to less of liver tissue. actually, this syndrome can also be seen as a special type of liver failure caused by non-soluble liver cell damage. soluble cell injury of liver is relatively common, such as the early hepatic failure in hepatocellular carcinoma after interventional therapy, some drug-induced alf, etc. liver failure caused by immune injury is more common in liver failure caused by autoimmune liver disease. liver failure caused by non-immune injury is more common in liver failure patients with severe cellular immunity damaged (hiv/ aids patients, chemotherapy patients) or immunity inhibited (in patients after organ transplantation). primary type is common in fulminant hepatic failure (fhf), the secondary type is seen in severe systemic diseases, such as severe sepsis and acute hemorrhagic. in fact, clinical liver failure is the result of combination of different proportions of various types of damage factors. based on the role of various factors in liver failure, it can be categorized. according to acute and sustained, the clinical course and the image changes, liver failure can be divided into different stages with some or certain factors dominated. we have divided it into two categories, necrosis type and the decompensation type. although this classification is based on practice and clinical management, large-sample analysis is needed in the future. no matter what the cause, liver failure may be divided into two major categories on the pathophysiology. one type is necrosis induced by hepatic inflammation; the other type is decompensation of liver cells. in particular, alf and salf are types of necrosis, aclf and clf are decompensated type. mixed type, both necrosis type and decompensation type, is possible, which treatment should be considered. the relevance of these two type of liver failure mainly reflects in treatment. necrosis type mainly focuses on treatment of the cause (as antiviral treatment for hbv infection and corticosteroid treatment for autoimmune hepatitis) and symptomatic support treatment (as anti-inflammatory treatment and integrated symptomatic support treatment). decompensation type mainly focuses on intensive treatment (as control infection for sirs and control bleeding for gastrointestinal hemorrhage). some pathophysiological processes such as hepatic encephalopathy (he) are in both type, but are different in different liver failure type. for example, cerebral edema is more prominent and progressive and less to do with high protein diet in he patient of necrosis type of liver failure, while is just the opposite in decompensation of hepatic failure. based on pathologic features and speed of progression, liver failure can be divided into four categories: alf, salf, aclf, and clf (tables 1.3 and 1.4). alf occurs liver failure syndrome characterized with varying degrees of hepatic encephalopathy (9):643-6 (article in chinese) [1] in 26 weeks except for liver cirrhosis. salf occurs liver failure syndrome in 15 days to 26 weeks. aclf is acute hepatic decompensation on the basis of chronic liver disease. clf refers to chronic hepatic decompensation characterized by ascites, portal hypertension, coagulation disorders or hepatic encephalopathy based on cirrhosis. diagnosis and classification of liver failure are the most controversial part, but has tended to unify in recent years. in 2007, professor roger william from the university of london proposed the same type criteria as chinese. the only difference is limited to 8 weeks for alf and no aslf. due to aslf belonging to alf, this part is not contradictory for two criteria. in recent years, a discussion of aclf proposed many times by sarin from india made aclf more valued and accepted. clf is also getting more recognition. recently, to clarify meaning and avoid misunderstandings, it was recommended that clf be converted into end-stage liver failure (eslf). although issues related to classification of liver failure have largely agreed, there are some differences in practical application. dispute over whether to set up subtypes (namely aclf/saclf) of aclf occurred during drafting the new version of guide. two suggestions of modification are: (1) with existence of cld, clinical manifestation of acute (within 2 weeks) and sub-acute (2 ~ 26 weeks) liver function decompensation occur; (2) with existence of cld, clinical manifestation of acute (within 4 weeks) liver function decompensation occur (this actually is the original version). the reasons are: (1) the classification of 2006 guide has only been published for 6 years, and it was not easy for it to be widely recognized home and abroad. it requires more time to accumulate experience in this field, so frequent revision are inappropriate; (2) as is generally accepted home and abroad, aclf mostly occurs within 1 month (4 weeks) after onset, while those as late as 26 weeks after onset are rare (the document of our department indicates the same result); (3) it still requires medical evidence and extensive clinical summary and proof to have the new diagnostic term "saclf" in english and chinese established and accepted; (4) clinical significance and importance of salf classification are neither prominent nor urgent. after multiple discussions, diagnostic classification in the 2012 guide applied the latter one (table 1.5). despite the differences, academia has become unified about the classification of liver failure in the world. the differences towards the convergence of: (1) in terms of naming and classification. naming has simply become to acute liver failure (including acute and subacute) and clf (include acute-on-chronic and chronic decompensation), and tend to be more simplified. aasld guidelines clearly stated that nouns used to differentiate the length of the course (such as hyper acute, acute, and subacute) had claimed not to use. (2) in terms of clinical diagnosis. because of many cause of liver failure, it is very difficult to achieve unity. the only way is to combine the clinical diagnosis (such as acute hepatitis) and the pathophysiologic diagnosis (such as alf). (3) if hepatic encephalopathy as a prerequisite for liver failure. currently, it is a prerequisite for alf, and not necessary for clf because hepatic decompensation is the main clinical manifestations. according to the severity of the clinical manifestations, liver failure can be divided into early, middle and late stage. 1. extreme weakness, severe gastrointestinal symptoms such as significant loss of appetite, vomiting and abdominal distension; 2. progressive jaundice (serum total bilirubin ≥171 μ mol/l or increased by ≥17.1 μ mol/l daily); 3. bleeding tendency, 30% < prothrombin activity (pta) ≤40% (or 1.5 < inr ≤ 1.9); 4. no hepatic encephalopathy or other complications. based on the early stage of liver failure, further develop to one of the following two: 1. below grade ii hepatic encephalopathy and/or obvious ascites and infection. 2. obvious bleeding tendency (bleeding point or ecchymoses), and 20% < pta ≤ 30% (or 1.9 < inr ≤ 2.6). based on the middle stage of liver failure, further aggravating, severe bleeding tendency (such as ecchymoses on injection site), pta ≤ 20% (or inr ≥ 2.6), achieve one of the following four: hepatorenal syndrome, upper gastrointestinal bleeding, severe infection and above ii° hepatic encephalopathy. considering the notorious difficulty to treat hepatic failure and its high mortality rate, special attention has to be paid to and active treatment has to be performed on patients showing the following early-stage clinical features of hepatic failure. 1. extreme weakness, severe gastrointestinal symptoms such as significant loss of appetite, vomiting and abdominal distension. 2. progressive jaundice (51 μ mol/l ≤ t.bil ≤ 171 μ mol/l), and increased by ≥17.1 μ mol/l daily; 3. bleeding tendency, 40% < prothrombin activity (pta) ≤50% (or 1.5 < inr ≤ 1.6). clinical practice has shown that, with or without history of chronic liver disease, there are patients with grade ii hepatic encephalopathy in a short period, with rapid development, poor prognosis. these patients should be regarded as fulminant type. meanwhile, in asia, including china, there are some patients with severe jaundice, ascites and bleeding as the main presentation, with relatively slow development and very poor prognosis, but without hepatic encephalopathy. these patients should be classified as subacute type. fulminant type must have a hepatic encephalopathy. however, it is not necessary for subacute type, which mainly characterized by severe jaundice and ascites. compared with severe hepatitis in china, fulminant type amounts to acute severe hepatitis and chronic severe hepatitis with acute onset, subacute type amounts to subacute severe hepatitis and chronic severe hepatitis with subacute onset. currently, a large divergence of these two types is about time, from 10 days to 8 weeks. according to clinical features of alf, alf can be further divided into fulminant type and subacute type interval for 4 weeks. however, according to more researches, fulminant hepatitis, characterized by massive necrosis of liver, brain edema, and hepatic encephalopathy, concentrated in 2 weeks, most of them in 10 days or less. taking into account the subacute type belongs to acute category, subacute are not be established in international classification. alf are defined as liver failure in 26 weeks. on the difference between acute and chronic liver failure, most chinese scholars depend on the past history, which be ignored internationally. the difference lies in this onset. acute inflammation, necrosis and chronic decompensated were classified as acute and chronic processes, respectively. most typical example is that patients with acute heavy syndrome of onset in hbv carrier were divided into alf by the scholars from hong kong, macao and taiwan. similarly, liver failure caused by hepatitis flares in chronic hbv carriers, reactivation of chronic hepatitis b, super infection with hdv and hbeag seroconversion are included in alf. it is inconsistent with classification methods in china. the reason lies in greater emphasis on the continuous development processes of hepatitis chronicity and severity in china, and focused on the acute effects this time abroad. some scholars suggest that considering the significant difference between clf and the other three types in clinical manifestation, it is worth discussing whether to list clf as a type of hepatic failure. we believe that significance and importance of clf classification are: (1) clf are similar to crf (uremia) in nephrology and chronic cardiac failure (congestive heart-failure) in cardiology. although their clinical manifestation differ significantly, the "coexistence of acute and chronic failures" is shared by failures of all those organs; (2) clf classification has been generally recognized at home and abroad, and the necessity of classification are further proved by the difference between clf and the other three types; (3) clf cases are relatively large in proportion (nearly 30%), which is still increasing (since the proportion of alf/salf are lowering); (4) complications of clf are common and are found in various forms, with bad prognosis; (5) in clf patients with correlation to hbv, virus replication are commonly found, which is closely related to decompensation. the efficacy of nucs are satisfying, which, if taken for a long term, can reverse decompensation, avoiding liver transplantation; it also increases support means in a fast rate, creating more chances for treatment. if the strict definition of acute and subacute liver failure as "no past history of liver disease" is executed, how to name the patients who had a history of chronic liver disease (caused by hbv from mother to child transmission in china)? as for alf and salf, rigorous definition for the past history of liver disease (including hbv carrying history) is necessary in china, and more interested is in this attack instead of the latent infection in the past, even a dominant attack in europe and america. in clinical practice, past history should not be ignored, because patterns of chronic hepatitis b reactivation vary. we summarize them into four types: (1) burst type: suddenly attack based on immune tolerance state, eventual liver failure; (2) recurrent type: repeated unequal flares, finally developing into liver failure; (3) occult type: no obvious attack, presenting with symptoms of decompensation; (4) document type: compensated cirrhosis, acute decompensation in certain situations (mainly due to sepsis and other infections). in burst type absence of history, or only carrier state, the past history can be ignored. in recurrent type, history of recurrent injury is important, which the significance lies in the extent of the occurrence, duration and consequences. because these factors determine the basis of injury to the patient's liver, the patients with mild liver disease have mild or even have no hepatic fibrosis and liver cirrhosis, otherwise, the symptoms will be serious and obvious. the former attack often leads to liver necrosis, the latter often lead to decompensation. the difference between occult type and document type is in the speed of decompensation; the former is slow and the latter is fast. in summary, although history has the certain reference value, pathophysiological changes in attack is main of necrosis or decompensation, or a combination of both. whether hepatic encephalopathy should be considered as a complication of liver failure is controversial, because many scholars have listed it as a prerequisite for liver failure, but in recent years, some patients do not necessarily have encephalopathy. from the complete course and early prevention and treatment of liver failure, it is necessary to incorporate non-encephalopathy type, but the effect and prognosis of the rescue treatment should be divided into the encephalopathy type and nonencephalopathy type, because they are different. hepatic encephalopathy is divided into a, b, c type in international guidelines. type a is acute hepatic encephalopathy (alfa-he), which does not include acute hepatic encephalopathy associated with chronic liver disease. some patients with a long-term hbv carrier were diagnosed with aclf or clf on the first time severe, especially in china. in fact, this type of patients with acute or subacute liver necrosis caused by alf. the mechanism of hepatic encephalopathy in this type liver failure is different from alfa-he, as well as treatment. liver failure classification has the greatest impact on treatment of hepatic encephalopathy, based on the following facts: (1) in acute phase of alf, fasting protein diet on the first day, unnecessary in the short term (4 days); however, chronic hepatic encephalopathy of clf don't have to fast; (2) the metabolism of branched chain amino acids (bcaa) in alf was reduced and increased in clf. this suggests that the former should not be added bcaa, and the latter can supplement the bcaa. (3) high blood ammonia in clf is more than in alf. the effect of clf was better than that of alf patients, but the effect was not good for deamination drugs. (4) because cerebral edema in alf is more than in clf, it is better to reduce the intracranial pressure in alf treatment, and in clf with poor efficacy; (5) as for type a and c according to the international consensus of hepatic encephalopathy are equivalent to the current alf and clf. severe cerebral edema has been found in alf and its mechanism is not clear. study on cerebral edema treatment have been found that hypothermia therapy can reduce the cerebral blood flow and brain edema. in 46th easl, larsen from affiliated hospital of university of copenhagen in denmark reported that therapeutic hypothermia did not support application in the treatment of patients with alf in a prospective, multicenter randomized controlled experimental study (the study was carried out in 2004-2010). the study results are different from previous studies. we believe that the reason is likely from the bias of group selection. in addition, there is a great difference of the mechanism of hepatic encephalopathy in patients with chronic hepatic failure and alf, so the response to hypothermia therapy will vary greatly. based on the existing research, hypothermia therapy has better effect on hepatic encephalopathy in alf patients caused by cerebral edema, and has bad effect on hepatic encephalopathy in the chronic decompensated liver failure caused by metabolic abnormalities. as for hepatic encephalopathy in aclf patients, the effect depends on the roles of cerebral edema in pathogenesis. therefore, it is recommended that the patients should be carefully screened to obtain a comparable result in the study of the hypothermia therapy in hepatic encephalopathy. we believe that, with the gradual elucidation of the pathogenesis of liver failure, the treatment measures will also be more targeted, the efficiency may be further improved. glucocorticoids (gcs) therapy in chronic active hepatitis b began in the 1960s to 1970s. however, compared with the control group, gcs did not show better effect. there have been reports that, after stopping the treatment of immunosuppressive therapy, early re-given long-term high-dose gcs can prevent severe hepatitis in hbv reactivation patients. however, this result had not been affirmed in the future clinical practice. although more application of gcs before 1980s, each effect is different. there is a negative trend in 1990s. we have analyzed, the effect may involve two major factors: one is the choice of indications, dose and duration of treatment; another is the prevention of adverse reactions and complications of gcs. according to the guidelines for diagnosis and treatment of liver failure (2006) in china, liver failure without viral infections, such as autoimmune hepatitis and acute alcoholism (severe alcoholic liver disease), etc. are gcs indications. at the early stage of liver failure caused by other reasons, in the patients with developed rapidly and no serious infection, bleeding and other complications, gcs may be appropriate to use. gcs can improve the survival rate of patients with autoimmune hepatitis and severe alcoholic hepatitis, and has been recognized by most scholars. for hepatitis flares in chb patients, if on the basis of combined application of nucs, gcs will inhibit excessive hyperactivity of host cellular immunity and excessive release of cytokines, and help preventing liver cell death. at the same time, the role of gcs in the treatment of chb and the specific usage, as well as the effect of combined treatment is still controversial. the reason is that the advantages and disadvantages of gcs are very prominent. the key of success or failure lies in the clinical skills. in recent years, the reports of gcs for the treatment of hbv related liver failure increased. there are three main reasons: (1) nucs can effectively resist hbv replication due to gcs; (2) application of proton pump inhibitors can effectively prevent gastrointestinal bleeding due to gcs; (3) increasing of infection prevention and treatment can effectively fight infection due to gcs. even so, the above three aspects of the problem have not been satisfactorily resolved. therefore, we put forward several viewpoints on the current application of gcs: first, to fully analyze the advantages and disadvantages, to consider the main function and purpose after the gcs application and the risk of adverse events before expanding the indications of gcs. secondly, both short course treatment (3-5 days) and long course treatment has drawbacks. the former may not be sufficient to adequately inhibit a strong immune response, and induce a stronger immune response after a sudden stop; the latter can induce bleeding, infection or viral resistance. finally, in a common clinical liver failure induced by hbv reactivation under immunosuppression, the mechanism is often unrelated with immune activation. the typical representative is fch, and the prognosis is extremely bad. it should be vigilant, focus on prevention. our department has treated a chronic hepatitis c (chc) patients after kidney transplant and taking large doses of gcs. severe fch was induced, resulting in liver failure and eventually died. in recent years, the hepatic stem cell therapy of liver failure was concerned, human stem cell transplantation in the treatment of clinical study on severe hepatitis/liver failure were carried out and the results were satisfactory. however, due to the difference of the patient's condition or stage, and most of which are case report, it is difficult to draw an objective conclusion. we have repeatedly reported that the human umbilical cord blood stem cells and bone marrow stem cells in vitro and in vivo can be successfully transformed into liver cells, but the biggest obstacle to its application to clinical practice is the limited number. it is difficult to repopulate the whole liver or at least part of liver compensatory function. in addition, considering the part liver failure patients with cirrhosis background, which clinical manifestations as aclf and clf in the practical application, there are the following issues: (1) it is difficult to provide effective growth and functional support for implanted cells in a diseased liver; (2) the original portal hypertension can be aggravated by portal vein implanted cells; (3) input cells by vein can obstruct the pulmonary vein, thus affect the pulmonary circulation; (4) if the implanted cells for allogeneic or xenogeneic tissue, may have compatibility problems; (5) when using gcs to prevent rejection, severe hepatitis patients are easy to infection and other related adverse reactions. finally, it is pointed out that there is a tendency of the academic circles to the understanding of liver failure for decades, that is, the existence of the chronic process is often neglected in the process of analysis, and vice versa. a typical example, until nearly a few years ago, many scholars in the world still do not recognize the existence of aclf and clf. chinese research papers on liver failure published very little in the international, the main reason is that the aclf and clf is not clearly defined. the papers are often rejected because the diagnosis of type does not conform to the international. in contrast, as previously mentioned in the definition of aki and hrs, the existence of alf (including salf) is ignored. another example: in the report of antiviral treatment of hbv related liver failure, some foreign scholars put forward that the liver failure should be changed to the decompensated liver cirrhosis, the reason is that acute hbv infection is self-limited. however, at present, there has been a clinical recognition that the hbv related alf is actually an acute episode of chronic carrying process and should be referred to as aclf. in chinese guidelines for the diagnosis and treatment of liver failure (2006), liver failure was divided into four types, mainly because of their different mechanisms and treatment (sometimes even the opposite). due to limitation of the space and data, we have not discussed infection, bleeding and other complications in relation to liver failure. these pathological processes are closely related to the classification, and should be studied further in relation to liver failure. in future, we should develop classification of liver failure according to the mechanism of liver injury with different causes, and provide the basis for clinical types of hepatic failure. in order to improve the level of diagnosis and treatment of liver failure, the mechanism based classification should be carefully assessed and evaluated. standard' of definite diagnosis, severity evaluation and treatment effect assessment, and it is irreplaceable compared with other examinations. histopathological evaluation of aechb and liver failure not only contribute to the definite diagnosis of severity of aechb and provide pathological evidence for effective clinical treatment of hepatitis b, but also is helpful to the early detection of histopathological proof of aechb by pathological examinations and of pre-warning function for clinical treatment of aechb. this section mainly focuses on pathological features of aechb and other types of liver failure. aechb, clinicopathological manifestation of aggravation of liver necroinflammation and disease deterioration of chronic hepatitis b, tends to be of poor prognosis without positive and effective intervention. its pathological characteristics mainly include: ballooning degeneration of diffuse hepatocytes, significantly increased focal necrosis, confluent necrosis, bridging necrosis, extensive and intensive interface hepatitis, massive or submassive necrosis, many neutrophil infiltrations in hepatic lobules and portal areas and moderate intrahepatic cholestasis. prominent hepatocyte necrosis is the pathological foundation of aechb and manifests extensive multifocal necrosis, confluent necrosis, bridging necrosis and other forms of necrocytosis. severe ones can even occur submassive and massive necrosis leading to the extreme form of aechb, severe hepatitis (liver failure). ballooning degeneration of diffuse hepatocytes is one of pathological characteristics of aechb ( fig. 1.1 ). the hepatocytes manifest sparse and granular cytoplasm, sometimes can be micro-bubble like. the degenerated hepatocyte is 2-4 times bigger than the normal hepatocyte. sometimes the ballooning degenerated hepatocytes can fuse and transform into multinucleated cells, and this lesion is similar to that of neonatal giant cell hepatitis when it is relatively extensive. the general performance of the liver is increased volume, tense capsular and cutting edge eversion due to tension. ballooning degeneration of hepatocyte is not specific histological manifestation of hepatitis band also can occur in liver tissues of hepatitis caused by factors such as alcohol or drugs. extensive and diffuse ballooning degeneration of hepatocyte can make the hepatocytic plate wider, and hepatic sinusoid is pressed to be narrower, causing microcirculation disorder of liver tissue and exacerbation of disease. liver lobular inflammation activity is enhanced, and apoptotic bodies and focal necrosis increased significantly when aechb occurs. hepatocyte apoptosis is the programmed necrosis and one of the major forms of hepatocyte death in hbv infection. histopathology manifests cell membrane shrinkage, deepened cytoplasmic staining, eosinophilic degeneration. free apoptotic bodies in liver sinus are large or the apoptotic cell fragments, sometimes containing nuclear fragments. focal necrosis is another form of liver cell necrosis and manifests as an interruption of the hepatocytic cords or replacement by focal lymphocytes and macrophages, with hepatic regeneration that often causes irregular arrangement of hepatocytic cords. this necrosis often inferred from the disappearance of the hepatocytes and the infiltration of inflammatory cells rather than what is actually seen under a microscope. swelling hepatocytes presented increased size and loosing cytoplasm, further develop to ballooning degeneration showed almost spherical in size and transparent cytoplasm, predominantly in the lower right confluent necrosis and bridging necrosis are the common histopathological changes, play important roles in the progression of aechb and are closely related to the adverse prognosis of hepatitis b. due to the larger necrosis range of confluent necrosis and bridging necrosis, even during repair stage after going through the active stage, the liver tissue often undergoes fibrous repair, resulting in liver fibrosis and hepatic lobule reconstruction, thereby causing liver cirrhosis. statistics show that about 18% of the patients with viral hepatitis who had bridging necrosis progress to cirrhosis. confluent necrosis is regional lytic necrosis of hepatocytes on a larger scale and is common in active stage or aggravation of viral hepatitis, or drug-induced liver injury, which often occurs around the central veins and inflammatory cell infiltration is not obvious. specific confluent necrosis can also be seen in other parts. take ferrous sulfate poisoning for example, confluent necrosis is more common in zone 1 of liver acinus, and when confluent necrosis expands to connecting vascular or portal area, bridging necrosis occurs ( fig. 1.2 ). bridging necrosis is large area hepatic lytic necrosis that connect the portal area to central area (p-c), portal area to portal area (p-p), and central area to central area (c-c). it can be caused by the expansion and confluence of interface hepatitis, or a one-time large-scale translocular necrosis. p-c necrosis: it starts on the periphery of the lobules, affects the central hepatic lobules when it expands, and forms bridging necrosis phenomenon. the currently acknowledged mechanism is as follows: the initial pathological change is serious periphery necrosis of hepatic lobules. with the aggravation of the disease, microcirculatory disturbance occurs in the lobules and causes the hypoxia, degeneration and necrosis of central area liver cells. p-p bridging necrosis: most scholars think it is caused by the expansion of interface inflammation, especially based on the fibrous septum, hbv load increasing significantly, immune response enhancing, activating the signal pathway of liver cell death. with the enhancement of the lesion activity, fresh and severe necrosis occurs. c-c bridging necrosis: it is usually seen in serious disease with bridging necrosis of hepatocytes, but mononuclear cells infiltration is rarely seen in the necrosis area and serum transaminase increases significantly (up to 1000 iu/l above). histological manifestations of bridging necrosis can vary due to the different stages of the disease. in the early stage, the liver parenchymal cells necrotize and then disappears, reticular framework residue accompanied by the infiltration of lymphocytes and macrophages. with the time extending, reticular framework collapses and forms the sparse interval crossing the liver tissues. when bridging necrosis is accompanied with reticular framework collapse, with hepatic necrosis and regeneration, disorder of hepatic lobules occurs. at this time, it is difficult to distinguish the fibrous septa between bridging necrosis and chronic hepatitis, and elastic fiber staining can help to solve this problem. the elastic fiber staining of bridging necrosis is negative, because elastic fiber formation often takes several months. interface hepatitis, formerly known as piecemeal necrosis, is one of the symbolic histological manifestations in the chronic activity of chronic hepatitis b. it mainly refers to single or small clusters of liver cells around the portal areas necrose and shedding, leading to worm-eaten defect of limiting plates. significant lymphocytes infiltration is commonly seen in and around the portal area. mononuclear cells extend to the hepatic lobules along the destructive limiting plates and encase the necrotic liver cells, resulting in the enlargement of portal areas ( fig. 1.3) . interface hepatitis increases significantly and extensive interface hepatitis occurs in aechb. the interface hepatitis area can exceed 50% of portal areas periphery and be more than a third of the depth of hepatic lobules, even causing bridging necrosis (p-p and p-c). because the limiting plate of the lobules is an important structure to maintain a whole hepatic lobules, interface hepatitis destroys the integrity of hepatic lobule structure. extensive and intensive interface hepatitis can often cause bridging necrosis and bridging fibrosis, and is an essential part of poor prognosis of aechb. massive necrosis and sub-massive necrosis are considered to be the basic pathological changes of severe hepatitis (liver failure) and major substratum for histologic diagnosis of liver failure. massive necrosis is the diffuse lytic necrosis of the liver parenchyma involving more than 2/3 of hepatic lobules. thorough and rapid liver tissue necrosis is shown with invisible necrosis process, and only reticular framework remained and is filled with red blood cells ( fig. 1.4) . sub-massive necrosis is diffuse liquefaction necrosis of the liver parenchyma involving 1/2-2/3 of hepatic lobules. reticular framework collapses and forms reticular fiber bundles, residual liver cells and bile ducts proliferate. massive necrosis and sub-massive necrosis will seriously affect the prognosis of patients with high fatality rate once they occur. the cause of massive necrosis and sub-massive necrosis remains unclear, and the possible causes include excessive virus replication, virus mutation, overlapping with other virus infection and microcirculation, etc. when extensive confluent necrosis, massive necrosis and sub-massive necrosis involve the entire hepatic lobule and even several adjacent hepatic lobule, causing a lobular or adjacent several lobular hepatocytes lytic necrosis, then the panacinar or multiacinar necrosis occur, which is the most severe form of necrosis of aechb. in panacinar and multi-acinar necrosis, with large range of liver cell necrosis, only a small amount of liver cells remain. residues of clump, rosetting, island or glands-like arrayed liver cells are commonly seen around the collapsed reticular framework after necrosis or loose fibrous connective tissue. due to distortion, normal structure cannot be recognized and sometimes can only be identified by portal area range around the necrotic area. cells proliferation can be observed in periportal area, arraying like bile duct structure and these cells can express hallmarks of hepatocyte and bile duct epithelial cells at the same time, which is considered to be the histological manifestation of liver stem cells (hepatic stem cell) activation and proliferation. infiltrating inflammatory cell types tend to be multiple, and the quantity varies. when there is less infiltrating inflammatory cells, the main cell type is macrophage, often containing brown pigment particles. notably, the liver biopsy might be error in diagnosing the necrosis of wide range of diffuse dissolved necrosis of liver parenchyma (above 2/3 lobule), with only the mesh stents remained the whole lobules and multi-lobules, and that is due to limited amount of liver biopsy specimens. for example, multi-acini necrosis occurs only in the area under the liver capsule, and liver biopsy pathological examination may overestimate the severity of illness. in aechb, types of infiltration inflammatory cells in the liver tissue are various, and most of them contain many neutrophils. it is different from obvious lymphocyte accumulation within liver parenchyma and portal area common seen in hepatitis b. usually, cd8 + t cells/cytotoxic t lymphocytes (ctls) are the major effector cells of the inflammatory response in hepatitis b. but when exacerbation occurs, the neutrophils in inflammatory cells infiltrating liver acinus and portal area increase significantly. neutrophils play an important role in innate immunity, and are the first inflammatory cells migrating to the lesion during inflammatory response. these neutrophils kill the invading pathogenic microorganisms through releasing protease and anti-microbial proteins, and producing reactive oxygen. meanwhile, neutrophils have important functions in activation and regulation of innate immunity reaction and adaptive immunity reaction and could release cytokines such as il-8 to participate in regulating adaptive immunity reaction. although cytokines produced by neutrophils are less than mononuclear macrophages, since neutrophils are the first inflammatory cell moving to inflammatory lesion, the immune regulator function of neutrophils is more important during the early or acute stage of immunity reaction. evidently increased neutrophils in liver tissues of aechb might be beneficial for eliminating infected cells, but it is also a "double-edged sword". accumulation of neutrophils may cause extreme immune response and excessive inflammatory reaction might lead to deterioration. therefore, when increasing neutrophils appear in liver tissues of hepatitis b patients, more attention should be paid to identify whether virus mutation, overlap infection, drug-induced liver injury occur and cause aechb. intrahepatic cholestasis (cholestasis), especially moderate or severe cholestasis, is one of the common histologic manifestations of aechb. intrahepatic cholestasis takes shape from the bile thrombus within the cholangioli around the central vein which is hard to be identified, forming cholestasis in expansive interlobular bile duct and large "bile lake" in hepatic tissue ( fig. 1.5) . microscopically, bile can be characterized by dark brown, green or yellow color, occasionally also can present the gray which is difficult to recognize. bilirubin is revealed as green in van gieson staining, which is helpful to pathological diagnosis of intrahepatic cholestasis. moderate and severe intrahepatic cholestasis often cause feather-like degeneration of hepatocytes, even intrahepatic cholestasis infarction. with the extension of duration of intrahepatic cholestasis, the inter-hepatocyte structure relation of 2-3 normal liver cells surrounding the capillary bile duct also changes. the number of liver cells around the bile duct increases, and the bile canaliculi expands, causing the cholestasis related rosette structure forms and the fusing multi-nucleus giant hepatocyte appears. kupffer cells with brown bile pigment can be seen in the hepatic sinusoid. remarkably, although intrahepatic cholestasis is often associated with clinical symptoms such as jaundice and risen serum bilirubin, the severity of intrahepatic cholestasis is not consistent with clinical symptoms and serum bilirubin level. severe hepatitis b (liver failure) is the most severe liver syndrome complex, which is characterized by poor clinical course and high mortality rate. over the years, scholars have continued to explore the definition, classification, diagnosis and treatment of liver failure, but so far no consensus has been reached. according to histopathological features and progression of the disease, china released guidelines on diagnosis and treatment of liver failure in 2006, which divided liver failure into four categories: acute liver failure (alf), sub-acute liver failure (salf), acute-onchronic liver failure (aclf) and chronic liver failure (clf). hbv infection is the most common cause of liver failure in china. acute liver failure is characterized by acute onset, and hepatic encephalopathy (above stage ii) often develops within 2 weeks of the onset, which results in high mortality rate. former knowledge about acute severe hepatitis mostly comes from the autopsy. nowadays, with the development and universal application of biopsy technique, and the further study on acute liver failure, we have better understanding of the development and process of necrotic lesions, and can predict prognosis and outcome according to the necrotic area and type. in alf, liver atrophy is significantly present in gross pathology inspection, especially for the left lobe. coverings shrinkage, thin edge, soft liver texture, section may be yellow or reddish-brown, and some area is red alternating with yellow, and the weight of the liver drops sharply to 600-700 g. histopathology emphasizes extensive and consistent liver cell necrosis caused by one-time strike, and most patients die in the short term. the morphology of liver tissues is relatively simple, manifested as massive or sub-massive necrosis of liver cells, dissociation of liver cords and hepatolysis. the regeneration of liver cells is not obvious, and surviving liver cells show clear ballooning degeneration. hepatic sinus expand and, congest with blood and occur hemorrhage. kupffer cell proliferates and sinusoidal mesh stent does not collapse or completely collapse. quantity of liver cell necrosis is closely correlated with prognosis. if the amount of necrosis is over 70%, mostly the patient will not survive. if the amount is less than 50%, the patient is expected to resume with rapid regeneration of hepatocytes. if there is diffuse small steatosis, the prognosis is often poor. concerning the hepatic regeneration of acute liver failure, former pathology emphasizes on liver cell and bile duct cell proliferation of sub-acute liver failure, and relatively neglects cell regeneration of acute liver failure. based on the authors' knowledge, in some acute liver failure cases, liver tissue demonstrates obvious bile duct-like or acinar-like regeneration within 4 days after onset. the regenerated liver cells were co-expressing albumin and ck18, ck19, indicating these cells have double markers of hepatocytes and biliary epithelial cells, which presumably come from liver pluripotent stem cells. the regenerated liver cells in acute liver failure have their unique characteristics that degenerated and regenerated cells co-exist in the liver. as the time of liver biopsy is different, the morphological change is also varied because of the rapid restoration. the usual dual-core, large nuclear or nuclear fission are rarely seen in regenerated liver cells; liver cell body swelling, transparency of cytoplasmic periphery and slightly basophilic center are commonly seen in regenerated liver cells. because of cell enlargement and transparent cytoplasm, it is often difficult to distinguish from serious ballooning degeneration; in some cases, it shows bile granules within the cytoplasm, bile thrombus within bile capillary with cell swelling and transparency symptoms, which is also similar to the feather-like degeneration caused by bile salt siltation. however, unlike feather-like degeneration which was scattered by small groups or disorderly arranged severe ballooning degeneration, cell enlargement often shows the pole adenoid arrangement, which is known as a sign of liver cell proliferation. the outcomes of continuous liver biopsy also prove its rapid regeneration. nayak et al. also proposed that hepatocyte swelling during the acute liver failure recovery stage indicates good prognosis. the continuous liver biopsy of 12 liver transplant centers in europe also confirmed that the appearance of liver cells enlargement after 12 days of partial liver transplant of acute liver failure with massive necrosis, cells linked to sheets, and lobular structure are basically recovered in 2 to 3 months. the vacuolation, cholestasis and duct-like structure presented by these regenerated enlarged liver cells will be gradually disappeared. the onset of salf is acute, and liver failure syndromes appear within 15 days to 26 weeks, mostly caused by delay of acute liver failure. in salf, liver atrophy is mainly presented as in gross inspection, and variable sizes of regenerative nodules, the yellow-green cutting surface due to cholestasis. histopathology manifests the new and old sub-massive necrosis of liver tissues, or bridging necrosis. in older necrosis area, reticular fibers collapse, or collagen deposit. survived liver cells may have varied degrees of regeneration, and are arranged in nodular. fine, small bile duct proliferation and cholestasis are commonly seen in the periphery lobe. the sinusoids congest in the early stage, collapse in mid-stage, and occlude in late stage. the histopathological distinction of salf and alf is based on the consistency of necrotic lesions. alf emphasizes consistency of necrotic lesions, that is, 'onetime strike', while the necrotic lesions of salf are mixed, caused by 'multiple attack'. in addition, differences also exist in aspects such as cell regeneration and extracellular matrix (ecm) expression between alf and salf. liver stem cells play an important role in the liver cell regeneration process of alf. the regenerated liver cells express dual markers of liver cell and bile duct cell, and are often orderly proliferated along mesh stents. whereas salf presents unipolar regeneration of liver cell and bile duct cell, and the regenerated liver cells is disorderly arranged. due to the different length of disease course, there is no obvious ecm deposit in alf, while salf presents in iii collagen-based ecm deposit. this type of liver failure often develops into post-necrotic cirrhosis. acute-on-chronic liver failure (aclf) refers to acute liver function decompensation occurring on the basis of chronic liver disease. liver gross manifestation of acute-on-chronic liver failure differs with the different stages of chronic liver disease. for instance, aclf occurring in the stage of cirrhosis is accompanied by hepatic cirrhosis nodules besides liver atrophy. the main histology of aclf is new and varied degrees of liver cells necrosis, hepatocyte focal and spotty necrosis, bridging necrosis, confluent necrosis, massive necrosis and sub-massive necrosis on the basis of chronic liver injury. the common chronic changes are as follows: fibrosis in collapsed reticular framework or periportal area with obvious extracellular matrix deposit, forming of large number of fibrous septa, sparse scars, or bridging fibrotic septa when distortion of lobule structures associated with disproportionate numbers of central veins and portal area, pseudolobule formed; twin-cell or multiple-cell of liver plate is commonly seen and the liver plate lose the radiated array, activated regeneration of liver cell caused the occurrence of tumor-like cell. chronic liver failure is chronic liver function decompensation caused by progressive deterioration of liver function on the basis of liver cirrhosis, with ascites, portal hypertension, blood coagulation dysfunction and hepatic encephalopathy as main symptoms. liver gross appearance of clf is significant liver atrophy and nodular liver cirrhosis. histopathological changes are mainly those of liver cirrhosis, including diffuse liver fibrosis, nodular liver cirrhosis with unevenly distributed liver cells necrosis. progression of hepatitis b is an interaction between hbv infection and body response. development of aechb is mainly caused by obvious increased viral load and/or decreased immune clearance. large number of hbv replication can activate hepatocyte death pathways, leading to serious liver inflammation, necrosis and aggregation of disease. additionally, hbv infection overlapping with hcv/hiv, or with etiological factors like drugs and ethanol, could also affect disease progression. fibrosing cholestatic hepatitis (fch), a new clinicopathological type, develops in stages of severe immunosuppression caused by various reasons, especially in hepatitis virus-infected patients lots of immunosuppressant after organ transplant. due to immunosuppressor, hbv replicates rapidly in the patients, leading to quick progression of hepatitis and progressive failure of liver function. the histopathological features of fch are as follows: fibrosis straps starching from the portal area to hepatic sinusoid and circumvoluting basal plates of biliary epithelial; obvious intrahepatic and hepatocytes cholestasis, bile embolism forms in small bile duct; hepatocytes ballooning degeneration with disappearance of cells; mass ground-glass hepatocyte; mild to moderate mixed inflammatory reaction ( fig. 1.6) . fch could quickly proceed to liver failure with blood coagulation dysfunction and hepatic encephalopathy, and mostly die in several weeks to months. due to common transmission, coinfection with hbv and hcv or hiv is not rare clinically. 5-20% of the chronic hbv infected patients carry hcv antibody and there are 7-20 million coinfectious patients all over the world. studies showed that hbv and notable intrahepatic cholestasis (black arrow) and ballooning degeneration and/or feathery degeneration (red arrow), a large number of ground glass hepatocytes (blue arrow), associated with inflammatory response (green arrow) hcv coinfection could promote the synthesis of collagen and promote disease progression to liver fibrosis. compared to the single hbv infection, hbv and hcv coinfection presents more severe liver fibrosis and inflammatory necrosis. studies demonstrated that hbv and hcv coinfection could promote chb progression, cause severe damage of the liver function and then exacerbation, increasing the probability of liver fibrosis, liver cancer and liver failure in chb patients. in hbv and hiv coinfection, hiv infection can affect the natural history of hbv and accelerate the development to end-stage liver disease and liver cirrhosis. immune deficiency induced by hiv infection fosters hbv replication, and even fibrotic cholestasis hepatitis in severe cases. histologically, chb caused by hbv and hiv coinfection had a severer fibrosis than that by simple hbv infection. cases of hepatitis b overlapping drug or alcohol induced liver damage are not rare. even antiviral drugs can cause aechb, and there is previous case report on hepatitis b patient died of acute liver failure induced by anti-hepatitis b virus medication lamivudine. abuse or nonstandard drug use and alcoholism have become the common causes of aechb. pathology manifests features of overlapping drug or alcohol induced liver injury on the basis of hepatitis b changes. for instance, in aechb caused by overlapping drug-induced liver injury, liver tissues present histological characteristics of hepatitis b accompanied with drug-induced liver injury, such as evident increased percentages of infiltrated eosinophils and neutrophils in liver tissues, confluent necrosis with less inflammatory cell infiltration in acinus three area, cholestasis of bile canaliculi and so on. in summary, aechb has its relative histopathological features. understanding of these pathological characteristics can not only help with clinical diagnosis and effective treatment, but also aid to prevent aechb. it is important to note that despite the value of histopathological examination in diagnosis, classification and prognosis assessment, considering the significantly decreased coagulation function of liver failure patients and liver biopsy examination has certain risk, hence more attention should be paid to indications of liver biopsy in clinic. laboratory tests for liver diseases is the important basis to help and ascertain the clinical diagnosis, and the important reference to evaluate disease severity, make classification, predict outcome and guide therapy. the laboratory tests may reflect the pathological change and the functional status of liver in time, and may provide the objective and detailed data as reference for clinical classification and evaluating therapeutic effects, so that clinical intervention and effective treatment can be performed successfully. the liver is a complicated organ and the laboratory test items of relevance to severe hepatitis b are many, there are various biochemical items reflecting liver function, including coagulation function, immune and inflammatory cells and genetic markers. in this section only those laboratory tests that are relevant to acute exacerbation of chronic hepatitis b and severe hepatitis b will be described. for nonspecific laboratory tests, the reader is referred to other more general pathology books and literature. serum bilirubin is not a sensitive parameter of hepatocellular injury, but a significant increase (commonly ≥ ten times of upper limit of normal value) is usually a specific manifestation of acute exacerbation of chronic hepatitis or liver failure, which is also necessary condition to diagnose severe hepatitis or liver failure. in the course of acute exacerbation of hepatitis both direct and indirect bilirubin rise markedly due to the disturbance of bilirubin metabolism and secretion because the injury and hypofunction of hepatocytes, paracholia, and the rupture of bile capillary and biliary duct. the main enzymes reflecting liver function are alanine aminotransferase (alt), aspartate aminotransferase (ast), γ-glutamyltransferase (γ-gt, ggt) and cholinesterase (che). enzyme protein content in the liver account for 66.7% of total liver protein. because the aminopherase content of the liver is 100 times that of blood, in a pathological condition, as long as 1% of the enzyme in the liver is released into blood and keep active, this will be enough to keep the activity of enzyme in the serum increasing at rate of double. alt is an enzyme with the highest increasing amplitude and highest positive incidence when acute liver damage is occurring, with activity in the liver 3000 times that of serum. there is a large range of activity in daytime, commonly higher in the afternoon than the morning. although the activity of alt is almost coincident with the degree of liver damage, the activity of the enzyme decreases rapidly when liver failure or hepatocyte necrosis becomes widespread, with significantly increasing levels of serum bilirubin, manifesting the disassociation of enzyme and bilirubin. alt mainly resides in the cytoplasm of the hepatocyte, whilst ast is found more in the mitochondria. when liver cellular necrosis and change of cell membrane permeability appear, there is more release of alt than ast, but in very severe damage of liver, mitochondria damage is witnessed with elevation of ast and significant elevation of ast/alt. che in the serum is mainly produced by liver, with its activity and synthesis decreasing when liver damage occurs. although the change of che is less compared to the aminopherase when liver cell damage occurs, it will drop dramatically when there is severe liver necrosis and liver decompensation, especially in liver encephalopathy. the half life of human serum albumin is 20-26 days. because the occurrence of low levels of serum albumin is usually a late marker, the albumin level cannot accurately reflect acute liver cell damage, especially during the acute exacerbation of chronic hepatitis b. serum globulin, especially the γ-globulin, is only elevated in particular chronic hepatitis situations such as decompensated cirrhosis and autoimmune liver disease. the half life of serum prealbumin is only 1.9 days, and its reaction is more rapid and sensitive, which can reflect liver cell damage earlier. serum prealbumin has special diagnostic value with acute exacerbation of liver especially acute and subacute severe hepatitis. total cholesterol is composed by cholesterol ester and free cholesterol in healthy people. when hepatocellular damage appears, its cholesterol esterification noticeably decreases. it has been reported that α lipoprotein decreased significantly in patients with liver failure and this indicates a poor prognosis, while the changes of other lipoprotein and serum triglycerides were not specific for the severe type of patients. it has been reported that patients with severe liver dysfunction usually have bile acid metabolism abnormalities, indicating that serum total bile acid is a sensitive indicator reflecting functional recovery of liver cell and improvement of pathogenetic condition, just like alt. detecting serum total bile acid plays an important role in predicting disease prognosis of severe liver failure and evaluation of therapeutic effect. plasma coagulation factor xii (hageman factor) can produce activating factor xii by surface activation, which can further activate kallikreinogen (also called prekallikrein, pk) to produce kallikrein, kallikrein then makes prokinin release bradykinin, which cause vasodilatation, increasing capillary permeability with decreasing blood pressure. because of the short plasma half-life of pk, its plasma content decreases rapidly during liver failure, so pk has important diagnostic value with acute severe type hepatitis and liver failure. it was reported that its content was 92 ± 20% in healthy controls, 30 ± 6% in survivors with decompensatory liver cirrhosis, while only 16 ± 6% in non-survivors. a content level below 23% predicts poor prognosis, with patients usually dying of liver dysfunction within 30-45 days. gst is a protein rich in the liver, the renal tubules and intestine cells in mammals. its major function is detoxication by combining with multiple metabolistic organics such as bilirubin, bromphenol, cholecystographic agent and epoxide. because of the high content of gst in the hepatocyte, its small molecular weight and short half-life (only 90 min), it can be released into the blood after hepatocyte necrosis with high concentration. thus, gst has become a good predictor of observing hepatocyte necrosis especially in patients with acute liver failure and fulminant hepatitis, not only for early diagnosis of hepatocyte necrosis, but also for predicting prognosis. other factors such as multiple circular lysosomal enzyme and serum hyaluronate are all significantly elevated during a course of liver failure. these markers can also be used for monitoring and diagnosis of the disease. severe hepatitis and liver failure caused by hbv infection and other pathogenic factors are not obviously different under laboratory examination, although they have different etiological factors, pathogenesis and clinical manifestation. the major liver function, including protein (especial various kinds of coagulation factors) synthesis, metabolism and detoxication, are the first to lose function, and so result in severe hemorrhage and hepatic encephalopathy. because of the difference on the production of coagulation factors and the steps they involve in, not all coagulation factor detections are suitable for detection of severe type hepatitis and acute exacerbation of hepatitis b. vitamin k dependent factors mainly includes prothrombin (factor ii), pre-convert in (factor vii), christmas factor (factor ix) and fibrin stabilizing factor (factor x). patients with severe type hepatitis can manifest vitamin k deficiency caused by bile accumulation exterior and interior of liver, ingestion reduction or diarrhea. factor vii with the shortest half-life (4-6 h) is influenced firstly, which cause prolonged prothrombin time (pt). factor ii is not sensitive on vitamin k deficiency, while factor ix and factor x are moderately sensitive. factor vii can be considered as a reliable indicator, and it has important clinical value on prediction of prognosis of acute liver failure because of its short half-life and less influence by other factors, such as inflammation, dic, fibrinolysis, etc. it was reported that when the level of factor vii was below 20% of normal controls, the probability of death increased significantly, with predicting value of 100% sensitivity and 77% specificity. human fibrinogen-like protein 2/fibroleukin prothrombinase is a mediator of inflammation produced by activated macrophages, which belongs to fibrinogen superfamily and can catalyze and convert prothrombin to activate thrombin directly, thereby starting the clotting process and promoting thrombogenesis. some studies have demonstrated that the level of human fibrinogen-like protein 2/fibroleukin prothrombinase, if expressed highly and specifically on pbmcs and liver tissue in patients with acute-on-chronic liver failure, can be correlated with disease severity. other clotting factors such as factor vii, xi, xii, i, c protein, plasminogen and platelet count are all decreased in various types of liver diseases, with no special changes on the course of severe-type hepatitis or acute exacerbation. so they are not suitable for evaluation of acute exacerbation of chb. some scholars considered that combination detection of antithrombin iii (at iii), hepaplastin test (hp) and thrombin time (tt) has important value on early predicting fulminant hepatitis. pt is mainly used to detect activity of factor vii, x, ii, v and i. it has three measurable methods: one is the prolonged pt, normal pt is 12 to 16 s, with abnormality above 3 s of normal control value; another is prothrombin activity (pta), which can be calculated by mathematical formula. normal pta is 80-120%, usually below 40% in liver failure. the third is international normalized ratio (inr), which can be calculated by certain correction factor: pt in patients/pt in healthy. inr is above 1.2 in abnormality, and it is generally over 1.5 in liver failure. pta and inr detection values have been included in diagnostic criteria of international and domestic liver failure. ptt is a screening test of intrinsic coagulation system. ptt can prolong when it is faced with factor vii, ix, xi and xii deficiency or factor i, ii, v and x reduction and increment of anticoagulant substances. because ptt can be prolonged in a variety of liver diseases, demonstrating that ptt is not necessarily specific for the diagnosis of liver failure. tt tests for the activity of plasma fibrinogen. tt can be prolonged when fibrin degradation product (fdp) is increasing, fibrinogenolysis activity increasing, or fibrinogen (fib) decreasing, or heparin-like anticoagulant substances occurrence. apart from tissue-type plasminogen activator (tpa) and plasminogen activation inhibitor-1 (pai-1), other proteins and molecules involved in the course of fibrinolysis are all synthesized in liver. so plasminogen, α2 antiplasmin and thrombin activation fibrinolysis inhibitor (tafi) all decreased significantly in severe liver disease such as decompensated cirrhosis. as a result of dysfunction of liver clearance, the tpa level elevates inversely, while with normal or less elevated pai-1 in patients with cirrhosis, can cause proportion disequilibrium and final hyperfibrinolysis. in patients with acute liver failure, because of large elevation of pai-1 as acute phase reactive molecule, the activity of fibrinolysis decreases. on the contrary, taf-1 decreases by almost 50%, which induces elevation of fibrinolysis activity. although synthesis of partial coagulation factors and clotting dysfunction usually appear during the course of acute exacerbation of hepatitis b and development of severe-type hepatitis/liver failure, with manifestation of prolonged pt/decreased pta/elevated inr, some investigators had observed conflicting results recent years. specifically, although the mean inr was 3.4 ± 1.7 in acute liver injury/acute liver failure patients complicated by hepatic encephalopathy, concentrations of factor v and factor vii also decreased, the mean values of the indicators mentioned above detected by thromboelastography (teg) were normal, and also five teg parameters were normal in 63% patients. we think that the reason of normal clotting function detected by teg in patients with ali/alf might be the normal value of platelet count and fibrinogen quantitation. furthermore, more platelet and factor vii can be produced, and the levels of anticoagulant protein (protein c, protein s and antithrombase) decrease, which also compensates for the defect of the other coagulation factors. in total, clotting parameters such as inr, etc. can be considered as valuable indicators for predicting prognosis, although they cannot be used to reflect hemorrhage severity in patients with ali/alf. ammonia has been considered as a precipitating factor of hepatic encephalopathy for more than 100 years. during severe liver dysfunction, carbamide synthesis is injured, and brain tissue becomes a major organ of ammonia detoxication. with glutamine synthetase, astrocytes in brain can convert glutamate to glutamine to remove accumulated ammonia in vivo by the amidation of ammonia. because the synthesis of glutamine consumes energy, the large consumption of atp can cause energy exhaustion. excess accumulation of glutamine in astrocytes induced by high blood ammonia can cause increasing osmotic pressure and brain cellular edema. this has been confirmed by mri, while with the recovery of liver antidotal function after liver transplantation, previous hepatic encephalopathy can be reversed. in patients with acute liver failure, the risk of cerebral hemorrhage increases rapidly when arterial blood ammonia is above 150 μmol/l. about 55% patients can have acute intracranial hypertension when arterial blood ammonia exceeds 200 μmol/l. bernal w, et al. in their study which involved 165 patients with acute liver failure, observed that the level of arterial blood ammonia on admission was an independent risk factor of hepatic encephalopathy and intracranial hypertension, and the sensitivity was 70% for predicting severe hepatic encephalopathy with arterial blood ammonia above 100 μmol/l. combining with the meld score can further increase specificity and sensitivity. the toxicity of ammonia is multiple. the accumulation of ammonia not only influences brain metabolism, injuries brain cellular organelle directly and indirectly, causes disequilibrium of brain internal inhibitory and excitatory neurotransmitter and injuries brain energy metabolism, but also changes a series of gene expressions that are important proteins maintaining brain function. autoregulation of cerebral blood flow is affected and the blood brain barrier broken down. latest studies have revealed that ammonia can injury many important functions of neutrophil, such as chemotaxis, phagotrophy and degranulation, and can also stimulate and produce large reactive oxygen species (ros), cause sirs, which then further aggravates the toxic effects of brain cells from ammonia resulting in a vicious cycle. the liver is an important site of amino acids metabolism. except for bcaa (leucine, isoleucine and valine) metabolism in skeletal muscle, almost all essential amino acid metabolism occurs in liver. because of this, patients with liver failure or cirrhosis, have large amounts of amino acids accumulate in the blood. fischer, et al. demonstrated that disequilibrium of plasma amino acids might be reason of encephalopathy, and further indicated that the molar ratio of valine+leucine+isoleucine and phenylalanine +tyrosine (bcaa/aaa) was closely related with the severity of hepatic encephalopathy. the analysis of plasma amino acids in animals with hepatic encephalopathy demonstrated that other concentrations of amino acids increased significantly except the concentration of arginine, which declined. analysis of brain homogenates from cases of fatal hepatic encephalopathy demonstrated that the concentration of aspartate, arginine and glutamate decreased significantly, and this was closely related with the severity of hepatic encephalopathy. other amino acids, especially the aromatic amino acids, such as tryptophan, phenylalanine and histidine, increased but with carrying amounts; thus, the concentration of the aromatic amino acids was closely related to the severity of hepatic encephalopathy, implying that these aromatic amino acids may play important roles in the pathogenesis of hepatic encephalopathy, although perhaps not as the primary driver. jiang y, et al. conducted a clinical study which enrolled 22 patients with acute hepatitis, 65 patients with chronic hepatitis, 22 patients with severe hepatitis and 47 cirrhosis patients. they observed the ratio of bcaa and aaa was normal in acute hepatitis, mildly lower in the chronic hepatitis (p > 0.05), significantly lower in the severe grade of chronic hepatitis (p < 0.001), and the lowest in the patients with severe-type or cirrhosis (p < 0.05). as for the child-pugh grading, the ratio of bcaa and aaa: c grade <b grade <a grade, with significantly different among the groups (p < 0.001 or p < 0.02). of the patients with severe-type hepatitis and cirrhosis, the ratio of bcaa and aaa in the patients with hepatic encephalopathy was lower than that without hepatic encephalopathy (p < 0.001); the ratio of bcaa and aaa was significantly lower in the non-survivors compared with survivors (p < 0.01 or p < 0.005). they concluded that detection of bcaa/aaa can reflect the degree of liver damage, the lower the ratio was, the worse the liver injury was, and with high risk to develop hepatic encephalopathy; the ratio might also have certain predictive value for prognosis. elevation of plasma free tryptophan has been correlated with aggravation of hepatic encephalopathy; while in patients with acute liver failure, serum methionine concentrations clearly increase, there is no overlap between acute hepatitis and acute liver failure, and this can be used for differential diagnosis between the two kinds of liver diseases. endotoxin is produced by gram-negative bacteria, and has many biological activities, which can cause endotoxemia, with multiple pathological changes, such as fever, shock, dic and granulopenia. critically ill patients even have acute respiratory distress syndrome (ards), acute renal failure (arf) and multiple organ dysfunction syndrome (mods)/multiple organs failure (mof). wang mr, et al. revealed that the incidence of endotoxemia is 64-100% in severe-type hepatitis patients, 46.5-75.9% in decompensated cirrhosis, 23.5% in compensated cirrhosis, and 36% in acute viral hepatitis. it has been considered that the main mechanism of endotoxemia in patients with cirrhosis and severe-type hepatitis is: (1) intestine mucosal nutritional disturbance, epithelial atrophy, shedding and ulceration, which cause injury of intestine mucosal barrier; (2) intestine mucosal congestion, edema and increased intestinal wall permeability; (3) alteration of intestinal flora resulting in translocation of bacteria from the intestinal tract and increasing endotoxin production and absorption; (4) low ability of clearance of endotoxin absorbed by intestine or endotoxin into systemic circulation from bypass circuit. most studies have demonstrated that endotoxemia and liver injury are interrelated; the liver injury can be aggravated when accompanied by endotoxemia. the detection of endotoxemia is beneficial for predicting clinical course, disease severity and prognosis. medium molecule substance was discovered in patients with uremia accepting hemodialysis, and these molecules have molecular weight of 300-1500 d. thereafter, it was confirmed to play an important role in hepatic encephalopathy. medium molecule substance can inhibit na + -k + -atp enzymatic activity of brain cells, which result in na + retention and cause cerebral edema. studies have confirmed that medium molecule substance in the blood increased when acute liver failure patient presented with hepatic encephalopathy, which indicated that an increasing amount of medium molecule substance was closely correlated with hepatic encephalopathy and could be used for predicting clinical severity and prognosis. as for the property of medium molecule substance, it has not been totally characterized. the clearance of human medium molecule substance is mainly by secretion and filtration from the glomerulus. according to clinic observations, the contents of medium molecule substance can be detected in the plasma when plasma creatinine (cr) concentration is above 397.8 μmol/l or creatinine clearance rate (ccr) is below 15 ml/min. lactic acid is an intermediate product of glycometabolism. the concentration of lactic acid can reflect glycometabolism, peripheral circulation, tissue blood-supply and oxygen-supply indirectly. large studies has demonstrated that total blood lactic acid levels are correlated with disease severity and prognosis. the higher the lactic acid is, the worse the clinical condition is, and also for predicting a poor prognosis. some studies have revealed that the arterial blood lactic acid has a low predictive value for liver failure caused by hepatitis virus infection, toxin and immunity, but a better predictive value for acute and super-acute liver failure caused by acetaminophen over-dose. however, this is controversial. schmidt le, et al. showed that the predictive specificity of lactic acid was not high, even for liver failure caused by acetaminophen. furthermore, lactic acid cannot be used as an indicator for of liver transplantation, and if used, must be done in association with the current king's college hospital (kch) criteria. procalcitonin (pct) is a precursor of human calcitonin, with composed of 116 amino acid residues, 13kd of relative molecular weight, and also has no hormone activity as a glucoprotein. pct mainly originates from the c cells of the thyroid. during pathologic conditions, multiple tissue and organs can become production areas, such as liver, lung and glandular tissue. the production of pct can be regulated by bacterial toxin and multiple inflammatory factors, and bacterial toxins are a major stimulating factor inducing production of pct. pct is the best ideal indicator for early diagnosis of systemic infection, and has high specificity. serum pct concentration is positively related with the degree of bacterial systemic infection, but can also descend to normal level gradually accompanied with control of bacterial infection and improvement of clinical condition. as for the cirrhosis patients with ascites, detection of serum and ascitic pct can be of benefit to the clinician for predicting early diagnosis and the curative effect and evaluation of spontaneous bacterial peritonitis (sbp). the pct levels can decrease significantly in cirrhosis patients with sbp upon effective antibiotics treatment. so, pct can be considered as a good predictive factor on diagnosis and differential diagnosis of cirrhosis ascites complicated by bacterial infection. it has been reported that the blood lactic acid and pct levels were elevated to different levels when facing patients with either acute liver failure or acute exacerbation of hepatitis b complicated by intestine endotoxemia. a persistently elevated compared to an or not decreasing level of lactic acid and pct are usually indicative of poor prognosis and therapeutic efficacy. combined detection of blood lactic acid and pct does have certain predictive value for acute exacerbation of hepatitis b. in patients with chb, peripheral blood tregs were positively related with serum virus load, while in patients with acute hepatitis b, peripheral blood tregs experienced process from low to high, and usually are normal in the acute, convalescence, and recovery stages respectively. for the patients their treg were rejected, the ability of ifn-γ secretion in the pbmcs increased significantly upon antigen stimulation. while the tregs isolated from the patients with hbv infection could inhibit proliferative response of hbv antigen treated with autologous pbmcs obviously, which might indicate that the peripheral blood and liver in the patients produced hbv antigen specific tregs. so, tregs not only play an important role on regulating immune response of hbv infection, but also influence the prognosis of the patients with hbv infection. natural killer cell (nk) and natural killer t cells (nkt cells) construct the first line of defense of body to defense pathogen invasion, which account for 13% and 4% respectively in peripheral lymphocytes, while higher with 37% and 26% in the liver. the content of nk cells account for 90% in liver lymphocytes. the accumulation of nk and nkt cells in the liver demonstrates that they have important function, which can directly eliminate virus by killing infecting cells or secreting cytokines. under the condition of virus specific igg antibody production, nk cells can mediate surface igg fc receptor (fcγriii), identify and kill target cells with complexed igg specifically by antibody dependent cell mediated cytotoxicity (adcc). furthermore, nk cells can also play a role in immunoregulation by secreting cytokines, such as ifn-γ, il-2 and tnf. nkt cells are a group of inherent t cells which can recognize lipoid and glycolipid antigen presenting by mhc-i molecules associated with antigen presentation, with cd1d molecule restriction. nkt cells manifest phenotype of both t cells and nk cells (tcr and nk1.1 [nkrp1c], ly-49). nkt cells have two subtypes: type i nkt cells, also called inkt cells, and type ii nkt cells, also called non-inkt cells. the important characteristic of inkt cells is to produce th1 and th2 type cytokines rapidly by activation of tcr signal, and also activate multiple other cell types, such as dc, nk cell, b cells, and conventional t cells rapidly by activation of tcr. so, inkt cells can influence adaptive immunoresponse and series of host defense reaction and pathological course effectively. tian et al. demonstrated that nk cells exist abundantly in normal liver, which account for 33% of lymphocytes within liver and can play important roles on natural immunoresponse of anti-hbv infection. nk cells within the liver can be directly activated by virus or indirectly activated by other cells, such as nkt cells and antigen presenting cells (apc), and exert antiviral effects by their natural cytotoxicity and production of high level of antiviral cytokines. also they can regulate the function of lymphocytes, such as t cells, b cells, apc, and co-ordinate an adaptive immune response. after the binding of fasl expressed by ctl and nk cells and fas expressed by liver cell, the intracellular region of fas protein binds with fas associated death domain (fadd) protein by its c-terminal death domain (dd), then fadd protein activates caspase and lead to apoptosis. malhi h et al. showed that the role of nk cells and nk t cells was a major factor leading to mass death of hepatocyte during the clinical course of viral hepatitis. dendritic cells (dc) are antigen presenting cells with the most powerful function not only playing an antiviral role by secreting cytokine, such as il-12 and ifn-α, but also by promoting immunoclearance by t lymphocytes activated via antigen presentation. furthermore, dc also can regulate the equilibrium of th1 and th2. so, the abnormity of number, phenotype and function of dc is bound to influence the outcome of hbv infection. some studies illustrated that during liver failure the number of pdcs and mdcs in the liver increased significantly; ifn-α produced by pdcs in the liver was correlated with the production of il-12 and il-10; the ability of ifn-α production by pdcs in the peripheral blood decreased and the degree correlated with disease severity, which indicated that the dcs in the peripheral blood had accumulated in the liver and activated when hbv associated acute-on chronic liver failure occurred. after dc activation, the role of antigen presentation was enhanced, and the immune response activated and even to a peak level. zh qian et al. observed that the dc of peripheral blood in patients with chronic severe hepatitis b manifested dysmaturity, dysfunction of cytokine excretion, especially the low secretion of il-12 which mediated cellular immunity, a high level of secretion of il-6 and tnf-α which mediated inflammatory reaction, and finally become an important factor of aggravating liver inflammatory reaction leading to severe hepatitis. th17 cell is a new discovered subgroup of cd4+t lymphocytes, with significantly different biological function, cytokine expression and differentiation compared with th1 and th2 cells. th17 cells can mobilize, recruit and activate neutrophil leukocytes by secreting cytokines such as il-17, il-6 and tnf-α, in order to mediate inflammatory reactions more effectively. zhang et al. detected the th17 cells in the peripheral blood and liver of chronic hepatitis b (chb) and hbv associated acute-on-chronic liver failure (aclf) patients. they observed the level of th17 cells and its associated cytokines increased in chb and aclf patients, and the increasing of th17 in the peripheral blood and liver is significantly positively associated with hbv dna load, serum alt level and histological index of activity. in vitro, il17 could promote mdcs and monocyte activation, increase its secreting ability of proinflammatory factors, such as il-6, tnf-α and il-23, which demonstrated that th17 might lead to aggravation of liver injury in chb patients, and th17 can be considered as one of the immunologic indicators reflecting clinical prognosis of hbv associated aclf patients. wu, et al. observed that the ratio of peripheral th17 in patients with ahb and severe hepatitis increased significantly compared with general patients with chb and health volunteers. th17 cells were significantly positively associated with serum alt increasing in severe hepatitis, while there was no correlation with hbv dna quantitation. furthermore, serum il-10 levels were significantly negatively associated with the ratio of th17 cells, which indicated that th17 cells correlated with the course of hbv and the severity of liver injury. ye et al. also observed that the ratio of secreting il-17 and secreting ifn-γ in the liver of the child-pugh c patients with hbv infection was significantly higher than that of child-pugh b; the numbers of the cells secreting il-17 in the patients with severe liver injury were more than the cells secreting ifn-γ; the degree of cell infiltration with il-17 secreting were significantly positively correlated with liver inflammation grading, expressing of il-8 in the liver and neutrophilic leukocyte infiltration. chen et al. observed that increasing serum ifn-γ levels was correlated with disease progression of chb. ifn-γ might be involved in inherent and acquired immunity after hbv infection; ifn-γ can be considered as a potential biomarker for predicting clinical severity of hepatitis b, especially for predicting the tendency of liver failure. il-10 is a suppressive cytokine which can be produced by th2 cells, kupffer cells and liver cells, with major functions of suppressing antigen presentation of macrophage, production of multiple proinflammatory cytokines and th1 response. zheng et al. observed that high expression of il-10 promoted active viral replication in chb patients, induced excessive immune response. il-10 can be used to evaluate disease severity and prognosis of severe type b hepatitis. serum il-10 levels decreased significantly in severe hepatitis b patients complicated by bacterial infection or overlap with other hepatitis virus infections, which indicated endotoxemia and concurrent viral infection play important roles on promoting occurrence of severe hepatitis b. tnf-α is mainly produced by active mononuclear macrophages. the concentration of tnf-α is low under normal conditions, with the function of regulating the immune response. persistent high level of tnf-α can induce liver apoptosis or necrosis. tnf-α has two surface receptors: tnfr1 and tnfr2. du et al. observed that levels of serum tnf-α, tnfr1 and tnfr2 increased significantly in patients with acute fulminant liver failure compared with subacute fulminant type, acute type patients and health volunteers, and levels of tnfr1 were even higher in nonsurvivors. another study showed that expression of tnf-α and tnfr was obviously positively correlated with apoptosis liver cells in patients with fulminant liver failure; tnf-α might lead to widespread liver apoptosis by inducing fas gene transcription and other signal transduction system. except for inducing apoptosis, tnf-α can also induce production or release of other cytokines, such as il-6, il-8, and so on, to promote intrahepatic inflammatory reaction and aggravate liver cell necrosis by cascade amplified action. ip-10 is a recently discovered cxc chemokine, mainly induced by ifn. ip-10 has strong function of chemiotaxis and activation on t cells expressing specific receptor cxcr3, and also plays important role on lymphocytic accumulating to inflammatory reactive site, which may be an important factor of inflammatory cell migration toward hepatic tissue and causing large area hepatic cell necrosis. detecting serum il-10 concentration can indirectly reflect expression of ip-10 in local hepatic inflammatory response. luo et al. observed that the levels of serum ip-10 in patients with severe hepatitis b were higher than that of chb group, indicating the higher degree of liver inflammation and injury, the higher serum il-10 levels. so, serum levels of il-10 might reflect the degree of inflammation in hepatitis b patients. trail is a new member of tnf α superfamily. a large number of studies indicated trail-r2/dr5 play an important role on apoptosis. the trail induced apoptosis pathway may play an important role in the pathogenesis of severe type hepatitis. trail receptor/ligand system can specifically kill cells infected by virus in hbv infection. vander sloot am et al. observed that affecting hepg2 cell strain by soluble trail produced by cultural monocyte after lps stimulation can cause apoptosis; higher levels of soluble trail, more obvious apoptosis, which demonstrated that trail induced hepg2 apoptosis was dose dependent. further studies have shown that serum soluble trail increased significantly in patients with severe type hepatitis and was positively correlated with serum lps concentration and severity of liver injury, which illustrated that decrease of lps degradation accompanied with the degree of liver injury can stimulate mononuclear macrophage and dc to express more trail, shedding strail also increased, cause liver apoptosis and aggravated liver injury after its binding with receptors on liver cells. interleukin-22 (il-22) is one of the major cytokines secreted by th17, and is a member of the il-10 cytokine family. th17 is one of the major cells secreting il-22. osteopontin (opn) is a secretary phosphorylated glucoprotein which contains rgd (arginine, glycine and aspartic acid) protein with an integrin binding region discovered in 1979. it was isolated from bone matrix by herring in 1983 who found that opn was a key cytokine which accumulates in immunocytes and initiates th1 cell immunity, and is involved in multiple pathological process of inflammatory reaction. some studies demonstrated that levels of peripheral blood opn were significantly high in patients with severe hepatitis b. it also confirmed that nkt cells can secrete opn, and opn also can enlarge activation of nkt cells, to further trigger neutrophil infiltration and activation. further studies on pathogenesis of opn in hepatitis demonstrated that the opn transgenic mice, after been treated with cona, manifested large necrosis of liver cells and monocytes infiltration, while with only minor liver injury on the controls. so it presumed that opn might cause immune disequilibrium of th1 and th2, induce large hepatonecrosis. in patients with fulminant hepatitis, opn increased significantly, indicating that opn can initiate the effect of th1 cytokine network such as il-18 and ifn-γ by an autocrine pathway, to further cause macrophage activation and severe hepatonecrosis. as a specific receptor of endotoxin, toll-like receptor 4 (tlr4) can recognize serum endotoxin, bind with lps/lbp/cd14, and recruit myd88, activate irak (il-1r correlated kinase) and mapkkk (mapk kinase kinase) family, induce activation of nf-κb, activate target genes transcription, release a series of cytokines, and cause hepatocytes damage. in patients with severe hepatitis, because of intestinal microbial population derangement, bacterial overgrowth and intestinal mucosa congestion, the ability of endotoxin production and absorption increases, which is accompanied by severe hepatonecrosis, immune dysfunction and a decrease in kupffer cells clearance, which further causes a reduction of exdotoxin and exdotoxin immunocomplex clearance, and finally gives rise to endotoxemia. lipopolysaccharide (lps) is a major component of gram-negative bacteria, with important physiopathologic function. tlr4 is a key receptor for lps via signal transduction. regulating expressing of tlr4 should control lps related inflammatory reaction. xu, et al. designed and constructed tlr4 sirna expression vector, evaluated mouse phagocyte raw264.7 gene silencing efficiency by transfection, and evaluated therapeutic efficacy of tlr4 gene silencing in the mouse hepatic injury model. it demonstrated that the expression of tlr4 mrna and protein decreased significantly in raw264.7 cells; tlr4 sirna can inhibit obviously the up-regulation of tnf-α and mip-2 treated with lps; the activation of p38-mapk and erk1/2 by lps can down-regulate by tlr4 sirna. further studies demonstrated pretreatment of tlr4 sirna can be of benefit to control lps inflammatory reaction, reduce hepatic lesion of g57bl/6 mouse treated by d-ga1 n/lps, and reduce mortality of mice with acute hepatic injury. some studies also showed that the tlr4 protein expression on the surface of pbmcs and mrna were obviously higher in patients with chronic hepatitis b compared with the healthy controls; levels of tlr4 in severe-type patient of chb were significantly higher than that of chb, which indicated tlr4 played important roles during the course of liver injury with hbv infection, and with obvious increasing accompanied with clinical severity. fan jg, et al. observed the expression of tlr4 increased accompanied with the degree of endotoxemia and the prolong of stimulus duration; lps can positively up-regulate expression of tlr4 and enlarge biological effect of lps. so the severe hepatonecrosis and weakening of kupffer cell deactivation can accelerate the occurrence of endotoxemia, while endotoxin also can regulate liver cell metabolism or influence tlr4 mediated immune reaction to aggravate the clinical situation. the two aspects interact quite closely. all the studies mentioned above indicated high expression of pbmcs tlr4 in patients with chronic hepatitis, especially with severe-type presentation. monitoring dynamic changes of tlrs might be benefit for treatment. nitric oxide (no) is a biological regulatory factor with an extremely short halflife, produced by l-arginine catalyzed by nos (no synthetase), with extensive biological functions. it is now believed that no has potential antiviral inductive activity, and also might be one of the factors mediating liver injury. the studies of mouse model with inos defection demonstrated that the mouse model can tolerate ctl induced immunologic injury during the course of inhibiting virus replication because of no dyssynthesis, and also opposed against injection of fatal anti-fas antibody, further caused no obvious hepatonecrosis after treated with anti-fas antibody. in severe-type patients with hepatitis, serum no levels significantly increase during the early stage, then no levels decrease significantly with extenuation of inflammatory reaction, liver function recovery in the convalescence phase. these studies revealed that no is related to the degree of inflammation and clinical severity, and can play an important role in the clinical course of severe-type hepatitis. some studies demonstrated that inos gene was related closely with viral hepatitis. the expression of inos gene was extremely low in normal hepatic cells. once invasion of hepatic cells by virus appeared, it can induce hepatic cells to activate inos gene expression and produce large amounts of no, not only to kill liver tumor and pathogens such as virus, bacterium, parasites, play broad-spectrum antiviral function, but also injury to adjacent normal liver tissue by cell toxic action, and cause liver cell death. gong fj, et al. presumed that pre-s2 protein can downregulate inos transcriptive activity by its trans-regulation, further inducing the decline of no production and blocking the clearance of hbv in hepatocytes by no, and finally accomplishing long-term hbv chronic infection. so, expression of inos can be considered as an important factor of inflammatory injury. neutrophil gelatinase-associated lipocalin (ngal) was considered as a novel troponin-like biomarker, which was discovered in 1993 by kjeldsen, et al. who studied neutrophil matrix metalloproteinase 9 (mmp-9) at that time. it was believed that ngal could be used to monitor acute kidney injury/acute renal failure. renal dysfunction is extremely common during the clinical course of severe liver disease such as decompensated cirrhosis and liver failure. serum cr levels can increase rapidly when acute renal failure occurs and is easily detected, while it is not easy detected or diagnosed when complicated by chronic kidney injury. furthermore, other diagnostic methods based upon glomerular filtration rate (gfr) are needed to detect clearance rate, and are not suitable for routine application. the detection of serum cr is simple, while is not accurate on detection of kidney injury based upon cirrhosis. gerbes al, et al. observed 22 cirrhosis patients with ascites whose serum cr below 1.5 mg/dl, compared the levels of serum ngal and cr. the patients was divided into two groups, the gfr was 69 ± 15 ml/min and 29 ± 10 ml/min, and with serum cr of 0.8 ± 0.2 mg/dl or 1.1 ± 0.3 mg/dl respectively, with no obvious difference. the estimated gfr was 106 ± 34 ml/min and 65 ± 17 ml/min respectively, also with no difference. while in the study, serum ngal was 50 ± 15 ng/ml and 136 ± 61 ng/ml, demonstrating significant difference (p < 0.01). furthermore, levels of urinary ngal is similar with the blood ngal, especially the patients in the group 2, with urinary ngal above 100 ng/ml. area under the curve (auc) analysis also revealed that ngal was better than serum cr assay when gfr < 50 ml/min, with auc = 0.98 (0.96 ~ 1.00) of the former, and 0.79 (0.66 ~ 0.92) of the latter (p < 0.05). macrophage inflammatory protein (mip) is a novel protein first discovered in 1988 by wolpe, et al. which can be divided into five subtypes: mip-1, mip-2, mip-3, mip-4 and mip-5. mip-2 has a binding site for heparin, which can interact with heparin dextran sulphate in endothelial extracellular matrix, and enhance adhesion of leucocytes and vascular endothelial cells. mip-2 is involved in the whole process of inflammation by chemical chemotaxis and activation of inflammatory cells, with neutrophils as specific target cells. mip-2 can also activate neutrophils specifically and has been considered as an important proinflammatory cytokine during the early stage of inflammation. chen z, et al. observed that the levels of mip-2 in severe-type patients with hepatitis b was significantly higher than other groups. persistent high expression of mip-2 can be used to predict acute exacerbation of hepatitis b and clinical severity of patients with hepatitis b. thymosinβ4 (tβ4) is one of the important actin regulatory molecules in human body. it can bind with globular actin (g-actin), inhibit production of fibrous actin (f-actin), reduce formation of microthrombi and microcirculation dysfunction in patients with liver failure, and inhibit occurrence and development of mof. tβ4 can also reduce the levels of free radicals, slow lipid peroxidation and inhibit production of proinflammatory factors. patients with liver failure usually manifest a large amount of mononuclear macrophage infiltration, secretion, releasing proinflammatory factors and aggravate systemic inflammatory reaction. liu y, et al. observed that the levels of serum tβ4 decreased significantly in patients with liver failure compared with the cirrhosis group, chb group and healthy controls. dynamic changes of serum tβ4 can be considered as a novel predictive indicator for prognosis in patients with liver failure. t cell immunoglobulin mucin 3 (tim-3) is a surface molecule correlated with function of t cells, which was discovered in 2002. the studies on mouse model revealed that tim-3 can be expressed on the surface of th1 selectively as a negative regulatory molecule; the interaction of tim-3 and its ligand plays a down regulatory role on th1 cellular mediated immune response. sabatos ca, et al. observed that the levels of peripheral tim-3 in chb patients increased significantly, and it indicated the expression of tim-3 can be induced by regulating treg whose activity was modulated during the chronic infection period in patients with chb, and then inhibit specific th1 cell responses. zhou xq, et al. observed that the levels of tim-3 was significantly higher than other groups in severe-type patients with hepatitis b, which can be used to predict and monitor the clinical severity of hepatitis b patients. with the progression of chb, serum inos and ngal all increased, with the highest levels in severe grade of chb, while decreased inversely in the serum of the severe-type patients with hepatitis b, which can predict acute exacerbation of chronic hepatitis b. it was reported that the crosspoint of persistent elevation of tim3 and mip2, and declining of inos and ngal, demonstrated important clinical significance on predicting acute exacerbation of chb. recent studies have demonstrated that genetic variation of hormone receptor gene was significantly different between chb patients with acute exacerbation and hbv carriers, which could explain the sexual difference on different clinical course of hepatitis b. clinical studies further demonstrated that male patients with hbv infection had higher incidence and ratio of chronicity; male patients with hbv infection had higher activity frequency, which indicated the sex hormone and its receptor might influence the process of hbv infection by regulating host immuno response and level of viral replication. it is now believed that immunoresponse of viral clearance might down-regulate male sex hormones and up-regulate female sex hormones. furthermore, steroid hormones might regulate replication of hbv mediated by androgen receptors. it has been proved that androgenic hormone can inhibit cellular and humoral immune reaction; estrogenic hormone can inhibit cellular immunoresponse and enlarge humoral immunoresponse inversely; progestogen can promote conversion from cellular immunity to humoral immuno reactivity. several studies have reported the action of estrogen receptor (esrs) in the diseases of multifactorial inheritance, and its roles have also been observed by geneticists and virologists. large number of studies have demonstrated that esr2 played important roles on regulation in patients with persistent hbv infection, and esrs genes were correlated with host hereditary susceptibility. because of low expression of esrs in hbv carriers, the reaction of immune system to sex hormone was insufficient, which make clearance of hbv difficult and can lead to hbv persistent infection. human esrs are divided into two types: estrogen receptor a (esr1) and estrogen receptor b (esr2). all the genetic variations can cause degression of estrogenic function, which may further cause different kinds of host hereditary susceptibility after hbv infection. of them, esr1 gene can mutate, with tendency of sex hormonal resistance. sex hormone mainly plays the role of binding esr1, and esr1 gene is the true minor gene with host hereditary susceptibility after hbv infection. deng gh, et al. observed the relationship between esr1 gene polymorphism and chronic persistent hbv infection in a large sample study. they found that the susceptibility increased significantly compared with the individual of esr1 29t/t genotype and the individual containing at least one 29c isoloci site (p < 0.001). linkage disequilibrium plotting analysis indicated that t29c polymorphism included a linkage disequilibrium area sited from esr1 promoter to intron 3, which indicated that the esr1 t29c convention originated from esr1 itself, and esr1 gene polymorphism in the chinese population was positively related with hbv persistent infection. the negative feedback regulation of hypothalamus-pituitary gland-genital gland axis can be performed by serum testosterone (t) and estradiol (e2) through central nervous system feedback, and control releasing of luteinizing hormone (lh) and follicle stimulating hormone (fsh) . some studies demonstrated that the levels of serum t and e2 decreased in male patients with hepatitis b cirrhosis, and the levels of lh and fsh increased correspondingly, which indicated liver injury might be an important reason of sex and hypophyseal hormone abnormality. until now, the studies on changes of sex and hypophyseal hormone in acute exacerbation of hepatitis b and severe-type hepatitis are still limited, while it is certain that precise detection of sex hormone and correlated hypophyseal hormone can reflect the severity of liver injury to a certain degree, and can be considered as a useful indicator evaluating the clinical course of hepatitis b, cirrhosis and its complication, hepatocellular carcinoma. it is now believed that the priming or control point of severe-type hepatitis occurrence and development is influenced by two aspects: virus (such as viral variation, viral protein and viral genotype) and host (such as cell immunity, cytokines and apoptosis), and of them, viral replication is the necessary condition and cause of severe-type hepatitis. some studies demonstrated that viral replication was relative vigorous during the early stage of severe-type hepatitis, which indicated viral factor occupy a central position in the occurrence and development of acute exacerbation of hepatitis b. the changes of host transcription factors and hbv genetic variation all possibly cause the changes of hbv transcription and replication, resulting in acute exacerbation of hepatitis b. hepatocyte nuclear factor 4 (hnf4) is a protein with the function of genetic transcription and modulation, and is mainly found in liver. long ce, et al. observed that the hnf4 binding sites, located in enh i/xp and enhii/c promoter, is an important factor regulating c promoter activity and 3.5 kb mrna transcription, and has an important role on hbv replication and expression with high tissue specificity. previous studies demonstrated the expression of hnf4 increased in patients with chb, and the level of hnf4 is positively correlated with hbv replication. furthermore, from a study on liver biopsy specimens using gene array in patients with hbv infection, honda m, et al. observed hbv infection can cause more gene transcription of hnf4, rxr/ppar and c/ebp, and possibly related with liver injury. so, the interaction of hnf4 and hbv genetic transcription and control might play an important role on the pathogenesis of severe-type hepatitis b. deng et al. studied cxcl10 snp variation on th1 immune response pathway, and observed that the polymorphism site g-201a existed in cxcl10 gene promoter region was significantly related with disease progression in male hbv carriers. an emsa study revealed that the g-201a site can change its binding ability with nucleoprotein; and reporter gene assays also revealed that this site can influence the transcriptional activity of cxcl10 gene promoter region; mrna real time quantitative experiments further revealed that the expression of susceptible allelotype -201g in pbmcs was significantly higher than allelotype -201a; elisa and immunohistochemical assays revealed a higher expression of cxcl10 protein in plasma and liver tissue of patients with chb that progressed than that of nonprogressive patients. all the studies mentioned above demonstrated that g-201a is a new functional polymorphism site, cxcl10 protein involved in the process of inflammation and necrosis in liver tissue of patients with hepatitis b, and influence the progression of chb. furthermore, yan z, et al. reported that mutation of ifn-γ gene can influence expression of ifn-γ, which is a predisposing factor of chronicity of hbv infection; fulminant hepatitis patients with poor prognosis had high frequency of occurrence of -1031c, -863a and tnf-βb2 allele in tnf-α gene promoter region; snp in il-10 gene promoter region was related with progression of chronic hbv infection; the frequency of haplotype expressed by down-regulated il-10 in the il-10 gene promoter region in patients with fulminant hepatitis was higher than the controls, while the frequency of haplotype gene occurrence by up-regulated il-10 expression was even lower. yan et al. also revealed the relationship between polymorphism of il-10 gene promoter in th2 immuno response pathway and chronic severe-type hepatitis, which provided new evidence on natural selection theory of il-10 promoter and sirs pathophysiology theory of acute liver failure. in a study of human fibrinogen-like protein 2 (hfg12) gene regulation mechanism and network, han et al. observed that hbc protein and hbx protein all had the function of hfg12 activation, while hbs protein cannot activate the gene. a series of promoter deletion experiments demonstrated that the regulation sequence of activating hfg12 gene existed between the sits of hfgl2 gene promoter −712 ~ −568, transcription factor c-ets-2 can bind with cis-acting element on hfg12 gene promoter, then activate the expression of the gene under the action of that viral protein; the phosphorylation level of p-jnk and p-erk was enhanced in patients with severe hepatitis b than healthy controls, which indicated jnk and erk signal pathways were activated. some studies in vitro revealed that jnk and erk signal pathways were activated respectively under the effect of viral protein hbx and hbc, and c-ets-2 expression and transposition were correlated with the activity of jnk and erk. all the studies mentioned above indicated that viral protein hbc and hbx can activate jnk pathway and erk pathway respectively, further activate transcription factor c-ets-2, shift into the nuclear, and finally up-regulate the expression of hfg12 gene by binding with cis-acting element of hfg12 gene promoter. tnf-α is an important proinflammatory factor induced during liver failure. mfg12/prothrombinase plays an important role on mouse fulminant hepatitis and acute on chronic liver failure. gao et al. constructed eukaryotic expression vector of mfg12 gene and tnfr1 gene, green color fluorescence fusion protein and its shrna interfere plasmid. it was more effective to improve the survival rate of mouse with severe type hepatitis using combination of fg12 and tnfr1 genes compared with single gene intervention, and improve serology and pathological changes of liver, which indicated that fg12 and tnfr1 genes might produce synergistic effect during the progression of severe-type hepatitis. hbv infection is a necessary condition and cause of occurrence and progress of severe-type hepatitis, while hbv gene transcription and replication are a key step of the hbv life cycle. beginning from the new point of genetic transcription, further exploring the effect and mechanism of acute exacerbation of hepatitis b on hbv genetic transcription and regulation, will provide important clue of host transcription factor, which can be useful to predict and monitor the occurrence, development of severe-type hepatitis, and to provide new targets for effective methods of treatment of severe-type hepatitis. genome-wide association study (gwas) has been available since 2005, and have become the main aspect of complicated genome association study. the strategy does not need to select candidate genes, but to make genome-wide associations and analysis by selecting directly ten thousand single nucleotide polymorphism (snp) sites and copy number variation (snv) sites over the total genome. compared with the strategy of a candidate gene, gwas was a significant improvement in statistical efficacy, and also avoided the bias of group stratification and randomness of gene selection. in recent years, some studies on the aspects of viral hepatitis and hbv related hepatocyte carcinoma has gained great breakthrough using these approaches. several independent and large sample gwas in usa, europe and japan demonstrated that only rs1297980 site near the il-28b gene had relationship with curative effect of interferon in the study on genetic correlation of ifn response in patients with hepatitis c (with p value of 10 −25 ). guo et al. observed that gene variation of hlp-dp gene can play great influence on clinical severity of persistent chronic hbv carriers. karlsen et al. also reported the relation between hla-ii gene and hbv infection. he fc and zhou gq made gwas of hbv related hepatoma, and they discovered a related site 1p36.22, where one of the three genes (kif1b, ube4b, pgd) might be predisposing gene of hbv related hepatoma. the studies on genetic predisposition of severe-type hepatitis b are relatively less. only a minority of studies have examined a candidate gene-disease associated study strategy, and their study populations were all from asia. recently, under the support of the national 973 program and key state science and technology project of infectious disease, chinese experts have taken affymetrix snp 6.0 array (including 900,000 snp sites and 900,000 cnv sites in the range of human total genome-wide) to make gwas in 1600 severe-type patients with hepatitis b and asymptomatic carriers. the project is now being analyzed, expecting to explore common genetic variance sites influencing severe-type hepatitis b under the point of host total genome. epigenetics mainly studies the change of herediable gene expression with no change of dna sequence. currently, epigenetic mutation mainly includes dna methylation, microrna (mirna) and chromatin remodeling. several functional studies also demonstrated that mirna participated and regulated the expression of multiple inflammatory signal factors. it was found that mir-150 and mir-223 might be linked with liver inflammatory activity of viral hepatitis. mir-150 mainly participated in regulating maturation of b lymphocytes, and also regulate the differentiation of th1 and th2 cells. mir-223 located in human x chromosome, and highly expressed specifically in granulocyte series cells. johnnidis, et al. observed the granular leukocytes increased twice in mir-223 gene knock out mouse, and these leukocytes were more sensitive when treated with antigenic stimulation outside (especial fungi); compared with mir-223 gene non-knock out mouse, the lung inflammation of mir-223 gene knock out mouse was more obvious, and the tissue injury induced by exdotoxin stimulation even more serious. the latest studies have discovered seven susceptible molecules correlated with acute exacerbation of hepatitis b: mir-478a (down-regulation in severe-type twins patients with hepatitis b), mir-7a (up-regulation in severe-type patients with hepatitis b), mir-16 (up-regulation in severe-type patients with hepatitis b), mir-122 (providing early warning signal on acute exacerbation of hepatitis b and disease severity), mir-1187 (has obvious decline tendency with the development of liver failure, and can be considered as biomarker for predicting clinical severity and acute exacerbation of hepatitis b), mir-155 (its expression decreases gradually accompanied with viral clearance, can be considered a biomarker of turnover of acute exacerbation of hepatitis b, and provide early warning signal on acute exacerbation of hepatitis b and treatment), mir-197 (down-regulation has predictive value to liver inflammation). it has been finally shown that detection of specific mirna correlates with liver inflammation, by using mirna array technique in severe-type patients, can provide novel target on early prediction of severe-type hepatitis. qi zx, et al. observed the difference of th1 and th2 cells chromatinization status in il-10 gene promoter region, which interfered with the binding of promoter and transcription factors, and influenced gene transcription of il-10. the distribution of methylation in patients with liver failure showed obvious differences between chb patients and healthy controls; concentrations of serum il-10 were negatively related with promoter methylation status, which indicated the methylation of il-10 gene promoter region might involve in the pathogenesis of liver failure, and it is also a reason of il-10 change. furthermore, some studies revealed that methylation of glutathione-s-transferase p1 promoter region can promote acute exacerbation of hepatitis b. of hepatitis b toll-like receptors (tlrs) mediated signal transduction pathway includes two forms: myeloid differentiation factor-88 (myd88) dependent form and myd88 independent form. tlr7, tlr8 and tlr9 mediated signal pathway belongs to myd88 dependent form pathway; tlr3 mediated signal pathway belongs to myd88 independent form or trif independent form signal pathway. in the myd88 independent form pathway, tlrs recognized ligand, then activate nf-κb and mapk, induce release of multiple cytokines, up-regulate costimulatory molecules such as cd80, cd86, et al., then finally activate specific immune system. tlrs and tlr4 in particular, play important roles on the mechanism of acute exacerbation of hepatitis b, which can better predict tendency of the disease, and can be considered as indicators for evaluating prognosis of severetype hepatitis b. tlr4 is a key receptor in endotoxin transmembrane signal transduction; it is also a rate limiting factor in lps transmembrane signal transduction of mononuclear cell and macrophage. some studies demonstrated that the expression of tlr mrna in severe-type patients with viral hepatitis was higher than the healthy controls; and it is higher during the aggressive stage than the critical stage in the nonsurvivors and it is lower during improvement than the critical stage in the survivors. furthermore, expression of tlr4 mrna is positively correlated with the levels of endotoxin. deng m, et al. observed that the severe-type patients with viral hepatitis had tendency to endotoxemia, with endotoxin binding with tlr4, then transmembrane signal activation of nf-κb through intracellular structure of the tlr4, further induced release of inflammatory factors, such as tnf-α and il-1, and produce secondary intrahepatic microcirculation disturbances. tlr2 is a pattern recognition receptor with extensive pathogen recognition abilities. tlr2 can recognize cell walls from multiple bacteria such as gram-positive bacteria, gram-negative bacteria, fungi, spirochaetes, mycobacterium tuberculosis and mycoplasma, and play important roles on acute and chronic infection. yan cg, et al. observed the expression of tlr2 of mouse with fulminant liver failure induced by d-gal/lps. they observed the high and persistent expression of tlr2 mrna, and accompanied with high expression of tnf-α, which indicated that tlr2 can up-regulate expression of downstream inflammatory response genes and release of cytokines, to involve in acute liver failure induced by d-gal/lps. metabolomics is a new technology, which studies various changes of endogenous metabolic products and biologic conditions in humans by observing biosystem changes qualitatively and quantitatively under various stimulations and changes of internal and external environment. the study object is the micromolecule compound with relative molecular mass < 1000 in the metabolic network. at present, the major techniques include nuclear magnetic resonance spectrum (nmr), gas chromatogram (gc), liquid chromatography (lc), capillary electrophoresis (ce), mass spectroscopy (ms), infrared spectrum and ultraviolet spectra. of them, nmr, gc-mc and lc-mc are used more extensively. yang et al. investigated the serological profile in acute hepatitis b patients with sudden aggravation of liver function by hplc-ms and pls analytical techniques. they observed the changes in individual atoms in lysophosphatidyl choline (lpc) and gcdca, which can predict early disease aggravation. feng b, et al. analysed the metabolic product in serum of rats with fulminant liver failure by gc-mc and pca software, and observed high content of 5-hydroxyindole acetic acid, glucose, β-hydroxybutyric acid and phosphate, which indicated the changes of these substances might become potential biomarkers of fulminant liver failure. yu, et al. set up a research platform based upon liver failure metabolomics by using gas chromatographic mass spectrometry (gcms) and super-high performance liquid chromatogram mass spectrometry technology, constructed a model for evaluating clinical severity of liver failure, and constructed serum specific metabolism spectrum by partial least squares discriminant analysis, which can be used to predict the prognosis, with sensitivity of 91.3%. mao et al. also derived similar results with a high positive diagnostic rate of 93.62% for liver failure, and also found that the changes of climax concentration of plasma citrate had potential diagnostic significance. li lj, et al. pointed out the low level of lysolecithin, high level of fatty amide and nonelevation of bile acid by using metabolomics were risk factors for predicting poor prognosis in liver failure patients with pretreatment and post-treatment by artificial liver support systems. by observing the trend changes of serum metabolic spectrum in patients with pretreatment and post-treatment with an artificial liver, we can predict the prognosis and assess curative effect of artificial liver. electrolyte disturbances were usually complicated in patients with liver failure because the patients typically have low renal blood flow and glomerular filtration rate (gfr). a majority of patients manifest hypokalemic, hyponatremia, hypomagnesiumemia and hypocalcemia. the occurrence of hyponatremia and its severity are positively related with prognosis in patient with liver failure. severe hyponatremia can cause acute low sodium syndrome and encephalopathy; and hyponatremia can also cause renal injury and promote or aggravate development of hepatic encephalopathy and hepatorenal syndrome (hrs), and increase mortality. some studies indicated that the mortality of the patients with severe hyponatremia (<115 mmol/l) is extremely high and up to 93.88%. in patients with acute liver failure, hypochloraemia can be caused by insufficient intake of chlorine, vomiting, large excretion of aldosterone and acidosis, while water intake insufficiency, renal failure, renal tubular acidosis and potassium chloride overdose can cause hyperchloraemia inversely. furthermore, hypophosphatemia is more readily detected than hypocalcemia in most patients. acid base disorders (abd) are very common in patients with acute liver failure, especially alkalemia. li xm, et al. presumed that arterial carbon dioxide pressure depression and respiratory alkalosis are frequent manifestations of acid base disorders. abd is not related with basic pathological changes of severe-type hepatitis, whilst blood ph value is an important factor influencing the survival outcome of severe-type patients with hepatitis. in the latest years, some scholars think that pre-estimation of compensatory formula (pcf) for abd has practical value on differential diagnosis of partial simple and duality abd. partial patients with acute liver failure complicated by abd can be diagnosed by anion gap (ag), potential hco 3 −· and cl − . elevation of ag is a reliable indicator of appearing potential metabolic acidosis, and is common in patients with hepatic encephalopathy, lactic acidosis caused by hypoxemia. during hepatorenal syndrome since reduction of acidum excretion of kidney ketoacidosis may appear. potential hco 3 −· is the hco 3 −· value exclusive of the masking hco 3 −· by high ag metabolic acidosis, which can raise the existence of ag metabolic acidosis and metabolic alkalosis with triplicity abd. a mixed metabolic acidosis can be discerned by combination of ag and blood ci. series of primary abd can overlap, even to develop duplex and triplication abd. for example, if the respiratory alkalosis prolongs, accompanied with increasing of potassium excretion by renal, and with iatrogenic factors to develop metabolic alkalosis, and further develop multiple complications combined with metabolic acidosis, which finally develop triplicated abd. triplication abd is usually the end stage of acute liver failure, with worst prognosis. so, prevention and active treatment of abd is an important parameter for rescuing patients with acute liver failure. liver biopsy is a good method to acquire pathologic changes of liver in multiple liver diseases including liver failure, which have important value on directly evaluating the severity of liver damage. liver biopsy is generally divided into percutaneous biopsy, transvenous biopsy, surgical/laparoscopic biopsy and plugged biopsy. of them, percutaneous biopsy is safe and simple, without special instrument or equipment, and being used extensively. transvenous biopsy is more suitable to the patients with large amount of ascites, clotting dysfunction, diminution of liver volume and increasing of liver hardness because of cirrhosis, difficulty to determine hypochondria because of obvious obesity, and with high hepatic vein pressure. the contraindication of liver biopsy are as the follows: (1) excessively prolonged pt, excessive declining of pta, which might cause haemostasis difficult even mild injury; (2) obvious cavity bleeding or large area of petechia; (3) patients with unconsciousness, who might not be able to fit the operation; (4) medium dose ascites, especially patients complicated by abdominal infection; (5) patients with obvious pleural effusion and severe heart and lung disease. it has been proved that liver biopsy is safe to a great extent under careful preparation, attentive operation, even in the liver failure patients with obvious clotting dysfunction. what we need to point out is that although liver biopsy has important value on recognizing liver tissue degeneration, necrosis and degree of inflammatory infiltration, the pathologic change of the biopsy tissue might not be able to reflect entire pathologic change of the liver because of the limited liver tissue obtained. so, it is very important for clinicians to collect and combine various forms of clinical data, laboratory and pathogenetic results from the patients, for getting an accurate judgment. the incidence of cerebral edema in severe-type patients is high, with ratio of 50 to 80%, and a quarter of them can appear cerebral hernia with poor prognosis. so, measurement of intracranial pressure plays important role on diagnosis and treatment of cerebral edema. traditional lumbar puncture manometry can measure intracranial pressure only one time; it cannot be observed that the changes of intracranial pressure dynamically and exactly. on the other hand, lumbar puncture on patients with acute intracranial hypertension might cause or aggravate cerebral hernia. furthermore, under the condition of cerebral hernia, cranial cavity and spinal cord cavity can not intercross, so the pressure detected by lumbar puncture cannot represent actual intracal pressure. so, persistent detection of intracranial pressure retrieves shortage of lumbar puncture, and cerebral ventricle catheterization is still an extensively used method, which is considered as gold standard of intracranial pressure monitoring by clinicians. intracranial hypertension is defined as persistent elevation of intracranial pressure with levels above 15 mmhg. according to the difference of intracranial pressure, intracranial pressure can be divided into four stages: (1) normal: 5 ~ 15 mmhg; (2) mild: 15 ~ 20 mmhg; (3) moderate: 20 ~ 40 mmhg; (4) severe: above 40 mmhg. if intracranial pressure is close to mean arterial pressure (map), which illustrates almost no brain perfusion, with nearly close to brain death. currently, 20 mmhg is defined as the critical value of applying treatment of anti-intracranial hypertension. complications of intracranial pressure measurement include infection, intracranial hemorrhage, iatrogenic acute intracranial hypertension and brain parenchyma injury. although laboratory parameters can reflect pathological changes and liver functions during the clinical course of severe-type hepatitis and acute exacerbation of hepatitis b sensitively and in time, and can provide detailed reference data for clinical typing, grading and curative effect evaluation, in total, the extensive and large numbers of indicators listed above are still under study and experimental evaluation, and their availability and utility need to be checked by more clinical practice. until now, laboratory parameters have been extensively used and applied clinically but are still limited. the laboratory parameters conventionally tested in the majority of hospitals are mainly pta, inr, serum bilirubin, alt/ast, pre-protein and blood ammonia. furthermore, some studies also reveal that serum afp, serum sodium and phosphate are closely related with prognosis of liver failure. although some indicators such as arterial blood lactic acid, pct, nagl and inos are closely related with severe-type hepatitis or liver failure, they also score as abnormal in other severe diseases or conditions such as septic shock and mods, so these parameters are not specific for severe-type hepatitis b and liver failure. it is the same on the indicators related with immunity or inflammatory cell or factors, with high sensitivity, while without specificity. other indictors related with heredity, metabolomics, genomics, proteomics and glycomics are still in research, or needing expensive instruments, and still difficult to the routine clinical practice in the short term. what we need to point out is that the various kinds of prognosis scoring systems and mathematical models constructed and tested in the latest years, such as royal school of medicine standard in united kingdom, model for end-stage liver disease (meld) and child-pugh scores, have higher accuracy and utility on predicting prognosis of severe-type hepatitis and liver failure, although some of them still need additional supplement and improvement in the future. because of diversity and individualization for particular causative factors, clinical types, clinical course, complications and clinical treatments, until now, there are still many challenges reflecting prognosis of liver failure. it is believed that more and more novel biomarkers will come into the clinic in the near future, and with development of studies of mechanism and clinical characteristics on acute exacerbation of hepatitis b, therapeutic efficacy of severe-type hepatitis and liver failure will be improved. pathogenesis of aechb is generally considered to include viral factors (hbv genotype, viral replication and viral mutation, etc.), host factors (host genetic characteristics, immune injury, cell apoptosis and necrosis) and interaction between these two aspects. currently, there are at least ten different hbv genotypes and several subtypes distributed all over the world (see table 1 .6) based on the differences of whole genome sequences >8% for the major genotypes or 4-8% at the sub-genotype level. genotype a is mainly distributed in northern europe (a2), west africa (a3), and sub-saharan africa (a1). in asia, genotype b and c are most common. genotype b have six subtypes (b1-b6), in which, b1 was found in japan, b2 ~ b5 were found in east asia, and b6 was found mainly in the arctic regions, such as alaska, northern canada and greenland. genotypes b2-b5 are also regarded as recombinants with genotype c. genotype c, including five subtypes (c1 ~ c5), is mainly distributed in east asia and southeast asia. genotype d, including subtypes d1 ~ d5, prevails in africa, europe, mediterranean and india. genotype e is only found in west africa. genotype f, having four subtypes, is found in central and south america. genotype g has been reported popular in france, germany and the united states. genotype h is found in central america [2] [3] [4] . genotype i was recently newly found in vietnam and laos, but this new genotype is under controversial [5] . the newly found genotype j in japan has a close relationship with the orangutan's genotype and human genotype c. population migration, promotion of antiviral therapy and host immune selection pressures result in increased risk of hbv gene recombination or mutation [6] . with the development of genetic testing methods, even more new genotypes could still discovered. due to the distribution differences of hbv genotypes, the study on the relationship between hbv genotypes and severe hepatitis b is quite limited. comparisons have only been conducted in a few genotypes. a study from united states on patients with hbv related acute liver failure suggested that outcomes of these patients were not associated with genotypes [7] . a multicenter study from japan involving 301 patients with acute hbv infection has compared 5 genotypes (ae [a2],ba [b2-5],bj [b1],ce [c2],cs [c1],dandg). multi-factor regression analysis indicated that subtype bj(b1) is one of the independent predictors for fulminant hepatitis. subtype ae(a2) is more related to hbv persistence but not self-limiting hepatitis b [8] . another multicenter study from japan showed that patients with genotype c accounted for 80 and 69% in patients with acute-on-chronic liver failure and acute liver failure, respectively. these rates are much higher than that in patients with chronic infection, suggesting that patients with genotype c are more likely to suffer from fulminant liver failure [9] . a study from china included 26 hbv carriers, 61 patients with chronic hepatitis b, 20 patients with aclf, 12 hbv related liver cirrhosis and 7 patients with hbv related hcc. data showed that genotype b (38.1%) and c (54.8%) were the main genotypes in these patients. compared with genotype b, genotype c was seen more frequently in those with severe liver diseases, was accompanied with high levels of hbv replication, indicating that genotype c is associated with high hbv replication and severe liver disease [10] . however, results from another study showed no difference in genotype composition among patients with chronic hepatitis b and those with chronic severe hepatitis [11] . in a study involving 487 hbv-infected pediatric patients, in which 217 patients had been treated with nucleos(t)ide analogues, genotype c2 and b2 were found to be the most prevalent subtypes (73.7 and 21.1%). compared with genotype b2, genotype c2 is more likely to cause severe hepatitis in hbeag positive pediatric patients [12] . the association between various genotypes and the pathogenesis of severe hepatitis needs further studies. hbv uses reverse transcription to copy its dna genome and lack of proof-reading capability permits the emergence of mutations in the genome. every day, approximately 10 11 viral particles are produced and released to maintain a stable level of virus in blood. the average mismatch rate of hbv polymerase is from 1:10 5 to 1:10 4 , potentially resulting in a large amount of mutants in the circulation [13] [14] [15] [16] . for single individuals infected with hbv, hbv genome mutation either naturally occurs naturally or is selected out by antiviral drugs or the change of internal host environment. the probability of hbv mutations varies in different regions of the whole genome. generally, mutations are more likely to occur in the basal core promoter (bcp), pre-c region and neutralization determinants of the viral envelope, but they can also be found in other regions. some of these mutations have important clinical significance, but most of them are silent mutation without biological significance. pre-c region encoding hbeag, is composed of 29 amino acids. the most common mutation is a guanine (g) to adenine (a) substitution at nucleotide 1896, which creates a premature stop codon at codon 28, and prevents the translation of the hbeag [17] . although synthesis of hbeag is inhibited, the hbv replication still continues, manifesting as hbeag negative hbv infection. hbeag expression is not necessary for viral replication, its role in the hbv life cycle remains unclear. in the immune system, hbeag may act as bait, which induces immune tolerance, especially in the newborn babies whose mothers have high level viremia. the hbeag-induced immune tolerance can prevent the attacks on the virus-infected hepatocytes by ctl on hbcag, thus the hbv-infected hepatocytes are not able to be cleared. the hindered synthesis of hbeag may facilitate the ctl to damage infected liver cells, which might be one of the mechanisms of severe hepatitis. it is reported in japan that patients, especially children, infected with this mutant, are more likely to suffer from severe hepatitis. other pre-c mutations include point mutations generating other termination codons, for example g1897a generating uga codons. point mutations may change the initiation codon of p25 or the specific amino acid for cleavage and insertion of key signal peptide, particularly between 1838 and 1839. all of these mutations can affect the hbeag production. the pre-c mutation can reduce hbeag expression, increase hbv replication and aggravate liver damage [18] . a number of studies have suggested the association of pre-c mutation and development of severe hepatitis. the hbcag encoded by c region, contains a t-cell-dependent/independent epitopes and induces the host humoral and cellular immune responses. the c gene mutation may cause deletion of the cell surface antigen, and consequently the lack of humoral immune response by the host against hbv. however, the cytotoxicity of ctls was not affected resulting in potentially massive necrosis of liver cells and eventually leads to severe hepatitis. core promoter directly activates pgrna transcription, and plays a central role in hbv replication. core promoter is composed of basic core promoter (bcp) and its upstream regulatory elements. the bcp region partly overlaps with the 3 'end of x gene and the 5' end of pre-c gene and can independently start the transcription of pre-mrna and pgrna. the core promoter mutation, which is associated with nt1758-1762 that are at the upstream of the starting point of pre-c mrna, reduces the synthesis of hbeag, but has no effect on hbcag. the development of severe hepatitis is often accompanied with hbv core promoter mutation, especially the 1762,1764 double set of mutations [19] . other common mutations in the core promoter region of patients with severe hepatitis include nt1768 c-t, nt1770 t-a and cluster mutations, including nucleotide insertion, deletion and substitution. the bcp mutations can induce changes of two codons (l130m and v131i) in x protein, and generate the hnf1 (hepatocyte nuclear factor 1) binding sites. insertion of 11 bases into the core promoter produces a new binding site of hnf1, which can enhance viral replication and lead to severe hepatitis [20] . in-vitro studies have confirmed that bcp trans-activating transcription causes x protein replacement, which downregulates the transcription of pre-c region and pgrna [21] . however, the double-mutant can upregulate the transcription of pgrna, increase hbcab production, thereby enhancing viral replication. previous studies have shown that a1762t and g1764a mutation are more prevalent in hbv genotype c, which partially explains the stronger pathogenicity of genotype c than genotype b. recent studies have shown that a1762t and g1764a mutations in hbv genotype b may be associated with severe hepatitis, but in hbv-infected pediatric patients, a1762t and g1764a mutations show no significant difference in genotype b and c. overall though, compared with wildtype hbv, bcp double mutation is more commonly associated with severe liver diseases, especially liver cirrhosis and hcc [22] . the mutations in pre c/bcp region may alter the biological characteristics of the virus, and induce the development of severe hepatitis through impacts on host immune responses, being more vigorous in hbeag-negative chb [23] . however, the biological significance of mutations in different sites or forms, the dynamic interaction between virus mutation and host immunity, the influence of different genotypes and viral quasispecies on mutation are still not fully elucidated. immune evasion or vaccine failure related pre-s point mutation or deletion mutation does not affect the viral replication. the pre-s1, s2 recombination, including deletion mutation and promoter mutation, have been regularly found in patients either with chronic or fulminant hepatitis. these mutations frequently occur after hbeag seroconversion or interferon treatment, suggesting the host immune selective pressure during their selection. the pre-s region mutations may play a role in hbv persistent infection, and may also cause liver damage. it has been reported that the pre-s2 mutation is related to fulminant hepatitis, and the pre-s mutation is a strong risk factor for the development of hbv-related liver cancer [24, 25] , presumably as a result of an accumulation of viral envelope proteins inside the cell.mutations of the surface antigen protein, particularly aa145 mutation (sg145r), result in conformational change of the major antigen epitope 'a'. this change disables the immunological recognition and surveillance of the host immune system for the mutant can also result in failure of the clinical vaccination and might be one of the precipitations for the exacerbation of hepatitis b [26] . the p gene mutation in key catalytic domains indicates the hbv resistance against nucleos(t)ide analogues (na). the current five nas in clinical application include lamivudine, adefovir, telbivudine, entecavir and tenofovir. the clinically extensive application of nas leads to rapid selective drug resistance. the selection of drugresistant mutation depends on the following factors: (1) the long half-life of hepatocytes and intrahepatic cccdna; (2) the capacity of hbv replication and mutation; (3) antiviral drug pressure; (4) genetic barrier to resistance. for example, the lamivudine resistance is closely associated with the hbv reverse transcriptase gene ymdd motif mutation, presenting as rt m204i and rt m204v mutation, with or without rt l180m mutation. the replication capacity of rt m204i/v mutants is weaker than the wild strains in the absence of drug. the rtl180m mutation can restore the hbv replication capability. in addition, the single or combined mutation of rt l80i, rt l82m, rt f166l, rt v173l, rt a200v and rt v207i may compensatorily restore the replication capability of rtm204 i/v mutant [27] . the resistance rate is up to 40% after 2-years of lamivudine therapy, and more than 76% after 5-year mono-therapy. the 1-year resistance rate of telbivudine is about 7%. the rtn236t site mutation (threonine substitute asparagine) is mainly seen in adefovir resistance, and the 5-year resistance rate is about 29%. the rta181t/v (valine/threonine substitution alanine) site mutation is found in all the above three antiviral drugs. entecavir resistance mutations include 204 and 180 substitutions, combined with substitutions at codons 184, 202 and 250. the 4-year resistant rate in patients initially treated with entecavir is less than 1%. for patients that have been previously treated with lamivudine, the resistance rate of entecavir increased significantly. so far, there have been no reports on the primary drug resistance for tenofovir mono-therapy. the rta194t mutation was found in the combination therapy of tenofovir and lamivudine [28] . the entire s gene is included in the p gene, and the rt region overlaps with s gene, thus the rt mutation may cause the s gene mutation. double mutations in these two regions can change the viral replication capacity. the hbv drug-resistance mutation occurs in patients with chronic hepatitis b may reduce the hbeag seroconversion rate, reverse the histological improvement, increase the disease progression rate, aggravate liver cirrhosis, and increase the death risk of liver transplant patients. rt mutations may also cause the s protein epitope changes and affect the hbs antibody and the function of ctl, suggesting its role in the development of liver failure [29, 30] . as salvage therapy for lamivudine resistance, lamivudine combined with adefovir dipivoxil has higher rate of viral suppression and lower rate of adefovir resistance compared with switching to adefovir dipivoxil monotherapy [31, 32] . tenofovir is a potent antiviral drug for lamivudine-resistance salvage therapy, and showed a better effect than switching to adefovir dipivoxil monotherapy [33] . in contrast, switching to entecavir is not an optimal choice for lamivudine-resistance [34] . telbivudine resistance is associated with the rtm204i mutation, and has crossresistance with lamivudine. therefore, telbivudine could not be an alternative for lamivudine-resistance [35] . treatment for telbivudine resistance is similar to that for lamivudine resistance [36] . treatment for adefovir-resistance is determined by the virus mutation types and antiviral medication history [37, 38] . lamivudine has been proved to be effective on inhibiting rtn236t adefovir resistance mutations [39] . in vitro data has also suggested the effectiveness of telbivudine. additionally, entecavir might be a reasonable choice for rtn236t mutants. patients with rtn236t mutation are suggested to (1) switch to or add entecavir; or (2) add lamivudine or telbivudine; or (3) switch to tenofovir. compared with that on hbv wild strain, lamivudine becomes less effective on a181v adefovir resistant strain. in vitro studies show that tenofovir has reduced sensitivity to the rta181t mutation. clinically, entecavir and tenofovir can effectively inhibit the replication of a181t adefovir resistant mutants [40] . patients with rta181t mutation are suggested to (1) switch to or add entecavir; or (2) switch to tenofovir. under this circumstance, lamivudine should not be suggested in case it increases the risk of cross-resistance [41, 42] . currently, there has been no data from large sample clinical trials that can guide the treatment for entecavir resistance. in-vitro studies and case reports suggested that adefovir and tenofovir are effective for entecavir-resistant patients. based on expert opinions, patients with entecavir resistance are recommended to add tenofovir or adefovir [43] [44] [45] . in summary, the virus mutation in above regions may be associated with the pathogenesis of severe hepatitis. however, the severity of hepatitis depends on key factors of virus and host. the same mutant may lead to different clinical outcomes in different hosts. thus except for virus mutation, factors including host immune status, cytokines and hla might also account for the development of severe hepatitis. tests for hbv infection hbv surface antigen/pre-s1 protein/pre-s2 protein hbsag is the major coat protein of hbv with antigenicity but not infectivity. in a broad sense, hbsag contains the major protein, middle protein and large protein. narrowly, hbsag simply refers to the major protein, which appears earliest and has the highest titer. thus it is considered as an important marker for early diagnosis of hepatitis b. for typical acute hepatitis b, hbsag appears during the incubation period, followed by the clinical symptoms and abnormal liver function in 2-6 weeks. hbsag stays in the blood for 1-2 months, and disappears in the recovery period. persistence of hbsag more than 6 months indicates the development of chronic hepatitis. hbsag can also be detected in hbv carriers and patients with hbv-related liver cirrhosis or liver cancer. a rapid reduction of quantitative hbsag within 3 months can predict the efficacy of antiviral drugs, but no changes in the quantitative hbsag level after 6 months is not considered as a good predictor [46, 47] . pre-s1 protein, which appears early and is significantly associated with hbeag and hbv dna, can be used as a marker of acute hepatitis b early in infection. the pre-s1 protein has a strong immunogenicity, and includes the important site where hbv attaches to and invade the hepatocytes, the sodium taurocholate co-transporting peptide (ntcp). it is also a reliable reflection of hbv replication level. the synergy from pre-s2 protein is also important for hbv invasion. most patients with acute exacerbation of chronic hepatitis, or chronic active hepatitis, or acute hepatitis developing to chronic hepatitis have persistent pre-s2 protein expression in serum. therefore, serum pre-s2 protein can be used to estimate the activity and infectivity of hbv in chronic patients [48] . pre-s1 and pre-s2 protein, which can induce and regulate humoral and cellular immune response of the host, provide important immune defense for eliminating virus in blood circulation and preventing healthy liver cells being infected. hbsab, which is a protective antibody, can eliminate the virus and prevent hbv infection. hbsab appears in the late stage of acute infection, just before hbsag becomes negative, and will gradually rise to the peak levels in 6-12 months. this antibody can last for a long time, but the titers will gradually decline after 10 years. a small number of cases do not produce hbsab after hbsag becomes negative. in acute hepatitis b infection, appearance of hbsab suggests recovery of the disease. patients with severe hepatitis often present with high titers of hbsab, which forms the immune complexes with hbsag, which can induce flares of hepatitis that lead to liver cell necrosis. after the hepatitis b vaccination, hbsab (>10 iu/l) means the successful vaccination and development of immunity. in the blood, hbcag is mainly located in the core of the dane particles or virions. the only small amount of free hbcag is also combined by high titers of hbsab and presents as immune complexes. thus, it cannot be detected unless treated by detergent. hbcag on the surface of hepatocytes is considered to be the main target antigen of the host ctls. hbcag is a direct evidence of hbv infection and replication, and also a marker for evaluating the efficacy of antiviral drugs. hbcag is strongly immunogenic, so that hbcab can be detected in most patients with hbv infection. hbcab often emerges in the early stage after infection, is present in high titer in blood and can persist for a very long time. titer of hbcab above 1: 100, together with abnormal alt level, can be used for the diagnosis of hepatitis b infection. for occult hepatitis b, high titer of hbcab is also valuable for the diagnosis. hbcab consists of igm and igg antibodies. anti-hbc-igm, which suggests hbvresulted liver damage, is the main evidence for the diagnosis of acute hepatitis b. it may become positive during the active phase of chronic hepatitis b and turn negative during remission. it also appears during the flares of chronic hepatitis b, mostly in a week after infection, and disappears within 6 months. anti-hbc-igg appears late but is sustained for many years or even a lifetime. in patients with acute hepatitis b, the titer of anti-hbc-igm is higher than anti-hbc-igg, while in those with chronic hepatitis b the situation is opposite. both antibodies show high titers in fulminant hepatitis. hbeag is a soluble antigen, which appears later than hbsag. sustained expression of hbeag suggests persistence of hbv infection. in patients with chronic hepatitis b, hbeag acts as an important immune tolerance factor leading to a low immune response to hbv infection. it is a valuable marker for evaluating the efficacy of antiviral drugs. seroconversion refers to the loss of hbeag and development of anti-hbe. approximate 2-15% patients have spontaneous seroconversion every year. studies have shown that spontaneous seroconversion occurs earlier in patients with genotype a, b, d and f than those with genotype c. appearance of hbeab demonstrates the decrease or termination of viral replication and low infectivity. recent studies showed that, after 1 year since the spontaneous hbeag seroconversion, viral load more than 2000 iu/ml increased the incidence of fulminant hepatitis. for hbv chronic carriers and patients with hcc, hbeab does not mean the recovery, or elimination. in contrast, hbv dna integration is often found in these patients. x protein is capable of transactivating the expression of numerous cellular and viral genes, and is vital for virus replication. x protein can be detected in some patients with chronic hepatitis, so it is used as an auxiliary diagnostic marker of hbv infection [49] . x protein plays a central role in hbv-related hcc progression and stimulation. thus, follow-up is necessary for patients active and persistent hbv replication. serum hbv dna is the direct evidence of active hbv infection, reflecting the level of viral replication and infectivity. the quantitative detection for viral genes is a very important marker for treatment decision, efficacy prediction and observation. long-term high load of hbv dna is an independent risk factor for predicting the development of liver cirrhosis and hcc. numerous studies have shown that viral load is the most reliable marker to predict the development of hcc [50] . the differences of hbv whole genome sequence are approximately 2-4%. the diverse variants that are genetically linked through mutation are known as quasispecies. quasispecies contain a large number of mutated genes serving as a reservoir for viral selection under the pressure of immune response and antiviral treatment. when changes occur in the environmental conditions, the quasispecies structure responds by rebalancing its composition. the predominant sequence may shift by selection of a variant that is better adapted to the new environment, in the classic darwinian process of survival of the fittest [51] . this feature gives the virus strong adaptability, and makes it difficult to prevent and control. study on the relationship between quasispecies and different clinical outcomes will provide valuable information for exploring anti-hbv treatment strategies. 1. quasispecies evolution under host immune pressure. in chronic hbv infection transmitted via perinatal transmission, the different immune phases of chronic hbv infection confer different environments on hbv quasispecies. thus, the characteristics of hbv quasispecies may differ. a preliminary study on the differences of full-length hbv quasispecies between mothers and their progeny showed that, after 30 years of evolution, the dominant sequence of hbv quasispecies became different between mothers and daughters. the characteristics of hbv quasispecies in various gene regions are different in mothers and daughters with different treatment responses or disease status. among these genes, the prec/c gene had the highest substitution rate [52] . under antiviral drug selection pressure, hbv mutants are selected from the preexisting pool of quasispecies and over time become the dominant species [53] . the probability of resistant mutations depends on the effectiveness of antiviral drugs. low potent drugs have almost no selective pressure on the virus, thus lead to low probability of viral resistance; on the contrary, drugs that completely inhibit viral replication also rarely result in resistance. only medium potent drugs have the highest rates of drug resistance. a study showed that during the 4-week lamivudine therapy, distinct patterns of quasispecies evolution are found between responders and non-responders; the structures of viral quasispecies tended to be simpler in responders, but more complicated (higher diversity) in non-responders. similar phenomenon was also observed during entecavir therapy. another study detected the full length sequence of resistant virus in lamivudine and adefovir sequential therapy, and found that variation of nucleotide or amino acid sequence usually occurs in hbv hbsag or rt region. using single strand conformation polymorphism (sscp) and dna sequence analysis, researchers found that the complexity of hbv quasispecies in patients with cirrhosis was more than those in patients with chronic hepatitis b, suggesting that complexity of hbv quasispecies is associated with disease status [54] . researchers from china had also used the same methods to analyze the difference of quasispecies complexity in s region among patients with chronic severe hepatitis b, patients with chronic hepatitis b and hbv carriers. it is found that the quasispecies complexity in the s region increases along with disease progression in chronic hbv infection. analysis on quasispecies in acute hepatitis b, chronic hbv carriers, chronic hepatitis b and chronic severe hepatitis by full-length hbv genomic clone and bioinformatics methods also discovered the positive correlation between hbv quasispecies complexity and disease severity [55] . on one hand, complex evolution of quasispecies may lead to persistent infection and continuous liver damage, and increase opportunities for the emergence of new hbv variants. on the other hand, enhanced virulence of mutated virus and change of antigen epitope may cause excessive immune response and severe liver damage. however, correlation between hbv quasispecies complexity and disease severity still needs dynamic large sample research to confirm [56] . the dynamic change of quasispecies during na antiviral therapy may be related to the antiviral efficacy and drug resistance. results from a study on patients receiving lamivudine for 48 weeks suggested that the baseline quasispecies heterogeneity is not associated with antiviral efficacy, and the changes of quasispecies complexity at an early stage may predict antiviral efficacy and drug resistance more accurately than the change of hbv dna level during lamivudine therapy. another study on dynamic changes of quasispecies in patients receiving entecavir antiviral therapy suggested that, compared to the partial responders, quasispecies complexity is reduced but dispersion is increased in complete responders after 4 weeks of treatment [57] . in these two studies, the quasispecies dispersion decreased in lamivudine responders but increased in entecavir responders after 4 weeks of treatment. this might be because entecavir has stronger antiviral effect than lamivudine, thus generates more selection pressure. in addition, entecavir can still induce complete response even when some mutations occur since it has a higher drug resistance barrier. therefore, the early changes in hbv quasispecies complexity may act as a predictor of sustained antiviral effect of nucleos(t)ide analogues. the resistant strains typically already exist before antiviral therapy, and become the predominant strains under selection pressure of antiviral drugs. a study showed that adefovir treatment for 240 weeks in patients with chronic hepatitis b selected resistant virus strains [58] . study on gene heterogeneity of hbv reverse transcriptase suggested that lamivudine monotherapy is liable to induce the quasispecies that affect response rate of salvage therapy with adefovir for virologic breakthrough in lamivudine-treated patients, and reduce the sensitivity to other nucleos(t)ide analogues [59] . under different drug selection pressure, non-responders have similar quasispecies evolution patterns, suggesting that this pattern may be associated with viral resistance mechanisms. from the perspective of quasispecies, drug selection pressure changes the relative ratio of viral populations, and leads to population drift. dna sequence analysis is the most direct and reliable method to detect gene mutations. it is also the gold standard to detect nucleic acid sequence mutation. however, the high cost of this method prevents its application in large sample research. dna sequencing, which can be divided into direct sequencing and cloning sequencing, is built on the basis of high-resolution denaturing polyacrylamide gel electrophoresis. direct sequencing detects the nucleotide sequence of dominant strains; clone sequencing method can find out changes of nucleotide in other strains. rflp is a technique that applies to detect variations in homologous dna sequences. it refers to a difference between samples of homologous dna molecules from differing locations of restriction enzyme sites, which may be naturally formed or brought in by pcr mismatch. the dna sample is broken into pieces and digested by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. mismatched pcr is a modified pcr, which changes one or a few nucleotide bases in designed pcr primers to make the synthetic dna meet special requirements. here, a restriction enzyme recognition sequence is introduced into the pcr amplified fragment in order to change the target dna restriction map and distinguish the mutants and non-mutants. nucleic acid hybridisation is the pairing of complementary single-stranded nucleic acids (dna or rna) to produce dna-dna or dna-rna hybrids. when the target dna obtained by pcr amplification combines with the probe labeled by radioactive or non-radioactive labels, any mismatch between the probe and target dna can be detected. this helps to distinguish the wild and mutant strains. nucleic acid hybridization applies to high frequency point mutation and is suitable for large sample detection, but the high requirement for hybridization temperature makes it difficult to popularize. the principle of sscp is based on conformational difference of single-stranded nucleotide sequences of identical length. this property allows sequences to be distinguished by means of gel electrophoresis so as to determine whether mutations exist. it applies to the single base substitution and screening of dna fragment mutation. however, sscp is neither stable nor practical [60, 61] . primer extension starts from the 3′ end in pcr amplification, and only succeeds when the 3′ end of the primer is an exact match to the template dna. based on this phenomenon, the primer with 3′ end containing mutated bases is used to detect the mutation of target dna. the 3′ base-specific pcr technology is simple but with low sensitivity, and the results are prone to be false-negative. the incomplete block of pcr amplification also leads to false-positive results [62] . melting curve analysis is an evaluation of the dissociation-characteristics of doublestranded dna during heating, the temperature at which 50% of dna is denatured is referred to the melting point [63] . in chronic hbv infection, the peak numbers of dna melting curve in patients with moderate and severe hepatitis are significantly more than those in hbv carriers and mild hepatitis; the peak number of melting curve in patients with severe hepatitis is significantly more than that in moderate hepatitis. the melt-curve analysis is less sensitive than sscp, but is more accurate on analyzing genetic variation. the strong operability and high cost-effective make it a preferable method for genetic variation analysis. the high demand for low-cost sequencing has caused the development of highthroughput sequencing-the next-generation sequencing, which includes massively parallel signature sequencing (mpss), solexa sequencing, solid sequencing, 454 pyrosequencing, heliscope single molecule sequencing, etc. these methods apply to genome sequencing, genome resequencing, rna-sequencing, chip-sequencing and epigenome characterization [64] [65] [66] . current dna sequencing methods under development include microscopy-based techniques, macromolecule and nanotechnology that can distinguish the base signal and directly read the sequence without the use of biological or chemical reagent. the third-generation sequencing methods include: (1) non-optical microscope imaging: the dna sequence can be read out if the resolution of image is high enough to differentiate the four kinds of bases on dna when visualizing the spatial linear arrangement of nucleotides. this idea is based on non-optical microscope at the atomic level, for instance, scanning tunneling microscopy (scanning tunneling microscope, stm) [67] . (2) nanopore dna sequencing: it is based on the readout of electrical signals occurring at the single stranded dna or rna molecules passing by alpha-hemolysin pores covalently bound with cyclodextrin [68] [69] [70] . those methods still need further validation and improvement. the pathogenesis of hbv infection is determined by the interplay between both virus and host. different outcomes after infection are related to different host immune responses and viral mutations. however, the biological significance of viral mutation has not been fully elucidated because of: (1) the limited samples and lack of comparison between different groups; (2) the overlap of each hbv genomic coding regions; (3) the limitation of detection technology. with the rapid development of detection technology, the large sample, long-term, multi-level studies will help to understand more about the host-virus interaction and potential mechanisms. zhi the indications for liver transplantation are changing over time. before 1980, the major indication for liver transplantation was primary malignancy of the liver, especially hepatocellular carcinoma (hcc) which significantly influenced the therapeutic efficacy of liver transplantation and postoperative survival. currently, not only are acute or chronic liver diseases nonresponsive to other medical treatment and surgery now the major indication for liver transplantation but also liver diseases that markedly affect quality of life. the major indications for liver transplantation are shown in table 1 .7. viral hepatitis has and continues to be a major public health problem in china. it is the most common liver disease and most common cause of liver failure. the proportion of patients with hepatitis b-induced liver failure among patients receiving liver transplantation in china is significantly higher than in other countries. hepatitis b has diverse clinical manifestations and may progress to chronic hepatitis, liver failure (including acute liver failure, acute on chronic liver failure, and chronic liver failure), hepatic cirrhosis (compensated and decompensated), and even hepatitis b-related liver tumors. whether liver transplantation is necessary for patients with hbv infection and the timing of transplantation must be carefully considered. early liver transplantation may increase patient risk and be an economic burden because patients could recover and even survive for a long time in the absence of liver transplantation. however, presently it is difficult to predict patient outcome to hbv infection. in contrast, if liver transplantation is performed in the late stages of disease, numerous complications of hepatitis b-induced liver dysfunction may either increase the risk of transplant surgery resulting in a poor prognosis. thus, for patients with severe hepatitis b, the indications for and the timing of liver transplantation are crucial and should be carefully evaluated. in general, the following conditions are indications for liver transplantation in patients with chronic hepatitis b: 1. obvious manifestations of liver failure including sustained elevation of serum bilirubin to >5 mg/dl; prothrombin time > 5 s longer than the reference range; plasma albumin <2.5 g/dl; and liver failure which is nonresponsive to active and/or symptomatic therapy (such as infusion of fresh plasma and albumin) or if the patient continues to deteriorate clinically despite optimal medical therapy. 2. when there are complications related to either liver failure or portal hypertension such as the presence of severe hepatic encephalopathy, disturbances of coagulation function, refractory bleeding due to rupture of esophageal or gastric varices, refractory ascites, repeated episodes of spontaneous bacterial peritonitis, or the development of hepatorenal syndrome. 3. when hepatitis b influences the quality of life including the development of severe lethargy, uncontrollable itching, metabolic bone diseases or the development of bacterial cholangitis and 4. the development of hepatocellular carcinoma. although there are some well defined indications for liver transplantation, the ultimate determination of the timing and specific indications for liver transplantation remains a challenge in clinical practice for hepatitis b patients with liver failure. the introduction of the model of end stage liver diseases (meld) and some derivative scoring systems are now frequently used to determine whether a patient should be listed for transplantation and the urgency of liver transplantation. in 1999, the mayo clinic proposed that meld should replace the child-pugh grading system for the determination of urgency of liver transplantation. meld is calculated using the following calculation: meld = 9.6 × log e (creatinine [mg/dl]) + 3.8 × log e (bilirubin [mg/ dl]) + 11.2 × log (inr) + 6.9 × (causes: 0 for biliary and alcoholic; 1 for others). meld was originally used for the evaluation of prognosis of hepatic cirrhosis patients receiving transjugular intrahepatic portosystemic shunt. as described above, the meld score is determined on the basis of total bilirubin, international standardization of bleeding time, serum creatinine, and etiology. compared to the child-pugh grading system, meld employs objective parameters, which are helpful for the comparisons of patients from different centers. while the meld score changes with the alteration of the liver disease, the child-pugh grade has only three levels and is unable to meet the requirements of accurate and objective evaluation. saab et al. investigated the prognosis of 404 patients receiving liver transplantation and found that the 1-year survival rate was 90, 89, 90, 79, and 69% in patients with preoperative meld scores of ≤10, 11-18, 19-24, 25-35 , and ≥36, respectively. however a higher meld score negatively affected the 1-year survival rate. for example patients with a meld score ≤24 had a 1 year survival rate of 88 whereas patient survival was reduced to 65% in patients with a meld score >24. despite the benefits and the widespread application of meld in clinical practice, there remain some imperfections in the evaluation of timing of liver transplantation and the assessment of prognosis and therapeutic efficacy. one of the major imperfections is the failure to consider complications such as infection, hepatic encephalopathy, hepatorenal syndrome, and disturbances in fluid and electrolytes which may significantly influence the prognosis and timing of liver transplantation but are not included in the meld scoring system. hence inclusion of sodium (na) in the meld scoring system: meld − na [meld + 1.59 × (135 − na)], imeld [meld + (0.3 × age) − (0.7 + na) + 100]; and meso [meld/na (mmol/l)] × 10 has improved outcomes of patients requiring liver transplantation. those scoring systems, however, also fail to consider other complications. including the development of hepatic encephalopathy or ascites and therefore, their applicability is limited in china. in china, liver transplantation was initiated relatively late compared with other countries; regulations regarding liver transplantation remain imperfect; and smooth communication among transplantation centers in different regions of the country are lacking. these drawbacks markedly influence the timely and fair distribution of donor livers. determination of waiting times for transplantation is not scientific. therefore, the comprehensive evaluation of a patient's condition of hepatitis b-induced liver failure, determination of the timing of liver transplantation, and optimization of the rational and fair distribution of donor livers needs to be carefully considered by chinese clinicians in the field of hepatology going forward if improvements are to occur. there are currently additional scoring systems which have been used by chinese clinicians. one of these was developed by professor ke wm in the department of infectious diseases of the affiliated third hospital of sun yat-sen university. in that system, hepatic encephalopathy, serum creatinine, prothrombin activity, serum total bilirubin, liver size (determined by ultrasonography), amount of ascitic and pleural fluid (determined by ultrasonography), and infection (peripheral white blood cell count, proportion of neutrophils, and inflammatory findings from thoracic imaging examinations) are taken into account and objectively and conveniently scored on a scale of 0-4 according to their severities as described in tables 1.8, 1.9, and 1.10. [72] these newer scoring systems and the meld scoring system can favorably predict the mortality of acute on chronic liver failure patients with hepatitis b. the area under receiver operator curves of scores determined with a new scoring system and meld scoring system was 0.960 [95% confidence interval (ci), 0.944-0.977] and 0.886 (95% ci, 0.852-0.920), respectively. there was no overlap in 95% cis between the two, and they were significantly different (p < 0.01) as illustrated in figs. 1.7, 1.8, and 1.9 ). wenzhou medical college in china subsequently proposed a scoring system in which the total score is calculated using the following equation: x = 1.4053 + 3.6017 × hepatorenal syndrome (hrs) + 1.2069 × lc − 1.1555 × hepatitis b e antigen (hbeag) − 0.1003 × alb − 0.042 × pta. that scoring system has been used in several centers for liver diseases, and its effectiveness and accuracy have now been confirmed in these centers. in summary, to establish a new scoring system on the basis of china's national status of liver disease and to further validate this system, large multicentered studies with a large number of patients is crucial before they are adopted in china. it is important that any new system developed under these conditions be subjected to rigorous study, standardization and scientific validation. for liver transplantations in patients with hepatitis b-induced liver failure, perioperative therapy is crucial, impacting the recurrence of hepatitis b virus reinfection following liver transplantation. in fact, perioperative therapy can make a major 10 fig. 1.9 receiver operator curve of a new scoring system and meld scoring system difference in patients with hepatitis b compared to patients with other liver diseases (e.g., tumor recurrence). it is well appreciated that antiviral therapy is necessary in hepatitis b virus patients as discussed below. the guideline for the prevention and treatment of chronic hepatitis b in china (2010) recommends that (1) for patients with hepatitis b planning to receive liver transplantation, oral lamivudine be administered within 1-3 months before liver transplantation when hepatitis b virus dna is detectable (100 mg q 24 h). hepatitis b immune globulin (hbig) should be administered during the anhepatic stage in surgery and long-term use of lamivudine and low-dose hbig (800 iu q 24 h for the first week postsurgical, 800 iu q 1 week, then 800 iu q 1 month). the dose of hbig and interval between two treatments [generally, trough hepatitis b surface antibodies (anti-hbs) are >100-150 miu/ml and anti-hbs is better if >500 miu/ml within 6 months of surgery) should be determined according to the anti-hb levels. for patients nonresponsive to lamivudine, other nucleos(t)ide analogues effective for resistance mutation should be used and for patients with a low risk for recurrence of hbv infection (i.e., hbv dna negative before liver transplantation and absence of hbv infection recurrence within 2 years after liver transplantation), lamivudine plus adefovir should be considered for the prevention of hbv infection recurrence. while patients are waiting for liver transplantation, an artificial liver support system (alss) may be of use as a bridge to transplantation. liver failure may significantly compromise the detoxication, synthesis, and metabolism activities, resulting in a significant accumulation of toxins and deficiency of some important factors (such as coagulation factors). unfortunately, some patients die while waiting for either their liver function to improve or for liver transplantation. alss have been generally used to partially substitute the liver's normal activities, such as clearing some toxins and supplement some missing factors, which is helpful for life maintenance while awaiting subsequent liver transplantation or spontaneous recovery. thus, for patients with liver failure secondary to hepatitis b, alss maybe an important therapeutic strategy while patients are waiting for liver transplantation. alss can be classified as a mechanical artificial liver, biological artificial liver, or mixed artificial liver. mechanical alsss mainly utilizes nonbiological materials to clear toxins in the body and supplement some missing factors. biological alsss use biological materials to substitute the liver's activities. the core feature of the biological alss systems is to simulate hepatocyte function. however, the source and biosafety, as well as their location (bioreactor), are important factors limiting the development of biological alsss. finally, mixed alsss employ the hemodiafiltration, plasma exchange, and hemoperfusion that are able to detoxify substances (nonbiological alss) and human or porcine "hepatocytes" in the bioreactor. currently, nonbiological alsss are the most widely used in clinical practice, and biological and mixed alsss are still undergoing investigation and have not been widely applied in clinical practice. in addition to its use before liver transplantation, alss post liver transplantation may be used to correct ongoing renal failure, brain edema, severe water and electrolyte disturbances, and severe infections. another factor to consider is that liver donors are rare. as a result, the inability to perform liver transplantation in a timely fashion results in reduced survival rates. under those conditions, pre transplant complications including multi organ failure may persist. in such cases, alss therapy may prove to be beneficial. for patients with hepatitis b-related liver failure, perioperative therapies are more important than in patients receiving elective liver transplantation (such as liver tumor patients without liver failure). liver failure causes a variety of complications that directly contribute to transplantation failure and a high-risk status after surgery. clinicians need to address problems such as portal hypertension, upper gastrointestinal bleeding, severe jaundice, ascites, spontaneous peritonitis, hepatic encephalopathy, hepatopulmonary syndrome, portal pulmonary hypertension, kidney dysfunction, hepatorenal syndrome (among others) through active treatment/management in specialized liver units. preserving liver function and maintaining a normal physiological status have been shown to reduce the risk of therapeutic failure in patients receiving liver transplantation due to hepatitis b-related liver failure. diagnostic and treatment guidelines for liver failure the clinical implications of hepatitis b virus genotype: recent advances new complex recombinant genotype of hepatitis b virus identified in vietnam possible new hepatitis b virus genotype when should "i" consider a new hepatitis b virus genotype? a genetic variant of hepatitis b virus divergent from known human and ape genotypes isolated from a japanese patient and provisionally assigned to new genotype clinical outcome and virological characteristics of hepatitis b-related acute liver failure in the united states influence of genotypes and precore mutations on fulminant or chronic outcome of acute hepatitis b virus infection mortality secondary to fulminant hepatic failure in patients with prior resolution of hepatitis b virus infection in japan profile, spectrum and significance of hepatitis b virus genotypes in chronic hbv-infected patients in yunnan, china. hepatobiliary pancreat dis int precore/core promoter mutations and genotypes of hepatitis b virus in chronic hepatitis b patients with fulminant or subfulminant hepatitis virologic and clinical characteristics of hbv genotypes/ subgenotypes in 487 chinese pediatric patients with chb quasispecies and its impact on viral hepatitis hepatitis b virus: molecular virology and common mutants a function of the hepatitis b virus precore protein is to regulate the immune response to the core antigen cytoplasmic expression of hepatitis b core antigen in chronic hepatitis b virus infection: role of precore stop mutants different hepatitis b virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis b e antigen seroconversion hepatitis b virus e antigen variants two core promoter mutations identified in a hepatitis b virus strain associated with fulminant hepatitis result in enhanced viral replication type, prevalence, and significance of core promoter/ enhancer ii mutations in hepatitis b viruses from immunosuppressed patients with severe liver disease probable implication of mutations of the x open reading frame in the onset of fulminant hepatitis b association between the various mutations in viral core promoter region to different stages of hepatitis b, ranging of asymptomatic carrier state to hepatocellular carcinoma number of mutations within ctl-defined epitopes of the hepatitis b virus (hbv) core region is associated with hbv disease progression pre-s2 defective hepatitis b virus infection in patients with fulminant hepatitis associations between hepatitis b virus mutations and the risk of hepatocellular carcinoma: a meta-analysis potential threat of drug-resistant and vaccine-escape hbv mutants to public health early changes of hepatitis b virus quasispecies during lamivudine treatment and the correlation with antiviral efficacy hepatitis b virus variant with the a194t substitution within reverse transcriptase before and under adefovir and tenofovir therapy restoration of replication phenotype of lamivudine-resistant hepatitis b virus mutants by compensatory changes in the "fingers" subdomain of the viral polymerase selected as a consequence of mutations in the overlapping s gene lamivudine resistance and other mutations in the polymerase and surface antigen genes of hepatitis b virus associated with a fatal hepatic failure case adefovir rapidly suppresses hepatitis b in hbeag-negative patients developing genotypic resistance to lamivudine adefovir dipivoxil monotherapy and combination therapy with lamivudine for the treatment of chronic hepatitis b in an asian population comparison of adefovir and tenofovir in the treatment of lamivudine-resistant hepatitis b virus infection two-year assessment of entecavir resistance in lamivudine-refractory hepatitis b virus patients reveals different clinical outcomes depending on the resistance substitutions present telbivudine, a nucleoside analog inhibitor of hbv polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir management of treatment failure in chronic hepatitis b resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the hbv polymerase selection of a hepatitis b virus strain resistant to adefovir in a liver transplantation patient susceptibility to antivirals of a human hbv strain with mutations conferring resistance to both lamivudine and adefovir impact of hepatitis b virus rta181v/t mutants on hepatitis b treatment failure management of drug-resistant chronic hepatitis b treatment of patients with lamivudine-resistant and adefovir dipivoxil-resistant chronic hepatitis b virus infection: is tenofovir the answer? successful treatment of an entecavir-resistant hepatitis b virus variant how to diagnose and treat hepatitis b virus antiviral drug resistance in the liver transplant setting prevention and management of drug resistance for antihepatitis b treatment is there an association between the measurement of qualitative hbsag and virologic response in chronic hbv infection serum hepatitis b surface antigen quantitation can reflect hepatitis b virus in the liver and predict treatment response serological and molecular diagnosis hbv induced hcc: major risk factors from genetic to molecular level new insight in the pathobiology of hepatitis b virus infection quasispecies structure, cornerstone of hepatitis b virus infection: mass sequencing approach clinical implications of evolutionary patterns of homologous, full-length hepatitis b virus quasispecies in different hosts after perinatal infection hepatitis b virus mutations associated with antiviral therapy hepatitis b virus s gene mutants in a patient with chronic active hepatitis with circulating anti-hbs antibodies hepatitis b virus genomes of chronic hepatitis patients do not contain specific mutations related to acute exacerbation molecular virology and the development of resistant mutants: implications for therapy evolutionary patterns of hepatitis b virus quasispecies under different selective pressures: correlation with antiviral efficacy complex dynamics of hepatitis b virus resistance to adefovir emergence of hepatitis b virus quasispecies with lower susceptibility to nucleos(t)ide analogues during lamivudine treatment optimization of nonradioisotopic single strand conformation polymorphism analysis with a conventional minislab gel electrophoresis apparatus evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene an improved allele-specific pcr primer design method for snp marker analysis and its application product differentiation by analysis of dna melting curves during the polymerase chain reaction next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solution next-generation dna sequencing methods applications of next-generation sequencing technologies to diagnostic virology fast dna sequencing via transverse electronic transport single-nucleotide discrimination in immobilized dna oligonucleotides with a biological nanopore rapid sequencing of individual dna molecules in graphene nanogaps detecting single stranded dna with a solid state nanopore peri-operative therapies of patients receiving liver transplantation due to severe hepatitis establishment of a scoring system for evaluating the severity of hepatitis b patients with acute-on-chronic liver failure key: cord-010088-s9tfvtao authors: nan title: oral abstracts date: 2013-11-01 journal: vox sang doi: 10.1111/vox.12100_1 sha: doc_id: 10088 cord_uid: s9tfvtao nan tadokoro k and satake m japanese red cross society, tokyo, japan it is estimated that there are 2 billion hbv-infected people including 350 million hbv-carriers in the world. it is highly endemic in south africa, amazon, and southeast-central asia. the genotype of hbv is geographically characteristic, e.g. genotype b and c are the major in east asia. hbv transmission remains the most frequent transfusion-transmitted viral infection despite the implementation of various screening tests applied in different settings. the residual risk is mainly related to donations either in the pre-sero (or pre-dna)-conversion window period or occult hbv infection (obi) where blood test is hbv-dna-positive and hbs-ag-negative. infectivity of hbv depends on the transfused blood (viral load, phase of infection, genotype, and anti-hbs in the concurrent blood) and immune status of the recipients (anti-hbs, immunocompetence). it was shown that infectivity is dependent on viral load. allain et al reported that ffps is more infectious than pcs or rbcs. the minimal infectious dose of blood in late acute infection phase in chimpanzee and chimeric mice is approximately 10 times higher than that of pre-acute phase. satake et al reported that transmission rate of obi-derived components with low titer anti-hbc was 1/33(3%), whereas that of anti-hbc-negative components was 11/22(50%), which was verified in the lookback programme conducted in japan. allain et al showed in the study conducted in europe that adjusted transmission rate of obi blood was 28%, and the rate was higher without anti-hbs(63.8%) and lower with anti-hbs(15.4%). discrepancy of transmission rate of obi-derived blood between above two reports might be related to the different cutoff levels of anti-hbc and presence or absence of anti-hbs. dna-positivity rate among obi-derived components is higher in those with the higher levels of anti-hbc and lower in those with the presence of anti-hbs. there has been no report of transmission by obi-derived blood with anti-hbs of 200miu/ml or more. screening test for hbv is different between countries. low endemic countries screen blood for hbs-ag, anti-hbc and mini-pooled nat, while highly endemic countries test for hbs-ag without anti-hbc, because high prevalence of anti-hbc-positive donation hamper securing necessary blood. japan as a moderately endemic country had tested for hbs-ag, mini-pool nat and anti-hbc/anti-hbs where anti-hbs of more than 200 miu/ml irrespective of anti-hbc and low anti-hbc with agglutination-inhibition titer of no more than 2 5 is qualified. transfusion-transmitted hbv cases related to window period donations declined by increasing the sensitivity of mini pool nat, whereas those related to blood with low titer anti-hbc remained stable with around 10 cases annually. in order to decrease such transmission japanese red cross implemented a novel strategy to eliminate all anti-hbc-positive donations with anti-hbs <200 miu/ml. considering the frequency of donations with low titer anti-hbc has decreased to 1.3%, loss of those donations was estimated to be covered by promoting donations, each country should establish its own hbv screening strategy considering the prevalence of hbv, residual risk of transmission, balance between safety and securing blood, and cost-effectiveness. implementation of individual donation nat and universal vaccination could further reduce further the risk of hbv transmission. our blood service is to motivate other population groups to diversify the age and gender composition of our donors. forty-two percent of the whole blood donors participate in blood donation only once a year, so we need develop programs to motivate them to re-join blood donation. also, we have to solve the annually recurring problem of blood shortages for transfusion during the winter and summer season. in the long term, our donor base is going to decrease gradually because of the low birth rate in korea and our rapidly aging population. therefore we should prepare a sustainable solution for a stable blood supply. aims: present methods to recruit blood donors based on age, gender and occupation groups for a stable blood supply. methods: to make blood donation more accessible to individual donors, fixed donation sites are continuously developed. facilities of our fixed and mobile sites are improved to provide safe and comfortable environment to donors. since 1999, we have been operating the 'registered donor system' for registered donors who agreed to donate their blood on a regular basis. to raise awareness of the importance of blood donation among youth, high-school students blood donor groups called 'red campaigners' and groups of university students called 'blood donation supporters' are actively participating in blood donation campaigns. to increase participation of the middle-aged group, agreements were made not only with enterprises and organizations but also with the government and public institutions. we have developed a computerized system for scheduling group donations according to demand and supply and for performance management. to recognize the necessity of blood donation, every 13th was designated as 'blood donation day' in 2012. to deal with donor complaints and requests, a customer relationship management center has been established. results: by increasing the number of fixed donation sites and making donation more accessible, rate of individual donations is getting increased. as of the end of june 2013, 250 agreements for blood donation had been signed with enterprises and organizations. every year more than 100 thousand donors agree to donate on a regular basis, and so far about 600 thousand donors have been registered in the 'registered donor system'. the campaign 'every 13th is blood donation day' has contributed to spread a positive perception about blood donation. conclusions: we have been very successful in engaging youth in blood donation. diversification of the donor group will take time and constant effort. however, with more participation of female donors and retention of our first-time donors, it will be possible to be self-sufficient in blood supply for transfusion and plasma fractionation. the provision of sufficient safe blood products to patients requiring transfusion is the common goal of blood transfusion services and the public expectation of absolute safety continues to be a challenge. although the focus of blood safety falls on laboratory testing, the role of pre-donation donor selection cannot be underestimated. transfusion-transmitted infections (ttis) are blood-borne microbes that can be spread from blood donor to recipient via transfusion. to prevent tti, donated blood must go through validated laboratory testing. in most countries, blood is tested for hiv, hbv, hcv, and syphilis. screening for other ttis may also be implemented in some countries after individual assessment of the prevalence of the infection in their general populations, for example, west nile virus in the united states. with the advance in testing technology, in particular the nucleic acid test, the window periods for the detection of various ttis have been significantly shortened. however, the risk of window donation still exists. furthermore, there are known or emerging ttis such as the variant creutzfeldtjakcob disease (vcjd) where there is still no suitable testing system for routine screening of donated blood. therefore, blood transfusion services have to continue practicing effective pre-donation donor selection to mitigate the tti risks. who recommends selecting voluntary, non-remunerated donors from low-risk populations for blood collection as the first step in reducing the risk of ttis. donor selection is usually conducted by a health history questionnaire to be completed by potential donors and a confidential interview. the questions asked should be effective in assessing whether the respondent's health status is suitable to donate and there is no tti risk factor. people who are deferred should be counseled and given the reason for and duration of the deferral. who has published a document titled 'blood donor selection -guidelines on assessing donor suitability for blood donation'. it recommends the following: national donor selection guidelines and criteria should be based on epidemiological and/or scientific evidence or, where evidence is limited or lacking, on best practice. donor acceptance and deferral policies for the prevention of tti should be based on up-to-date information on the local epidemiology of infections, the markers screened for, the availability of suitable blood screening and confirmatory assays, and the technologies in use. national donor selection criteria should define conditions of acceptance and deferral for each criterion. adequate resources, including a sufficient number of qualified and trained staff, should be made available for the consistent and reliable assessment of donor suitability for blood donation. quality systems should be in place for donor selection, including selection criteria, staff training and documentation. blood transfusion services should establish mechanisms for monitoring and evaluation to assess the implementation and effectiveness of donor selection criteria. in conclusion, pre-donation selection is essential to protect the safety and sufficiency of blood supply, and safeguard the health of recipients and donors. background: blood components may be contaminated by a variety of commensal, pathogenic or environmental bacteria during collection, manufacture or storage. the outcome of transfusion is dependent on the ability of the specific strain to multiply to clinically-relevant titers during storage, the pathogenicity of the strain and the patients' situation. platelets in particular are stored in conditions conducive to bacterial growth and septic reactions to these products are the most frequently documented infectious risk of transfusion. aim: the objective of this update is to review estimates of risk of bacterial sepsis and contamination of platelets, and recent findings describing interventions designed to safeguard patients. methods: this update will use recent reports and unpublished data to describe our current understanding of the role of bacteria in transfusion safety. results: historical data suggests that~1:1-3000 platelet products are contaminated with bacteria, and septic transfusion reactions occurred in 1:15-100,000 transfusions. clinical awareness of the danger and a 2004 aabb standard to 'limit and detect bacteria' in platelet products have driven the development of safety systems. the last decade has seen substantial progress in the implementation of optimal skin disinfection techniques and sample diversion strategies to reduce contamination, and many centers implemented either bacterial culture testing or pathogen inactivation processes to reduce the risk. culture testing can reduce but cannot eliminate the risk of exposure to contaminated components and sepsis. the majority of contaminated products do not cause adverse reactions, however, at the time of collection and manufacture the only means to prevent serious and fatal reactions is to ensure that that the component is functionally sterile. pathogen inactivation technologies show variable efficacy at killing bacteria, suggesting a need for strict adherence to the manufacturers suggested protocols to ensure optimum performance. alternatively, assays performed on the day of transfusion prevent the transfusion of high concentrations of bacteria that are associated with the most severe adverse reactions. conclusions: bacterial contamination and sepsis remain the greatest infectious risks of transfusion. enhanced testing or pathogen inactivation should be implemented to ensure patient safety. the australian red cross blood service, sydney, australia background: pathogen reduction technologies have been developed as a means of reducing the risk of transfusion transmission of blood-borne pathogens. published literature indicates that systems currently in use or under development effectively inactivate a range of pathogens in platelets and plasma, with the exception of some non-lipid enveloped viruses, bacterial spores and prions. development of pathogen reduction technology (prt) is ongoing, and many countries have adopted prt as part of their routine blood component processing. the aim of this update will be to review technologies currently in use or under development for treatment of labile blood components during processing. the impact of prt on blood component quality as well as ongoing challenges will be reviewed. platelets: there are currently three systems for prt treatment of platelets. these are the mirasol tm system (terumobct), the intercept blood system tm (cerus corporation) and the theraflex uvc tm system (macopharma). the intercept and mirasol systems are used widely in blood centres in europe, asia and the middle east for the treatment of both platelets and plasma, whereas clinical trials of platelets treated using the theraflex uvc system are ongoing. in vitro data suggests prt treatment leads to some loss of platelet function. however, haemovigilance reports from sev-eral countries where intercept treated platelets are transfused indicate no change in component usage, and indeed a reduction in transfusion related adverse events. similarly, there have been no reports to date of serious adverse events relating to clinical use of mirasol-treated platelets. plasma: the intercept and mirasol systems can be used to treat both plasma and platelets, and the theraflex system utilises methylene blue (mb) with visible light for prt treatment of plasma. there are some losses of coagulation factors following treatment with each of these systems. theraflex mb plasma has been transfused world-wide since 1999, and haemovigilance data indicates there is not a higher incidence of allergic reactions or other adverse events with mb-treated plasma. red cells/whole blood: commercial prt systems for red cell or whole blood components are under development. cerus corporation has continued development of a second-generation s-303 system for red cells, demonstrating in vivo recovery after 35 days storage. a mirasol prt system for treatment of whole blood is also being developed by terumobct. preliminary in vitro quality and in vivo recovery and survival data indicate that this technology may eventually become available, but further development and clinical trials are still required. challenges: concerns still exist regarding long term effects on patients receiving prt treated blood components, particularly the potential toxic effects of residual products following photochemical treatment. ongoing post-marketing surveillance and clinical trials are required to address these concerns. ideally, prt should provide proactive protection against emerging pathogens and reduce the need to introduce additional pathogen testing, minimise bacterial contamination and potentially replace processing steps such as gamma irradiation. the challenge for blood services is to understand and rationalise the costs, risks and benefits of prt compared to removing any of these tests or processes, whilst aiming to provide the highest level of blood safety. steps in getting a paper published/abstract accepted 1b-h4-01 no abstract available. 1b-h4-02 how to get your abstract accepted and how to present it daniels g many abstracts are submitted to the isbt for presentation at international and regional congresses. all abstracts are refereed by a large panel of international experts, who decide which abstracts should be accepted and which ones should be rejected. they also decide which of the accepted should be presented orally or as a poster. most of the submitted abstracts are accepted, though there is plenty of room for improvement in the quality of many of those abstracts. in this session i will describe why some abstracts are rejected and discuss how you might avoid this pitfall. i will also discuss some methods for improving your abstracts so that they represent the quality of the work you are describing and enhance the reputation of yourself and your institution. once your abstract has been accepted you will have to present it at the congress. if it is accepted for poster presentation, you will have to make a poster. the easiest way to produce a poster is to design it on a single powerpoint slide and then send the file to a company that will turn it into a poster on paper, or even on cloth for easier transportation. if your budget does not run to that, there are cheaper ways, such as printing it on single pages of a4 with the text printed in a very large font. the best posters do not have too much written information (bullet points are often best), contain diagrams and/or pictures, and are colourful and visually attractive. if, however, your abstract is accepted for oral presentation, then you will have to give a talk, usually of 10 min, and then be prepared to answer questions. this may be a daunting prospect, especially if you are not experienced in public speaking, and particularly if english is not your first language. i will discuss techniques for improving your presentation skills, both from the points of your spoken presentation and your visual aids, which will usually be a powerpoint presentation. if you are thoroughly prepared before you stand up in front of an audience, you will feel more confident and, consequently, less nervous. devine d publication of research findings and novel concepts in the biomedical literature is the mainstay of knowledge mobilization. the communication of scientific work as papers follows a well-established framework. a clear understanding of the nature of this framework and how to assess one's own work against it is critical to successful acceptance and subsequent publication of manuscripts. in this session, we will review the standard framework for scientific communication. communicating your findings is a form of scientific story telling. one must clearly explain why it was important to write the paper so that the reader will be interested and want to keep reading it. generally, an author must capture the interest of a potential reader right at the abstract which is sometimes the only way a reader sees the paper if they have discovered the work using a search engine such as medline. in the main body of the paper, the author must clearly explain how the study was designed and carried out with careful attention paid to any important details and to the statistical analysis, if appropriate. results must be presented in a manner that is clear and easily interpreted by the reader. then the work should be discussed in the context of other work in the area, emphasizing the novel findings. in this session, we will also address the following questions: how do i know if i have enough information to write a paper? where should i try to publish the paper? how should my paper be put together to give it the best chance of being accepted? what happens to my paper after i submit it? if it is returned to me with comments from the reviewers, what should i do? what is my next step if my paper is rejected, either without review or with review? we will also consider how to prepare papers in a language other than one's native language. the various sorts of scientific communications (original papers, review articles, reports, letters to the editor) will be discussed. the session is intended to provide general guidance for the publication of papers in the biomedical literature rather than be specifically focused on publication in the society's journal vox sanguinis. the membership survey commissioned by the isbt central office in 2012 gave a good insight into what members and non-members expect from the society. the main message was that members and potential members would like more educational and training resources to be available. many respondents requested that isbt should write international guidelines and develop standards. developing international guidelines when so many are already available and when countries and regions have different requirements would be a titanic task. the isbt board decided that the society should put together a repository (library) of guidelines, standards and regulatory documents that are already currently available. the repository is now ready and launched during the 24th regional congress of the isbt in kuala lumpur. for practical purposes the repository contains recent guidelines in the english language that are freely available, however following consultation with experts some manuals are included that are either relatively old or are not yet freely available on line. approximately 250 documents are in the repository from around 25 countries. the repository is a work in progress and further documents will be added in other languages. the repository is available via a link on the isbt home page www.isbtweb.org to the isbt academy e-learning portal. it can be accessed by country and by subject and a search facility is available. the guidelines are organised by six main subjects; donor, clinical, laboratory, quality/haemovigilance, processing and regulatory. there are sub topics for each main subject. isbt anticipates that you will find the repository a useful resource. background: blood services in africa operate at different levels of development from those comparable to the first world to those operating at a basic level with just hospital based blood banks and no national coordination. in recent years, countries have made efforts to improve their blood services based on voluntary non-remunerated blood donation. major challenges include low knowledge levels, inadequate funding, weak regulatory framework and poor quality systems. many international standards are too stringent for most economies in sub-saharan africa. to address these challenges, the africa society for blood transfusion (afsbt) started a program to develop blood transfusion standards relevant to africa. the afsbt step-wise accreditation standards: these standards were drafted by an afsbt task team for accreditation with guidance from the aabb and input from a team of experts. they were initially based on the who aide memoire for blood safety. an evidence-based decision-making process, where possible, was used to modify existing requirements or create new specific requirements. the goal of the standards is to provide a benchmark for accreditation of facilities and to maintain and enhance the quality and safety of blood transfusion in africa. the development process started in 2009. there is one standard with 3 progressively more rigorous steps of achievement as follows: level 1: minimum quality and operational requirements level 2: intermediate quality and operational requirements level 3: full accreditation at international standard a facility chooses the level to be assessed at. however accreditation at any grade is attained if facilities meet standards of the grade but also comply with specific requirements in section 11 which deals with legal and regulatory requirements, blood supply, equipment and supplies and clinical use of blood and blood products. application of the standards: they apply to facilities that perform any or all of the following functions: mobilization, recruitment and selection of blood donors; blood collection; components preparation; blood group and serological testing; compatibility testing; storage, handling, transportation and distribution of blood products. requirements do not apply in cases where the blood transfusion service is not responsible for an activity e.g compatibility testing. guidance document: there is a guidance document which clarifies and enhances understanding of the requirements of some of the standards. other standards are straight forward and do not need guidance. the guidance document is being updated as queries are received from the field. training: to facilitate meeting the requirements of the standards, there is a training committee, tasked with supporting training for blood services. a full training program is currently being developed. piloting: the standards were piloted in namibia and malawi. the namibia piloting was completed in 2012 while the malawi piloting will be completed in 2013. comments from these pilot sites have provided valuable input into the standards development process. conclusion: stepwise accreditation program is relevant in encouraging improvements for developing blood transfusion services. accreditation standards need to be commensurate with local needs. training is very important in helping developing blood transfusion services attain accreditation status. blood transfusion has become an integral part of modern healthcare. when it is required, it is an essential element of therapy, helps safe lives and improves quality of life. however, it is not without risk. being of human tissue origin, this risk is related to its source as well as the process involved in its provision. to ensure that blood transfusion is safe and does not cause harm to patients, these risks have to be monitored, evaluated and managed appropriately. therefore it is essential to develop system of ensuring safe blood supply and safe transfusion. quality systems should be developed covering the whole transfusion chain. policies, standards and guidelines are important tools. to ensure that the system is effective, there are various mechanisms to monitor, evaluate and analyse in order to bring about improvement. these include developing indicators, quality and clinical or transfusion audits, quality assessment programmes and haemovigilance programme. indicators can be used to monitor transfusion practice such as prevalence of transfusion transmitted disease among blood donors, crossmatch transfusion ratio, expiry fate of blood components and transfusion error. these indicators not only measure safety but also efficiency of the blood service. in countries where the blood supply is not consistent, the rate of blood requirement not met is an important indicator to monitor. performance of laboratories providing blood can be measured using quality assessment programmes. this is an important element that links the source of blood to the patient. audits covering all processes and procedures ensure that the quality and safety of blood is maintained. while these processes and procedures are controlled in ensuring safety and quality of the blood supply, the process involved in the transfusion process is sometimes not controlled by documented procedures and poorly supervised. auditing blood usage which is more complex and laborious provides an important mechanism for ensuring that this precious national resource is utilised efficiently. guidelines whether national or international are required against which these audits are performed. transfusion audits looks at the quality of care of patients besides the use of blood. it analyses the process of diagnosis, the decision making in treatment and the use of available resources. over the last two decades, haemovigilance has evolved and expanded worldwide. it focuses on adverse events that occurred throughout the transfusion chain, analysis of the facts and provide an avenue for corrective actions to be taken to prevent further recurrence. initially, its focus was more on adverse events that occurred to patients, it is now used to monitor adverse events relating to blood donors and blood donating process. in resource limited economies these tools can be used effectively when a stepwise approach is adopted. the aim is to create a culture of professionalism in delivering quality of care to patient with efficient use of available resources. however, its benefit can be extended to influence change and improvement in healthcare in general. only when deficiencies are demonstrated that request for resources can be shown to be justified. serological methods have the advantage of being fast, simple, and determining the expression of antigens directly. however, genotyping and molecular diagnostic methods have their advantages in determining subtypes and variants, as well as in detecting other rare blood groups. commercial blood group genotyping kits have been widely used, not only in clinical blood banks but also in transfusion services. these commercial kits mainly aim to analyze coding genes of abo and rh blood group systems. common alleles found in local populations are included in these kits. at present, many kinds of molecular diagnostic methods are employed in detecting blood group alleles by transfusion laboratories in china. these methods include multiplex pcr, multiplex pcr combined with pool system, sequencing, and gene chip technology. considering the genetic background of several rare phenotypes with clinical importance in chinese persons, one multiplex pcr system was developed to detect fy a , s, and ok a antigens, and the other system was developed to detect di b , k, and js b antigens. using the existing multiplex pcr-ssp assays, only one sample can be detected in a single pcr tube because the primers used in these assays are specifically devised for corresponding high-frequency alleles, and aim to screen for the negative results. therefore, the positive results would mask the negative results by adding more samples in one pcr tube. to improve the efficiency of screening, recently we established a novel method combining the multiplex pcr-ssp assays with the dna pooling strategy. the primers were designed to amplify the corresponding low-frequency snp sites. in each multiplex pcr-ssp assay, every dna pool is tested for the presence of the low-frequency snp sites. a pool is released for further processing when the positive results were found in any site. after then, each individual dna sample of the positive pool was detected respectively to determine the positive sample. finally, pcr-ssp methods were used to determine whether the positive result was caused by homozygous or heterozygous alleles. normally, the homozygous low-frequency alleles would lead to rare phenotypes. at the same time, the control system based on site-directed mutagenesis solves the problem of the lack of experiment controls due to unavailable negative or positive dna control samples. with large scale screening of population samples and diagnosis of various clinical special samples, the relationship between blood group coding genes and the expression of proteins is being clear. the frequency of multiple blood group alleles has also been investigated. the specific molecular events found in asians and chinese populations are revealing the difference among races and regions, as well as the expression diversity of blood group alleles. 1c-h6-02 diagnosis and treatment of autoimmune haemolytic anaemia autoimmune haemolytic anaemia (aiha) occurs as a result of antibodies directed against self-red blood cell (rbc) antigens, leading to the premature destruction of rbc. rbc destruction may occur within the vasculature, often mediated by the membrane-lysing complex, which is generated upon complement activation or occur extravascularly, within the reticulo-endothelial system that expresses fcî¥r and c3 receptors, that bind antibody and complement coated rbc. the laboratory hallmark of aiha is a positive direct antiglobulin test (dat) although it has to be recognized that occasional cases of dat-negative aiha may occur. secondary causes for aiha such as an underlying autoimmune disorder, infections or malignancies should be considered as part of the investigation process for aiha. immunohaematology investigations for aiha should always include a dat using monospecific anti-igg and anti-c3d. on occasions, further testing with anti-iga or -igg subtypes may be necessary. while the dat provides information on bound rbc antibodies, the indirect antiglobulin test (iat) using screening and antibody identification cells will provide information on the specificity of circulating antibodies. often this will give a pan-reactive pattern although it is not unusual to identify coincident autoantibodies with defined red cell specificity. negative iat with screening and identification cells in the presence of a positive dat should raise suspicion of drug-induced aiha. in patients who may have been potentially alloimmunized, further procedures should be performed to exclude the coincident presence of alloantibodies as failure to recognize alloantibodies may result in an immediate or delayed haemolytic transfusion reaction if red cell transfusions are initiated. elution and auto-or alloadsorbtion techniques are useful to separate allo-and autoantibodies. complete red cell phenotyping should be concurrently performed to aid in identifying antibody specificities. heavily antibody-coated red cells may be difficult to phenotype with some anti-sera, in which case, molecular based red cell genotyping should be initiated. molecular based red cell typing is also particularly useful where the patient has recently been transfused and the initial red cell phenotype of the patient is not known. the primary management of the patient with aiha is amelioration of the underlying condition and suppression of immune mediated haemolysis. this is usually achieved with administration of steroids or immunosuppressive agents. splenectomy may be an effective second line treatment. rituximab is increasingly becoming a promising treatment option in patients who are steroid-refractory and not suitable for splenectomy. other treatment modalities for the refractory patient include intravenous immunoglobulin and danazol. red cell transfusions in patients with aiha should be undertaken carefully although they should never be denied blood transfusions because of inability to find compatible units. as far as possible, phenotype matched red cells, cross-match compatible with the patient's autoadsorbed serum should be transfused. if coincident alloantibodies are identified, antigen-negative red cells will need to be selected. clinical immunology and transfusion medicine, university & regional laboratories, lund, sweden what is the value of discussing case studies? they provide us with a forum for sharing our combined experience and permit the development of ideas and techniques as well as the possibility to see a given situation from another perspective. cases that illustrate the following will be discussed: (1) the presence of polyagglutination in a patient with a bacterial infection in europe and the usa, polyagglutination is rarely seen since monoclonal blood grouping reagents are the norm; however across asia, a broad spectrum of blood typing reagents are used and polyagglutination maybe encountered. (2) investigation of an antibody to a low-prevalence antigen in a case of haemolytic disease of the foetus and newborn antibodies to low-prevalence antigens occur not infrequently. how can they be investigated and what should be considered? (3) antibodies to a high-prevalence antigen the discussion will consider how to resolve such a case in laboratories with different levels of resources. what should be considered in different clinical situations? the goal is provide useful tips for investigation of difficult serological cases and information on how to proceed when the investigation is beyond the resources of the laboratory. haemovigilance is an important element of blood safety. it aims to identify, monitor and prevent adverse reactions, incidents and adverse events related to transfusion for both donors and patients (from 'vein to vein'). various local, regional and national haemovigilance models exist, which reflect the range of health systems and blood systems in different countries; for example, some haemovigilance systems are coordinated by professional bodies, some by blood suppliers, and some by health authorities (health departments or regulators). participation may be voluntary or mandatory, and may differ depending on whether all events or only serious ones are reportable. some systems capture events with all levels of imputability, whereas others record only those which are confirmed or highly probable. 'near miss' events are captured by some systems, and many valuable lessons can be learned from these cases. from an early stage of haemovigilance reporting it has been identified that processrelated problems are a major cause of serious transfusion complications. these include 'incorrect blood component transfused' events, where the blood component was intended for another recipient (frequently due to errors in patient identification at the time of collection of the pre-transfusion sample, or at the time of bedside administration), or did not meet the patient's special needs (such as a patient with a red cell antibody who did not receive the required antigen-negative unit). human and system factors, such as lack of awareness or training, working environment, interruptions and inadequate communications between clinical teams, or between the clinical teams and the transfusion laboratory, are very important contributors to these events. investigation of transfusion reactions, incidents and events at the hospital level is essential to identify clinical consequences, contributing factors and to develop and implement plans to prevent recurrence. reportable incidents should be notified to the haemovigilance programme. transfusion safety officers, transfusion nurses and similar roles have been introduced in many countries and they play important roles in haemovigilance, especially at the hospital level. adequate medical and transfusion laboratory support for hospital haemovigilance activities is also essential for success. hospital transfusion committees should oversee haemovigilance activities and reporting, and should ensure that hospital senior management is aware of and responds to serious reactions and events, especially where systems issues are identified to be contributory. at an international level, isbt's working party on haemovigilance brings together isbt members with an interest in haemovigilance. isbt works closely with ihn, an international collaboration of regional or national haemovigilance programmes, and other partners. ihn operates the istare database for international data sharing and benchmarking. haemovigilance reporting can identify priority areas for action (either where the events have serious clinical consequences, and/or occur frequently) and can help identify and monitor the implementation of solutions. an important feature of haemovigilance programs is the sharing of experiences and results. haemovigilance reports can both provide valuable feedback to clinical teams and hospitals locally, as well as share experiences nationally and internationally to improve patient outcomes. wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: haemovigilance systems capture data on adverse reactions and infections in recipients of blood transfusions as well as on errors and incidents in the transfusion chain. the objective is to analyse them and make recommendations for improving transfusion safety. many haemovigilance systems also collect data on complications in blood donors with a view to monitoring and improving blood donor safety. standardised definitions are necessary for classifying and comparing data in all these domains and at all levels. method: since 2004, at international meetings of blood transfusion professionals, members the international haemovigilance network (ihn) and the haemovigilance working party of the international society for blood transfusion (isbt) have collaborated in developing and validating definitions for non-infectious transfusion complications, errors and incidents in the transfusion chain as well as adverse reactions in blood donors. from 2012 contacts with other groups including the who have been put in place to ensure wide consultation as well as awareness and use of the definitions. results: standardised definitions have been published for recipient adverse reactions, for complications of blood donation and for a limited number of types of incident in the transfusion chain. the isbt haemovigilance working party and ihn are committed to ensuring that the definitions remain up-to-date and that revisions and improvements are conducted with wide consultation of professionals in relevant organisations worldwide. the haemovigilance working party and working party on transfusion-transmitted infections are collaborating on the development of definitions and criteria for assessing suspected transfusion-transmitted infections. conclusion: internationally agreed definitions are available for registration and surveillance of complications of blood donation and most types of adverse reaction in patients receiving blood transfusions. for errors and incidents in the transfusion chain, further work is necessary to improve comparability of data between hemovigilance systems. 1d-h7-03 distler pb and ashford p critical to patient safety is the capability of rapidly tracing a medical product of human origin (mpho) from donor to recipient and vice versa. traceability requires that each product be uniquely identified in order to provide a clear, unambiguous path. historically, uniqueness was defined only in the context of a single organization. for example, a blood product identifier was unique only to the blood bank that assigned it. because some medical products of human origin (mpho), especially cells and tissues, are frequently distributed across international borders, it is becoming increasingly important that identifiers of mpho need to be unique not only within an organization, but globally as well. in 2010, the world health assembly urged member states 'to encourage the implementation of globally consistent coding systems for human cells, tissues, and organs as such in order to facilitate national and international traceability of materials of human origin for transplantation.' more recently who has recognized the need for common strategies for global governance of all mhpo, including the global use of isbt 128, to ensure unique identification, optimal traceability, and interoperability between countries and across all mpho for both routine and emergency use. this requires a globally consistent coding system that can provide: a mechanism to allow distinct items to be uniquely identified and consistently characterized to all participants within the system, the means to allocate identifiers in a manner that avoids duplication, and the information infrastructure on which effective traceability can be built. isbt 128 is an international terminology, coding, and labelling system that supports the assignment of unique identifiers to support global traceability of mpho. it is currently in use by many blood banks, tissue banks, and cellular therapy facilities around the world and the who global forum on blood safety has recognized promotion of the use of isbt 128 as a priority for action in improving quality management and haemovigilance. 1d-h8-01 the university of tokyo, tokyo, japan antibodies directed to human platelet antigens (hpa) play important roles in the pathogenesis of neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). presently, six hpa biallelic systems, namely hpa-1 to -5 and -15, which are involved in immune mediated thrombocytopenia, are characterized. there are important ethnic differences in the frequency distribution of these hpa systems, the incompatibility of the hpa-1 system being the mostly involved in thrombocytopenic conditions in caucasian, whereas in japanese, the hpa-4 system is the mostly involved. the frequency distribution of hpa systems reported in other parts of asia seems to be different from caucasian as well as japanese, especially related to hpa-1 and -4, respectively. in addition to hpa, antibodies to human leukocyte antigen (hla), blood group abo, and human neutrophil antigens (hna) have also been shown to be involved in immune mediated thrombocytopenia. in fact, the majority of the cases of ptr are dependent on anti-hla antibodies, and anti-hpa antibodies comprise only a small proportion. the identification of the causative antibody is very important for the implementation of preventive/therapeutic measures for ptr, such as the selection of hla-and/or hpa-compatible platelets. on the other hand, the involvement of anti-hla antibodies in the pathogenesis of nait is questioned, but cases in which the causative antibody cannot be determined still remain relatively high. thus, for the implementation of preventive and therapeutic measures for the immune mediated thrombocytopenia, the detection and identification of the causative antibody is essential. the standard methods applied varies among the regions, the monoclonal antibody-specific immobilization of platelet antigens (maipa) and the platelet immunofluorescence test (pfit) being the preferred methods in the us and europe, whereas in japan, the mixed-passive hemagglutination is the most applied one. neither of the methods alone, however, is able to detect all the clinically significant antibodies, thus, improvement of the available methods as well as the development of new technologies is required. considering the ethnical differences of the hpa frequency distribution, we considered important to develop the research of this field also in asia, and for this purpose, the isbt platelet immunobiology working party, asia regional (isbt piwp-ar) was launched in 2010, during the xxxist international congress of the isbt in berlin, which aims the sharing of knowledge and improvement of technology in asia. a training course on platelet immunology methods and genotyping was provided in tokyo in 2010, and the first workshop of the piwp-ar was organized in taipei in 2011. in may 2013, the second training course on platelet immunology methods and genotyping was organized in guangzhou, china, and the 2nd workshop of the piwp-ar is going to be held in kuala lumpur, malaysia, during the 24th regional congress of the isbt. the presently available methods for the antigen/antibody testing, the problems related with antibody detection, and the activities of the platelet working party will be presented. nanning institute of transfusion medicine, nanning, china background: immunization against human platelet antigens (hpa) is associated with a number of clinical syndromes, including neonatal alloimmune thrombocytopenia (nait), platelet transfusion refractoriness (ptr), post-transfusion purpura (ptp), and other platelet immune disorders. the detection and identification of the clinical relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia. aims: the aims of this study is to investigate the characteristic of the detection of clinical relevant platelet antibodies in the asian population, and to evaluate the ability for the detection and identification of platelet antibodies in the platelet immunology laboratories in asian countries. methods: total of 378 cases that were diagnosed and studied as naitp (70), ptr (305) and ptp (3) in asian platelet immunology laboratories were reviewed. of the 378 cases, 167 were japanese in japan, 142 were chinese in china, 30 were in india, 24 were chinese in taiwan, china, 5 were in south korea, 4 were in thailand, 3 were in kuwait, and 1 case each for oman, lebanese and palestinian. the specificities of platelet antibodies in these cases were investigated and compared with the data from western country's laboratories. the methodology of detection and identification of antiplatelet antibodies in asian labs were also reviewed. results: among 378 cases, the immune thrombocytopenia associated antiplatelet antibodies were two cases of anti-hpa-1(in kuwait), 2 of anti-hpa-2 (1 in china and 1 in japan), 5 of anti-hpa-3 (three in japan, one in china and one in taiwan, china), 5 of anti-hpa-4 (all in japan), 4 of anti-hpa-5 (1 in china and 3 in japan), 1 of anti-hpa-7new(antihit a )(in japan), 1 of anti-hpa-15 (in japan), 2 of anti-hpa-21bw (in japan), 309 of anti-hla(101 in china, 147 in japan, 5 in korea,3 in thailand, 23 in taiwan, china and 30 in india), 1 of anti-a (in japan) as well as 15 cases of anti-cd36(nak a ) isoantibody (eight in china, three in japan and one case each in thailand, oman, lebanese and palestinian). thirty one cases of antibodies could not find the specificities (30 in china and 1 in kuwait). the methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (maipa), mixed passive hemagglutination (mpha) assay, platelet immunofluorescence test (pift), modified antigen capture elisa (mace), and solid phase red cell adherence (spaa) have been used in asian laboratories summary/conclusions: platelet alloantibodies are found with variable frequencies in different ethnic groups in asian population. anti-hpa-4 is mainly found in japanese individuals, while anti-cd36 (nak a ) isoantibody that occurred in cd36 type i deficient individuals is most frequent found in asian population especially in chinese population. only two cases of anti-hpa-1 antibodies were found in asian population (all in kuwait), while anti-hpa-1 is the most frequent antibody associated with severe complications in caucasian populations. however, anti-cd36 isoantibody is of a risk factor of immune thrombocytopenia in asian population, as the incidence of cd36 deficiency is 3-11%. the human platelet antigens (hpa) are a group of polymorphic antigens, expressed relatively specific on platelets, capable of eliciting an immunological response with development of alloantibodies. hpa directed alloantibodies have been implicated in neonatal alloimmune thrombocytopenia (nait), post-transfusion purpura and refractoriness to platelet transfusions. twenty-seven hpa are recognized, of which antibodies to both the given alloantigen and the antithetical alloantigen has been reported for hpa-1 to 5 and hpa-15. the remaining hpa are designated with a 'w' as an alloantibody to the antithetical antigen has yet to be reported. most hpa polymorphisms are a result of a mis-sense single nucleotide alteration and are readily detectable using standard molecular typing methods. dna based population wide genotyping have revealed considerable variation in hpa allele frequencies among different ethnic groups. the 'b' forms of hpa-1, -2, -3, and -5 are common among caucasians while hpa-4b is extremely rare. in the chinese however, hpa-1b is extremely rare while uncommon in malays. hpa-4b and hpa-21bw meanwhile are more commonly seen among asians compared to caucasians. consequent to this, alloanti-hpa-4b is the most common cause for nait in the asian population as compared to alloanti-hpa-1a among caucasians. several cases of nait due to anti-hpa-21bw have also been reported in asia. in contrast, nait due to anti-hpa-6b is rare despite the alloantigen being more common among asians. although platelet transfusion refractoriness is more commonly associated with hla antibodies, anti-hpa antibodies have been implicated in some cases. management of nait and platelet transfusion refractoriness include the supply of antigen negative platelet units. a platelet donor registry with a critical mass of hla and hpa typed blood donors is therefore necessary for effective management of these conditions. ready availability of low-cost high-throughput snp genotyping platforms allow for establishment of large hpa genotyped donor pools. knowledge of hpa genotype prevalence in the local population as well as its implications on nait and platelet refractoriness is however crucial before deciding on donor screening strategies, careful considerations would also need to be made with regards to the cost-effectiveness of such ventures in light of alternative management strategies and local incidence rates of nait. clinical -improving patient outcomes 2a-s01-01 setting up a patient blood management programme wood e 1 , engelbrecht s 1,2 and robinson k 2 1 transfusion research unit, monash university, melbourne, australia 2 australian red cross blood service, adelaide, australia patient blood management (pbm) aims to minimise unnecessary transfusions, and also to ensure that if transfusion is required it is managed appropriatelyby individualising care so that patients receive what they need when they need it. pbm is comprehensive and patient-centred, with active participation by patients and a multidisciplinary approach from the hospital team to achieving these aims. 'three pillars' of pbm have been suggested, to optimise a patient's red cell mass, reduce bleeding and improve tolerance of anaemia. in the perioperative setting, important elements of pbm include attention to medical, surgical and anaesthetic interventions and techniques to improve haemostasis and reduce blood loss. where significant intraoperative blood loss is anticipated, use of cell salvage techniques can be very valuable. pbm concepts also apply outside the perioperative setting, and the broad principles can be applied to a wide range of clinical settings, including obstetrics, trauma, critical care and haematology/oncology and other medical settings (e.g. gastroenterology). an effective hospital pbm programme requires planning and communication, with a stepby-step approach to implementation, identifying priority areas for action, engagement, education and training of staff, and on-going monitoring against plans to demonstrate progress and identify areas for improvement. feedback to staff on progress provides a sense of achievement and helps engagement. adequate resources are required, including from medical, nursing and laboratory staff from a range of disciplines who have important contributions to make in clinical practice, education and training, and audit and review. minimisation of unnecessary transfusions saves money for hospitals and the community, and other resources such as staff time, and therefore offsets the costs of establishing and maintaining a pbm programme. effective implementation requires change at individual and organizational levels, and therefore support of hospital executive management, local health authorities and the blood supplier are also very valuable. oversight of the program can be by the hospital transfusion committee or a pbm programme committee, but the particular structure and governance arrangements should be developed to suit local needs. general practitioners can play key roles in preparing patients for surgery by identification and management of anaemia, as well as other pbm activities. ultimately an effective pbm programme can optimise care and outcomes for patients, make better use of limited and precious blood supplies, and reduce risks and costs. trauma is a leading cause of death around the world, with haemorrhage accounting for more than a third of the preventable mortality, with the majority of these deaths occurring within the initial 24 h. massive blood transfusion is generally defined as the replacement bytransfusion of more than 10 units of red cells over 24 h. massive transfusionprotocols (mtps) have evolved over the past decade and are especially importantin facilitating the early delivery of copious amounts of blood products topatients who have major injuries and severe haemorrhage. various studies havealso demonstrated that with mtps, there is a more efficient use and lesswastage of blood products. however, for trauma patients, stopping thehaemorrhage and resuscitating the patient does not only involve the expedientdelivery of red cells to the injured patient. mtps have also emphasized theneed for a more balanced ratio of delivery of blood products, includingplatelets and plasma, to patients who sustain massive blood loss and havedeveloped acute traumatic coagulopathy (atc). often times, mtps also stress theimportance of the consideration of use of haemostatic adjuncts, such astranexamic acid, activated factor vii, level one transfusion units and the useof blood warmers to reverse the potential effects of hypothermia with sustainedtransfusions of large amounts of blood products. ultimately, in tandem withdamage control resuscitation, which allows permissive hypotension whilstsecuring haemostasis, mtps have been shown to also improve survival of theseseverely injured patients. a more recent paper describes the development of amassive haemorrhage protocol to aid in the identification of patients who wouldbenefit from the mtp and this may be the next step in the evolution of a workprocess by which resuscitation for severely injured patients may be optimized. background: rh blood group antigens are highly immunogenic and transfusion of rh d positive blood in rh d negative recipients is avoided. platelets do not express rh antigens, however they may contain significant amount of contaminating red cells to illicit an immune response in the patient. due to limited shelf life of platelets and inventory issues, rh d positive platelets which are not visibly contaminated with red blood cells maybe transfused to rh d negative patients including children and females of child bearing age. there has been increased focus on whether rh immunoglobulin should be routinely administered after such transfusions. in saudi arabia no clear guidelines exist on the administration of rh immunoglobulin after d positive transfusions in d negative patients. aim: the purpose of this study was to determine whether the transfusion of rh d positive platelets to patients who are rh d negative, results in d alloimmunization and whether rh immunoglobulin should be routinely administered in such patients. methods: eligibility criteria for inclusion in the study included the following: transfusion of rh d positive platelets, no anti d detectable before transfusion, no previous exposure to rh d positive blood components, and results of follow-up testing of anti-d in patients serum available. the patients blood group and antibody screening was done using liss-iat gel technology (diamed). results: one hundred rh d negative patients who received rh d positive blood components were identified. out of this 63 (63%) patients received rh d positive platelet transfusions. in 47 (74%) patients out of this, the results for post transfusion antibody screening were available. the mean age of the patients was 46.5 years and included 30 males and 17 females. average number of rh d positive platelets transfused per patient was 11.3 units and total number of platelet units transfused was 523. anti d was detectable in 4 (8.5%) patients post transfusion and included one male and three female patients. of the female patients one was a 3 year old child who received 50 units of random donor platelets and second a 21 year old women who presented in the emergency department as a case of trauma. the third female was 54 year old post-menopausal woman. conclusion: we conclude that the chances of developing d alloimmunization after receiving rh d positive platelets is generally low. however keeping in view the antenatal complications which can arise in the future in females due to this alloimmunization, it is highly recommended that rh immunoglobulin be administered to all rh d negative women in the reproductive age group and female children who receive rh d positive platelets. background: hemovigilance is a quality process which takes into account all the activities of a blood transfusion chain with the aim to improve quality and enhance safety of blood transfusion. we have implemented a transfusion feedback reporting mechanism in our hospital as a part of hemovigilance. the current study aims to collect and analyze this data to improve our transfusion system. aim: to systematically analyze the transfusion process from issue of blood components to completion of the transfusion material and methods: the transfusion feedback forms received back at the transfusion medicine department during a 3 months period were systematically analyzed for documentation related to patient identification, product identification, documentation, completeness, etc. results were analyzed statistically for specific co-relation with patient's location, time of transfusion and type of component transfused. results: of 3474 blood components were issued during the study period. transfusion feedback form were received for 2000 (57.5%) blood components, as follows: prbc-800 (40%), platelets-651 (32.5%), ffp -441 (20.7%). patient identification number /wristband check was not done in 25 (1.25%) cases. pre-transfusion verification of blood group, patients name and patient's identification number was done in 1963 (98.15%) cases. cross checking of component unit with request form was missed in 16 (0.8) cases. pre-transfusion and post-transfusion monitoring of blood pressure was documented in 698 (34.9%) episodes, monitoring of pulse in 696 (34.8%) episodes and patient's temperature was monitored in 681 (34%) episodes. signature of nurse was missing in 86 (4.3%) and that of medical officer in 11 (05%) of the form. adverse transfusion reaction was documented in 1/2000 forms, whereas the transfusion reactions notified at the blood bank during the same period were 2. of 553 (27.65%) transfusions were carried out during non routine work hours. the mean time between the issue of the components and start of transfusion was 28 min for rbc components, 21 min for platelets and 16 min for ffp. patient identification and monitoring and product identification related non-compliances significantly correlated with out of routine transfusions (p = 0.002). conclusion: though overall compliance with established procedure for transfusion was evident, activities related to unit identification, patient monitoring and identification and reporting of adverse reactions were not well documented. this emphasizes the need for ongoing training of nursing staff and medical doctors in safe blood administration practices. introduction and method: matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (maldi-tof ms) is an ideal tool for high-throughput blood group genotyping. using this technique, this swiss (bts zurich) -german (sequenom gmbh, hamburg) cooperation aims to genotype swiss blood donors for blood groups rh, kell, kidd, duffy, mnss (n = 6000), hpa and hna (n = 3000) and high frequency antigens (hfa, n = 36,000) within 3 years. specificities are grouped into single multiplex reactions (mpx, n = 10) with up to 17 snps tested simultaneously in one tube. results: using the kel-jk-fy mpx, more than 4000 donor dnas have been tested and compared to serological pre-values. concordance between geno-and phenotypes reached 100% for k/k, 99.98 for kp a 99.93% for jkand 99.23% for fy a/b/x/0 . only one discrepancy each for kp, jk and fy could be attributed to genetics, the others were erroneous serotypes revealed by genotyping. genetic discrepancies were three new variants: kel*02.03(r700g)nulla kp a relative, jk*b(mutation n.d.) and fy*b (g261r)null. serology for js a/b of some few kel*02.06 positives confirmed validity of genotyping. numbers of detected known kel*mod and null, jk*null and fy*null (gata) alleles were 3, 2 and 51, respectively. call failures (no result) were observed in less than 2% of all mpxs. genotyping for rhd, rhce (5 mpx), gypa and b (mnss, 1 mpx) on more than 4000 samples, delivered results with discrepancy rates for mnss comparable to above, and better rates for rhdce. call failure were at approximately 2% again. reproducibility, robustness and analytical accuracy of the technique allowed measurement of gene copy numbers with relevance for rhd zygosity estimation and detection of the gypb deletion in u negatives. rhd category, partial, weak, del and null alleles and the genetic correspondents of vw, mg, mi(a), he, and uvar were observed. genotyping for hpa and hna (1 mpx) showed expected results among 2300 samples with the exception of hna-1a, b and c, where frequent duplications and deletions of fcgriiib pose difficulties for all genotyping approaches in general. in hfa ('rare') genotyping (2 mpx), currently, more than 13,000 blood donors were analyzed, and delivered: 29 kk, 2 kp(a+bã�), 48 js(a+bã�), 41 kel11+kel17+, 20 lu(a+bã�), 3 lu14+lu08ã�, 52 yt(aã�b+), 17 co(aã�b+), 11 kn (aã�b+), 98 lw(a+b+), and >10 others. specificities for vel negativity and scianna have been included into hfa typing, recently. conclusion: analysis for kell, kidd and duffy showed that genotyping worked qualitatively better and to costs comparable to serology. consequently, genotyping kell, kidd and duffy instead of routinely performing a second round of serotyping as mandatory for donors in switzerland, is recommended. ahead of comparable suggestions with regard to rh and mnss, a more detailed statistical analysis of existing raw data is needed. however, genetically identified donors with rare blood phenotypes, e.g. such as yt(aã�b+), are already selected for respective transfusions and are a strong indicator for the value of the presented project. the serological data suggest that retention of the 208-210 tcc (51s in glycophorin bs) codon from the gypb pseudoexon, prior to the gene conversion insertion of gypa sequence and coding for serine in the hybrid glycophorin, is the basis of 'anek-like' activity for both hybrid glycophorins. it should be considered that these antisera react with a new kipp-related mns system antigen. genetic studies revealed the same crossover for gp.kipp and gp.yak that have been independently reported in conference abstracts, suggesting that these are in fact the same hybrid glycophorin. background: with the increasing knowledge of the genetics of blood group antigens, molecular immunohaematology is gaining popularity. molecular immunohaematology refers to the use of genotyping to encode red blood cell antigens, representing an indirect method used to predict one's phenotype. there are certain advantages of genotyping, namely, typing of red blood cells with autoantibodies, prenatal testing and patients with multiple transfusions. molecular methods are also useful in phenotyping donors as it enables the prediction of numerous phenotypes in one single test. however, there are several misgivings about the cost of molecular methods used to genotype antigens. aim: to evaluate and compare the cost of conventional serological phenotyping and molecular genotyping. methods: a batch of 30 donor samples was typed using both serological and molecular methods. the test kit used for molecular methods, from gen-probe, included the typing for the following antigens -kell (k, k, kpa, kpb, kpc, jsa and jsb), kidd -(jka, jkb, jk), duffy (fya, fyb, fyx, fygatasil) , mns (m, n, s, s, s-s-uvar), rh (c, c, e, e) and dombrock (doa, dob). negatives that are obtained using this method were confirmed using serology. serological typing was performed with available antisera according to the various manufacturers' instructions. this includes kell (k, k), kidd (jka, jkb), duffy (fya, fyb), mns (m, n, s, s) and rh (c, c, e, e). the cost for the different tests were tabulated and compared. the cost includes labour, consumables and equipments. results: the results show that molecular method of typing red blood cells is more expensive than the traditional serological method. the cost of consumables is comparable for both methods. the consumables make up about 80% of the total cost for serological methods, and about 82% for molecular methods. the cost of labour is about 9% for serological methods and <1% for molecular. equipment cost contributes to <1% of the cost using serological methods and about 14% using molecular methods. conclusion: molecular methods may seem more expensive, about two times the cost of serology but results are obtained faster and are less labour intensive which could prove to be an advantage when phenotyping samples in large quantities, especially for donor phenotyping. molecular immunohaematology is a relatively new process, thus kits for these methods are expensive at the moment. as development and adoption of genotyping progresses, consumables and equipment costs are expected to become more affordable. serological methods do have their limitations even for patient testing, especially for patients with autoantibodies and patients who have had multiple transfusions. molecular methods may serve useful in overcoming these limitations. background: transfusion dependant patients often develop multiple antibodies to red cell antigens on exposure to red cells and require antigen-negative red blood cells for further transfusions. finding suitable red cell units for such patients is often difficult and time consuming, requiring phenotyping of large numbers of red cell units in inventory. the increasing cost and scarcity of anti-sera also makes this an expensive exercise. aim: in order to improve provision of phenotype-matched blood for such patients, we studied the feasibility of large-scale snp-based genotyping of common blood groups for establishment of an antigen-negative red blood cell inventory. methods: genomic dna was extracted from donor blood samples using an automated platform. samples were subjected to snp-typing for common local polymorphisms of rhce (ccee), fy (fy a /fy b ), jk (jk a /jk b ), mns (s/s) and co (co a /co b ) using taqman 㢠snp genotyping assays in 384-well plates at 5 ll final reaction volume, on a lightcy-clerii 480 real-time instrument. identification of the cc polymorphism was by probes targeted to the 48c>g and 307t>c of the rhce allele while probes targeted to 676c>g was utilised for ee detection. for the remaining alleles, probes were designed only to detect the most common mutation for the polymorphism within an east-asian population. results were analysed and integrated into the blood donor software system. results: the 48c>g probe designed for identification of c was unsuccessful with poor discrimination between genotype calls. the remaining probes showed satisfactory discrimination between genotypes, with 462 of the 505 (91%) samples analysed, fully genotyped for the five alleles studied. relatively rare genotypes were successfully identified using this strategy: ccee (15/505, 3.0%), fya-/fyb+ (19/505, 3.8%), ss (5/505, 1.0%). the co2 allele responsible for co b was identified in the heterozygous state in four of 494 evaluable samples giving an allele frequency of 0.4% in our population. the estimated reagent and consumables cost for sample dna preparation and snp testing was usd12.00 compared to usd27.15 for phenotyping using commercial anti-sera. we did not factor in the cost of capital acquisition of instruments for genotyping as we used facilities which were common for other molecular tests in the hospital. conclusions: results of this study indicate that snp genotyping would be a costeffective strategy for screening and establishment of an antigen-negative red cell inventory and genotyped whole blood donor pool. the cost of genotype screening was effectively reduced by use of small final reaction volume and extensive use of automation. in addition, genotyping allowed us to identify local prevalence of blood groups which were previously unidentifiable due to lack of commercially available anti-sera. background: over the past 20 years the molecular, biochemical and serological basis of almost all blood groups have been determined, highlighting the different frequencies of antigen, related to different ethnic groups. since october 2012, in our transfusion center (asl caserta) in order to have a blood bank that ensures the different transfusion needs, extended erythrocyte typing is practiced on periodic donors with specific features such as age, group and rh phenotype. aims: the aim of our study was to test the validity of the molecular method of erthrocyte phenotype typing with the serological technique by comparing the results obtained by each procedure. we also compared each method in regards to reliability, ease of use and reproducibility of results. methods: we selected 250 donors aged <45 years old, group o and a, rh homozygous phenotype. samples from each patient were tested by serological typing in solid phase (capture r immucor) and then dna was extracted and each sample was tested by molecular typing in microarray (bioarray solutions immucor) results: see table 1 . table 1 : summary/conclusions: the results show that the frequencies of more immunogenic antigens (k, fya, jka) reflect the specific and ethnic frequencies of the donors. we emphasise the absence of donors having an antigenic structure that is defined as rare (that is present with a frequency of 1:1000 according to american rare donor program (ardp), council of europe, international donor panel (idp), international society of blood transfusion (isbt), council of europe, japanese red cross). we only found one case of a donor expressing weak fyb which is due to mutation in fy 265c> t. the results were confirmed in 99.6% of cases by serological technique. however for the phenotype fyb weak, the result was discordant in serology, where the result was fyb-. in conclusion we can confirm the validity and the need to use both techniques in order to obtain a reliable and reproducible result. the molecular technique is able to identify mutations in particular genes, especially in specificity whilst the serological technique excels in sensitivity and speed but fails to confirm the data obtained. background: pathogen inactivation of blood components promises a new layer of transfusion safety, enhancing existing strategies and providing proactive protection against emerging infectious risks. processes for plasma and platelets are well established in many blood centers and those for red cells and whole blood are in development. clinical acceptance of new technologies depends on the demonstration of product safety, a minimal effect on product efficacy both in vitro and in vivo, and the range and degree of pathogens inactivated. aims: to review the major processes of pathogen inactivation of plasma and cellular blood components with a focus on the range and degree of inactivation of relevant pathogens, especially with regards to platelet pathogen inactivation. methods: this review will use recent reports and unpublished data to describe our current understanding of the potential efficacy of pathogen inactivation in improving transfusion safety. results: the major indication for platelet pathogen inactivation is bacterial contamination, a persistent problem despite multiple innovations to minimize and detect contamination. pathogen inactivation processes need to cover a wide range of possible bacterial concentrations and species. there have been few published reports using the current commercially available systems: optimized in vitro testing documents the ability of the terumo bct mirasol tm , cerus intercept tm and macopharma theraflex tm uv systems to effect a 10 2 ->10 6 log reduction of various bacterial species. most systems are less effective at inactivating bacterial spores, a particular problem as bacillus spp. is common platelet contaminant. testing the level of pathogen inactivation under clinical conditions at very low and high concentrations of bacteria reveals further weaknesses in pathogen inactivation strategies. conclusions: clinical trials of pathogen inactivated platelets have focused on the safety and clinical efficacy of pathogen inactivated treated platelets, but little has been reported on the efficacy of pathogen inactivation to reduce the risk of infection transmission. blood centers should focus on this aspect of efficacy as they decide whether to implement, and to favor those commercially available systems that best meets the clinical need for pathogen protection. for pathogen inactivation (pi) using amotosalen and uva light induces the formation of covalent adducts and interstrand crosslinks between amotosalen and nucleic acids, thus preventing dna replication and rna translation. pi technology has been adopted into routine use in some european countries and is under fda review for licensure in the us. current documentation of pi efficacy relies on illuminator sensors that measure the uva light dose delivered. an indirect methodology is utilized for the validation and qc of the process in some centers that measures the amount of amotosalen consumed during illumination in platelets and before removal with the compound removal device. the% amotosalen remaining is a direct measurement of the uva light delivered and photochemical conversion of amotosalen. however, a functional qc method that measures a direct target within the treated blood product has not been introduced into clinical use. residual leukocytes, platelets and potentially plasma contain mitochondrial dna (mtdna) which is a collateral target of the pi process. aims: the goal of this study was to quantify the impact of intercept treatment on platelet-derived mtdna by real-time pcr. methods: to evaluate the feasibility of detecting pi-induced mtdna modifications by real-time pcr, we spiked purified human leukocyte dna into human plasma, 35% plasma/65% intersol, or pbs. each sample (3.6 ml) was treated with 150 lm amotosalen and 3 j/cm 2 uva (n = 2). control samples were either untreated or treated in the absence of amotosalen or uva. dna was extracted from each sample (0.2 ml) in duplicate and assessed by measuring the inhibition of real-time pcr amplification in duplicate wells using mtdna-specific primers and sybr greenbased detection. amplification of sequences ranging in size from 73 to 1065 bp was evaluated over 45 cycles of amplification. subsequent to the initial feasibility tests, a pilot validation was performed by treatment of platelets in 35% plasma/65% inter-sol (30 ml). six different platelet units were tested as outlined above for inhibition of amplification of endogenous mtdna sequences. results: treatment of purified dna with amotosalen plus uva resulted in 1.3 log to >6.0 log inhibition of pcr amplification (results shown in table) . the extent of pcr reduction roughly correlated with the size of the amplicon, as well as with the type of diluent in the following order: pbs > 35% plasma > 100% plasma. exposure of platelets to amotosalen and uva showed an average of 2.5 log to 3.6 log reduction in pcr signal, with increasing inhibition observed for larger amplicons. in all cases, no pcr inhibition was observed in the absence of amotosalen and/or uva. conclusions: a quantitative real-time pcr assay specific for mtdna is capable of documenting pi-induced collateral nucleic acid modification in platelets. based on this work, this assay can be developed further for use as a quality control method for pi efficacy. background and aims: pathogen reduction technology (prt) provides a proactive approach to improving transfusion safety for platelet concentrates (pcs). however, prt treatment is known to exacerbate the effects of the platelet storage lesion, leading to increased platelet activation and secretion of immunomodulatory factors. little is known regarding how prt-treated platelets may affect the cells of the recipient's immune system once transfused; therefore the aim of this study was to examine the cytokine responses of a recipient's inflammatory cells after exposure to prt-treated platelets using an in vitro whole blood model of transfusion. methods: two abo/rhd matched buffy coat derived pcs were pooled and split to form matched pairs on day-1 post-collection (n = 11). one unit was treated with the mirasol prt tm system (terumo bct), while the other unit remained as an untreated control. all units were stored at 22â°c with agitation and samples were taken on day 2 and 7 post-collection for 'in vitro transfusion' experiments. to represent a transfusion in vitro, 10% v/v platelets were incubated with abo/rhd-matched fresh whole blood, with or without lipopolysaccharide (lps; 1 mg/ml) for 6 h at 37â°c/5% co 2 . protein secretion was inhibited using brefeldin (2 lg/ml) to allow detection of intra-cellular cytokine production in monocytes (cd45 + /cd14 + ) and neutrophils (cd45 + / cd16 + ), using multi-colour flow cytometry. the fold change of cytokine production was calculated by comparison to a 'no-transfusion control' (whole blood without addition of platelets). data was analysed using a one way anova with post-hoc tests for pair-wise comparisons. results: in the absence of lps, both prt-treated and untreated platelets stored for 7 days significantly increased monocyte mip-1b expression (1.5-fold; p = 0.047), whereas exposure to day 2 platelets did not result in any significant differences in mip-1b expression. as expected, lps stimulation significantly increased monocyte production of both il-12 (5.0-to 7.4-fold) and mip-1b (1.8-to 2.4-fold). however, lps-induced monocyte il-12 production was significantly reduced by exposure to prt-treated or untreated platelets stored for 2 days (prt-treated: 3.0-fold, untreated: 2.5-fold; p < 0.0001) and 7 days (prt-treated: 4-fold, untreated: 2.1-fold; p < 0.0001). furthermore, il-12 production was significantly lower following exposure to day 7 prt-treated platelets compared to untreated platelets (p = 0.006); however there was no significant difference following exposure to day 2 platelets. lps-induced mip-1b production was not significantly differentfollowing exposure to either day 2 or day 7prt-treated or untreated platelets. exposure to platelets (untreated or prt-treated) did not significantly modulate monocyte production of ip-10, mcp-1, mip-1a, il-1a, il-1b, il-6, il-8, il-10, or tnf-a. co-culture of platelets and whole blood did not result in any significant changes to cytokine expression in neutrophils. conclusion: using an in vitro whole blood transfusion model, we have demonstrated that exposure to prt-treated platelets stored for 7 days results in significant changes in the il-12 production by monocytes. these changes may reflect the way prt-treated platelets interact with immune cells upon transfusion. therefore, the effect of stored prt-treated platelets, especially in recipients with underlying inflammation, should be further examined. backgrounds: hepatitis c virus (hcv) infection is one of the major causes of chronic hepatitic disease. hcv has six genotypes and more than 80 subtypes. the epidemiology of hcv subtypes vary with different geographic distribution. understand the subtypes prevalent in certain area will help to understand the transmission modes and the spreading trend of hcv and thus help to make effective precautionary measures. hcv subtypes are also closely related with clinical therapeutic effect. it is important for guiding clinical therapy and prognosis and predicting the possible burden of hcv infection and treatment in the future. aims: to investigate and compare the prevalence of hcv subtypes in clinical patients and blood donors in guangdong china. methods: of 191 samples ofclinical patients and 222 samples of blood donors whose hcv rna were positive were collected from guangdong province. hcv ns5b gene was amplified by rt-nested pcr and then sequenced. hcv subtypes were assigned by constructing phylogenetic trees with mega5 software. moreover, spss16.0 software was applied to compare the difference between these two groups. results: of 191 clinical patients, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 2 (1.05%), 127 (66.49%), 17 (8.90%), 5 (2.62%), 5 (2.62%), 34 (17.80%) and 1 (0.52%), respectively. of 222 blood donors, hcv genotype 1a, 1b, 2a, 3a,3b, 6a and 6n was 1 (0.45%), 92 (41.44%), 1 (6.76%), 19 (8.56%), 11 (4.95%), 83 (37.39%) and 1 (0.45%), respectively. the proportion of hcv 1b was higher in clinical patients than in blood donors(v 2 = 25.866, p = 3.66e-07), while the proportion of 3a and 6a subtypes were higher in blood donors than in clinical patients(v 2 = 6.602, p = 0.010; v 2 = 19.398, p = 1.06e-05). one possible cause was the transmission modes varied with different hcv subtypes. hcv 1b is more related with blood transfusion while 3a and 6a are more relevant to intravenous drug abuse and sexual behavior. with the anti-hcv screen implemented from 1993, the risk of hcv infection by transfusion is diminishing. the other reason was the average time from hcv infection to serious pathological lesions is about 20 years and hcv 6a was transmitted into guangdong later than 1b. conclusions: in guangdong province, hcv 1b and 6a were the predominant subtypes in clinical patients and blood donors. the proportion of hcv 1b, 3a and 6a subtypes were significantly different between clinical patients and blood donors. the reason may relate with the time of hcv transmission into guangdong area and the hcv propagation modes. blood safety and increased public health initiatives to reduce hcv infection from those in these high risk groups to the general population remain a priority. background: in japan, we routinely evaluate the presence of transmission of hbv, hcv and hiv in all transfused patients at three months after the last transfusion. although the sensitivity of detection of hepatitis b virus (hbv) in blood donation improved during recent years, transfusion transmitted hbv infection is left as a serious problem. the japanese society of transfusion medicine and cellular therapy (jstmct) conducts the nationwide questionnaire survey about the clinical blood transfusion activities, supported by ministry of health, labour, and welfare, every year. in this survey, we can collect detailed characteristics of patients, who showed a positive result of post-transfusion hbv-marker-test. aims: the aim of this study is to elucidate the cause of hbv positive in transfused patients. materials and methods: data concerning to transfused patients with positive hbvmarker were collected using results of the nationwide questionnaire survey in 2007-2011. in this questionnaire, if there is a patient showing a positive result of hbsag and/or hbvdna evaluated by post-transfusion-test, it is requested that detailed patient's characteristics, including a total amount of transfusion, disease (hematologic or non-hematologic), therapeutic methods (surgery, use of anticancer drugs, use of immunosuppressive drugs, use of molecular target drugs, blood stem cell transplantation), and results of hbv-marker-test prior to the first transfusion, should report to the jstmct office. a number of hbsag and/or hbvdna positive patients were 234 cases in 2007-2011. among them, 19 patients were not eligible because of the absence of results of hbv-related markers. finally, a total number of 215 patients (36, 37, 41, 43, 45 patients in 2007, 2008, 2009, 2010, 2011, respectively) were enrolled for the present study. results: all the eligible patients showed positive results of hbsag and/or hbvdna in samples obtained from three months after the last transfusion. we classified the cause of these results into five categories according to results of hbv-markers tests prior to the first transfusion. (i) if a result of hbsag and/or hbvdna performed prior to transfusion is positive, a patient is categorized as the hbv carrier group. (ii) a patient showing both a negative result of hbsag and positive results of anti-hbs and/or anti-hbc are categorized as the hbv past-infection group. (iii) if a patient shows no hbv-related marker prior to the first transfusion and patient's hbvdna is identical to donor's hbvdna, we categorized as the transfusion transmitted infections (tti) group. (iv) if a patient does not show any hbv-related marker prior to the first transfusion and hbvdna is not detected in donor's blood by single nat, we categorized as the unknown cause group. results were summarized in table 1 . conclusion: although the positive result of hbsag and/or hbvdna of transfused patients has been considered the sequel of blood transfusion, we showed that hbv reactivation was also an important cause of it. to distinguish hbv reactivation from transfusion transmitted infection, we have to perform hbv-marker-test prior to the first transfusion, or we should freeze pre-transfusion patient's serum. plenary session: it's all about red cells 2b-pl1 the english dictionary provides several definitions for the word 'myth'. one definition is, 'a widely held but false belief'. this seems suitable for the purposes of this presentation. but who decides what is false? as we shall see in the course of this presentation, some 'myths' of blood groups may not be myths at all, and some established facts may indeed be myths. perhaps a more scientific definition would be, 'a widely held but unproven belief'. abo, the original and most important blood group in transfusion and transplantation medicine, has engendered many 'myths'. these have mostly arisen through associations between abo groups and other characteristics such as personality, dis-ease, psychological traits, and ideal diet. although many may indeed be myths, or even fabrications advanced for political reasons or financial gain, some are not. for example, the statistical associations between abo type and thrombosis, where a biochemical basis involving clearance of von willebrand factor from the blood by enzymatic cleavage appears to be affected by abh glycosylation. in rh, the first myth was that the human antibody, now called anti-d, was the same as the antibodies made by immunising rabbits with rhesus monkey red cells. hence the name rhesus, now rh, for the blood group system. in the 1940s, early serological work with rh antibodies led to two genetic theories, involving either one or three rh genes. both theories have now been rejected because molecular genetics revealed two rh genes. but was the three-gene theory really wrong, when the boundaries of genes were not understood at the time? since the 1960s it has been commonly understood that there are two types of variant d antigens: weak d and partial d. policies for transfusing patients with these variants have often been based on this dichotomy. but is this a myth? the difficulty we have in defining the terms weak d and partial d suggests that it might be. it is always tempting to dismiss anything that we don't agree with as a myth. as wiener, one of the discoverers of the 'rhesus' antigen, wrote several papers on 'blood group mythology', doing just that. scientists should beware of falling into this trap. perhaps 'myth' is a term best avoided in science. five new blood groups -what next? the past 2 years has seen the discovery of five new blood groups. at the isbt meeting in 2012, fors, jr and lan were ratified as blood group systems and since then, the molecular basis of the vel blood group antigen has been elucidated, and the complement regulator protein, cd59 has been shown itself to be a blood group antigen. these last two discoveries will no doubt lead to their elevation to blood group systems. how has this happened? it turns out to be a mixture of old and new techniques. rapid advances in molecular biology and in our understanding of the human genome have opened new fields of discovery within human blood groups. the development of comprehensive snp arrays, exome sequencing and rapid sequencing techniques, e.g. next-generation sequencing, has provide us with tools for rapid discovery. combining these with sophisticated algorithms for database mining has resulted in the identification of the molecular bases behind the hitherto unresolved, clinically relevant blood group antigens jr a and vel. however classic biochemistry and subsequent peptide and dnasequencing still play an important role and lie behind the (simultaneous) discoveries of jr a and vel, but also of lan and fors. a rare cd59-deficient patient produced an alloantibody to a high-prevalence antigen that was shown to be targeted at the cd59 protein, which was confirmed by routine dna-sequencing. the jr a and lan antigens were assigned to already well-investigated abc-transporter proteins (abcg2 and abcb6 respectively) whose presence on the red blood cell (rbc) had not been established previously and for which the function on rbcs is still not known. fors antigen was shown to result from the reactivation of the human pseudogene gbgt1. this enzyme builds the carbohydrate forssman antigen on sheep and dog rbcs but is normally inactive in humans. a mutation that reactivated the enzyme explained the unusual a pae phenotype in two families. vel was shown to be carried on a hitherto unknown protein on the rbc, smim1. the function of the protein remains unknown although the protein is highly conserved across all species, which is both intriguing and hints at a fundamental function. absence of cd59 whose function in complement regulation is well-understood, resulted in production of an alloanti-cd59, demonstrating immunogenicity of this protein for the first time. these simultaneous discoveries have shown that regardless of the route of identification, assignation of orphan antigens to their blood group 'home' continues at a rate unparalleled since the 1990s. as the '-omics' fields identify new erythroid genes and proteins, and next generation sequencing permits rapid genome sequencing of individuals, we can anticipate that many more currently unassigned antigens will find their genetic and molecular home. 2c-s04-01 teo d blood services group, health sciences authority, singapore, singapore an emerging infectious disease (eid) is defined as one that has appeared in a population for the first time, or that may have existed previously but is rapidly increasing in incidence or geographic range. in 1992, an institute of medicine report predicted continued emergence and re-emergence of microbial pathogens, facilitated by changes in human populations, environment and infectious agents. east and southeast asia, with 30% of global population, has a reputation as a hot spot for eid. within the region, the forces of rapid social, economic and environmental change have resulted in factors such as urbanization, deforestation, agricultural intensification, rapid population growth and mobility which contribute to the increased exposure and efficient transmission of new pathogens. the emergence of severe acute respiratory syndrome (sars) exactly 10 years ago has dramatically transformed individual and national awareness and capabilities for identifying and responding to regional eid threats. the re-emergence of highly pathogenic avian influenza a(h5n1) virus in 2004, isolation of novel bat-associated reoviruses in 2006, emergence of artemisinin-resistant malaria and discovery of a tick-borne bunyavirus associated with fever and thrombocytopenia in 2009 are some examples of eid emerging within the region since sars. at the time of writing, the situation involving human cases infected with avian influenza a(h7n9) virus is evolving, and there has been a recent report of human infection with avian influenza a(h6n1) virus as well. additionally, there are imported eids such as influenza a(h1n1) virus, west nile virus, and the present cause for concern in middle east respiratory syndrome coronavirus. climate changes in recent years have also accelerated the increase in incidence and range of existing diseases such as dengue and chikungunya. 40% of the world's population is now at risk of dengue, with the majority living in the asia-pacific region. the scourge of dengue is sufficiently high in the region for the association of southeast asian nations (asean) in 2011 to designate 15 june as asean dengue day. hepatitis e is another infection that is widespread in some parts of the region, and reports of silent infection in blood donors are cause for further study. the impact of eids on the blood supply may be direct through the potential for transmission through transfusion, the effect on blood donor attendance and eligibility and the effect on blood demand. blood products such as hyper-immune plasma preparations may be useful treatment options in some eids. there is a need for constant surveillance and the capacity to identify, assess and manage eid risks to the blood supply. in recent years, the strengthening of regional and international partnerships and the availability of new diagnostic tools has improved our ability to respond to infectious threats. nonetheless, the volatile and ever-changing nature of eids will remain a constant challenge to the vigilance and response capabilities of the transfusion medicine community. summary: the residual transmission risk is relatively high in hbv followed by hcv and hiv-1. this finding is not new as malaysia is a country of medium seroprevalence for hbv which ranges from 1.5% to 9.8% in the general population, but is relatively low in blood donor population (0.04%). the obi nat yield was higher than acute phase wp in hbv-nat yield. these obi positivity have shown inconsistent nat results on repeat testing and also low viral loads ranging from <12 to 317 iu/ml. in contrast for hiv and hcv nat yields, their viral loads were consistently high (34,220-424,310 cp/ml and 42,780-5,980 ,350 iu/ml respectively). implementation of id-nat is probably the best option to improve blood safety especially for the detection of low viral load such as in obi and sero-negative wp donation in malaysia scenario. blood centers reported to kcdc. repository samples of these donors were tested for anti-hav igm/igg and hav-rna. if any of these test result was positive, the recipients of the blood components generated from these donations were traced. transfusion records of hospital were reviewed to identify recipients of blood suspected to be contaminated with hav. recipients were contacted by telephone. if the recipients agreed participating in the investigation, laboratory test was performed. results: from may 2007 to december 2011, 15 donors notified the blood center of having been diagnosed with hepatitis a. the median interval from donation to diagnosis in donors was 18.2 days (table) . eleven (73%) of these 15 were male donors, and the median age was 28 years (range 19-42 years). the pcr for hav rna was positive in all of 15 depository samples of these donors. none of the repository samples showed positive result of anti-hav igm. a total of 44 blood components (rbc 15 units, pc 14 units and ffp 15 units) were delivered to hospitals. twenty six products were transfused to 26 patients, and the rest 18 blood components (4 rbcs, 1 pc, and 13 ffps) were recalled immediately and discarded. twelve recipients (46%) were already expired. fourteen recipients agreed to participate in the lookback procedure through testing. twelve recipients did not showed viremic for hav, however two recipients showed positive either on the test for anti-hav igm or hav-rna. these two recipients who were 35 and 37 years old developed symptoms on 46 and 48 days after blood transfusion respectively. they were treated for hepatitis a successfully. summary/conclusions: recipients who had anti-hav igg were not infected with hav, even though they were transfused with blood suspected to be contaminated with hav. however ttha cases were those who did not have anti-hav igg and all of them were 30s. in korea, most people under 40 years old are susceptible to hav because of low immunity. there is a risk of transfusion transmitted infection, if recipients received blood contaminated with hav. 2c-s04-04 cable rt 1 , pistorius c 1 , andersson m 2 , maponga t 2 , lopez t 2 , preiser w 2 and tedder r 3 1 hav seroprevalence was highest in the black donors (86%), lower in coloured donors (62%) and lowest white donors (36%). all were hav igm negative. there was an age-related increase from 44.8% (52/116) in those 16-25 years old to 81% (47/58) in those >46 years. although no active hev infection was identified by pcr on pooled samples, 25% of donors were hev igg positive. rates were highest in whites at 33%, followed by coloured at 23% and lowest in black at 19%. again prevalence increased with age from 12.1% in those 16-25 years to 48.3% in those >46 years. discussion: the results show a lower hav seroprevalence in the white population compared to the black and coloured population groups, likely to be due to socioeconomic living conditions such a contrast is striking given the introduction of democracy in south africa almost 20 years ago. the reduction in anti-hbc (as a marker of past hbv infection) with age is reassuring, suggesting that hbv vaccination is impacting hbv population prevalence. the pattern of hev exposure appears to implicate a zoonotic transmission route rather than being related to socio-economic circumstances. given the subclinical nature of hev infection in healthy donors, larger studies are urgently needed to establish the prevalence of active infection in blood donors. background: hbv demonstrates remarkable genetic variability, with eight genotypes and more subgenotypes. in addition, mutations in the polymerase region may lead to drug resistance and changes in the pres region (including deletions and mutations such as t31c and t53c) and prec/bcp region (mutations including a1762t/g1764a, g1896a, t1896a, c1766t, t1768a) are associated with higher risk of hepatocellular carcinoma (hcc). aims: to study the hbv subgenotype distribution and analyze the changes in pres region, prec/bcp and the polymerase region of hbv in chinese blood donors. methods: of 245 blood samples were selected randomly from hbsag positive blood donors from five blood centers in china. the pres plus s region or the whole genome of hbv was amplified and sequenced and hbv subgenotype was determined. the amino acid sequences of the polymerase region were aligned and the mutations related to drug resistance were determined. the nucleotide sequences of pres region and prec/bcp region were aligned and the mutations related to hcc were determined. distribution of genotype, subgenotype, and mutations by different regions were examined using chi-square statistics. results: of 200 samples (81.6%)were subgenotyped successfully. the predominant subgenotypes were b2, c2, d1 and a1 which accounted for 50.5%, 19.2%, 5.3%, and 3.4% respectively. deletions and mutations (t31c and t53c) in pres region were found in 28 (28/200, 14.0%) samples. five of these 28 samples (2.5% of all samples) have deletions and no deletions specific to genotype d was detected. the prevalence of mutations in pres region was significantly higher in genotype c than in genotype b (p < 0.001). mutations in polymerase region were found in 14 samples (7%, 14/ 200), most of which were related to resistance to adefovir and lamivudine. mutations in prec/bcp region were found in 68 samples (29.8%, 68/228). the prevalence of hbeag was significantly lower in samples with mutations in prec/bcp region than that in the samples with no mutation (p = 0.02). more a1762t/g1764a mutations were found in c than b genotype while the opposite was observed for g1896a mutation (p's < 0.01). conclusions: subgenotype b2 was the most frequent strain circulating in hbv infected chinese blood donors, followed by c2. hcc related mutations were found less in pres region but more in prec/bcp region in blood donors. the prevalence of mutations in pres region and a1762t/g1764a mutations was higher in genotype c than in genotype b while the opposite is the case for g1896a mutations. this is consistent with the distribution of hcc related mutations in general hbv carriers in china. since all donors in this study reported not having received hbv treatment, it is not clear whether drug resistance mutations occurred spontaneously in the hbv-infected blood donors or were acquired by the donors from hbv infected patients who underwent antiviral therapy. 2c-s05-01 a fresh look at measuring quality in blood components devine d confidence in the quality of blood components produced for transfusion, particularly with respect to their safety and efficacy, is a necessity for clinicians and patients alike. the assurance of blood product quality is dependent upon the collection of data that can demonstrate products are within specification. however, the linkage between confidence that an individual blood component unit will perform as expected and the conduct of quality testing is imperfect. this begins with the manner in which we conduct these tests. although we describe our practice as 'quality control', it is, in fact, a type of process control testing in which we test a small proportion of our production inventory to ensure that the process was conducted properly. this testing is often conducted at product outdate, long after problem products have been issued and used in hospitals. in addition, the standards used to assess blood components often have 'wiggle room'. for example, the north american standards for the number of platelets in a whole blood-derived platelet concentrate require that at least 75% of tested products meet or exceed the required platelet count. in practice, this means that up to 25% of individual platelet components issued to hospitals may have fewer platelets than the user specification requires. there are other examples in component quality standards of this same phenomenon. in an utopian blood production laboratory, there would be real time quality control measures that would be made prior to release of a unit to the hospital blood bank or transfusion service, and these measures would be highly predictive of product efficacy. as a community, we have considerable work to do to get to this utopian ideal. first we must identify better product characteristics to use as standards for blood component production. modern science, especially in the field of cell biology, has made huge strides since quality standards were first introduced some 50 years ago, yet we have not applied these advances to quality assessment for blood products. those studying blood component quality have begun to collect data sets that will help to inform this change over to new standards. production methods are only one way to impact component quality; another is the actual features of the donors themselves. this biological variation includes not only well known phenomena such as the range of platelet counts in normal healthy humans or the distribution of plasma factor viii or fibrinogen levels, but also appears to extend to storage characteristics of components made from individual donations. this presentation will review the state of the science of product quality and the regulation of blood products, including new information arising from clinical trials, and the application of modern scientific methods such as proteomics and metabolomics to the broad question of blood component quality. background: blood transfusion has been shown to be associated with poorer surgical outcomes such as higher incidence of infection, higher mortality, and increased number of serious adverse events. microparticles (mp) released in packed red cells (pc) in storage have been suggested to be mediators for transfusion-related complications. however, the underlying mechanism for mp release during storage is mostly unresolved. aims: to examine mp released in pc in storage for procoagulant and proinflammatory activity and define the role of residual platelets in pc in generation of mp during storage. methods: (i) leukoreduced (lr) and non-lr (nlr) packed cells (pc) were stored according to blood bank standards and sampled at day 0, 10, 20, 30, and 40. assay of mp was by flow cytometry using mab to label cd235a, cd45, cd41, and cd62e. thrombin generation (tg) and cd11b expression were used as measures of procoagulance and proinflammation, respectively. (ii) the impact of platelets was further evaluated by reconstituting lr pc with increasing concentrations of platelets at day 0. results: (i) multiple species of mp were released in a time-dependent manner. using nlr pc, we found that, relative to day 0, red cell mp (rmp) were increased by 2.59 at day 20, and by 8.59 by day 40. small amounts of mp from leukocytes (lmp), platelets (pmp), and endothelia (emp) were detected, generally <20% as many as rmp. levels of pmp rose rapidly from day 0 and peaked at day 20. lmp began rising at 20 days, increasing to 1.59 at day 30 and 2.49 at day 40. emp changed little over 40 days. (ii) comparing mp in nlr vs lr pc. as expected, pmp and lmp were higher in nlr pc. unexpectedly, however, the rate of rmp production was <50% as fast in lr vs nlr pc. the levels of rmp were found to be significantly associated with residual platelet counts. therefore, we investigated the possible role of platelets in rmp production. (iii) effects of residual platelets on rmp release. when lr pc were incubated with increasing numbers of platelets (0-200,000 per ll), rmp as well as pmp generation increased. rate of increase of rmp was closely associated with platelet count and storage time. this shows that residual platelets catalyze rmp generation. (iv) procoagulant and proinflammatory activities. mp-mediated thrombin generation and cd11b expression increased from day 0 to day 40 in both lr and nlr pc, but in lr pc, it was only 30-50% magnitude of nlr pc. the time course curves did not match any specific mp species. conclusions: procoagulant and pro-inflammatory mp were generated in a timedependent manner. the new finding is that residual platelets markedly augment release of rmp, which is a known indicator for storage lesion. the benefits of leukoreduction may be due to reduction of platelets and mp production in addition to reduction of wbc. reducing platelet count in pc may be beneficial in reducing storage lesion and transfusion related complications. background: it has been reported the soluble cd40 ligand (scd40l, scd154) that was accumulated during platelet storage induce polymorphonuclear leukocyte (pmn) mediated damage of human pulmonary microvascular endothelial cells(hmvecs), was a potential cofactor in developing the transfusion-related acute lung injury (trali). however the amount of scd40l was only slightly elevated in the recipient by transfused blood components as it was fully diluted in the recipient's blood circulation. the mechanism by which cd40l exert its effect is still needs to be elucidated. aim: to determine the effect of platelet derived microparticles (pmp) on pmn mediated hmvecs damage, and its correlation with pmp bounded cd40l. method: the pmps were isolated by centrifugation of the platelet-free plasma from 10 apheresis platelet concentrates (a-plts) at 20,000 g for 1 h. the pmps were counted by flow cytometric analysis, followed by western blotting that was performed on isolated pmps. the scd40l was assayed with elisa. the priming of the formyl-met-leu-phe (fmlp) activated pmn respiratory burst was measured with the hydrogen-peroxide production. a two-insult in vitro model of pmn-mediated hmvecs damage was used to investigate the effect of pmp. result: the pmp priming activity to pmn are correlative with pmp accumulations and their level of scd40l during 5 days storage (correlation was significant at the 0.05 level); pmn respiratory burst are declined by removing pmp with 0.1 lm pvdf membrane filtration or depletion pmp with cd154 monoclonal antibody combining magnetic dynabeads pan mouse igg; the lipopolysaccharides(lps) activated hmvecs were damaged more significantly by pmp isolated from 5-day stored a-plts compared with 1-day stored a-plts (p < 0.05). conclusion: platelet-derived microparticles carry concentrated cd40l signal, promote pmn mediated hmvecs damage, may be relative to developing of trali. background: continuous efforts has spared on improving the quality of platelets harvested from plateletpheresis. little is known about the contribution of donors on the variation of platelet quality, particular the effect of frequent platelet donation on donor's platelet function. aims: aim of this study is to investigate the effect of frequent platelet donation on the state of in vivo platelet activation in platelet donors. material and methods: of 107 whole blood donors and 335 platelet donors with vary donation history from 1 to 74 times (mean at 11.5 ae 12.7) were recruited. they were stratified into three subgroups according to their previous plateletpheresis history (g1: 0-1 time, g2: 2-10 times and g3: >10 times). blood sample were collected from each participant for the determination of plasma levels of soluble p-selectin (sp-selectin), marker of platelet activation and total platelet p-selectin (tp-selectin), as well as platelet count and platelet indices. results: following the increase of donation times, sp-selectin levels were steady increase (g1: 20.50 ae 5.76, g2: 23.21 ae 6.70 and g3: 25.33ae7.67 ng/ml respectly, p = 0.001) and mean platelet volume (mpv) was decrease (g1: 9.29 ae 0.89, g2: 9.17 ae 0.84 and g3: 9.02 ae 0.91 fl respectly, p = 0.039) as estimated by the analysis of covariance adjusted for sex and gender. no significant changes in tp-selectin, platelet count, platelet distribution width (pdw) were observed. further multivariate regression analysis including variables of abo blood groups as well as donation history, sex, age indicated that increased plateletpheresis donations are positively associated with the elevated sp-selectin levels in blood donors (t = 4.16, p < 0.0001). conclusion: our data suggested that frequent plateletpheresis result in the increased level of sp-selectin in platelet donors, implicating a higher state of platelet activation in vivo in frequent platelet donors. the potential effects of frequent plateletpheresis on the quality of platelet harvested and the donor complication are worthy of attention. background/aims: structural and functional changes in erythrocytes occur during ex vivo storage, including the accumulation of bioactive substances in the supernatant of red cell concentrates (rccs). many of the constituents of the supernatant fraction, which are potential mediators of transfusion-related complications, may be reduced by washing of rccs. with emerging paediatric clinical data supporting a beneficial effect of rcc washing prior to transfusion, the aim of the current study was to characterise the effects of rcc age and post-washing storage on erythrocyte structure, function and the accumulation of bioactive substances in paediatric-sized washed rccs. methods: two units of abo/rhd-and age-matched rccs (either 1-or 4-days old; n = 11 each) were pooled and equally split to obtain matched pairs (day 0). one unit was washed with 0.9% saline by repeated centrifugation then resuspended in 100 ml sag-m, while the other remained unwashed. subsequently, both rcc units were divided equally to produce 4 units of paediatric-sized washed or unwashed rccs. all units were stored at 2-8â°c and samples were taken on days 0, 1, 2, 7, and 14 of storage to measure metabolic activity and quality of rccs, as well as the concentration and activity of bioactive substances in the rcc supernatant. the overall effects of washing and storage were compared using repeated measures anova with posthoc paired t-tests as required, with p < 0.05 considered significant. results: the washing process resulted in reductions in red cell count (9.3%), haemoglobin (9.2%) and haematocrit (5.9%) compared to unwashed rccs. overall, washing and subsequent storage of 1-and 4-day old rccs significantly reduced the ph (p < 0.0001), lactate production rate (p < 0.0001), and 2,3-diphosphoglycerate concentration (p = 0.046). although the atp content of the rcc decreased during storage, it was not changed by washing (p = 0.570). haemolysis in the rccs was increased by the washing process, but remained <0.15% on day 14 for all products. extracellular potassium was significantly reduced by washing (p < 0.0001), but increased during storage in both washed and unwashed red cells (p < 0.0001 for both). washing significantly reduced the number of microparticles in the supernatant of both 1-and 4-day-old rcc compared to unwashed rccs (p = 0.01 and 0.012 respectively). however, the microparticle number in the supernatant of all rccs increased during storage. washing of both 1-and 4-day old rcc also markedly reduced the supernatant concentration of monocyte chemoattractant protein-1, scd62p, rantes, anaphylatoxins (c3a, c4a, and c5a) and iga to levels below or near the limit of detection. incubation of cultured human umbilical vein endothelial cells (huvecs) with supernatant from unwashed rcc led to endothelial cell activation, with increased cell-surface expression of e-selectin and vcam (p < 0.0001 for both). however, little or no activation was observed when huvecs were incubated with supernatant from washed rcc. conclusion: although washing affected some aspects of the in vitro quality of rccs, it effectively reduced the concentration and activity of bioactive substances in the supernatant of rccs, leading to reduced endothelial cell activation. such a reduction may be clinically beneficial in selected patient groups. blood services group, health sciences authority, singapore, singapore many of the critical issues associated withbiobanking have been effectively addressed in blood banking. blood transfusion therapy with its emphasison traceability has developed robust systems for inventory and product release. the ethos of proper quality management hasalso been an integral part of blood banking. the lessons learnt have been applied into the biobanking of cord blood, stem cells, tissue and organs. biobanking includes both banking of tissuefor research only as well as public cord blood banks that play a vital rolesupporting clinical stem cell transplantation. the growth of regenerative medicine will only increase the scope, variety and numbers in biobanking. similarly, the discovery of induced pluripotent stem cells (ips) and itspotential myriad applications has highlighted the central role of biobanking inboth diagnostics, research and therapeutics. principles and key considerations in biobanking including biospecimenprocurement, consent, processing, preservation and traceability will beaddressed. introduction: cell therapy generally includes the extraction, processing, manipulation and implantation of characterized cells effectuating specific functions in a patient. however, adjacent fields like tissue banking or tissue engineering should be incorporated. all together have donor selection and validated core procedures in common. production cycles are carried out in gmp clean rooms. furthermore, quality control includes assays which are common in transfusion medicine. it might be tempting to speculate, that cell therapy is closely related with transfusion medicine and requires minor adaptions. the big moat surrounding cell therapy: but there are huge differences: cell therapy is a domain of specialists rooted in patient care with profound knowledge about specific pathologies and how to target them. cell therapy derives from individual clinical needs and rarely is a prefabricated procedure. this is in stark contrast to transfusion medicine, which focuses on standardized products for any patient in need. today, complex regulations for cell therapy surpass those in transfusion medicine. this distracts clinicians in a cell therapy program. this may aspire transfusion medicine specialists to engage in cell therapies. however, only a small niche is left for blood centers, as two major trends arise: one is the more or less 'academic gmp' setting, utilizing the hospital exemption status. the other is the commercial arena, where companies produce standardized cell therapies to achieve maximized market shares. 'academic gmp' entities promote individualizedmainly autologoustherapies, which require a constant flow of financial assets to keep underused clean rooms running. therefore it is serious to ask why and where blood centers should engage in cell therapies. strategy first: transfusion medicine has been heavily influenced by external factors such as viral safety, blood usage, cost pressures and low resources. the term 'transfusion medicine' misleads outsiders to suppose, that it deals with transfusions, nothing else. a wise strategy must include a change in the mind-set of all. this is the most crucial issue as many clinicians are needed to collaborate and co-develop cell therapies. without deeply rooted partnerships it is impossible to establish sustainable cell therapy programs in a blood center. furthermore, a thorough evaluation includes possible products, quantities, as well reimbursement schemes. cell therapy is one of the most expensive treatments and financial assets must be secured first. second: establishing a cell therapy facility: a mock-up facility, without clean room status is highly recommended. processes will be developed, staff is enabled to learn and define procedures, before a cell therapy unit is planned. planning and establishing a cell therapy unit is very complex and specific expertise is scarce. a basic prerequisite is a project manager carrying out final decisions with a profound knowledge about processes interacting with different technologies (hvac, controls, microbiology). cell therapy units fail if project management has flaws and deep involvement of engineering with medical expertise is ignored. a cell therapy unit is an endless, stressful, path riddled with expensive failures, but rewards in the long run a blood center with exciting future prospects integrating grateful clinicians and patients. any kind of accreditation, either by regulatory authorities or any professional entity, like jacie/fact, is an important milestone in the time line of a new cell therapy and a possible showstopper of a long, expensive and enduring project. honest and thorough preparations before accreditation should start before an application may be sent. three phases are distinguishable: keeping the basics on track: running a cell therapy unit is a high wire task. fundamental knowledge about the medical background, processes, quality control, specifications is interwoven with engineering and controlling tasks. it is inevitable for anyone working in this environment to know about air quality, hygiene, staff education and additional technical features. especially controls and the design of engineering and its qualification should be documented continuously. maintenance, re-qualifications, adjustments in the control-system of a clean room facility offer chances to learn the interplay of systems. build a sufficient knowledge base and freeze the process: protocols for cell therapy are often introduced on primary events and work well in first shot experiments. as further patients are included it is very probable, that the whole process and specifications will be modified and sometimes fundamentals and documentation are out of focus. altering and scaling methods, processes and assays may or may not change the product or its intended use. slippage is often not detected and therefore first steps aim at the build-up of as much information as possible. firstly, current literature has to be collected, reanalyzed and mirrored onto the own processes. then the regulatory framework has to be analyzed. the main question is, how the product fits into the regulatory system. is it an atmp or non-manipulated cell product, is it a blood product or a pharmaceutical? pros and cons about alterations should be meticulously discussed and alternative procedures highlighted in this phaseespecially those which are already accredited. it will be very probable that the same inspectors, who have inspected similar cell therapy units, know about alternative procedures and will raise comparative questions. after building up the knowledge base it is advisable to finish a risk assessment focused on the intended use in patients and to revamp the process. after adjusting all methods, processes and assays the whole production has to be frozen. further changes are prohibited and the documentation has to be refreshed. the last test trial: in the last phase before accreditation all aspects of quality management have to be finished. risk assessments should focus on the safety and effectivity of the cell therapy. donor/patient eligibility criteria, quality control of incoming cells and tissues, production processes and their internal quality control criteria as well storage conditions have to be well documented and validated. under certain circumstances a file of clinical studies has to be prepared. all documents and clinical studies should be reviewed, regulatory aspects should be clear. a preparation project gives better chances to pass the last milestone before patients can be treated on a regular cell therapy. 2d-s07-01 burnouf t plasma fractionation is a complex biotechnological process exhibiting unique features compared to downstream technologies used for recombinant proteins, vaccines, and animal-derived antisera. in human plasma fractionation, by contrast to other biological products, multiple end-products (typically 5-10 or more) are obtained from the same manufacturing pool. some of the targeted proteins are present in plasma in large amounts, as is the case for albumin and immunoglobulin, whereas, by contrast, others, such as coagulation factors and anticoagulant proteins are in trace amounts. with the emergence of selective hemotherapy, plasma fractionation has, over the years, turned into a highly integrated protein separation process carefully designed to isolate various proteins under optimized conditions of yield and purity. current plasma fractionation methodologies combine diverse protein purification tools based on 'crude' precipitation techniques and refined chromatographic procedures. some proteins are stable and not prone to degradation, while others, with specialized functional activity, are fragile and prone to enzymatic degradation, activation, or aggregation. contamination of plasma products with harmful residual plasma protein impurities (such as activated coagulation factors or proteases) can lead to serious adverse events in some patients. in addition, while a few plasma products, like albumin and immunoglobulin, can be formulated as liquid preparations, all others products are freeze-dried to ensure long-term stability, which increases cost and technical difficulties. the diversity of protein products made from plasma explains why plasma fractionation facilities have complex design. the manufacturing lines of the various fractions should be strictly segregated from one another. in addition, the risk of viral contamination of plasma pools requires that each product be subjected to several (typically two or more) dedicated viral reduction treatments, the goal being to gradually increase the degree of viral safety along the downstream process. production zones should therefore be physically segregated to limit risks of cross-or downstream contaminations, adding to the complexity of the plant design, flows of product, personnel and wastes, and working procedures. the plasma fractionation industry has a long history from the years 1940's, when cohn and co-workers developed a sequential cold ethanol precipitation process. this method which evolved over the years, remains the core fractionation process, albeit integrated with cryoprecipitation and multiple chromatographic steps. combined with modern viral removal treatments, the current fractionation process ensures therapeutic protein products of established quality and safety, at more or less acceptable yields. the current safety record of plasma products, which contrasts with that of earlier product generations, somehow represent an impediment to the emergence of new fractionation technologies. novel plasma fractionation processes based on integrated chromatographic steps, membrane electrophoresis, aqueous two-phase system, mini-pool fractionation in disposable equipment, are being developed at pilot-scale and represent interesting alternatives. to reach the market, such technologies should be integrated with robust viral reduction steps and proven to achieve at least equal, if not superior, products yield, quality, safety, and productivity to justify the regulatory load, clinical trials, and licensing of what would be regarded by most regulatory agencies as new plasma products requiring full validation. effective specific antiviral agent is generally not available for emerging infectious disease agents such as sars-coronavirus and middle-east-respiratory-syndromecoronavirus. passive immunotherapy with plasma or plasma derived hyperimmune globulins have been used for treatment or prophylaxis against many exotoxin mediated bacterial or viral diseases such as viral hemorrhagic fever and 1918 influenza despite the lack of data from randomized control trial. antigenically shifted influ-enza a virus causes pandemics and antigenically drifted viruses are associated with seasonal outbreaks. poultry to human transmission of avian influenza a h7n9 and h5n1 virus can cause acute community acquired pneumonia with 25 to 50% mortality. risk factors including extremes of age, pregnancy, underlying medical illness and low serum igg2 are associated with severe pneumonia with delayed clearance of viral load and excessive proinflammatory response. though treatment with neuraminidase inhibitors within 48 hours after onset of symptoms should be effective, those with severe disease and respiratory failure usually present later than 5 days after symptom onset. during the 2009 pandemic of h1n1 influenza, we harvested convalescent plasma from a small percentage of recovered adults sufficient for a case control study for treating severe cases during the pandemic in hong kong. plasma supply is constrained by plasmapheresis capacity during most stages of the epidemic. between august 26 to october 31, 2009, a total of 9101 persons were successfully contacted. a total of 1309 screening and 619 whole blood donation appointments were made. in the former 786 (60.0%) attended screening but only 301 could donate plasma by apheresis because of failure to meet blood donation eligibility criteria, failed laboratory tests, insufficient neutralization antibody titers, and inability to make the apheresis appointment. for those who opted for whole blood donation, 379 (61.2%) had attended and donated. 10.5 l (21 units) and 276 l of convalescent plasma with sufficient neutralization antibodies titers was collected for passive immunotherapy as convalescent plasma or h-ivig production respectively. we recruited 93 patients with severe h1n1 2009 infection already put on neuraminidase inhibitors and requiring intensive care. twenty patients (21.5%) received convalescent plasma. mortality in the treatment group was significantly lower than the demographically matched control nontreatment group (20.0% vs 54.8%; p = 0.01). convalescent plasma treatment was associated with significantly lower day 3, 5, and 7 viral load, compared with the control group (p < 0.05) and corresponding lower serum levels of il6, il10, and tnf (p < 0.05). between 2010 and 2011, 35 patients were randomized to receive h-ivig (17 patients) or ivig (18 patients). no adverse event related to treatment was reported. serial respiratory viral load demonstrated that h-ivig treatment was associated with significantly lower day 5 and 7 posttreatment viral load when compared to the control (p = 0.04 and p = 0.02 respectively). the initial serum cytokine level was significantly higher in the h-ivig group but fell to similar level 3 days after treatment. subgroup multivariate analysis of the 22 patients who received treatment within 5 days of symptom onset demonstrated that h-ivig treatment was the only factor which independently reduced mortality [or:0.14, 95% ci, 0.02-0.92; p = 0.04]. background: in recent years, with the global spread of the west nile virus (wnv), dengue virus (denv) and chikungunya virus (chikv) that are transmissible by mosquitoes, concern has arisen regarding their entry to japan. furthermore, chikv as well as wnv and denv are potentially transfusion-transmissible, posing a serious risk for transfusion medicine. of these, wnv and denv belong to the flavivirus genus, as does the japanese encephalitis virus (jev), and they have similar antigenicity. since most japanese people are periodically vaccinated against jev, there is a possibility that anti-jev antibodies cross-react with wnv and denv and induce a protective immune response. however, because wnv and denv have similar antigenicity to jev, there is concern that the anti-jev antibodies are present in japanese plasma against wnv and denv owing to antibody-dependent enhancement (ade) in fccr-expressing cells. furthermore, if the anti-jev antibodies present in japanese plasma have high ade activity, these antibodies may act in an infection-enhancing manner rather than an infection-neutralizing manner against wnv and denv in vivo. aims: using intravenous immunoglobulin (ivig) prepared from pooled plasma from japanese donors, we evaluated its neutralizing activity and ade activity against these viruses for the purpose of estimating the potency of the japanese individuals to protect themselves against these viruses. methods: neutralizing activity (tcid 50 ) and ade activity were compared among three types of ivig, nisseki polyglobin n (made in japan), gammagard (made in germany) and sanglopor (made in the usa). tcid 50 was calculated from the results of cytopathic effect (cpe) assay using vero cells as target cells. ade activity was measured by plaque assay using bhk cells and fccr-expressing bhk cells as target cells. results: nisseki polyglobin n showed a significantly higher neutralizing activity against jev than gammagard and sanglopor. the neutralizing activity of nisseki polyglobin n against wnv was approximately a log reduction factor of 2.3 higher than that of sanglopor. furthermore, the neutralizing activity of nisseki polyglobin n showed approximately the same neutralizing activity as gammagard against wnv. none of the ivig preparations showed significant neutralizing activity against denv or chikv. nisseki polyglobin n showed only marginal ade activity against any of the viruses. conclusion: although the neutralizing activity of plasma from japanese individuals is not known, it is suggested that the japanese population as a whole has a potency to protect themselves from infection by wnv to some extent, probably due to the cross-reaction of anti-jev antibodies to wnv as a result of jev vaccination and natural infection. therefore, if wnv invades japan, a pandemic may not occur and the risk of wnv infection by blood transfusion may be low. it was suggested that plasma from the japanese individuals has almost no neutralizing activity against denv and chikv. nisseki polyglobin n showed only marginal ade activity against wnv and denv, suggesting the low possibility of the anti-jev antibodies present in japanese plasma acting as infection-enhancing agents against wnv and denv as a function of ade. background: platelet-rich-plasma (prp), platelet gel (pg), and platelet lysate (pl), are used in regenerative medicine to stimulate the healing of soft and hard tissues. in addition to their tissue regenerative properties, platelet materials are claimed, mostly through anecdotal observations, to limit post-surgical inflammation and decrease pain. the anti-inflammatory properties are thought to be due to the release of platelet components, including transforming growth factor-b1 (tgf-b1) and hepatocyte growth factor (hgf), which are known to inhibit some inflammatory pathways in vitro. however, there is a large diversity in the type of platelet fractions used in clinics. they differ significantly in protein composition and content of proinflammatory and anti-inflammatory molecules and may therefore not be equally effective in controlling inflammation. one needs to elucidate the factors responsible for the possible anti-inflammatory properties of platelet materials to standardize the preparation and clinical use of these products when an anti-inflammation effect is clinically beneficial. aims: to investigate the potential anti-inflammatory effect of various plasma/ platelet fractions using an established in vitro model of raw 264.7 mice macrophages stimulated by lipopolysaccharide (lps), and studying the production of nitric oxide (no), inducible nitric oxide synthase (inos), and cyclooxygenase-2 (cox-2). methods: apheresis platelet concentrates (pc) were obtained from three donors and separated within 3 days into pc, platelet poor plasma (ppp), platelet gel releasate (pg), frozen-thawed platelet lysate (pl), and solvent-treated platelet lysate (s/d-pl). proteins were determined, sds-page patterns obtained, and growth factors quantified by elisa. raw 264.7 cells were grown in medium supplemented with 10% of fetal bovine serum (fbs) or plasma/platelet fractions, with or without lps (500 ng/ ml added after 24 h of culture). cell growth and cytotoxicity were checked by cell count determination and mtt assay. no was determined in cell culture supernatants by colorimetric assay and inos and cox-2 in cell extracts by western blot. prp from mice and quercetin, a known anti-inflammatory compound, were used as controls. results: pc and s/d-pl had the highest mean tgf-b1 content ranging from approximately 100-200 ng/ml, and ppp the lowest (5-10 ng/ml). cell count analysis and mtt assays showed consistent cell growth and viability in all conditions evaluated but were slightly lower in the presence of lps and quercetin, as expected. there was no no, inos, cox-2 detected in absence of lps stimulation. the plasma and platelet fractions were all found to reduce no production and inos expression, when compared to fbs, after lps stimulation. interestingly, inhibition of no production and inos was generally more pronounced with s/d-pl. cox-2 synthesis was lower in the presence of s/d-pl compared to other plasma/platelet fractions and higher with pg. the mice prp did not exert any stronger anti-inflammatory action in this mice cellular model suggesting absence of species specificity. conclusions: the plasma and platelet fractions evaluated exert, to various degrees, an anti-inflammatory effect mostly revealed by inhibition of no production and inos. impact on cox-2 synthesis is less obvious. the fact that s/d-pl exhibits stronger global anti-inflammatory activity requires further studies. 2d-s08-01 tani y japanese red cross kinki block blood center, ibaraki, japan rare blood is generally defined as one that occurs at a frequency of 1:100~1000 individuals or less, and it is sometimes difficult to provide such blood types to patients because of their rarities. the japanese red cross (jrc) society lists 46 rare blood phenotypes that are divided in two categories as shown in table 1 . the rare blood types listed in category i occur much less frequently than those listed in category ii. we screen for rare blood cells using monoclonal antibodies (moabs). since 1987, our blood center has established 93 moabs (32 human and 61murine), and has provided them to the other blood centers. many of igg moabs are available on the machine such as beckman coulter pk-7300 blood grouping analyzer by saline or bromelin method by cross-linking with anti-human or anti-mouse igg. in addition, we routinely screen for antigen negative-cells (rhc, c, e, e, jk a , jk b , fy b , di a , m, le a , s to which antibodies are believed to be clinically significant). thus, more than 10,000 donors with rare blood phenotypes (category i, 748; category ii, 9314) are registered in japan, but the number of donors with some category i blood types are insufficient. we freeze rare blood, particularly category i types, and domestically and sometimes internationally supply these units of blood. since 1977, a total of 576 units of rare blood with phenotypes di(b-), d-, jr(a-), ko, jk(a-b-), (para-)bombay, p, en(a-), m k m k , lan-and rhnull have been supplied to 23 international countries. thus, the jrc contributes to the international panel of donors of rare blood type (idp) which is maintained by the international blood group reference laboratory (ibgrl) in bristol, uk. the idp provides information on the location of rare blood donors when they cannot be found in their respective countries. the jrc has encountered difficulties in finding rare donors with rhnull, p k , m k m k , en(a-), u-and ge-phenotypes. we also joined the isbt working party on rare donors which handles all matters related to rare blood. our rare blood donor program is successful because of international cooperation. 2d-s08-02 the china national rare blood group screening program started in 2008. the program has screened more than 1,500,000 donors in thirteen regional blood centers by using large-scale serological tests, gene diagnosis, and other different specific screening technique. now, more than 1300 donors with rare blood phenotypes have been found. including rh null, d --, wrb-, lu(a-b-), jk(a-b-), vel-, lan-, coa-, k 0 , dib-, s-as well as yta-. the primary target for the project is to screen the negative blood antigens whose conjugational antigens have high frequency, and the blood groups which is easy to produce antibodies and the negative antigen in the system is very rare in chinese population (for example, fya-, whose frequency was 1/400 in chinese han population). a professional website for rare blood groups (http://www.chinarareblood.cn)was set up and serviced in jan, 2010. the information of blood donors with rare blood types is registered into the national registering system for blood donors with rare blood types by professional technician from organizations join the program. the information includes the specificity of the rare blood types, other common blood groups, personal data of donors, and the information of contacts. except the network administrator, other visitors could only see the specificity of the rare blood type and the number of the rare blood in rare blood bank storage. application for the rare blood products should communicate with contacts through administrator. according to the standard of the pretransfusion test in china, all the donors and recipients must be identified the abo and rh systems and done the cross-match test, in addition, all the donors must pass the test for contagious marks. nowadays, more and more chinese blood centers use nucleic acid technique to detect hiv and hcv. the results of infection test must be added to the information of the blood products at every time for collecting the rare blood. in recent 2 years, 21 units (1 unit = 200 ml) blood products with different rare blood types have been used in clinical treatment. background: anti-emm is a rare specificity detecting the high-prevalence red blood cell (rbc) antigen emm (isbt 901008). five cases were reported by daniels et al, (transfusion, 1987; 27:319) and two by reid et al, (transfusion 1998; 38 (suppl) :101s). six of these were in untransfused males and anti-emm had not been implicated in a transfusion reaction, most likely because the patients were not transfused. case study: a 58-year-old, untransfused, japanese man with group a, d+, datnegative rbcs, urgently needed transfusion due to an abdominal stab wound. pretransfusion testing using gel column agglutination and peg-iat, demonstrated an antibody reactive with all panel cells, but non-reactive with autologous rbcs; anti-le a was detected by papain methods using gel column. unavoidably, one bag of crossmatch-incompatible le(a-) rbcs had to be transfused. thirty minutes after transfusion, he experienced a drop in blood pressure and hematuria. however, as his hb level was 5.5 g/dl, another two rbc bags were transfused, and his vital signs became stable. on day 3, he was transfused one rbc bag without a hemolytic transfusion reaction. on day 6, after receiving 30ml of rbcs, the patient vomited and had cola-colored urine (t-bil 6.1 mg/dl, ldh 912 u/l) and the transfusion was stopped. following transfusion, his rbcs reacted in the dat: 1+ on day 1, 2+ on day 3 and negative on day 7. thereafter his anemia improved gradually by iron medication and he was discharged on day 24. aims: to identify the antibody in the patient's plasma that caused the transfusion reaction. methods: serological testing was performed by standard methods. result: the patient's plasma reacted in saline at 4c (2+), by albumin iat (2+), peg-iat (2+), and papain-iat (3+) with all panel cells, and by peg-iat with 30 samples lacking high-prevalence antigens; the autologous rbcs were non-reactive. testing his plasma against phenotypically-similar enzyme or chemically treated rbcs showed that the antigen detected was resistant to bromelin, papain, ficin, trypsin, a-chymotrypsin, pronase, dtt, aet, and acid. two examples of emm-rbcs were non-reactive and the antibody was identified as anti-emm. the reactivity of enzyme and chemically treated rbcs is consistent with anti-emm. his rbcs reacted with antibodies to 17 high-prevalence antigens, but could not be confirmed as emmbecause anti-emm was not available. the anti-emm was igg1 and igg3 with a titer of 16 by peg-iat before transfusion rising to 128 on day 10; his serum demonstrated hemolysis after day 6. no other underlying antibodies were detected in the patient's plasma alloadsorbed to remove the anti-emm. conclusions: we report the first japanese case of anti-emm and the first to cause an acute hemolytic transfusion reaction (ahtr). in previous reports, six people with anti-emm were untransfused men and one was in a woman whose transfusion history was unknown, suggesting that the antibodies may be 'naturally-occurring'. the anti-emm reported here, also in an untransfused man, also may be considered a 'naturally-occurring' antibody. similar to the first reported example of anti-emm, the antibody reacted, albeit weakly, at 4c. our case suggests that anti-emm is clinically-significant as the patient experienced an ahtr. 2d-s09-01 background: to recruit blood donation volunteers and provide stable blood supply according to demand, it is more important to change the overall social perception than to carry out one-time event or short-term campaign. the social understanding of blood donation is formed as valuable and honorable service over certain level in korean society. nevertheless, there still are many people who don't even try to participate in blood donation because of fear, health concern, and expectation for reward. to change this culture and social awareness, it is important for the present and future blood donors to have a perception that the blood donation is the sharing one's life and a genuine service which helps other people for nothing. aim: the main purpose of this article is to introduce korean red cross' recent efforts (operation of the red campaigner and blood donation supporters, the construction of virtual blood donation experience center, the blood donation promotion program) to change the blood donation culture and widen the donor base in korea and to present their effect and improvement direction. (1) this article is comprised of the case study and analysis on the operation of red campaigner and blood donation supporters, the construction of virtual blood donation experience center and blood donation promotion program (2) this article outlines the red campaigner and the blood donation supporters and the related programs and addresses their significance in addition, describes the effect and direction for improvement, presenting research related data. (3) this article outlines virtual blood donation experience center construction and presents the exhibition in it and it suggests the effect and possibility on the authority of the research case that the education with fun has more considerable impact than learning by rote. result: to change current culture and increase positive understanding about blood donation, korean red cross is making various efforts. the red campaigner, consisting of high school students and the blood donation supporters, consisting of college students, aim to influence the youth, the potential blood donors, to have a positive understanding of blood donation and to carry out continuous and organized publicity of its importance and safety. in addition, korean red cross is making a progress in the construction of the virtual blood donation experience center which is going to be completed by the end of 2013. we expect that we can achieve the educational purpose that sends a message that the blood donation is a volunteer work to save life and make future donors to recognize the blood donation as an object of not fear, but interest. finally, 'the blood donation promotion program' which began in 2009 is designed to encourage for general groups or organizations to participate in the blood donation campaign and to create the voluntary blood donation culture. conclusion: various projects operated by korean red cross contribute to widen blood donor based by changing blood donation culture in korea and are expected to make continuous contribution. but these projects have a limitation that the partici-pant is restricted and continuous participation isn't progressing in terms of national participation. continuous and positive endeavor to foster these projects as a national campaign should be encouraged. although it is possible to increase the blood donor temporarily through one-time event when blood shortage recurs, widening the donor base by changing blood donation culture should be the fundamental solution. the changing blood donation culture and donor understanding may not be optimistic for a short-term blood shortage problem but will be useful to make conditions that expand the donor base and increase voluntary donors in the medium to longer term. understanding our future donors is of critical importance to blood collection agencies worldwide. not only do we need to know who they are, but also why they do or do not engage in the required behaviour of blood (product) donation. the surge in psychological research into blood donation in the last decade has provided significant insight into understanding the psychological factors underpinning the commencement and continuation of blood donation. this review will summarise the state of our current understanding of knowing how to effectively recruit the non-donor and the complexities that lie ahead for all involved. at the descriptive level, and stemming from the systematic application of various psychological theories and models within blood donation contexts, we now know more than ever about the key factors that non-donors report impact on their blood donation intentions and behaviour. from this, certain psychological elements such as perceived control or self-efficacythe individuals' self perception that they can cope with donationhave emerged as key determinants of donor recruitment. drawing on these results, research psychologists have worked collaboratively with blood collection agencies to develop and evaluate recruitment materials designed to target these central constructs. both laboratory and in field trials have been undertaken which have consistently shown positive effects. for example, participants receiving these materials are more likely to volunteer to donate blood than those receiving standard recruitment materials. the collaboration of researchers and blood collection agencies has furthered understanding and recruitment efficacy generating measurable, operational deliverables. however, this collaborative research has also served to highlight the challenges that lie ahead both in terms of the diversity of our non-donor population as well as the limitations of our current theoretical models. further, there is an increasing need for large scale randomized controlled field trials to systematically evaluate interventions designed on the basis of psychological research. while these needs may provide substantial challenges for researchers and blood collection agencies alike, the promise of psychology in providing the 'who' and 'how' to effectively recruit remains. background: developed countries rely solely on voluntary non-remunerated donors to ensure adequate blood supply but ageing has brought in significant pressure on the health care system including blood supply. in hong kong, blood demand has recorded a 17% increase in blood demand from year 2008 to 2012 with almost 60% blood being transfused to patients aged >60. therefore, sourcing for more blood to meet demand is one of the most urgent issues in blood service. in this report, we report how we successfully modeled donation preference into the development of a new collection site to leverage the blood demand. material and methods: demographic information of all donors with successful donations was retrieved from blood bank computer system for the years 2004-2007. statistical analyses were performed to determine how frequent they came back to donate, whether residential location affected their donation frequencies and lastly identify potential district in hong kong to build a new donor center against the donor and general population distribution. results: of 775,690 donations made by 191,302 male and 201,446 female donors were analyzed. on average 75.8% of donors donated only once during the calendar year but donors were more likely to make repeated donations at donor centers than mobile venues. significantly more donors would come back for second or more donations at donor center (36.1% vs 20.1%, p < 0.05). male were more likely to come back for repeated donations than female (43.3% vs 30.9%, p < 0.05). a total of 466,121 donations made at donor centers were further studied. upon matching with their residential address, distance from donor centers was shown to be a determining factor on their choice of donation through linear regression analysis. reduced donation frequencies were observed with increasing distance from donor centers. regression analysis then identified several districts where many donors had to travel a long distance to the nearest donor center. the district with the highest expected increase in donations, adjusted by the expected growth rate, was then chosen as the site for building the new donor center. a fixed donor center was so selected at yuen long district and open to donors by 29 july 2011. by the end of year 2011, 6160 donations (consisting of 3047 males and 3113 females) were made with more than 88.1% collection given by donors residing at yuen long and the nearby district, tuen mun. when the whole year figure was reviewed in 2012, 16,990 donations were collected which already exceeded the planned collection target of 15,000 by year 3. interestingly, same 88.1% donations were given by donors residing at these two districts. conclusion: despite being a small city, the retrospective analysis of donation behavior has provided valuable information in the service planning in hong kong. the new donor center was able to reach the planned third-year collection target by 15 months earlier. further work is being done by using the more recent data to identify the next optimal collection sites for future expansion under most best cost effective way. singapore red cross, singapore, singapore background: singapore is moving towards providing more fixed blood donation sites with the aim of enhancing donation experience and encouraging repeat donations. it was recognized that the choice of location and an understanding of the human traffic in the vicinity are elements of success. a 2-prong approach was undertaken to: collect information on the footfalls, the social profile and demographics in the designated location. collect information from potential donors on their preferences and sentiments in relation to operating hours and outreach channels for marketing communications. method: a month-long study was conducted in the vicinity by observing and counting the human traffic, the crowd density at various exit/entry points at various timings, the flow and direction of the general foot traffic, overall make up of the surrounding district such as types of corporate, civic & religious organizations operating in the vicinity and number of educational institutions. in addition, an on-site survey to determine potential donor preferences and sentiments in relation to operating hours, tactical outreach and design mechanism of the blood centre was also conducted to help develop a tactical marketing communications strategy. the results indicated that during week days, about 85% of people visiting that vicinity were youths aged 16 to 25 (50.5%) and young adults aged 26 to 39 (33.4%). the main purposes of the visit were for work related activities (22%), attending school (19.1%) and shopping (38.1%). on weekends, 76% of these age groups visited the vicinity for leisure activities or to church. another 24% who visited there were adults age 40-60 years old. most of the respondents had a specific destinations, and most of them indicated lunch time and after office hours as their preferred donation periods. a tactical marketing communications strategy targeting youth and young adults was developed to meet the behavior and preferences of the target group. social media platforms such as online mobile app advertising and locational media buys were employed. in addition, partnerships were developed with nearby educational institutions and churches to host road shows and blood donation awareness activities to engage youths and to foster fun and excitement in the social media atmosphere for the blood collection center. the strategies undertaken proofed favorable as the daily attendance at the new blood collection center has surpassed its baseline target collection of 50 units of blood a day within the 3 months of its operation (jan 2013); and now, has a daily average collection of 60 units of blood. 3a-s10-01 setting up haemovigilance programme from the very first step background: national haemovigilance programmes wherever established have yielded significant data to implement blood safety initiatives. settting up a national haemovigilance programme requires meticulous planning and the following issues need to be addressed; whether reporting will be voluntary or mandatory, what is to be reported and by whom, reporting formats and data submission, resources to sustain the programme, staff training and responsibilities. india is a country of 1.18 billion people, 2700 blood centres, one-third each in public, charitable and private healthcare sectors and annual blood collection of 9 million. given the diversity of blood centre management, setting up such a national programme is a complex task. aim: to set up a national haemovigilance programme in india. method: the ministry of health and family welfare (mohfw), govt. of india had launched the pharmacovigilance programme of india (pvpi) in july 2010, with oversight by the indian pharmacopoeia commission (ipc). adverse drug reaction (adr) monitoring centres were setup in 90 medical institutions in the country and trained staff was recruited, for data collection and submission. after the successful launch of pvpi, the mohfw, initiated the haemovigilance programme, distinct from pvpi with the co-ordinating centre at national institute of biologicals (nib), but in close collaboration with ipc. the broad organizational structure of the programme is as follows; reports will be generated in the medical institution based blood centressubmitted online to the haemovigilance national co-ordinating centre at nibreports will be reviewed by the national advisory committee which will make recommendations to the national co-ordinating centre, ipc for onward transmission to the regulatory authoritythe central drugs standards control organisation to formulate safety related regulatory changes and communicate to blood centres. the programme was formally launched in december 2012. terms of reference of the national advisory committee are: to finalize transfusion reaction reporting form (trrf) for the country. to give expert opinion for collection, collation and analysis of data and development of software for the same. to monitor the quality of data collected. to develop training modules and guidelines for implementation of haemovigilance programme. results: based on recommendations of the committee, the initial focus is on reporting serious adverse reactions as defined by the working party of the international society of blood transfusion, reporting is voluntary and a guidance document and trrf have been made available to the medical institution blood centres, which are the designated reporting centres. the reporting is online through a software -haemo-vigil accessible on the nib website. each centre has been given a unique username and password for login. the security of data submitted through the software has been validated. reporting commenced from february 2013 and till date 434 reports of adverse reactions have been submitted. the data is yet to be analysed. a series of awareness workshops are planned countrywide, five have already been organized. specific funds have been allocated by the mohfw for this programme. conclusion: a well structured programme of haemovigilance has been initiated in india and is now in a phase of development. 3a-s10-02 background: congenital haptoglobin (hp) deficiency being homozygous for a deleted allele of the hp gene, hpdel, was identified in a japanese pregnant woman who had experienced severe anaphylactic transfusion reactions (trs) after infusion of red blood cells (rbcs) and human serum albumin in 1998. in addition, the allelic frequency of hpdel was calculated to be 0.015 by a genetic study of a limited number of the japanese individuals, suggesting that hp deficiency might distribute among the japanese population as a phenotype of serum hp. aims: in this report, we present the results obtained from a hemovigilance survey carried out between 1998 and 2012, in which hp deficiency was identified among japanese patients who had experienced nonhemolytic trs (nhtrs), and those obtained from a screening of hp-deficient japanese healthy blood donors. materials and methods: patients with nhtrs: a total of 19,675 patients who had experienced nhtrs, reported by hospitals to the japanese red cross society between january 1998 and december 2012, were examined. healthy blood donors: a total of 272,068 blood donors who visited the japanese red cross blood centers in the tokyo area between june and august 2010 were examined. testing for identification of hp deficiency: (i) serum hp concentration was determined using peak-rate nephelometry with the detection limit of 6 mg/dl followed by simplified sandwich elisa with the detection limit of 3 lg/dl. individuals who showed a negative result of elisa were assessed as hp-deficient. (ii) the presence of the hpdel allele was analyzed using an allele-specific pcr method. testing for anti-hp antibody: the anti-hp antibody produced in all the hp-deficient individuals was measured by elisa and western blot analysis. it was further analyzed using ouchterlony or surface plasmon resonance technology in some cases. results: thirty-one individuals were identified as hp-deficient among the 19,675 patients who had experienced nhtrs (0.16%). all the patients, except three who could not be tested, were homozygous for the hpdel allele. they were transfused blood products [pc, 17; ffp, 1; rbcs, 9; mixed, 4] . nineteen of them (61%) experienced anaphylactic trs accompanied by severe hypotension and the other patients (39%) experienced milder nhtrs. all the patients except one had a history of transfusion. the anti-hp igg antibody was detected in 28 patients (90%). in addition to the igg antibody, the anti-hp ige antibody was detected in 20 patients (64%). hpdeficient individuals were identified among japanese blood donors with a prevalence of 105/272,068 (0.039%). all the donors were homozygous for hpdel, except one who was heterozygous for hpdel, hpdel/hp2. the anti-hp antibody was not detected in 104/105 (99%) of the hp-deficient donors. conclusions: the higher prevalence of hp deficiency caused by hpdel -homozygosity producing the anti-hp antibody among the patients with nhtrs than in the normal donors (p < 0.001), suggests a higher risk of such trs in hp-deficient patients. hp-deficient individuals are present among normal healthy donors with a prevalence that is expected from its allele frequency. they might be expected as suitable donors for hp-deficient patients to prevent hp-related anaphylaxis. hlaing aa background and objective: collection of the correct blood sample from the correct patient is a vital step in the process of safe blood transfusion. transfusion laboramethods: a study on all reports of mislabeled and miscollected specimens from january 2010 to december 2012 was undertaken and the results were analyzed. mislabeled samples were defined as samples that were incorrectly labeled at the time of collection and miscollected samples were 'wrong blood in tube' samples due to patient misidentification. errors resulting in discrepancies in blood group between the current blood sample and historical records were identified by program flagging during validation of blood group results. these discrepancies were resolved by requesting a second sample from the patient collected by another person. some errors detected at the ward level, were reported by the staff member who had sent the blood sample. workload data for group and screen samples received during 2010-2012 was collected from the annual transfusion laboratory records. using these data, ratio of errors from mislabeled and miscollected samples, to number of group and screen samples received was calculated. results: between january 2010 and december 2012 a total of 162,999 samples were received for abo grouping. of 77 incidents were recorded relating to errors in either sampling or labeling. the overall sample error rate was 0.047% or 1 in 2116 samples. of 52 cases resulted from wrong labeling during collection, 17 cases were due to patient misidentification, five were errors that had occurred during the initial request, in two incidents the cause could not be identified and one labeling error occurred in the laboratory. all errors in labeling resulted from failure to check the pre printed name label with the label on the patient's identity band. in 14 incidents, labeling was performed away from the bedside, in 11 cases name labels of a different patient were found in the correct patient's medical file, in 25 cases labels were taken from wrong patient's file and two errors were due to using prelabeled tubes. of 17 patients had been misidentified and blood taken from the wrong person. root cause for these errors was not following hospital polices in patient identification and sample collection. sixty-four percent of the errors occurred out of normal working hours, mainly during the night while the rest 36% had occurred during normal hours. conclusion: we conclude that mislabeling and miscollected sample errors represent a potential for mistransfusion in our institution. the rates of mislabeled and miscollected samples can be used as key performance indicators in this important step in the clinical transfusion process. this baseline data will be used in formulating standards of performance for sample collection and patient identification and, for implementing risk -reducing strategies. background: haemovigilance is a surveillance programme dedicated for the practice of blood transfusion. it is an important part of the quality system of the blood programme. haemovigilance programme in malaysia was initiated as a national programme in 2003 under the ministry of health (moh). since its inception in 2003 the programme has evolved and has become an integral part of our transfusion service. all adverse events and near misses were reported to the national coordinating haemovigilance unit at the national blood centre (nbc) using a standardised form and predefined criteria. these were compiled and analysed into an annual report for the national background: the process of blood transfusion from blood collection and processing to issue and bedside transfusion of blood components involves several areas of human participation. human error is therefore inevitable in this chain of events. transfusion laboratories have long focused their attention on quality control methods and quality assessment programmes dealing with analytical aspects of blood testing. however, there is enough evidence to suggest that the steps most prone to error are in fact in the pre and post analytical phases. various international accreditation bodies now require clear and effective incident reporting protocols to enhance measures for error trapping and error avoidance. aims: this study aims to quantify and characterize transfusion errors in a joint commission international (jci) accredited tertiary care centre in india. methods: all reports of transfusion related errors, registered in the blood bank or outside, between january 2008 till december 2012 were reviewed and categorised into pre-analytical, analytical and post-analytical events. the process adopted at our centre for assessing transfusion related events at the patient's side uses widely tested criteria of: (1) incident reporting (2) root cause analysis (3) identification of corrective actions results: during the study period 89,156 requests for blood and blood components were received and total of 1,98,505 blood components were issued within the hospital. a total of 16,834 reported errors were analysed. pre-analytical errors comprised a large majority (13,676; 81.24%), most of them being errors of inadequate patient information on request forms (10976; 65.2%) followed by sampling errors (1756; 10.4%). analytical errors comprised (563; 3.3%) and post analytical errors accounted for (2595; 15.4%) of the total errors reported. there was no incidence of acute haemolytic transfusion reaction or direct patient harm during the study period but on several occasions near miss events were reported which, if missed could have background: in australia, we rely on non-remunerated, voluntary donors to provide sufficient blood to meet patients' needs. for fresh components, the australian red cross blood service (blood service) is unable to import components for routine use, so is 100% self-sufficient. hospitals and pathology laboratories are under increasing pressure from government/s to improve value for money for blood and blood products, which is resulting in extra demands being placed on the blood service, especially in relation to lower age at issue and a continuing trend to hold more group o stock and less of the other groups, especially ab. with a typically seasonal inventory pattern for red cells, the blood service has focussed on closer management of blood inventory. aim: the aim of the inventory program was to improve reliability of blood coming into the supply chain and therefore improved reliability in delivery to customers. this is measured by average and variance in the number of whole blood collection packs being receipted at the processing centre. the aim was to reduce the variability in this metric, which would naturally lead to decreased inventory holding requirements, greater control and efficiency, and increased reliability and service to the customers. order fulfilment is another measure used to demonstrate improvement. methods: in order to manage blood inventory effectively, the first step was to introduce a minimum and maximum inventory level, by blood type, by state. this provided a transparent target to aim for. the bands were calculated by firstly setting a minimum inventory level using traditional supply chain safety stock calculations. the next step was to develop a 12 week inventory forecast, using a number of planning assumptions. one of the core assumptions is the number of appointments booked in the lead up to a donation. in order to improve reliability, minimum tar-gets were agreed at 3 months out (re-booking time) through to one week out. a 'traffic light' style appointments portal was developed to provide improved visibility of appointment levels for each collection mode and by state. results: results show that the quarterly standard deviation of blood coming in to inventory has improved from 1711 to 1285 in the last four financial yearsa 25% reduction. in addition, order fulfilment has improved from 82% to 95%, demonstrating that, with improved planning systems and processes, it is possible to manage inventory effectively. the results are demonstrated in the two graphs attached. summary/conclusion: the blood service in australia set a goal to improve reliability of fresh components, in particular, red cells entering finished goods inventory, to improve order fulfilment and provide service excellence to customers. by implementing robust and disciplined planning and reporting systems, reliability has improved which shows that there are methods available to improve the effectiveness of inventory management for blood components. 3a-s11-02 wooi-seong k one of the fundamentals of a blood collection center that procures, processes, stores and supplies blood and blood components to hospitals or other blood banks is an effective and sound management of blood inventory. as blood supply is dynamic, blood supply management requires continuous monitoring and interfacing between blood procurement and inventory management and with hospitals. in an effort to provide adequate, safe and equitable blood supply from voluntary non-remunerated blood donors in the face of increasing demand and decreasing donor population, blood collection centers are also challenged with blood shortages, which unless managed, could impact the healthcare delivery and negatively affect patient care. in order to provide a consistent and reliable blood supply blood centres will have to resort to creative and innovative measure. malaysia, a unique multicultural and multiethnic country celebrates significant religious and historic events as well as commemorations. as such, malaysia observes numerous national and state holidays. in fact, malaysia is ranked as the seventh country in the world in the number of observed holidays. by virtue of its geographical location, malaysia is not exempt from natural and man-made disasters, the most severe being seasonal monsoon floods and flash floods. these and the poor response to blood donation campaigns as a result of 'balik kampung' phenomenon during major holidays due to mass exodus of malaysians to their hometowns, contribute to acute seasonal blood shortages in blood collection centers around the country as well as within the region. adopting a proactive approach to blood shortages includes embracing new measures to recruit and retain blood donors, establishing a blood forecast system, developing a strong network among blood collection centers, being transparent with the blood inventory levels which will lead to greater trust and increased confidence in bts and having a contingency plan. at national blood centre (nbc), the blood action team was formed in 2010. it is a multidisciplinary team comprising of members from the donor education and publicity, donor recruitment, blood procurement, component and processing and inventory divisions as well as medical officers, transfusion medicine specialists and consultant. meetings are held regularly and this has greatly improved the communication interdepartmentally, and has fostered a team whose members are committed to improving blood supply management and preventing blood shortages through discussion and brainstorming sessions. also, blood forecasting is carried out as far ahead as months in advance. the blood stock forecasts are also communicated to blood banks from public and private hospitals which are supplied by nbc, a measure to increase transparency. since the implementation of these measures, nbc has successfully and effectively overcome the annual seasonal blood shortages for the last 3 years. evidently, blood shortages are largely preventable by adopting a proactive approach. 3a-s11-03 . the c/t ratios were calculated and analyzed for each major department. nbc and hkl had continuously introduced several interventions to reduce c/t ratios during the period of this study. results: the overall c/t ratio for hkl had been reduced from 2.2 in year 2005 to 1.9 in year 2012. the four major departments in hkl that showed high reductions in c/t ratio for the same period were obstetrics and gynecology (6.4 reduced to 2.5), surgical (2.6 reduced to 2.1), orthopedic (2.6 reduced to 2.1) and neurology (3.8 reduced to 2.2). in this study, interventions that had contributed to the drastic reduction in c/t ratio were compliance to the maximum surgical blood order schedule (msbos) which was periodically updated within each department, effective communication between clinician and blood bank staff, and continuous medical education (cme) for house officers and clinicians. the active hospital transfusion committee (htc) and hospital transfusion team (htt) also played an important role in reducing the c/t ratio by creating awareness among the paramedics and medical officers regarding the judicious use of blood and blood products, and regular monitoring and audits of the whole transfusion process starting from blood sampling to monitoring of patients during and after transfusion. summary/conclusions: this study showed that several interventions that have been introduced by hkl and nbc such as continuous medical education, compliance with updated msbos, active role of htc and htt, and effective communication between clinician and blood bank staff have successfully reduced c/t ratio in four major departments in hkl. this successful achievement needs continuous monitoring and evaluation to ensure consistency. this can also be a role model that is shared with other hospitals to ensure that the c/t ratio is within the set target. 3a-s11-04 fusion) were collected. during second step, a modeling and simulation were used to define the optimal rcl inventory for the metropolitan area. average rc cell shelflife of regional inventory as well as the number of transfused rcs were calculated. in addition it was hypothesized that an efficient turnover of rc inventory will result in inventory reduction and relatively fresh blood for the transfusion reducing the blood utilization and frequency of transfusion among non-surgical patients especially those with chronic transfusion. results: dynamic inventory management and application of inventory index at regional level (four referral hospitals providing direct health care services to 1.4 and specialized services to 2.0 million population) reduced the regional rc inventory by 32% (1100 rcs to 750 rcs; optimal hospital inventory index of 7.5). this change in inventory was accompanied by a reduction in shelf-life of transfused red cells at 36% (average shelf-life of transfused rcs reduced from 3.45 to 2.21 weeks). the total annual rc utilization and specific categorical data of patients prior to and after the implementation of bump (2010 and 2012) included in table 1 . conclusion: optimization of rc inventory by application of inventory index improved the pattern of regional blood utilization. red cell utilization among chronically transfused patients was decreased by 20% (average). chronically transfused hematology and renal patients showed the highest reduction on rcu (21-26%, p value < 0.0001). that was no change in amount of surgical transfusions. non-surgical (medical) category showed a mild reduction (4%) but statistically was not significant. the results indicate that implementation of bump could significantly improve red cell utilization among chronically transfused patients. this change may also result in reduction of transfusion associated adverse reactions and long term complications like as iron overload. 3a-s12-01 no abstract available. 3a-s12-02 burnouf t 1 , tzeng ys 2 , deng sc 2 , wang ch 2 , tsai jc 3 and chen tm 2 1 taipei medical university, taipei, taiwan 2 tri-service general hospital, taipei, taiwan 3 tatung university, taipei, taiwan background: approximately 15% of diabetic patients develop chronic ulcers, and 25% of those may undergo foot amputation. activated platelet gel, which contains growth factors, has been proposed as an adjunct to promote healing of small diabetic foot ulcers. for large un-healing diabetic ulcers, skin graft is usually needed. we have demonstrated that single-donor allogeneic platelet gel and fibrin glue improve skin grafting to achieve successful persistent healing of large ulcers [1]. however, it is not known whether autologous platelet gel can be beneficial in this application. aims: to evaluate in a prospective study the safety and efficacy of using autologous platelet gel to enhance skin graft take in non-healing diabetic lower extremity ulcers. methods: approval was obtained from the institutional review board of our hospital. eight consecutive diabetic patients aged 25-82 with nine non-healing lower extremity ulcers (median size of 50 cm 2 ; range 15-150 cm 2 ) were enrolled. their median duration of diabetes and ulcer was 10.6 years (range 5-25 years) and 6.5 months (range 3-24 months). none of the patients had received conventional skin grafting in the past. prp was prepared from 100 ml of venous blood using sep-ax-vgr protocol (biosafe sa, switzerland). autologous thrombin was prepared by activating 10 ml of plasma (tgd-001; merries international inc., taiwan). skin ulcer was debrided to remove the infected and necrotic tissues and covered with moist saline dressing. daily dressing change without additional treatment was performed. the wound was sprayed after 7 to 10 days with equal volumes (5 to 7 ml) of autologous prp and autologous thrombin to form the platelet gel within 5-10 s. thin-splitthickness skin graft with multiple slits was then applied on the wound bed and fixed with staples or cat-gut sutures. each patient was placed on antibiotics during the course according to wound cultural results. bolster dressing with sofa-tulle were used to avoid post-graft hematoma formation. negative pressure wound therapy (vac) was not used in this study. results: there was no adverse reaction during the study. eight out of nine skin grafts took well (88% healing rate). the interval between skin graft and complete wound healing ranged from 2 to 3 weeks in the eight successful cases. no ulcer recurrence was noted during the 2-19 months follow-up period. the non-successful case was an attempt to treat an ulcer that was deep to the periosteum of calcaneus bone. free tissue transfer would have been required, but the patient refused the microsurgery, due to age and medical condition, which led to skin graft loss. conclusion: this study shows for the first time, to our knowledge, the possibility to use platelet materials in combination with skin graft procedures to treat large nonhealing diabetic ulcers of lower extremity recently, human platelet lysates (pl) rich in growth factors were shown to replace fbs for ex vivo expansion of various cells, but whether they can be used for cec expansion is unknown. aims: to evaluate the possibility to isolate and propagate cecs ex vivo using a xenogeneic-free, recombinant growth factor-free medium supplemented exclusively with human pl. methods: pl was prepared by cacl 2 activation of apheresis platelet concentrates from three volunteer donors, and centrifuged to obtain a fibrin-free supernatant that was heat-treated (56â°c/30 min; hpl) or not. pl was characterized for proteins, platelet growth factors (pdgf-ab, bdnf, egf and vegf) and chemical composition. cecs were obtained from over 10 bovine corneas (bcecs) using standard procedures and grown in a dmem-f12 medium (containing sodium bicarbonate, selenium, and antibiotics) supplemented either with (i) 5% fbs, 0.5% dmso, 2 ng/ml rhu-egf, 5 lg/ml insulin, 5 lg/ml transferrin, and 1 nm cholera toxin (termed '5% fbs medium'), or with (ii) 2.5%, 5%, 7.5%, or 10% pl or hpl as the only source of protein nutrients and growth factors. cells were grown in duplicates in 6, 24 or 96-well plates at 37â°c in a controlled atmosphere containing 5% co 2 , with medium changes every two days. viable cells were counted for 7 days and cell viability was determined by mtt assay. bcec phenotype was determined by immunostaining using anti-phospho-connexin 43, anti-na/k atpase alpha-1 subunit, anti-zo-1 and purified anti-n-cadherin. anti-mouse and anti-rabbit igg fitc were used as the second fluorescent antibodies. results: pl or hpl contained 55-60 mg/ml total proteins, and a range of approximately 30-40.5, 23.1-32, 0.44-1.82, and 0.24-0.39 ng/ml of pdgf-ab, bdnf, egf, and vegf platelet growth factors, respectively. cecs could be expanded in a med-ium supplemented with 2.5-10% pl or hpl. interestingly, better cell bcec morphology and adherence was found when using hpl compared to pl. cell growth and mtt equivalent to that of the '5% fbs medium' could be achieved only using 10% hpl. in addition, bcecs could be isolated from bovine corneas and subsequently expanded using the dmem/f12 medium supplemented with 10% hpl. bcecs expanded in the hpl-medium maintained their typical morphology, adherence, transparency and phenotype. conclusion: bcecs can be isolated and expanded ex vivo in a growth medium supplemented solely with human platelet lysate material. although further studies using cec from human origin are mandatory to confirm these conclusions, such findings open a possible new paradigm for gmp-compliant, clinical-grade ex vivo propagation of cec and regenerative therapy protocols of human corneal endothelium. platelets are the smallest and second most abundant circulating cells in the blood and their primary role is to maintain the integrity of the vasculature. when blood vessel injury occurs, platelet adhesion and activation receptors recognize subendothelial matrix proteins such as collagen and this can initiate a coordinated series of reactions leading to the formation of a fibrin clot to arrest bleeding. it appears, however, that in addition to hemostasis, platelets also have important inflammatory and immunological functions. as early as the 1960's, reports began to demonstrate that platelets may play an active role in the stimulation and regulation of immune responses. for example, platelets can store and secrete several pro-and anti-inflammatory chemokines (e.g. platelet factor 4 and rantes) and cytokines (e.g. interleukin-1b and transforming growth factor-b) that can affect local immune responses such as chemoattracting neutrophils to sites of tissue damage. on the other hand, platelets may be able to directly regulate adaptive immune responses via their ability to express and secrete cd40/ cd40l co-stimulatory molecules. more recent reports have also suggested that depending on their activation state, platelets may be able to either suppress cd8+ t cell responses or under certain circumstances, present mhc class i associated peptides to activate cd8+ t cells. these studies have suggested that platelets represent a critical link between innate and adaptive immunity. platelet mhc class i expression may also have a detrimental role by conferring tumor cell resistance against immune attack. of perhaps greater interest, platelets have been shown to express the entire family of tolllike receptors (tlr) and this may allow them to act as circulating sentinel cells that first encounter bacterial products for presentation to the innate immune system. in particular, surface expression of platelet tlr4 enables platelets to present lipopolysaccharide to mononuclear cells and neutrophils which modulates their phagocytic capabilities and this has implications for the development of immune platelet disorders. furthermore, tlr9 appears to be contained within a unique platelet granule underneath the cell surface that can be expressed by platelet activation. thus, elucidating the role of platelets in sepsis and a better understanding of the apparent central role that they play as immune cells may be important for the potential development of efficient therapeutic modalities against infections. this lecture will highlight the many characteristics of platelets as immune-like cells will discuss how platelets may be the major controllers of immune responses. macquarie university, sydney, australia malaria remains a major health problem in most of the tropics, and is especially burdensome in economically underprivileged areas. our ability to reduce the high rates of morbidity and mortality due to malaria are hampered by wanning efficacy of current antimalarial drugs and the spread of insecticide-resistant mosquitoes. we desperately need a greater understanding of how the plasmodium parasite succeeds in invading and growing within red cells, how the host responds to an infection, and importantly, the protective mechanisms employed by the host to combat the infection. platelets regulate blood haemostasis, but are now also regarded as an important component of the body's early innate defense against invading microbial pathogens. recently, my laboratory discovered that platelets are able to protect against a malaria infection. in mouse models of malaria, survival to a chronic infection is reduced when platelet levels are artificially depleted. purified human platelets directly bind to p. falciparum-infected red cells in culture and kill parasite within. our current work is exploring how platelets can kill intrerythrocytic malaria parasites. i will present our current understanding of the platelet and red cell molecules involved in the killing mechanism. these include the platelet cytocidal molecule, platelet factor 4 (pf4) and the erythrocyte duffy-antigen molecule, which binds pf4 and mediates the platelet killing effect. the critical requirement of duffy has lead us to propose that platelet-mediated protection against p. falciparum infection is compromised in individuals homozygous for the common duffy-antigen negative allele. 3c-s13-01 graduate school of medicine, the university of tokyo, tokyo, japan although bleeding is a major side effect of heparin, which is used for treatment of thrombosis, heparin also causes a prothrombotic adverse drug reaction called heparininduced thrombocytopenia (hit). hit is caused by the development of platelet-activating antibodies (hit antibody), which is directed against the heparin and platelet factor 4 (pf4) complex. these reactions accelerate platelet activation and coagulation, leading to thrombosis. thus, if hit is strongly suspected clinically in cases of thrombocytopenia and thromboembolism that occur during or after heparin therapy, it is vital to stop all heparins and start administering an alternative antithrombotic drug immediately. in japan, a test to screen for hit antibody (the automated immunoassays based on two types of chemiluminescent immunoassay and a latex-enhanced immunoturbidimetric assay) was approved as a clinical laboratory test in the medical insurance system, in september 2012. only the latex agglutination test is now widely used clinically because of its simplicity, convenience and cost-effectiveness. however, these immunological methods, including enzyme immunoassays (eias), which detect binding of antibodies to immobilized pf4/heparin complexes, may not be employed suitably. the immunological hit tests are useful in diagnosing hit because of their high sensitivity; however, they also often cause overdiagnosis of hit. the value of the selected cut-offs is the key element in ruling out hit. consequently, hit should be confirmed through laboratory detection of platelet-activating antibodies by using functional assays for the hit antibody; it must also be diagnosed based on careful consideration of the clinical picture. in order to diagnose hit properly, our laboratory asks clinicians to assess the pretest probability of hit by using the scoring system (the '4t' scoring: thrombocytopenia, timing, thrombosis, and other explanations). furthermore, our expert staff ensures that the diagnosis is correctly performed, since hit has not been fully recognized in clinical practice in japan as compared to in western countries. the functional assay for hit antibody has been regarded as the gold standard for diagnosing hit in patients in spite of its disadvantages. the platelet activation test procedure is cumbersome, the tests are technically challenging, and limited to specialized laboratories. additionally, the most important requirement for the test is the selection of platelet donors with high reactivity to the platelet activation antibodies. accordingly, in japan, there are very few places where the functional assay is conducted, whereas many institutions still assess patients with only the immunological assay. in our laboratory, the heparin-induced platelet aggregation method is performed, as isotopes such as radiolabeled serotonin release assay should not be commonly used in routine laboratories. in an attempt to improve the sensitivity of the functional assay, we developed two methods for increasing hit antibody reactivity in donor platelets. one is the cooling donor platelet method, used for improving reactivity, and the other a way of donor selection by using monoclonal hit antibody. further studies are necessary to introduce a simple assay method in ordinary laboratory testing to detect platelet-activating antibodies. 3c-s13-02 autoimmune or immune thrombocytopenia (itp) is an acquired bleeding disorder with a low platelet count mediated by immune-mediated mechanisms. this condition is seen in patients with various associated diseases, such as systemic lupus erythematosus, and can also occur without an underlying disease. production of igg autoantibodies to platelet surface glycoproteins, such as gpiib/iiia and gpib, is the hallmark of the disease. it has been thought that anti-platelet autoantibodies promote platelet clearance in the reticuloendothelial system, but recent findings indicate that anti-platelet antibodies also suppress megakaryogenesis, resulting in impaired platelet production. the diagnosis of itp continues to be one of exclusion. several antigen-specific assays for detection of anti-gpiib/iiia and anti-gpib antibodies are reported to be useful in identifying itp patients, but these assays require complicated procedures such as platelet solubilization, and use of commercially unavailable monoclonal antibodies. to solve these problems, we have developed an enzyme-linked immunospot (elispot) assay for detection of circulating b cells secreting igg anti-gpiib/iiia and gpib antibodies, which is a sensitive, specific, and convenient method for evaluating the anti-platelet autoantibody response. in addition, reticulated platelets and circulating thrombopoietin (tpo) are useful in evaluating platelet production status. these findings led us to propose preliminary diagnostic criteria for itp based on a combination of itp-associated laboratory findings, including erythrocyte and leukocyte counts, anti-gpiib/iiia antibody-producing b cells, platelet-associated anti-gpiib/iiia antibodies, percentage of reticulated platelets, and plasma tpo. although the etiology of itp remains unknown, complex dysregulation of the immune system is observed in itp patients. based on a series of experiments using cd4+ t cells reactive with gpiib/iiia derived from itp patients, we have proposed a 'continuous pathogenic loop' model as a mechanism that explains ongoing antiplatelet antibody response in itp patients. this model includes b cells that produce anti-platelet antibodies, reticuloendothelial macrophages that phagocytose opsonized platelets via fcc receptors and present platelet-derived antigenic peptides, and platelet-reactive cd4+ t cells that exert their helper activity upon recognition of the antigenic peptides. once this pathogenic loop is established, anti-platelet antibody production would go on endlessly. recently, regulatory systems that control this pathogenic loop are attracting a great deal of attention. a series of studies in itp patients have found that foxp3+ regulatory t cells are reduced in circulation, bone marrow, and spleen, and are deficient in their suppressive function. in addition, a critical role of regulatory t cells in preventing the anti-platelet autoimmune response has been demonstrated in mice deficient in regulatory t cells, which spontaneously develop anti-platelet autoantibody-mediated thrombocytopenia. in addition, our recent analysis indicates that the eradication of helicobacter pylori leads to up-regulated expression of inhibitory fccgriib on macrophages, resulting in the attenuation of the pathogenic loop. therefore, therapeutic strategies aimed at interrupting this pathogenic loop would inhibit anti-platelet autoantibody production and subsequent increase in platelet count. in fact, current treatment regimens for itp, including corticosteroids, splenectomy, and rituximab, are able to suppress the pathogenic loop. interestingly, tpo mimietics have a potential to induce peripherally induced regulatory t cells, resulting in suppression of the pathogenic loop. 3c-s13-03 seguchi s 1 , maeda t 1 , kanaumi y 1 , kawamura s 1 , kodama m 1 , kawai t 1 , okazaki h 2 and miyata s 1 1 national cerebral and cardiovascular center, osaka, japan 2 the university of tokyo hospital, tokyo, japan background: heparin-induced thrombocytopenia (hit) is a devastating immunemediated thromboembolic complication of heparin therapy. heparin administration can cause conformational changes in platelet factor 4 (pf4), resulting in the production of anti-pf4/heparin antibodies. a subset of these antibodies can activate platelets and monocytes (hit antibodies), leading to thrombocytopenia and a thrombininduced hypercoagulable state. up to half of hit patients suffer from arterial or venous thrombosis. if platelet concentrates are transfused into hit patients, it is conceivable that the transfused platelets can be activated by the same mechanisms that affect the patient's own platelets and trigger the onset of new thromboembolism or exacerbate hit-associated thromboembolism. thus, platelet transfusion is thought to be contraindicated in acute hit patients. however, it remains uncertain whether platelet transfusion is a risk factor for thrombosis in hit patients since only a few studies have investigated this issue systematically. aim: the goal is to clarify whether platelet transfusion increases the risk of thrombosis in hit patients. methods: we constructed a nationwide registry for hit with the approval of the ethical review committee. between august 2008 and may 2013, 329 patients from 185 hospitals clinically suspected of having hit were retrospectively included in the registry with clinical information such as changes in platelet count, timing of heparin administration, episodes of transfusion, thromboembolic events, and the results of serological assays for hit antibodies. hit was definitely diagnosed by the detection of anti-pf4/heparin igg with platelet-activating properties at a therapeutic heparin concentration, but not at a high heparin concentration or with anti-fccriia antibodies. the assay was performed using washed platelets prepared from hit antibody-sensitive healthy volunteers at a reference laboratory. we examined patients who received transfusions of platelet concentrates after hit was suspected. results: of the 329 patients, 85 patients were ultimately diagnosed with hit (25.8%). optical density values of anti-pf4/heparin antibodies detected by elisa were significantly higher in hit patients than in non-hit patients (2.3 ae 0.95 vs 0.36 ae 0.53 for igg/a/m, p < 0.001; 1.68 ae 0.80 vs 0.25 ae 0.37 for igg, p < 0.001). the incidence of thromboembolic events was significantly higher in hit patients (51.8%) than that in non-hit patients (28.3%; p < 0.001). among the 85 hit patients, 20 patients received platelet transfusions after the onset of hit. only two of them experienced a thromboembolic event after platelet transfusion, one within 24 h and the other after 9 days. notably, neither patient was being treated with a thrombin inhibitor at the time. the incidence of thromboembolic events in hit patients who received platelet transfusions was not significantly higher compared to hit patients who did not receive platelet transfusion or non-hit patients who received platelet transfusions after the suspicion of hit arose, respectively. conclusions: to our best knowledge, this is the first systematic report that clarifies the clinical impact of platelet transfusion on the occurrence of thromboembolic events in acute hit patients whose diagnosis was confirmed by a washed plateletactivating assay. even in acute hit patients who possess platelet-activating antibodies, the transfusion of platelet concentrates does not appear to increase the risk of thromboembolism, especially while on thrombin inhibitor therapy. 3c-s13-04 lu p, ling b and li rs background: transfusion platelet matches with antigenic similarity would evoke less allorecognition and immune activation. strategies have been based on the theory that selection for hla-a and hla-b cross-reactive groups (cregs) compatible donor as well as abo/hpa-matched donor will predict good increment in platelet corrected count after platelet transfusion. aim: establish large-sized platelet donor registry with hla class i,hpa,abo-typed to meet the needs of immunized patients with platelet transfusion refractoriness. evaluate the effectiveness of platelet transfusion therapy in ptr patients. progressive management to ptr patients maintain a long-term platelet transfusion strategy. methods: to establish platelet aphaeresis donor registry in shanghai, 1931 repeat donors were typed for hla-a, -b and hpa-1,-2, -3, -4, -5, -6 and -15 using standard pcr-ssp method. eighteen patients with hematologic or oncologic diseases which refractoriness to platelet transfusions from random donors who are receiving 36 units of apheresis platelet products transfusion were studied. results: eighteen patients(eight male, 10 female)showing platelet refractoriness to random donor platelets [1 h corrected count increment (cci) <7500 ml/m 2 , percentage of platelet recovery (ppr) <20%] before. patients phenotyped for both hla-a,b and hpa-1,-2, -3, -4, -5, -6 and -15. apheresis platelets from donor registry in shanghai matched to patients abo, hpa and hla-a and hla-b cross-reactive groups (cregs) are transfused. ten patients ( show 24 h ppr >20%. the mean 1, 24 h cci and ppr values from the best donors were significantly higher than those from random donors they transfused before. conclusion: the use of hla-a,-b and hpa,abo-compatible aphaeresis platelet improves posttransfusion 1, 24 h cci values and percentage of platelet recovery in refractory patients. transfusion with hla-a,-b and hpa,abo-matched platelets is mandatory to reduce the risk of bleeding in ptr patients. refractoriness to platelet transfusions developed at least in 50% of the patients we observed and to maintain a long-term platelet transfusion strategy. establish large-sized platelet donor registry with hla class i, hpa, abo-typed may be needed to circumvent platelet-specific antibodies of unknown specificity in all chronically transfused patients. the optimal strategy for platelet substitution in immunized patients remains a challenge. 3c-s13-05 xia w 1 , xu x 1 , ye x 1 , fu y 1 , deng j 1 , liu j 1 , ding h 1 , chen y 1 , shao y 1 , wang j 1 , li h 2 and santoso s 3 1 guangzhou blood center, guangzhou, china 2 department of biotechnology, guangdong food and drug vocational college, guangzhou, china 3 he institute for clinical immunology & transfusion medicine, justus-liebig univ., giessen, germany background: immunization against cd36 leads to the production of anti-nak a antibodies associated with fetal/neonatal alloimmune thrombocytopenia (fnait), platelet transfusion refractoriness (ptr) and post-transfusion purpura (ptp). however, no data regarding the clinical relevance of cd36 immunization is currently available for chinese population. study design and methods: platelets and monocytes derived from 998 healthy blood donors were typed for cd36 deficiency using flow cytometry. in addition, four patients with suspected fnait (one case) and ptr (three cases) were investigated. nucleotide sequencing was performed to identify the mutations underlying the cd36 deficiency. transfection in mammalian cells (hek-293t) with cd36 mutated constructs was conducted to confirm these results. anti-nak a antibodies were screened by the use of platelet solid-phase kit (pak-plus, gti diagnostics). results: of 18/998 blood donors failed to express cd36 on their platelets surface. in 5/12 individuals no cd36 expression was detected both on platelets and monocytes, suggesting that the frequencies of type i cd36 deficiency (platelets and monocytes) and type ii cd36 deficiency (platelets only) were approximately 0.5% and 1.3%, respectively. nucleotide sequencing analysis of type i cd36 deficient individuals revealed eight different mutations; four of them were not described so far. however, 1228-1239del attgtgcctatt and 329-330delac appeared to be the most common mutations related to type i cd36 deficiency in south chinese population. further analysis showed that the presence of anti-nak a antibodies in one healthy donor (donor 1) as well as in three cases of ptr (patients 2-4) and one case of fnait (patient 1). these results could be confirmed by immunoprecipitation using biotinylated platelets and by antigen capture assay with stable transfected cd36 cell lines. in all ptr patients, transfusion with platelets derived from cd36 negative donors resulted in good increment (24 h, ppr >20%). table 1 shows the mutations found in these five gpiv defective individuals. conclusions: more than 0.5% of cd36 type i deficient individuals are at risk to be immunized through blood transfusion or pregnancy in china. in this study, we could demonstrate that this immunization is of clinical relevance for the development of ptr and fnait. therefore, testing of anti-nak a antibodies should be considered in suspected immune mediated thrombocytopenia. a national registry of cd36 deficient blood donors should be established to maintain bleeding disorders associated with anti-nak a antibodies. since immunization against cd36 is conceivable for other asian populations an international network within laboratories in south asian region should be established in the future. 3c-s14-01 do we really need ffp? the evolving role of pf24 and pre-thawed plasma devine d fresh frozen plasma (ffp) is defined as plasma frozen within 8 hours of collection. while this product maintains a high functional activity of both coagulation factors and anticoagulant proteins, there has been recent movement in some jurisdictions away from reliance on ffp. in many blood systems, an increasing role for plasma frozen within 24 h of collection (fp24) is seen. such plasma shows little difference in functional protein levels when compared to ffp, with the exception of fviii levels which a show time related decay of activity. even factor viii loss can be consistently minimized if whole blood is held on controlled rate cooling plates prior to preparation of fp24. in addition, the activity profiles of coagulation proteins in fp24 prepared in routine production closely resemble those of commercial pooled plasma products. taken together, these observations have led many blood systems to move from the exclusive use of ffp to a mix of inventory of ffp and fp24, if not to the complete removal of ffp from their menu of offerings. the preparation of cryoprecipitate has also been a driver for the retention of plasma frozen within 8 h of collection. since the most common labeling of cryoprecipitate has focused on the content of both fibrinogen and factor viii, in part owing to original role of the latter in the treatment of hemophilia a, collection of ffp has persisted as the starting material for cryoprecipitate production. in jurisdictions where hemophilia or other factor viii deficiencies are treated with factor concentrates, the labeling of cryoprecipitate to emphasize its antihemophilic factor activity is no longer warranted. as data began to accumulate on fp24, similar studies began to appear that investigated the effect of prolonged cold storage of plasma that had been thawed. this led to the introduction in some jurisdictions of the extension of the allowable period of use for thawed plasma from 24 h to up to 5 days, if stored at 4â°c. such practice is increasingly widespread and there is no evidence that patients receiving such products are compromised. from the perspective of health resources management, the use of both fp24 and pre-thawed plasma reduces discard of products or prevents the use of these products in non-group specific recipients. with the advent of massive transfusion protocols which may require pre-thawed plasma at the ready, it also allows better use of relatively scarce but high demand products such as ab plasma. this presentation will focus on a review of the relevant studies of plasma quality for ffp, fp24 and pre-thawed plasma. we will review the appropriate uses of these different components as well as groups of patients for whom specific products should be restricted or supplemented. 3c-s14-02 background: in 2008, the australian red cross blood service (blood service) began a programme of process improvement aimed at maximising the manufacture of clinical plasma components from male donors, which is a key mitigation to the risk of trali (transfusion related acute lung injury). the challenge of sourcing all clinical plasma from male donors is exacerbated in australia due to its adherence to the council of europe guidelines that stipulate a maximum allowable time between collection and freeze of apheresis-derived clinical plasma of six hours, which is considerably more stringent than for most other blood services despite the greater tyranny of distance that exists in australia. aim: the aim of the programme was to achieve a result of 100% of clinical plasma sourced from male only donors. methods: work began in early 2008 to gather detailed quantitative data that linked information on donor panel to collection centre to production facility, and that could be broken down by blood group and by day of the week. this was then formulated into a suite of reports, highlighting opportunities and variance in performance. based on those reports, a cross-functional team designed a range of improvement initiatives across disciplines such as transport, systems enhancements, donor acquisition and processing, such as: incoming blood donation shippers were marked with colour coded labels that notified the receiving production facility of clinical suitability. this assisted with prioritisation and workflow management. additional deliveries of blood from collection centres to processing centres. a range of targeted campaigns and marketing collateral were produced to attract male donors to apheresis plasma donation donor centre collection staff were trained to convert male ab donors over to plasmapheresis donation activity. changes to progesa to prevent manufacture of female plasma were made (after a time). results: the first report in july 2008 showed that the blood service were issuing 87.71% male clinical plasma; the group ab rate was 57.86%. the results improved dramatically by november 2010 in groups o, a and b (99.64%, 99.72% and 99.62% respectively), allowing for progesa to be configured to prevent the routine manufacture of female plasma from those groups, whilst still allowing supervisor over-ride. by march 2012, the results in group ab had improved to 97.77% (chart below) and the directive was given to manufacture male clinical plasma only as routine. january 2013 was the first month ever where 100% of all clinical plasma was sourced from male donors only. in 2012/13, 99.98% of clinical plasma was male onlythe 0.02% constituted short lead time requests for iga deficient plasma. subsequent to a system change that allowed for a donor identification marker for iga deficient donors, inventory levels of this sub-product have increased by 34%, negating the need to turn female production on to accommodate sporadic demand. summary/conclusion: the multi-disciplinary efforts over an extended period of time have resulted in the practical removal of the risk of trali in australia. this is an achievement that many thought impossible and one that many other blood services have been unable to attain. results: quality control (qc) parameters were measured in the prepared blood components and were listed in table 1 . by standardising bc volume and haematocrit in the primary separation, recovery of red cells and plasma was optimised in both the red cells in sagm and plasma units while wbc and rbc contamination levels in pc and plasma were maintained low. all parameters were well within the blood component specifications set out in the council of europe guide (16th edition), the standard adopted by hkrcbts. qc parameters of the pathogen reduction-treated platelet concentrates in pas so produced were also within the hkrcbts blood component specifications. low contamination with red cells and white cells were demonstrated and the ph range was acceptable after 5 days of storage at 20-24â°c. the new t&b production method for the separation of 450-ml quadruple wb and the preparation of intercept platelet concentrates in ssp+ pas was successfully developed and can be applied to the production of high quality blood components in preparation for clinical evaluation of the pathogen reduction-treated platelet concentrates. 3c-s15-01 blood donors are healthy volunteers who give whole blood or blood components by apheresis for altruistic motives. they should be managed in a way that ensures high standards of care. nevertheless, there are recognized adverse reactions that can occur during blood donation. the overall incidence of complications directly related to blood donation is 1%. they are generally more common in women, in younger and in first-time donors. although the incidence seems to be small, it is of great importance considering the large number of people giving blood each day worldwide. adverse blood donation reactions can generally be minimized or avoided by appropriate donor selection and care, and appropriately trained staff. vasovagal episodes and soft tissue injuries (bruises/haematomas at the venepuncture site) are the most common donor reactions. the majority of these are minor and donors usually recover quickly; however, these reactions can be of concern to donors and reassurance should be provided. other reactions include nerve injury and arterial puncture which, although less frequent, may require medical care outside the blood service and may lead to prolonged symptoms or incapacity. staff should be trained to recognize and manage such adverse reactions, including the provision of first aid. the incidence of bruising should be monitored so that further venepuncture training may be provided to staff as necessary. iron deficiency in regular blood donors has been a top donor health and safety concern in many countries. as each donation causes a loss of 213-236 mg of iron, repeat donation can lead to a continuous depletion of body iron stores. studies have shown that donation frequency had the greatest impact on iron deficiency and further risk factors were lower weight and female gender. to address this issue, many blood services encourage donors to take iron-rich food and/or give them iron supplements. adverse reactions such as delayed faint may occur after the donor leaves the donation venue. donors should be advised to inform the donor centre staff of any ill-effects they suffer after donating. a system for the reporting and investigation of adverse donor events and reactions should be in place as part of the donor haemovigilance system. all adverse events and reactions in donors should be identified, documented and reported. these data should be regularly analysed for possible corrective and preventive actions. the goal of donor haemovigilance is to reduce the occurrence of adverse events and reactions and improve the outcomes both for donors and patients. for various reasons, even today, donors often do not receive detailed information on the blood donation procedure and possible complications. not only do blood services have the ethical duty to inform donors of possible adverse events to enable them to give informed consent and take action for preventing adverse effects, protecting the safety of donors is also important for donor retention because a safe and good donation experience ensures donors will return regularly. 3c-s15-02 wiersum-osselton jc trip national hemo-and biovigilance office, the hague, the netherlands background: without blood donations and the availability of blood transfusion, many important therapeutic advances could not have been achieved. donor hemovigilance is the systematic monitoring of adverse reactions and incidents in the whole chain of blood donor care, with a view to improving quality and safety for blood donors. method: this 'global update' draws on work by the international haemovigilance network and international society for blood transfusion haemovigilance working party, experience in the netherlands, as well as a pubmed search using terms blood donor and adverse reaction. results are discussed for vasovagal reactions, needlerelated complications, long-term morbidity, donor iron status and frequent apheresis. results and discussion: the occurrence of vasovagal reactions is associated with young, female donors, lower body weight and estimated blood volume, first-time donor status. a reduction of vasovagal reactions has been documented with use of a water drink before donation, muscle tensing, social distraction and lower collection volume for donors with small estimated blood volume. needle injury is relatively frequent as a cause in cases of long-term morbidity; needle injury is associated with traumatic phlebotomy and in some cases nerve damage is documented. repeated whole blood donations lead to reduction of body iron stores and in some cases anaemia. some blood services adjust donation intervals to avoid or reduce this, while others have or are considering a policy of iron replacement therapy. fewer studies on acute complications in plasma and other types of apheresis have been published. preliminary studies of bone density and protein levels in non-commercial frequent plasma donors have not substantiated any specific hazard despite theoretical concerns of calcium or protein depletion. international collaboration in strengthening donor vigilance definitions and data analysis may in future increase potential for study of risk factors and measures to improve donor care worldwide. conclusion: donor vigilance is gaining international interest and has increased knowledge of risk factors for vasovagal reactions associated with blood donation. there remains a need of research and of developing preventive measures, including prevention and treatment of needle injury as well as possible long-term effects of frequent donation. assuming that these donors were newly infected, it is crucial for bts to monitor the prevalence of this category of donors in order to strategize specific measures to these targeted groups to improve blood safety. aims: this study aimed to profile blood donors who donated during the hiv serological window period and to identify the risk factors of these blood donors. methods: past donor records of blood donors who had donated blood during the hiv serological window period (nat ultrio and discriminatory detected and negative for both anti-hiv and p24 antigen) at nbc or at blood mobiles organized by nbc from november 2007 until july 2013 were retrieved and analyzed using spss 15.0. results: a total of 18 donors were nat detected and negative for anti-hiv and p24 antigen (none in 2007, 2 in 2008, 2 in 2009, 1 in 2010, 6 in 2011, 4 in 2012 and 3 in 2013 introduction: in pakistan, the predominant reliance for blood supply is on the replacement donors, as sufficient numbers of voluntary blood donors are not available. an increase in the proportion of voluntary donors following the promotion of the concept of voluntary non-remunerated blood donation (vnrbd) will enhance safety and will also help to shift the responsibility for arranging blood availability from the patients to the health care system. objective: the objective of the current study was to promote vnrbd through a public awareness campaign (pac) based on a thorough analysis of the knowledge, attitudes and practices (kap) of a key segment of the society, i.e. 18-25 years old college and university students. material and methods: a cross-sectional, descriptive study was conducted over a period of three months (jan-mar 2012). multi-stage random cluster sampling approach was followed and college and university students were targeted through 20 university based blood donor organisations (bdos) out of a total 56 bdos identified. all the participants voluntarily participated in the study and informed consent was obtained orally. a pre-tested questionnaire comprising of 28 questions related to knowledge, attitudes and practices was applied. the questionnaire was kept anonymous and each question included multiple options or statements. statistical analysis was conducted by the assistance of statistical package for social sciences (spss) software version 17. results: a majority (65%) of the students had heard about blood donation through family/friends and a minority (9%) through the internet, although 45% preferred internet as spare time activity. majority (+85%) of the students had access to the internet and mobile phone. more than 50% of the respondents had donated blood: 22% donated for family, relatives or friends, 4% donated voluntarily as an act of altruism, and 25% donated voluntarily once and then stopped donating, but 55% of these respondents still considered themselves as volunteer blood donors. 35% indicated that important people in their environment had an influence on crucial decisions that they made. motivation for blood donation was a desire to help other people (73%), 28% followed friends invitations, in 21% cases respondents family or friends had received blood transfusions, 16% followed the example of fellow students. restriction for blood donation: 40% generally feared donation, 25% had a specific fear of the needle, 20% had no confidence in the public (health) sector, 25% condemned blood selling practice, 14% had no confidence in the donation procedure (hygiene), 13% experienced parental discouragement. conclusion: to overcome the apprehensions and fears of the donors it is important to provide adequate information about donation to potential donors. this strategy will help convince family replacement donors to become vnrbd and also recruit healthy individuals to become a vnrbd. the approaches and strategies for this transition can be based on the findings of the study. the reported preference for internet as leisure time activity suggests that internet can be utilized as an important tool for information dissemination in a pac, for which detailed study is required. 3d-s16-01 murphy mf platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. there are immune and non-immune causes of platelet refractoriness. the main immune cause is hla alloimmunisation which occurs predominantly in females with a history of pregnancy. other immune causes include hpa alloimmunisation, abo incompatibility, platelet autoantibodies and drug-related platelet antibodies. the incidence of alloimmune platelet refractoriness due to hla antibodies has declined due to leucocyte-reduction of blood components and more aggressive treatment for patients with haematological malignancies and other cancers. in current practice, platelet refractoriness is mainly due to shortened platelet survival associated with non-immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, dic and splenomegaly. if there are poor responses to hla-matched platelet transfusions, the reasons should be sought including hla incompatibility which is most likely to occur in patients with unusual hla types with few well-matched donors, non-immune platelet consumption, and hpa and abo incompatibility. further serological investigations including testing for hpa antibodies may be used to differentiate between these possibilities. depending on the results, the appropriate management could be the use of abo-identical or hpa-matched platelet concentrates if the specificity of the hpa antibodies can be identified. platelet crossmatching may be helpful in some patients with non-specific hpa antibodies. the management of patients with hla and/or hpa alloimmunisation and no compatible donors may be very difficult. there is no evidence that alloimmunised patients benefit from incompatible platelet transfusions which do not produce an increase in the platelet count, and prophylactic platelet support should be discontinued. if bleeding occurs, platelet transfusions from random donors or the bestmatched donors, despite being incompatible, may reduce the severity of haemorrhage although increased doses of platelets may be required. other management approaches such as the use of high-dose intravenous immunoglobulin, splenectomy, and plasma exchange have not been shown to be effective. the management of patients with non-immune platelet consumption is similarly problematic. the usual practice is to continue with daily platelet transfusions as prophylactic platelet support, but it is not known whether this approach is effective, or whether platelet transfusions should be discontinued or the dose of platelets increased. 3d-s16-02 managing bleeding in cardiac surgery: despite major advances in the management of perioperative blood conservation, transfusion rates in cardiac surgery remain very high, with large variations among individual centres. among all major surgical procedures, cardiac surgery with cpb still consumes a large part of the available blood supply. in england indicated that 10-15% of the blood units supplied by the national blood service is used in cardiac surgery units. in the usa, nearly 20% of blood transfusions are associated with cardiac surgery. during the early history of cardiac surgery, patients received large amounts of allogeneic blood. in the 50's, most operations were performed to correct congenital heart disease. during the 60's and 70's, the introduction of satisfactory valve prosthesis and direct grafting for atherosclerotic coronary artery disease led to rapid growth in the scope and number of patients having open heart surgery. in the 80's pharmacological methods to reduce bleeding were introduced and the focus of blood conservation was expanded to include blood components as well as red cells. with increasing application of cardiac surgery in acutely ill older patients with more comorbidities as well as the increasing safety of blood supply have contributed to an increasing incidence of allogeneic transfusions. not surprisingly, physicians, surgeons and anaesthesiologists have shown a great interest in the promotion of safe and effective alternatives to the transfusion of allogeneic blood in cardiac surgery. perioperative risk factors for allogeneic transfusion can be regrouped in three main categories: factors affecting the patient's preoperative rbc mass, factors affecting the perioperative blood losses, and factors affecting the transfusion practice. the ability to predict a patients risk for transfusion allows modification of patient management with the goal of decreasing allogeneic transfusions. using the trac and trust scoring system predicts candidates likely for transfusion. diminished rbc mass appears to be one of the strongest predictors of transfusion. the acceptance of a lower postoperative haematocrit (in ijn the hct on bypass is 20-25% and post bypass is >25%) or haemoglobin concentration represent an important element in current blood conservation practice. the decision to transfuse a patient cannot be based only on haematocrit concentration. optimizing preoperative rbc mass involves the early detection of anaemia and its correction before surgery. preoperative autologous blood donation can be used to conserve allogeneic blood. besides economic concerns, one essential argument against pad is the lack of sufficient time because of the uncertainty of waiting list. erythropoietin has also been used to augment pad in elective cardiac surgery. acute normovolaemic haemodilution (anh) aims at reducing allogeneic blood exposure through a reduction in the net red blood cell mass lost during or just after surgery. perioperative cell salvage (cs) also aims at reducing allogeneic blood exposure through a reduction in perioperative blood loss. antifibrinolytics (ltranexamic acid or epsilon aminoaproic acid) or serine protease inhibitors (aprotininnow unavailable) may reduce excessive fibrinolysis and platelet dysfunction. the use of activated f vii has been reported in intractable bleeding post cardiac surgery. 3d-s17-01 the university of tokyo, tokyo, japan antibodies against human neutrophil antigens (hna) are involved in the pathogenesis of immune neutropenia, such as neonatal alloimmune neutropenia (nan), refractoriness to granulocyte transfusions, and transfusion reactions, such as febrile non-hemolytic transfusion reactions, and transfusion-related acute lung injury (trali). the hna systems are assigned to five antigen groups, namely hna-1 to 5. hna-1, -2, and -3a alloantibodies have been implicated in the pathogenesis of trali, and especially hna-3a alloantibody has been found in the severe cases requiring artificial ventilation or with fatal reactions. besides alloantibodies to hna-1, -2 and -3, those against hna-4a and hna-5a have been implicated in nan. the identification of the causative antibodies is essential for the diagnosis as well as for the prevention of these disorders. the detection of hna antibodies has been mainly dependent on cell-based assays so far. among them, the granulocyte agglutination test (gat), the granulocyte immunofluorescence test (gift) and the monoclonal antibody immobilization of granulocyte antigens (maiga) are the most commonly applied. according to the isbt working party on granulocyte immunobiology, the combination of gat and gift is presently the best means of hna antibody detection. gift is usually more sensitive than gat, however, hna-3a antibodies associated with severe cases of trali are better detectable by gat. in gat and gift, the presence of hla antibodies with broad specificities may affect the detection of hna antibodies. on the other hand, the maiga assay allows the differentiation between hna and hla antibodies. these classical methods, however, require fresh neutrophils from hna-typed donors. also, these assays are time-consuming, which makes them not appropriate for the large-scale antibody screening. in our lab, we modified the mixed-passive hemagglutination (mpha) assay, the method largely applied in japan for platelet antigen/antibody detection, for the detection of hna antibodies. recently, alternative assays have been developed, including elisa with recombinant hna (rhna), and immunofluorescence tests with transfectant cells of hna (hna-1, -2, -4, and -5). more recently, the molecular basis of hna-3 antigen has been elucidated, and stable cell lines expressing hna-3 antigens became available. these cell lines seem to have low background, and do not express detectable levels of hla antigens, which make the identification of hna antibodies easier. additionally, kits that use luminex microbeads coated with hna antigen are being developed. these kits, however, do not include hna-3 antigens. these new technologies significantly help improving the detection and identification of hna antibodies, and allow the large scale screening of hna antibodies, contributing for the reduction of the risk of the pathological conditions associated with hna antibodies, especially trali. however, these new technologies significantly increase the cost of the tests. presently, although many assays have been developed, the standard hna antisera are not necessarily available in every lab, which makes their validation difficult to be conducted. thus, the collaborative study among the various labs, by exchanging the available antisera, and comparing the test results, is essential for the improvement of this field. 3d-s17-02 one of the main sites where pmns carry out vital to surveillance functions is in the lungs. the large surface area of the lung is needed for gaseous exchange but lungs also present a vital direct mechanical barrier to the external environment. to patrol and protect this interface, about 28% of the body's total pmns are located in the pulmonary microvasculature. illness may increase the number of lung pmns as well as change their phenotype from quiescent to primed. in trali, the transfusion of blood products with either pmn reactive antibodies or biological response modifiers can activate this concentration of primed pmns to produce an augmented respiratory burst. this causes injury to the pulmonary microvasculature and consequently the symptoms of trali. circulating antibodies to pmns also can compromise their numbers and function. pmns carry human neutrophil antigens (hna) as well as class i hla, which can become targets for pmn reactive antibodies. the granulocyte immunofluorescence test (gift) and granulocyte agglutination test (gat) are primary tools for investigating these pmn reactions, as they are able to detect reaction of hna as well as some hla class i antibodies. immune neutropenias: alloimmune neonatal neutropenia (ann) occurs when a neonate's pmns are destroyed by transferred maternal antibodies developed against an inherited paternal neutrophil antigen. this is similar to haemolytic disease of the newborn, but importantly can occur with the first pregnancy. in early childhood, some children develop severe neutropenia as a result of pmn auto-antibodies. although the pathogenesis of such chronic benign autoimmune neutropenia is still not understood most of these autoantibodies demonstrate specificity for the hna system. passively acquired autoimmune neutropenia, wherein pmns are destroyed by maternal pmn auto-antibodies crossing the placenta are a rare finding. hna specificity is unlikely. autoimmune neutropenia in adults is either primary, secondary to another autoimmune disease or drug related. it can present a clinical and diagnostic challenge as many adults invariably have alloantibodies to neutrophils and the patient's neutrophil count is too low to make a definitive identification of a self reactive autoantibody. hna specificity is extremely rare. the severity of trali and immune neutropenias demand rapid and precise diagnosis with reliable neutrophil serology. the isbt granulocyte immunobiology working party maintains a list of granulocyte immunobiology reference laboratories around the world. 3d-s17-03 when seven sera from 778 donors were screened for neutrophil specific antibodies, 9% samples showed positive reaction. these results, however, could not be confirmed by gift and gat. conclusions: in this study, we found alloimmunization against hla class i and ii in 4.6% male,~4.3% nulliparous and~13.9% parous females. in contrast, alloimmunization against hna was not detectable in this cohort. these results indicate that the use of plasma containing blood products from parous females without hla antibodies pre-testing may increase the risk of trali reaction. although alloimmunization against hna seems to be a rare event in china, further observation is necessary to exclude the necessity of hna antibodies screening in our blood products. it is becoming clear that the ccn family of extracellular proteins play an important role in the health and function of several cells of the hematopoietic lineage. ccn2, also known as connective tissue growth factor, ctgf, has recently been found to be in high abundance in platelets and released upon activation, an effect inhibited by aspirin, suggesting a role in blood clotting and/or wound healing. on the other hand, ccn3, also known as nephroblastoma-overexpressed, nov, has been found to play an important role in hematopoietic stem cell health and function. in fact, treatment with ccn3 has recently been shown to promote hematopoietic potential, a discovery with dramatic clinical potential. initially using bioinformatics, our laboratory has discovered a signaling pathway that connects these two discoveries and appears to be a key functional node within the development of blood cells. specifically, we have found that the myeloid zinc finger protein 1, mzf1, is a transcription factor that trans-activates both ccn2 and ccn3, in distinct cell types. for example, we have shown that mzf1 can stimulate ccn2 production and secretion in stromal fibroblasts, which is then taken up by megakaryocytes and loaded into platelets. this is the first time that ccn2 loading into developing platelets has been directly achieved and observed in vitro. secondly, we have discovered that mzf1 also regulates the synthesis of ccn3 in several hematopoietic cell types. putting these results together with previous data suggests a new and immediately testable clinical treatment. it is known that both vitamin d (calcitriol) and vitamin a (all-trans retinoic acid) stimulate transcription of the mzf1 gene. (we also have new data exploring the mechanism and suggesting other pharmacological ligands). we have confirmed that treatments with either vitamin a or d activate this pathway and results in increased production of ccn2 in stromal fibroblasts, which in turn results in enhanced loading of ccn2 into developing platelets in vitro. similarly, we have observed that both vitamin a and vitamin d induce ccn3 expression, through mzf-1, and we are currently testing if this will lead to enhanced hematopoietic potential. this work could impact the efficacy of blood donation and transfusion, bone marrow transplants, and the treatment of bleeding and clotting diseases as well as lymphomas and leukemias. 3d-s18-05 background: foxp3 + t regulatory cells (tregs) consisting of natural and induced treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. while recent studies demonstrated that natural tregs are instable and dysfunctional in the inflammatory condition, induced tregs (itregs) may have a different feature. furthermore, it was reported that tolerogenic dendritic cells (tdcs) could expand itregs in vitro and this action designed to correct defects in numbers or functions of itregs may be therapeutic in the treatment of autoimmune diseases. in this study, the suppression efficacy of tgf-beta-induced tregs expanded by tdcs in vitro and in mouse model of autoimmune arthritis was determined. method: in vitro, first, cd4+ cd25ã� t cells were purified from splenocytes of d1 mice and stimulated by anti-cd3/cd28 in the presence of tgf-b1 for 5 days, which were termed 'itreg'. and tdcs derived from bone marrow of d1 mice were induced by gm-csf, il-10 and tgf-b1 and harvested after 10-day cultivation. then, itregs were expanded by tdcs at the ratio 5:1 and collected after 4 days (termed 'itreg tdc '). the phenotype, proliferation, suppression of cd4t proliferation, induction of foxp3 + tregs from foxp3ã� t cells and suppression of th17 cell differentiation were assessed. for in vivo experiments, the animal models of ra were established. in this model, arthritis was induced in d1 mice after immunized with bovine type ii collagen (cii) on day 0 and day 21, termed collagen-induced arthritis (cia). and 1 9 10 6 itregs or itreg tdc cells were transferred <iv. into cia recipient mice when cia onset. control mice were treated with pbs. clinical and histopathologic scores, cytokine and anti-cii antibody secretion in serum and cd4 + th subsets were analyzed. results: after 5-day induction in vitro, the percent of foxp3 + itregs increased from 9.8 ae 1.5% to 54.9 ae 2.5%. and these itregs could be expanded effectively by tdc in additional 5-day co-incubation, which continued to express higher levers of foxp3 (82 ae 3.6%) and increased 4.17 ae 0.17 fold. compared with itregs, the itreg tdc cells showed stronger inhibitory effect, and could prompt cd4t cells converting to foxp3 + itreg and resistant to th17 cell differentiation more effectively. during the 4-weekobservation-period, a more remarkable anti-arthritic activity, improved clinical scores and histological end-points were found in the itreg tdc -treated group than those in itreg-treated group. serological levels of tnf-a, il-6, il-17 and anti-cii antibodies were also significantly lower and tgf-b production were higher in cia mice following adoptive transfer of itreg tdc cells as compared with itregs. moreover, the itreg tdc treatment resulted in an increase in the number of tregs (cd4+ foxp3+) and a decrease in the percentage of th17 cells (cd4+ il-17+) more obviously. conclusion: these results indicated that itregs expanded by tdcs showed more effective suppression in vitro, and reduced more markedly the severity and progression of cia than itregs. this reduction was associated with modulating inflammatory cytokine and anti-cii igg secretions to lower levels and polarizing the treg/ th17 balance to treg more obviously. this study highlights the potential therapeutic utility of itregs expanded by tdcs in autoimmune arthritis and should facilitate the future design of itreg tdc immunotherapeutic strategies against ra. in may 2010 the world health assembly passed a resolution, wha 63.12, on the availability, safety and quality of blood products. this urges member states to 'take all necessary steps to establish, implement and support nationally co-ordinated bts with the aim of achieving self-sufficiency'. the resolution applies to both fresh blood components and plasma derived medicinal products (pdmps). who defines self-sufficiency as meeting national needs for six products from blood and plasma collected locally. these comprise red cells, platelet concentrates, fresh frozen plasma, and factor viii, immunoglobulin and albumin solution. the definition and overall goals were established by a who expert consensus statement on 'achieving self-sufficiency based on vnrbd' developed at a meeting of who experts in geneva switzerland in 2011. blood services develop in line with the health care system that they support. typically this involves a transition from a supply for whole blood mainly in the context of emergency management of trauma and child birth to the implementation of blood component therapy. blood component production will likely lead to an excess of recovered plasma. effective utilisation of this requires access to plasma fractionation facilities. if this can be achieved then this will reduce reliance on commercial pdmps and ensure maximal utilisation of the donated whole blood. increasing demand for pdmps will potentially lead to a requirement for introduction of plasmapheresis programmes if the goal of self-sufficiency is to be maintained. there are a number of significant barriers to both utilisation of recovered plasma and the implementation of effective plasmapheresis programmes. individual countries will need to consider these as part of their long term strategic planning process. the main barriers for low and medium hdi index countries are access to suitable plasma fractionation facilities. few countries will be able to justify creation of a national fractionation plant. regional co-operation and development of facilities will be needed. secondly plasma for fractionation is seen as a critical material in the manufacture of a medicine. quality system requirements, including full compliance with a code of good manufacturing practice will be a pre-requisite for acceptance of the plasma as suitable for fractionation. there is an urgent need to develop support programmes to raise standards to achieve this. in contrast the main barrier for most high hdi countries is cost and the easy availability of pdmps produced by commercial countries from paid plasma. the safety profile of pdmps is currently excellent since the integration of a number of specific viral inactivation steps in the manufacturing process overcomes any infectious burden associated with payment for plasma. the critical question is then how a country can assure ongoing supply at a reasonable cost. ethical principles will play an important role in defining the strategy and individual countries should be able to define their own approach without unnecessary intervention and competition from the commercial plasma industry. 4a-s19-02 background: blood transfusion service of nepal red cross society was established in the year 1966. since the beginning nrcs has been running the service exclusively with assurance of government of nepal. in 1991 the government of nepal elaborated the mandate with clear policies and declared nepal red cross society as the sole authority to conduct the national blood program through out the country. at present it consists of 86 centers in 62 districts. national leadership and oversight of management and quality standards is the responsibility of the central blood transfusion service, who in partnership with regional and district centers and aims to provide a safe and secure supply of blood/blood products to all needy in the country. in the initial years the service was made possible through collection of blood from professional donors but since 1982 collection of blood was emphasized from voluntary non-remunerated donors only. aims: blood service is provided according to the need base and with the expansion of health services, establishments of medical colleges, government hospitals, private hospitals and nursing homes the demand of blood is rapidly going up through out the country. nationwide more than 250,000 units of blood were served to the needy and around 60% of it is served through the central blood transfusion service. results: with the view of providing safe blood and bloodproducts to the needy patients all collected blood is routinely tested for hiv, hbsag, hcv, syphilis besides grouping, cross-matching and anti-d antibody identification. blood components service is limited in few centers. conclusion: blood program is run through cost recovery basis with material and nominal service charge nationwide. no defined budget is allocated for blood program. there is on and off support provided from nepal government, un agencies, national societies and international non government organizations. currently, the total revenue raised for the blood program is insufficient to meet the total cost of production of blood and blood products. 4a-s19-03 background: blood transfusion services in cambodia face a number of significant challenges in providing a supply of safe blood and blood products sufficient to meet the clinical needs of patients. these challenges include access to adequate and sustainable financing, trained staff, sufficient voluntary non-remunerated blood donors (vnrbd) and appropriate equipment and facilities. aims: a 2011 programme funded by the us presidents emergency plan for aids relief (pepfar) and managed by the us centres for disease control (cdc) was initiated by the cambodian based us cdc office, in a collaborative partnership with the nbts and ministry of health (moh). specific blood program needs were identified and presented in a logical task order framework. the task order required support at both the national and provincial level across the whole blood sector; and priorities three key deliverables: comprehensive assessment of the country's current practices and infrastructure including national policy, legislation, collection, testing and the clinical use of blood. vnrbd recruitment strategies/protocols. a new 5 year national strategic blood plan. methods: the selected technical assistance partner (ta) developed a proposal that addressed the areas of the task order, and utilised a number of strategies to deliver the technical program; including: a critical initial design phase with an introductory visit to map prior local and development partner work and draw on in-country. seeking ongoing engagement and advocacy from local thought leaders. tailoring assessment and strategy planning tools to local requirements. use of inclusive communications with all stakeholders. strengthening of local governance infrastructure (blood safety working group) . the 1 year program resulted in development and delivery of an assessment report on cambodia's blood transfusion services, a knowledge, attitudes and practices survey and a five year national strategic blood plan (nsp background: indonesia has recognized that blood transfusion plays a key role in their modern health care; yet ensuring the availability of safe blood is a challenge for a population of over 250 million living on 6000 islands with limited health care resources with a high background prevalence of hepatitis b virus (hbv) of 9.4%, human immunodeficiency virus (hiv) of 0.2%, and hepatitis c virus (hcv) 0.4%. aim: a health economic study was performed by the irc to evaluate the economic impact of individual donor (id) nat on the burden of disease (hbv, hcv, hiv) associated with transfusion transmitted infections (ttis) and understand the comprehensive costs to the ministry of health (moh) of providing id-nat-tested blood. method: a health economic analysis was performed using a markov method, which predicts the probability of an outcome based on a chain of outcomes and defined transition probabilities. the two arms of the analysis compared the testing costs and treatment costs of: (i) serology alone, and (ii) serology and nat. testing costs were defined as the total costs to the indonesian moh of purchasing the reagents used in conducting either serological testing alone or serological and id-nat testing on donor blood units. the treatment costs were defined by the outcomes associated with each of the three viruses (hbv, hiv, hcv) and the likelihood of various clinical outcomes for patients infected by transfusion. indonesia-specific data drawn from peerreviewed published sources was used to populate the model. in cases where published local data was not available, proxy published data from other geographies and data collected from interviews with local transfusion experts were used. cost information for id-nat and serology testing was sourced from test manufacturers. results: previous effectiveness of nat testingin annual collection of 2.1 million donations determined that id-nat could prevent 3187 infections missed by serology screening. if id-nat assays were not available ttis represents a calculated usd $73 million in treatment costs for the indonesian healthcare system. the introduction of id-nat could saveusd $27 million per year (or~20% savings) due to reduced treatment costs by preventing ttis. the estimated savings exclude costs associated with lost productivity and litigation. conclusion: sincethe initial analysis, additional testing has been performed in variousdemographic locations and it is now estimated that 8500 whole blood units andup to 15,000 blood components could be interdicted with id-nat. these new datarepresent even more favorable economics to the moh. while id-nat represents an additional cost tokeep blood as safe as possible, the incremental cost comparison of serology andid-nat compared to serology testing alone shows a minimum estimated savings ofusd $27 million. based on these results, id-nat blood screening appears to be aclinically and cost-effective way to prevent transmission of infection to patients. 4a-s20-01 yee-sin l tan tock seng hospital, singapore, singapore arbovirus is an acronym for a group of viruses that are transmitted by arthropod vectors, commonly mosquitoes and ticks. person-to-person transmission of arboviruses is not common. however, transmission can occur through transfusion of blood and blood products, and organ transplantation, if the virus is present in the donor's blood or organ. the majority of arboviral infections in humans are asymptomatic. if symptomatic, the symptoms generally occur 3-15 days after exposure. clinical features of arboviral infections can be classified into four groups: acute central nervous system illness, acute benign fever with or without exanthem, hemorrhagic fever, and polyarthritis. there have been published reports of transfusion-transmitted infections of a number of arboviruses, including west nile and dengue viruses, as well as colorado tick fever and tick-borne encephalitis viruses. the risk of transmission depends largely on the burden of the disease in a particular geographical location, the proportion of asymptomatic infections in which clinical diagnosis is not possible, and the availability of diagnostics to detect low levels of viruses in the blood. west nile virus (wnv) entered the united states (us) in 1999 and has spread across the entire north american continent. wnv is now the most significant arboviral infection in the us. one out of every five exposed individuals will develop illness and 1 out of 150 may suffer from severe infection with significant neurological sequelae. in 2002, 23 transfusion-transmitted wnv infections were described. since 2003, all blood donations in the us have been routinely screened for wnv rna using wnv nucleic acid test (nat). since the implementation of screening, 13 transfusion-associated transmissions of wnv have been documented (mmwr 2012) . dengue is the most common arboviral infection in the world. the world health organisation (who) estimates that 50 million dengue infections occur annually and approximately 2.5 billion people live in dengue endemic countries (who 2009 ). the ratio of asymptomatic to symptomatic infections vary widely. singapore reported three cases of transfusion-associated dengue infections involving one blood donation episode. an asymptomatic repeat blood donor informed the national blood bank that he had fever the day after blood donation. by then, packed red cells and other blood products from his donation had already been transfused to three recipients. all three recipients were infected; two of the recipients' blood and the donor's stored serum sample were tested positive for dengue serotype 2 virus by pcr. wilder-smith et al. estimated the risk for transfusion-associated dengue transmission in singapore to be 1.6-6/10,000 blood transfusions in the year 2005, assuming a ration of asymptomatic-to-symptomatic infections of 2:1 to 10:1. subsequently, teo et al. reported the practice of deferring blood donation from recently returned travellers from dengue endemic areas. the massive outbreaks of chikungunya fever in the indian ocean and southeast asia again raise concerns about blood safety. although there has yet to be a reported case of transfusion-associated chikungunya virus infection, such a transmission route remains a distinct possibility. liumbruno gm et al. reported an economic loss of approximately 2 million euros, resulting from the suspension of blood donations at the peak of the chikungunya outbreak in italy in 2008. 4a-s20-02 australian red cross blood service, perth, australia background: arthropod-borne viruses (arboviruses) are an expanding global threat to human health. among them dengue (denv) and west nile virus (wnv) have both been documented to be transfusion-transmissible and transfusion is a plausible route of infection for several others including chikungunya virus (chikv) and ross river virus (rrv). addressing the transfusion risk associated with arboviral outbreaks poses new challenges. unlike established viral threats to the blood supply (e.g. hiv, hcv and hbv) in which the risk is generalized to the population-at-large and chronic infection is common, arboviruses are characterised by focal and often seasonal outbreaks of acute, predominantly self-limiting infection. thus, unlike traditional viral threats the risk is not 'continuous' in nature complicating risk mitigation strategies. aims: to describe possible risk management strategies for arboviral transmission including; geographical donor deferral/restrictions on labile component manufacture, donation testing and pathogen inactivation (pi) which can be applied individually or in combination. methods: how strategies are applied depends on the nature of the risk i.e. whether internal (i.e. local outbreak) or an external (i.e. potentially viraemic donors returning from an outbreak affected country). geographical donor deferral can take several forms ranging from deferral of donors returning from precisely defined 'outbreak risk' areas (e.g. wnv deferral for travel to n. america) to a 'blanket' deferral of all donors returning for a defined period after return from overseas (e.g. the netherlands). while this can be an effective risk mitigation strategy it can lead to the deferral of significant numbers of donors and the impact on sufficiency needs careful consideration. in some cases (e.g. for local dengue outbreaks in australia) this can be partially addressed by restricting manufacture to 'plasma for fractionation' since pi within the fractionation process satisfactorily addresses the transmission risk. triggering implementation of deferrals for specific outbreaks is problematic because a high proportion of arboviral infections are asymptomatic so asymptomatic donor viraemia poses a risk before the first symptomatic case is apparent. instituting a deferral only during the 'transmission season' is complicated because of the difficulty in precisely defining the beginning and end donors for major transfusion-transmitted infectious diseases (ttids; hiv, hbv and hcv) has evolved from performance of serologic assays only to include nucleic acid testing (nat) for detection of window phase and occult infections in most developed and some developing countries. nat screening has also been implemented for other viruses known to be transmitted by blood components and/or plasma derivatives (parvovirus b19, hav), and for several emerging/expanding infectious disease (eid) agents that threaten blood safety in some regions (e.g., wnv in the us, hev in japan, and dengue virus in puerto rico). despite this dramatic progress, eid threats continue to be identified and cause pressure to further enhance the safety of transfusions. the concept of a 'global village' has emerged, which reflects the fact that a potential blood-borne infectious agent present in any region of the world could 'traffic' to other regions overnight. there are increasing examples of zoonotic transmissions of infectious agents, with the potential for rapid adaptation to humans and subsequent spread to blood donors and recipients. there is also intense research to identify new blood born infectious agents using novel molecular discovery strategies. although recent examples of putative new agents have proven to be non-pathogenic (hgv/gbvc, ttv and sen-v), not transmitted at significant rates by transfusions (hhv-8), or contaminants (xmrv), every newly discovered agent requires serious investigation to assess its relevance to transfusion safety. this presentation will highlight recent progress in developing approaches for systematic assessment of eid risks and for applying rational approaches to respond to new pathogens. in an effort to proactively assess transfusion safety threats in the united states and canada, the transfusion-transmitted diseases committee of the aabb has systematically reviewed relevant data and prioritized the risks of individual established and emerging disease agents; this program includes aabb website accessible fact sheets for~100 agents which are updated as new information becomes available or new agents of concern are identified. recently a research program has been started using data of id-nat users for analyses of the efficacy of various testing strategies this allows comparison of the yield and risk reduction obtained by nat and serology assays in countries with differing incidence and prevalence rates of the three major viral infections. furthermore the aabb eid sub-group and the european cdc have developed 'toolkits' for assessing the risk of eids with respect to potential impact on blood safety and availability, and for evaluating and validating the effectiveness of proposed interventions. the toolkit process, including methods to monitor eid agent emergence, identify and recognize a transfusion-transmission threat, processes to quantify risk, and the appropriate management of such threats using formal decision analysis methodologies, should be considered for implementation by all blood systems. the main challenges for hospital transfusion are patient safety, the effective use of blood, robust methods for documentation and monitoring practice, good blood stock management and low wastage, good staff training, and the rapid availability of blood for those patients who need it urgently. patient safety is paramount, but haemovigilance schemes worldwide continue to demonstrate that errors in the transfusion process result in patient morbidity and mortality. this talk will indicate where progress has been made in improving transfusion safety in hospitals in the uk, and use the implementation of an electronic transfusion management system in oxford as an example of improving patient safety. the project involved the development of an end-to-end electronic transfusion process involving barcode patient identification, handheld computers at the bedside and electronically controlled blood fridges linked to the blood transfusion laboratory information system. it was taken through pilot stages between 2001 and 2006 through to its full implementation across the acute hospitals in oxfordshire in 2006/07, and its maintenance and further development since then. there was an improvement from 11.8% to 100% of staff following the process for correct bedside patient identification; no abo incompatible red cell transfusions or serious wrong blood events in over 10years; a reduction in wrong blood in tube events; reduced nursing (one nurse rather than 2 and half the time to administer blood) and laboratory workload; and more rapid delivery of urgently required red cell units (from a median of 18 min to 45 s). the electronic system provided a simple mechanism for compliance with uk/eu regulatory requirements for the traceability of blood, and the documentation of transfusion and training. feedback from both staff and patients was positive. our group wrote a national specification and provided the material for a national recommendation for hospitals to adopt the electronic transfusion process, but wider implementation by other hospitals has been slow. an additional module into the electronic transfusion process is being implemented to provide decision support for doctors ordering blood to minimise inappropriate use of blood as part of a patient blood management programme. there are considerable barriers to improving the safety of transfusion practice in hospitals, but they can be overcome by 're-engineering' the process with the support of a multidisciplinary team with shared, achievable and measurable objectives, engaging with senior hospital management, and having a determination to see the development of a new process through to completion and its implementation as routine practice. whats new with trali? in the last 20 years there have been substantial efforts to minimise or eliminate trali. despite these activities, in 2012 the fda still reported trali as the top cause of transfusion-associated mortality. the pathophysiology of trali follows a two event mechanism. the patient's illness leads to activation of the pulmonary endothelium with the sequestration of primed neutrophilsthe first event. the transfusion is the second event, and leucocyte antibodies or biological responsive modifiers (brms), present in the blood product, activate the microbicidal arsenal of the primed neutrophils to cause injury to the pulmonary microvasculature and consequently acute lung injury (ali). hence, trali may be antibody-mediated or brm-mediated. antibodies to human neutrophil antigens (hna), hla class i and class ii have been associated and implicated with trali. interestingly, a large prospective case-controlled study of over 400,000 transfusions reported that recipients with cognate antigens to hna and hla class ii antibodies had increased risk of trali but found no statistical significance with hla class i (toy et al. 2012 blood) . that hna-3a antibodies are associated with more severe trali indicates that antibody specificity is another determinant of trali injury (reil et al. 2008 vox sang) . additionally, murine models demonstrated that antibody titre also determined the severity of injury (fung et al. 2010 blood) . while the use of male predominant plasma has significantly reduced the incidence of antibody-mediated cases, trali still occurs likely due to brms. brms are generated during the routine storage of blood products. a meta-analysis of cardiac and trauma patients concluded that older blood is associated with a significant risk of death (wang et al. et al. 2012 blood) . given the success of male predominant plasma in managing the risk of antibody-mediated trali, ongoing efforts to reduce the risk of trali are likely to focus on brm-mediated trali. developing a better understanding of the how specific brms may contribute, either individually or in combination, to the development of trali, and how brm concentrations vary between donors, will be crucial to the future development of strategies to manage the risk of brm-mediated trali. they were all thais and their forebears have lived in northeast thailand for at least two generations. hna-1,-3, and -4 were typed by the pcr-sequence specific primer (pcr-ssp). results: the gene frequencies of hna-1a, -1b, -1c were 0.697, 0.302 and 0.001, respectively. hna-1 null allele was found in one sample. the gene frequencies of hna-3a and -3b were 0.780 and 0.220, respectively. hna-4a and -4b gene frequenfrom asian populations aims: to assess whether symptoms consistent with dengue are more common in transfused patients who test positive for denv rna compared to patients who do not. methods: in 2012 and rio de janeiro, patients who were transfused were enrolled and interviews inquiring about symptoms (fever, headache, bone or joint pain, body or muscle pain, vomiting, rash, painful eyes, and bruising) were conducted between 3 to 7 days, and about 30 days following transfusion. inpatients and outpatients were included. blood samples were collected at the time of first interview. subsequently, coded samples were tested for denv rna using the hologic/novartis tma assay. we used two definitions for denv disease: the who clinical definition consisting of fever with at least two contemporaneous symptoms, and a more general definition of having â�¥3 symptoms of any kind. we calculated odds ratios (or) and 95% confidence intervals (ci) for symptomatic infection comparing transfused denv rna+ to rnaã� patients. results: of 595 patients with samples and interviews 3-7 days following transfusion 54% were inpatients while it is possible that viremia was missed in some recipients who had symptoms consistent with dengue, we did not find evidence of significantly increased rates of symptomatic infection in denv rna+ compared to rnaã� recipients. investigation into whether any of these patients acquired denv through transfusion and mie prefectures) where there are many immigrants from latin america including japanese-latin americans. methods: we conducted a questionnaire survey on chagas disease for the subjects who were borne or grew up in latin america. since jan. 8th 2013 we have performed testing for anti-t. cruzi for donors from whom informed consent for epidemiological study was obtained. tests used were ortho t. cruzi elisa assay and an immunochromatographic assay donors who were aware of chagas disease and kissing bug were 67% and 56%, respectively. nobody has ever been bitten by kissing bug. 14% of the donors had been tested for anti-t. cruzi in their country. four donors had a family member with chagas disease and they were all brazilian. four donors had been diagnosed as having a cardiac or digestive system disease before. all of the 287 blood samples obtained from the subjects were anti-t. cruzi-negative except for one false positive result. conclusions: none of the subjects surveyed were anti-t. cruzi-positive. most of latin american blood donors in tokai region of japan are brazilian and about twothirds of them were younger than 40 years. we consider that the risk of transfusion background: leukocyte antibodies presence in the blood product(s) was responsible for the development of immune mediated transfusion related acute lung injury (trali) in recipients. several studies had demonstrated that hla antibodies (class i and class ii) as well as hna antibodies (hna-1, -2, and -3) were capable to induce trali reaction. among them, hla class ii and hna-3a antibodies seemed to be the most frequent antibodies responsible for severe trali reaction. therefore, screening of hla and hna antibodies in our female blood donors may reduce the risk of trali. however, the prevalence of leukocyte antibodies among parous female blood donors in china is currently not available. study design and methods: blood samples were collected from 142 nulliparous females, 343 parous females and 453 male donors. pregnancy history was obtained from consenting donors. hla class i and ii antibodies were screened by the use of lifecodes lifescreen deluxe (lmx), and antibody specificities were determined with lifecodes lsatmclass i & ii (single antigen). antibodies against neutrophil antigens hna-1 and -2 were screened by the labscreen assay (one lambda, inc) and rapid elisa detection (bayat et al, 2009; werth et al, 2013). hna-3 antibodies were analyzed by antigen capture assay (bayat et al, 2012). the specificity of positive samples was reanalyzed by the granulocyte immunofluorescence test (gift), the granulocyte agglutination test (gat). results: screening of the total cohort of 938 donors (453 males and 485 females) showed higher prevalence of hla antibodies production in female compared with male donors (18.9% vs 4.63%). we found in 61 out of 485 females donors alloantibodies against hla class i (12.58%) and hla class ii (10.10%). analysis among female additional immunosuppression may be used in patients with low adamts13 due to autoantibodies. aims: to investigate differences between idiopathic and secondary ttp with regard to clinical presentation, management and outcomes. methods: the australian ttp registry was established in 2009, and expanded in 2012 to include all tma. of 34 hospitals currently participate in the registry. of 103 cases in 94 patients were registered from 18 sites; eight were excluded for incomplete data, leaving 95 episodes in 86 patients. results: median age was 46 years (15-83 years), 53% were female, 16% were relapses. of 51% were idiopathic and 49% had â�¥1 identified precipitant, including infection 23%, autoimmune 18%, malignancy 12%, medication 14%, pregnant/postpartum 4%, solid organ transplant 3%, and allograft 3%. adamts13 levels were available in 66 cases (37 idiopathic and 29 secondary). adamts13 was <10% in 22/37 (59%) idiopathic and 9/29 (31%) secondary cases. precipitants in cases with adamts13 <10% included infection (n = 5), autoimmune (n = 5), medication (n = 1), and pregnancy (n = 1). neurological, gastro-renal, bleeding and thrombotic presentations were similar in idiopathic and secondary cases. data on plasma therapy were available in 88 cases (table 1) . of 84/88 (95%) received pex, by centrifugation in 28/84, membrane filtration in 25/84 (30%), and unknown method in 29/84 (35%). plasma infusions were used in 11/88 cases (without pex in 4). data on additional therapy was available for 89 cases (table 2) . rituximab was given in 12/45 cases with â�¥1 precipitant, including infection (n = 5), non-haematological malignancy (n = 1), haematological malignancy (n = 3), autoimmune (n = 5), medication (n = 2), and pregnancy (n = 1). death rates were lower in idiopathic than in secondary cases (8% vs 25%, p = 0.05). conclusion: our data confirm that very low adamts13 does occur in cases with secondary causes identified. clinical presentation did not differ between idiopathic and secondary cases. plasma therapy was similar in both groups and additional immunosuppression was not only used in idiopathic cases, but also in secondary cases. mortality remains high, and more data to support improved diagnostic and therapeutic approaches are needed. background: living donor liver transplantation (ldlt) is a curative treatment for patients with end-stage liver disease. ldlt is associated with major blood loss and allogenic blood transfusions. although transfusion practices in adult liver transplant (lt) has been widely studied, the same in pediatric lt has not been well documented in india. aims: the purpose of the study was to evaluate the intraoperative transfusion requirements in pediatric ldlt recipients (<192 months) against the diagnosis, preoperative clinical and laboratory variables. methods: the data was collected retrospectively from blood bank and patient records. demographic and important preoperative variables namely clinical diagnosis, peld score, hemoglobin (hb), platelet count, inr, albumin, and fibrinogen levels were reviewed. data on intraoperative transfusion of packed red cells (prc), fresh frozen plasma (ffp), platelet concentrates (plt), cryoprecipitate was computed as ml/kg bw. massive transfusion (mt) was defined as >80 ml/kg of blood components during the surgery. analysis was done using spss software version 16. median levels were compared between mt and non-mt group using mann whitney test. results: between august 2010 to july 2013, 60 pediatric ldlt were performed in a single center in south india. thirty five (58.3%) of them were females and 25 (41.7%) males; 32 (54%) of the recipients blood group were o positive, 14 (23%) b, 9 (15%) a and 5 (8%) ab. recipient characteristics are given in table. transplant indications were biliary atresia and cirrhosis in 28, metabolic /hereditary liver disease in 24, hepatic tumors in 5 and acute liver failure in 3 recipients. of 56 (93.3%) patients received prc, 40 (70%) ffp, 17 (28.3%) plt and 34 (56.4%) cryoprecipitate. sixteen (26.7%) patients received massive transfusion (mt) with a median peld score of 22 (ã�7 to 43) compared to 9 (ã�9 to 46) recipients without mt (p < 0.005). also, mt group had significantly lowered median levels (preoperative) compared to non-mt group, viz. hb (8.6 vs 10.3 mg/dl), platelet count (96 vs 186 9 10 3 per mm 3 ), fibrinogen (137 vs 248 mg/dl), and a higher bilirubin (16.5 vs 3.7 g/dl). transfusion requirements of ffp was higher in acute liver failure (53 ae 17.3 ml/kg) compared to metabolic liver disease (11.1 ae 2.2 ml/kg) and biliary atresia (19.5 ae 4.2 ml/kg); (p = 0.001). conclusion: to conclude, massive blood transfusion requirement in pediatric recipients during ldlt was associated with higher peld score, and more deranged preoperative hematological and coagulation status. in depth analysis of recipient disease status, controlling for the effect of surgical interventional variables on larger samples are recommended to develop predictive models of transfusion therapy. conclusions: this study is the first report of hna gene frequencies in ethnic northeast thais. it could be used for the risk prediction of alloimmunization to hna and estimation of alloimmune neutropenia and trali in the ethnic northeast thai population. 3d-s18-01 distler pb 1 , slaper-cortenbach i 2 and ashford p 1 1 iccbba, san bernardino, united states of america 2 university medical center utrecht, utrecht, the netherlandsbackground: standardized isbt 128 terminology is used by cellular therapy organizations in many countries. as products evolve and new products are created, terminology is required to support the new products. changes to the terminology are managed by the cellular therapy coding and labeling advisory group (ctclag), a committee of experts representing international professional cell therapy societies, technical experts, and regulatory liaisons. since the early nomenclature was devel-oped, the ctclag has approved classes and terminology for very innovative products, including some for which therapeutic benefit has yet to be clearly demonstrated. because the term 'therapeutic cell' was used in the terminology, this became a great concern to the us fda, even to such an extent that the use of isbt 128 for cellular therapy products in the usa could be problematic. aims: the ctclag recognized the concerns raised by regulators and determined it needed to revise nomenclature to address these concerns and to be consistent with isbt 128 nomenclature used in related fields. methods: the ctclag held a face-to-face meeting and reconsidered the use of tc (therapeutic cells) terminology for products and proposed new nomenclature for the problematic terms. a draft of new nomenclature was developed and made available for public comment as well as review by the boards of ctclag sponsoring organizations (aabb, apbmt, asbmt, asfa, ebmt, fact, isbt, isct, jacie, nmdp and wmda). following this review, terminology was updated. results: major changes are:(1) the class name will comprise the type of cells and, where appropriate, the source (eg. 't cells, cord blood').(2) hyphenated class names will be replaced (3) tc and therapeutic terminology will be replaced (4) modifiers will be replaced with attributes providing the same information (5) new attributes will be added the terminology remains compatible with the single european coding system. summary/conclusions: changing terminology will create rework for facilities that have implemented isbt 128 and delay for those in the process of implementation. however, it was felt the revised terminology will provide a strong foundation for consistent nomenclature as new products are developed and address regulatory concerns. an appropriate timescale for implementation of the revised terminology in facilities already using isbt 128 will be developed. this presentation will describe the revised terminology and explain the reasoning supporting the changes.3d-s18-02 kordelas l 1 , rebmann v 2 , ludwig a-k 2 , radtke s 2 , beelen dw 1 , giebel b 2 and horn p 3 1 department of bone marrow transplantation, university hospital essen, essen, germany 2 institute for transfusion medicine, university hospital essen, essen, germany 3 university hospital essen, essen, germanybackground: graft-versus-host disease (gvhd) is a major cause of morbidity and mortality after allogeneic stem cell transplantation. a number of studies reported positive impacts of systemically applied mesenchymal stem cells (mscs) for preventing or treating acute gvhd. in contrast to the initial paradigm that mscs intercalate into injured tissues and thus reduce tissue damage, it is now widely assumed that mscs secrete a number of immune-modulatory factors, which impair inflammation and thus help to suppress gvhd. exosomes are secreted cell organelles, which exert immune-modulatory properties. these small membrane vesicles are released by a huge variety of different cell species, including mscs. methods: here, we enriched exosomes from bone-marrow derived mscs of four different unrelated stem cell donors and compared their immune modulatory properties in vitro. next, we administered immunosuppressive msc-derived exosomes in escalating doses into a 22-years female gvhd patient. this patient suffered a severe and therapy-refractory cutaneous and intestinal gvhd grade iv. we monitored the clinical effects on an in-hospital basis and correlated this with the levels of inflammatory cytokines measured in the patient's plasma. results: we show that even though all propagated msc lines released exosomes, exosome-enriched fractions differed in their potential to modulate immune responses in vitro. administration of the exosome-enriched fraction with the strongest immune suppressive in vitro effect into the gvhd patient was well tolerated and appeared to be safe. during the course of the exosome therapy a clear reduction of the proinflammatory cytokines il-6, il-8 and il-17 was observed in the patient's plasma. in line with that, the clinical cutaneous and intestinal gvhd symptoms improved significantly and the dosage of the immunosuppressive agentsparticularly of steroidscould be reduced. in total the patient was stable for 5 months. interpretation: msc exosome-enriched fractions exert immune suppressive functions in vitro and in vivo. since the in vivo administration seems to be safe, msc exosome administration appears as a promising new treatment option for steroid refractory gvhd patients. key: cord-347710-ff64y6ef authors: wan, qianya; song, dan; li, huangcan; he, ming-liang title: stress proteins: the biological functions in virus infection, present and challenges for target-based antiviral drug development date: 2020-07-13 journal: signal transduct target ther doi: 10.1038/s41392-020-00233-4 sha: doc_id: 347710 cord_uid: ff64y6ef stress proteins (sps) including heat-shock proteins (hsps), rna chaperones, and er associated stress proteins are molecular chaperones essential for cellular homeostasis. the major functions of hsps include chaperoning misfolded or unfolded polypeptides, protecting cells from toxic stress, and presenting immune and inflammatory cytokines. regarded as a double-edged sword, hsps also cooperate with numerous viruses and cancer cells to promote their survival. rna chaperones are a group of heterogeneous nuclear ribonucleoproteins (hnrnps), which are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. hnrnps involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. dysregulation of stress proteins is associated with many human diseases including human cancer, cardiovascular diseases, neurodegenerative diseases (e.g., parkinson’s diseases, alzheimer disease), stroke and infectious diseases. in this review, we summarized the biologic function of stress proteins, and current progress on their mechanisms related to virus reproduction and diseases caused by virus infections. as sps also attract a great interest as potential antiviral targets (e.g., covid-19), we also discuss the present progress and challenges in this area of hsp-based drug development, as well as with compounds already under clinical evaluation. stress proteins (sps) are a diverse group of proteins that are synthesized at increased levels when cells are exposed to either intracellular or extracellular stressful stimuli. they exhibit protective effects against stresses. stress proteins include heat shock proteins (hsps), rna chaperone protein (rnps), and proteins mainly function in the endoplasmic reticulum (er): peptidyl-propyl isomerases, protein disulfide isomerases (pdis) and the lectin-binding chaperone system. 1 sps are ubiquitously expressed in all kind of cells, triggering signal cascades for neutralizing and eradicating the stresses occurring both intracellularly (e.g., pathogen invasion) and extracellularly (e.g., starvation, stimulation by cytokines/chemokines or hormones). responses triggered by sps can either activate pathways to promote cell survival or initiate cell death (i.e., apoptosis, necrosis, pyroptosis or autophagic cell death) for eliminating the damaged cells to protect a particular organ/ tissue under given conditions. it is widely noted that the dysregulation of stress proteins is associated with a variety of human diseases, including cardiovascular diseases, neurodegenerative diseases (e.g., parkinson's diseases, alzheimer disease), stroke, human cancers and infectious diseases. in this review, we focus on their functions and update findings involved in infectious diseases, particularly, the diseases caused by viral infections. heat shock proteins in 1962, an italian geneticist ritossa inadvertently elevated the incubation temperature of drosophila larvae and discovered an increased gene transcription of unknown proteins. he nominated these protein as hsps. 2 further studies have revealed a large number of hsps, which form a big family and are ubiquitously expressed in cells. based on the molecular weight, hsps are classified into different families, including hsp100s, hsp90s, hsp70s, hsp60s, hsp40s, and some small hsps . [3] [4] [5] hsps belong to the largest family of chaperones. the hsp expression is rapidly induced when cells meet physiological or environmental attacks such as starvation, high temperature, hypoxia or hyperoxia, pathogen invasion, malnutrition and exposure to chemicals or uv, etc. they form a network to promote or stabilize the correct folding of substrate protein to gain its functional/active conformation (fig. 1 ), although they may not associate with the substrate protein in the final structure. 3, 6 hsps are important factors in regulating cell survival, differentiation and cell death. accumulating evidence shows that some hsps participate in not only innate cellular immunity but also antigen presentation in adaptive immune response. 7, 8 hsps also serve as potential biomarkers for some diseases. it has been shown that the increase of hsp70 in plasma is associated with heart failure, 9 and the elevated hsp27 level in human peripheral blood mononuclear cells is related with coronary artery disease (cad). 10 hsp90s. hsp90, an abundant chaperone in all eukaryotic cells, controls a variety of critical signalling pathways in eukaryotic cells. 5, 6, 11 hsp90 is an atp-dependent chaperone with different isoforms, including 1) hsp90α (hsp90aa1, or hspc1), hsp90α-a2 (hspaa2, or hspc2) and hsp90β (hspab1, or hspc3) locate in cytoplasm. 2) glucose regulated protein grp94 (hspc4 or gp96) locates in the er. 3) trap1 (hspc5) locates in mitochondria. 12 among them, hsp90α and hsp90β account for the greatest proportion in humans. hsp90 contains three regions: an atpasedependent hydrolytic domain in the n-terminal region, a middle linker region, and a dimerization domain in the c-terminal region. 5 like other hsps, hsp90 binds non-native substrate peptide to prevent its aggregation and degradation. when hsp90 binds atp, a transient dimerization of the n-terminal domain allows the substrate peptide binding to hsp90. then the atp hydrolysis and energy release lead to a conformational change of the n-terminal domain that facilitates the correct folding of the substrate petite ( fig. 1) . 5, 6 besides, hsp90 is also involved in telomere maintenance, apoptosis, and cell cycle progression, etc. 6, 13 it is well known that hsp90 not only interacts and contributes to rna polymerase assembly and nuclear import of some (−) ssrna viruses (e.g., pb2 of influenza virus), but plays crucial roles in the folding process of viral capsid proteins and virion assemblies as well. 14 cochaperones of hsp90. cochaperones of hsp90 regulate hsp90 functions at many aspects. cdc37 (also called p50) delivers kinase to hsp90 and inhibits its atpase activity; carboxyl terminus of hsp70-interacting protein (chip) functions as e3 ubiquitin ligase; hsc70/hsp90-organizing protein (hop, also called sti1) inhibits dimerization of the n-terminal domain; and the activator of hsp90 atpase 1 (aha) and p23 participates the maturation of substrate peptides. 11, 15 hsp70s. hsp70 is a subfamily of hsps' superfamily with~70 kda molecular weight. it accounts for the majority of molecular chaperones in cells. 11 the members of the hsp70 family mainly include: (1) hsp72 (hspa1a), hsp70-2 (hspa2), hsp70b' (hspa6) and hsc70 (hspa8) are commonly located in the cytosol; (2) grp75 (hspa9) is located in mitochondria; (3) grp78 (hspa5) is associated with the er. 16 hsp70 consists of two domains: a 44-kda nucleotide-binding domain (nbd) which can be divided into four subdomains (ia, ib, iia, and iib) in the n-terminal region and a 28-kda substrate-binding domain (sbd) composed of c-terminal α-helical (sbdα) and n-terminal β-sheet (sbdβ) subdomains in the c-terminal region. 17, 18 as a critical component of cellular protein surveillance, the atp-dependent molecular chaperone protects cells from damage caused by stress and takes part in a number of folding processes, including folding of newly synthesized polypeptides, recognition and refolding of misfolded or aggregated proteins, solubilization or degradation of proteins, transporting proteins, assembly or disassembly of oligomeric protein complexes, and the regulation of certain natively folded proteins. 5, 13, 19, 20 the functions of hsp70 are not limited to host protein folding. its functions are considerable during viral infection. the members of hsp70s exhibit quite different roles in the course of virus life cycle. for example, hsp72, hsp70b' and hsc70 participate in the hcv viral entry, virion assembly and translation of the viral genome. grp78 in er is associated with the homeostasis of viral proteins and prevents the overload of viral proteins in host cells. in hepatocytes, the elevated grp78 stimulates innate immunity to restrict or eliminate hepatitis b virus (b) replication. 21 grp75 interacts with the ns5a protein of hcv in mitochondria. 22 accumulating evidence shows that hsp70 interacts with viral components of human cytomegalovirus, rabies virus, respiratory syncytial virus, human papillomavirus, herpes simplex virus. chaperone cycle of hsp70. the chaperone cycle is mediated by the n-terminal nbd, which regulates the binding of hsp70 with substrates through switch of two states. the first state is the atpbound state with low affinity for substrate binding, i.e., a high association and dissociation rate of the substrate peptides to the sbd. the second state is atp hydrolysis that switches to the adpbound and nucleotide-free state. at this state, the substrate exchange rates are low while the affinity to substrates is high. the chaperone activity of hsp70 mostly relies on atp hydrolysis. the basal atpase of hsp70 is normally low unless it is stimulated by the substrate peptide itself. it takes 20-30 min for a molecule of atp to be hydrolysed per hsp70 molecule at 30°c. as a result, it is necessary for some cochaperones to encounter with hsp70 atp to induce atp hydrolysis and help the increase of the affinity for substrate peptides. 19, 23 cochaperones of hsp70. the most crucial cochaperones of hsp70 are members of the j-domain proteins (jdps) family and nucleotide exchange factors (nefs) family. previous studies focused on the function of hsp70 machinery and led to the development of a "canonical model" for its mode of action. the model contains two steps. first, the unfolded peptide substrates bind jdps of the hsp40 family; then the substrates are delivered to hsp70 that stimulate the hsp70's atpase activity. simultaneously, jdps prevent the aggregation of unfolded proteins. second, nefs work as substrate release factors that assure the substrate to fold into the correct and active conformation. in this way, the cochaperones strongly facilitate the function of hsp70. therefore, hsp70 generally does not work individually but cooperates with cochaperones. 23, 24 hsp60s. hsp60s are large cylindrical oligomers with two back-toback rings. 19 the non-native proteins of the central cavity in each ring are folded into the native protein through an atp-dependent process. 25, 26 hsp60s are classified into two subfamilies. group i is mainly in prokaryotes, while group ii appears in eukaryotic cytosol and some archaea. 27, 28 the most well-studied one in group i is the groel-groes chaperonin system in the cytosol of bacteria. groel is an about 57 kda protein with two rings arranged back-to-back; and 7 subunits form a tetradecamer structure. groes is the cochaperone of groel. 11 the two rings-tetradecamer structure appears in two fig. 1 the general chaperone cycle of heat shock proteins. initially, unfolded client protein bound to the hsp70-hsp40 chaperones interacts with the hsp90. atp binding to hsp90 induces the client proteins transfer from hsp70 to hsp90. later, the conformation of hsp70-hsp40 chaperones will be released. finally, the hydrolysis of atp induces additional conformation changes leading to the client protein release forms include asymmetric (1 groel: 1 groes) and symmetric (1 groel: 2 groes) complexes, which are described as "bullet" 14, 29, 30 and "american football" 31 shaped respectively. the groel-groes chaperonin undergoes allosteric regulation dependent on atp and which completes the protein folding function. the polypeptide binds to the hydrophobic sites of one of the seven subunits of groel and changes conformation upon atp binging and hydrolysis. with the help of cochaperone groes, the polypeptide finishes its folding process. 32 in contrast to the groel-groes system, the mammalian homolog hsp60/hsp10 system is less studied. hsp60 is thought to be imported into the mitochondria and converted into its mature form with a molecular size of 58 kda. [33] [34] [35] hsp60 exhibits important roles in facilitating protein folding, transportation and proteostasis in mitochondria. 36, 37 group ii chaperonins include the archaeal thermosome and eukaryotic cct (chaperonin-containing tcp1, or called tric), which are oligomers with eight to nine subunits with molecular weight 57-61 kda in each ring. compared to group i, group ii chaperonins show different allosteric movements by atp binding. 11, 19 hsp60 family is known to participate in viral life cycle at various stages from viral attachment to the replication of the viral genome. hsp60s are essential for host cell immunity regulation. some viral proteins require hsp60 for its translocation within host cells. pb2 is a subunit of influenza a viral rna polymerase, which mostly locates in the nucleus but also appears in mitochondria. 38 when the virus infects the host cells, pb2 is responsible for maintaining the function of mitochondria. pb2 interacts with mitochondrial antiviral signaling protein (mavs) to downregulate intracellular immune response by decreasing the level of ifnβ so that the invading virus can easily escape from the defence of host cells. 39 hsp60 takes the role of transporting pb2 from cytosol to mitochondria in the host cells. besides, hsp60 shows great regulatory functions on innate immunity by inducing proinflammatory cytokine release, such as tnf-a, il-6, and il-1b, etc. 40 small heat shock proteins. small heat shock proteins (shsps) are a group of small proteins with a low molecular weight ranging from 15 to 40 kda. 4 there are 10 members in the shsp family and some are ubiquitous including hsp27 (hspb1), hspb5 (αb-crystallin, or αbc), hsp20 (hspb6), and hsp22 (hspb8), while the others are tissue-specific including hspb2 (myotonic dystrophy protein kinase, or mkbp), hspb3, hspb4 (αa-crystallin, or αac), hspb7 (cvhsp), hspb9, and hspb10 (sperm outer dense fiber protein, or odf). 41 shsps can exist as monomers, dimers or even large multimeric complexes in the cells. 42 the structure of shsps is different from the other hsps due to their less conserved sequences. the basic structure of shsps is a conserved α-crystallin domain (acd) flanked by two non-conserved domains including the n-terminal sequence (nts) and the c-terminal sequence (cts). among these domains, acd becomes the characteristic of different shsps. 43, 44 shsps play crucial roles in several physiological processes regarding stress tolerance, apoptosis, aging, and longevity. [45] [46] [47] [48] the phosphorylation together with the n-terminal wdpf motif helps shsps to form homo-or hetero-oligomers. 49, 50 the oligomerization is the hallmark of shsps for supporting their quite different activities. phosphorylation favors small oligomer formation while dephosphorylation provokes a shift toward large oligomer formation. 51 oligomerization dynamics is crucial for chaperone activity because it gives rise to the possibility to format different homo-and hetero-oligomers, each with different binding properties to chaperone a broad range of substrates. 41, 52 for instance, the phosphorylated species are required for actin dynamics. small phosphorylated dimers/tetramers bind f-actin to regulate actin polymerization. 53 among different shsps, hsp27 has been broadly studied. hsp27 exists in all tissues though it is known to mainly express in cardiac, skeletal and smooth muscles. 54 its importance has been demonstrated in cell differentiation, cell survival, cellular innate immunity, viral protein translation, and intracellular virus transport, etc. [55] [56] [57] same as the other shsps, hsp27 shares a highly conserved α-crystallin domain. 41, 58, 59 hsp27 is capable of oligomerization and phosphorylation. there are three serine residues 15, 78, and 82 can be phosphorylated by different kinases including p90rsk, pkg, mapkap kinases, etc. 55 the phosphorylation of hsp27 is a reversible process. the dephosphorylation contributes to the formation of large size oligomers. 60, 61 hsp27 can not only form large homo oligomers up to 800 kda; but it can also cooperate with other shsps (e.g., hsp20) to form heteromeric structures. 56, 58 hsp27 is upregulated and activated upon infections. 62, 63 the elevated hsp27 activity promotes either cytoskeletal stability or cell motility, 64, 65 and prevents apoptosis. 66 hsp27 is required for il-1-induced expression of the pro-inflammatory mediators il-6, il-8, and cyclooxygenase-2. [67] [68] [69] hsp27 is also linked to various signalling pathways involved in the development, differentiation, and cell growth. 70, 71 the long-term and high-level expression of hsp27 stimulated by variety of stresses (such as hbv or ebv infections) enhances carcinogenesis, cell survival, stemness of cancer cells, cancer metastasis, tumour formation and drug resistance. transcriptional regulation of the hsps. heat shock factors (hsfs) display great contributions in regulating the transcriptional activation of hsp genes. in all invertebrate animals, only hsf1 is responsible for the transcriptional activation. in vertebrates, four members of hsf family (hsf1-4) regulate hsp expression. 72 among them, hsf1 is the most critical one. the fibroblasts from hsf1 −/− mice undergo apoptosis upon heat stress because of no hsp transcription. 73 upon stress conditions, the originally monomeric hsf1 in the cytoplasm could trimerize and translocate into the nuclei to promote the hsp expression by binding on the heat shock elements (hse) in the promoter region. 74 protein disulfide isomerase protein disulfide isomerase (pdi) is a multifunctional oxidoreductase and chaperone that catalyses the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (er). during disulfide bond formation, cysteine residues at the cghc active site of pdi accept two electrons from the cysteine residues in polypeptide substrates, leading to the reduction of pdi and oxidation of the substrate. then pdi transfers the electrons to an acceptor to start another cycle of disulfide bond formation. 75 in addition to pdi's catalytic function as a thiol-disulfide isomerase, it also exhibits molecular chaperone properties for glycosylated protein quality control. 76 erp57 (pdia3, grp58) is possibly the most thoroughly studied pdi family member that shares a similar structure consisting of four domains (namely a-b-b'-a') and possesses two localization sequence-an er retention signal (qdel), and a nuclear localization signal (kpkkkkk). unlike other pdi family members that directly bind the substrates for their reductase or isomerase activities, the b domains of erp57 have a high affinity to associate with calreticulin (crt) and calnexin (cnx), which would help to recognize and recruit polypeptide segments of the glycoproteins. 77 if the protein is not correctly folded, udpglucose:glycoprotein glucosyltransferase (uggt) would be recruited to reglycosylate the proteins, allowing them to be recognized and re-associated by erp57/crt/cnx complex. 76, 78, 79 considering the essential roles of pdis in the oxidative folding and chaperone-mediated protein quality control, they are now linked to a growing range of diseases including those are caused by virus infection. proteins that interact non-specifically with rna and resolve the non-functional inhibitory structures are usually referred to as rna chaperones, which have distinct roles without common sequences or motifs. 80, 81 they participate in a large number of cellular processes, including chromatin remodelling, transcription regulation, rnp assembly and stabilization, rna export, histone-like nucleoid structuring, intracellular immunity, and viral rna replication and translation. rna molecules mostly rely on welldefined 3d structures to fulfill their functions. however, the process of rna folding is very complicated. 82 the multitude of possible rna base-pairings together with the high stability of rna duplexes would give rise to a large number of alternative secondary and tertiary structures that are thermodynamically as stable as the functional, native structure. 83 rna chaperones promote rna folding by accelerating the escape from kinetic folding traps and prevent rnas from being trapped in nonfunctional conformations. [84] [85] [86] so far, no protein has been characterized whose primary function is to resolve nonspecifically misfolded rnas in cells. 80, 81 hnrnps are a group of heterogeneous nuclear ribonucleoproteins. they are essential factors for manipulating both the functions and metabolisms of pre-mrnas/hnrnas transcribed by rna polymerase ii. more than 20 hnrnps have been identified to date. hnrnps contain common rna binding motifs like arginine glycine boxes (rgg boxes), rna recognition motifs (rrms), hnrnp k homology (kh)-domains and zinc finger (zf)-domains (khzf domain). 87 well-defined functions of this family include transcription regulation, pre-mrna splicing, 3′-end formation, mrna packaging, rna transport, translational regulation, rna silencing, dna repair, and telomere biogenesis. they also have the ability to shuttle between nucleus and cytoplasm, therefore could transiently help to form rnp complexes in nucleus and also participate in rna metabolism in cytoplasm. 88 a large collection of hnrnps are involved in virus activities, most of which were first identified using viral rna-protein binding assays, followed by functional assays. 89 the importance of stress proteins one of the main functions of stress proteins is to maintain cellular homeostasis. under pressure, stress proteins are hyperactive to release the pressure. hsp27, hsp70 and hsp90 accumulate to a very high level in quite a few types of cancer cells. [90] [91] [92] although the mechanism underlying the increase has not been fully understood, it suggests that the fast increased hsps respond to the folding stresses. with tumor progression, the accumulating oncogenic proteins need powerful protein folding ability. under long-term stresses, hsps participate or promote carcinogenesis, cell survival, anti-apoptosis, angiogenesis, cancer cell stemness, invasion and metastasis. 93 however, hsps and rna chaperones are downregulated in nearly all age-related neurodegenerative diseases including alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, and several polyglutamine diseases such as huntington's disease and different forms of spinocerebellar ataxias 94, 95 (fig. 2) . downregulated rna chaperones lead to disorder of rna metabolism; 94 while the attenuated hsps result in a failure of the protein quality control (pqc) system to adequately handle the folding or timely degradation of proteins in neurologic disease. 95 both mechanisms cause protein aggregates, the hallmark of age-related neurodegenerative diseases. in this review, instead of paying much attention to these topics, we would only focus on the main biological functions and target values of stress proteins in human diseases caused by pathogen infections, particularly by virus infections because the welldeveloped antibiotics have already controlled the bacterial infection very well since 1950s. viruses infection causes a variety of diseases that are highly associated with dysregulation of stress proteins (fig. 2 ), e.g., respiratory symptoms, 96, 97 gastroenteritis, [98] [99] [100] [101] hemorrhagic fevers. 102, 103 critically, it has been demonstrated that neurodegenerative diseases as well as neurobehavioral disorders are the consequences of loss of neurons and axons in the central nervous system with ageing. it is evidenced that these diseases are possibly caused by chronic neuropathic viral infections. 104 stress proteins are involved in many steps of virus infection process, including virus entry, uncoating, replication, gene expression, as well as virus assembly and releasing as steps 1-5 shown (fig. 3 ) some viruses replicate in the nucleus, while stress proteins take part in the virus protein and/or rna nuclear import/export processes as setp a-c shown (fig. 3 ). the summary of the relationship between stress proteins, virus infection as well as host cell response is listed in table 1 . in this section, we would review and present new findings on hsp90 family proteins in the life cycles of different viruses including rna virus, dna virus and retrovirus. in addition, we also discuss their functions in virus-induced cellular response. the function of hsp90 family in rna virus infection virus entry. in the case of rna virus, hsp90 is critical for the entry of enterovirus a71 (ev-a71), 107 japanese encephalitis virus (jev) and dengue virus (denv). 108 ev-a71 entry is significantly blocked when cells are pre-treated with hsp90 inhibitors or hsp90-specific sirnas. 107, 109 since both denv virus and jev belong to the flavivirus, the entry of the these two viruses differently utilize hsp90 with the support of hsp70s in both neuroblastoma and microglial cells. 95, 108 denv depends much more on hsp90 to enter into cells as compared with jev since anti-hsp90 antibodies or hsp90 inhibitors block the majority of denv entry 110 but only a small portion of jev entry. 111 additionally, both hsp90 and hsp70 are associated with membrane lipid rafts in response to denv infection. 110 however, in denv infected liver cells (hepg2), neither hsp90 nor hsp70 works as the receptor to enable denv internalization, 110 indicating alternative entry mechanisms in different cell types. it is likely that the receptor functions of hsp90 and hsp70 are replaced by other unknow molecules. virus replication. hsp90 protein facilitates virus replication in several aspects. first, hsp90 works as a classic chaperone protein to stabilize virus proteins. hsp90 stabilizes paramyxoviruses polymerase and l protein, as well as assists virus replication. inhibition of hsp90 could hamper virus replication and shorten the half-life of l protein in vesicular stomatitis virus (vsv), human parainfluenza viruses-2 (hpiv-2), human parainfluenza viruses-3 (hpiv-3), simian virus 41 (sv41), or respiratory syncytial virus (rsv) infection cells. 112, 113 similarly, hsp90 is shown to maintain the stability of chikungunya virus (chikv) non-structural proteins (nsps) including nsp3 (a protein essential for rna synthesis) and nsp4 (rna-dependent rna polymerase, rdrp); 114 and the protease of hcv nonstructural protein ns3. 115 second, hsp90 modulates virus polymerase activity to enhance virus replication. taking hcv as an example, hsp90 indirectly modulates the hcv polymerase ns5 activity by maintaining the stability of kinase phosphoinositide dependent kinase l (pdk1), an upstream kinase of ns5 phosphorylation kinase prk2. 116 besides, mediated by fkbp8, hsp90 forms a complex with ns5 and directly regulates ns5 activity. 117 hsp90 inhibitors suppress viral replication by disrupting the hsp90-ns5 complex formation. 117 third, hsp90 manipulates the proper location of virus polymerases. during influenza virus infection, hsp90 interacts with the viral rdrp subunits polymerase basic protein-1 (pb1) and -2 (pb2) to form a complex, and then co-translocates into nucleus. 14, 118 in this process, hsp90 maintains the stability of pb1 and pb2. after entry into nucleus, hsp90 dissociates from the hsp90/pb1/pb2 complex and forms a new functional complex with polymerase acidic protein (pa). 119 extending study shows that the state of hsp90 acetylation is strictly regulated by histone deacetylases 6/8 while some viruses (such as influenza, sv40, hbv etc) also enter into nucleus of host cells. they may undergo other steps in their life cycle which are labelled as a-c: virus nucleus import, nucleus export, and virus rna processing. during the process of virus infection, hsp90, hsp70, hsp60, hsp40, hsp27, and pdis participate in the virus entry and uncoating steps. hsp90, hsp70, hsp60, hsp40, hsp27 and rna chaperones take part in the virus replication step. hsp70, hsp40 and rna chaperones are required in virus gene expression step. hsp90, hsp70 and hsp40 assist virus assembly. hsp70 and rna chaperones contribute to the virus release. while hsp90 is also important in virus nucleus import and export. hsp70 plays a role in the virus nucleus import. and rna chaperones play a major role in virus rna processing, including replication, initiation of translation, stabilization and decay 485, 486 (hdac6/8) in the influenza rdrp nuclear import stage. hdac6/8 inhibitors efficiently limit the polymerase nuclear import and suppress virus replication. 120 in the course of influenza infections, hsp90 expression is stimulated through mtor/p70s6k signalling. 121 our recent studies show that hsp90 also exhibits significant importance on ev-a71 replication through pim1 signalling (unpublished). it has been shown that ev-a71 infection elevated both the mrna and protein levels of pim1. 122 the elevated pim promoted ev-a71 replication while knockdown of pim1 decreased ev-a71 replication. knockdown of hsp90β decreases 60% of virus replication 12h post infection (p.i.), while the secreted virions decrease by approximately 80%, indicating the crucial roles of hsp90β in both virus replication and secretion (fig. 4a-c) . other researchers reported that hsp90β is responsible for ev-a71 assembly which may be the reason that hsp90β attenuates the secretion of ev-a71 virions in our study. 107, 109, 122 notably, hsp90β contributes to virus replication more than that of secretion. our data also shows that knockdown or knockout of hsp90β decreased the proteins expression level of ev-a71 structure protein (fig. 4d , e). and hsp90 inhibitors 17-aag, geldanamycin (ga) and ver50588 all dramatically inhibit ev-a71 protein expression (fig. 4f ). among them, ver50588 display the strongest inhibitory effect which has not been reported before. we predicted that hsp90β is a potential target of pim1 (fig. 4g ). to address this hypothesis, we conducted experiments by overexpression pim1 and knockdown of pim1. accordingly, the phosphorylated status of hsp90β is increased and decreased (fig. 4h,i) . more importantly, knockout of hsp90β by crispr/cas9mediated gene editing almost completely abolishes the effects of pim1 signaling on ev-a71 replication (fig. 4j, k) . viral protein maturation, virion assembly, and release. during the viral protein expression and maturation, hsp90 works as a classic chaperone to monitor the proper folding of viral proteins. hsp90 modulates the maturation of hcv nonstructural protein 2/3 (ns2/ 3) kinase. 123 hcv ns2/3 is cleaved into two separate proteins right after translation, a key step of ns2/3 protein maturation. hsp90 strictly regulates the proper folding of newly synthesized ns2/3 protein. 123 hsp90 and its co-chaperone p23 form a complex to assist the proper folding of capsid precursor polyprotein p1 of poliovirus, rhinovirus, and coxsackievirus; 124 while the inhibitor ga reduces the maturation of p1, leading to immature p1 degradation in proteasome. 124 during the virion assembly, hsp90 interacts with capsid vp1 protein of noroviruses and the termini of the murine norovirus 1 genome. 124, 125 this interaction not only stabilizes vp1, but ensures the viral genome to be encapsulated into capsids as well. 124 hsp90 interacts with and stabilizes influenza neuraminidase (na), a major surface glycoprotein involving in virion release. 126 more importantly, it emphasizes the hsp90-na complex formation on promoting cell survival, leading to more virus production. 126 the function of hsp90 family in dna virus infection virus entry. the entry of dna viruses includes steps of crossing over cell membrane and nuclear import. hsp90 is mainly shown the ability to assist the nuclear import of virus. the nuclear transport of many viruses depends on the microtubules (mt) and mt-dependent molecular motor dynein/dynactin complex. 127 virus strictly modulates the status of tubulin acetylation, a critical event for the transportation of viral components. [128] [129] [130] in hsvinfected cells, hsp90 co-localizes with acetylated tubulin and capsid protein vp5. 131 hsp90 inhibitors disrupt its binding to the acetylated tubulin, thereby inhibiting the nuclear transport of hsv capsid protein. 131 during hbv infection, the glucocorticoid receptor shows a strong possibility to enhance the nuclear import of hbv. 132 hsp90 facilitates glucocorticoid receptor redistribution from the cytoplasm to the nucleus. 133 virus replication. during dna retroviral replication, hsp90 mainly contributes to regulating and maintaining the reversetranscriptase (rt) activity. taking hepatitis b virus (hbv) as an example, the reverse transcription is an essential step to generate viral genomic dna in type vii viruses. the beginning of reverse transcription is the recognition and interaction of rt with an rna signal (the packaging signal, ε) on the pre-genomic rna. 134 it was identified that hsp90 is an essential host factor that facilitates duck hepatitis b virus (dhbv) replication by interacting with viral rt. 135 treating with hsp90 inhibitors or monoclonal antibodies (mab) sufficiently block rt-ε binding. 135 hu et al. demonstrated that rt-ε interaction depends on hsp90's atp hydrolysis activity. 136 two independent regions in the terminal protein (tp) and the rt domains of polymerase separately bind with hsp90 at the n-terminal and c-terminal fragments, and both domains are essential for ribonucleoprotein (rnp) and protein priming. 137, 138 although a model is established to show how hsp90 bridges the two separate rt domains of polymerase together to enable the formation of an rnp complex with the hbv rna; 137 there are still some fundamental questions to be addressed. firstly, whether the hsp90 chaperones or hsp70 chaperones are essential for the rt-ε interaction. stahl et al. believed that hsp70 chaperones are much more important for the rt-ε interaction. they proposed that hsp90β another hsp90 family member, is shown a critical regulator in stabilizing and activating rt, allowing its preferential binding to the pregenomic rna during hbv replication. 143 the replication of most dna viruses occurs in the nucleus, where virions form in replication centres. therefore, the proper location of viral proteins is quite important for virus replication. hsp90 is also found to regulate the location of virus dna polymerase in virus-infected cells. after treated with hsp90 inhibitors, hsv polymerases is mislocalized from the nucleus to the cytoplasm and subsequently degraded in a proteasomedependent manner. 144, 145 similar to hsv, the nuclear translocation of dna polymerase of ebv also requires hsp90. 146, 147 during the polymerase transportation, the polymerase catalytic subunit balf5 forms a complex with bmrf1 in the assistance of hsp90β. hsp90 inhibitors effectively block the translocation of viral dna polymerase. 146, 147 virus gene expression. hsp90 is important for virus gene expression both at the transcription and translation levels. the transcription of hsv immediate-early α (ieα) genes is initiated by the transcription factor complex, which is composed of octamerbinding transcription factor 1 (oct-1), host cell factor 1 (hcf-1) and viral protein 16 (vp16). 148, 149 in the complex, vp16 is the major virus-encoded transcriptional activator that controls the efficiency and level of viral gene transcription. in the transcription process, hsp90α is shown to maintain the stability of vp16 by keeping it from degradation in a macroautophagy-mediated manner. 150 similarly, hsp90 also regulates the transcription of human cytomegalovirus (hcmv) immediate-early genes through activating akt and nf-κb signalling pathways, which are critical for major immediate early genes transcription. 151, 152 at the translation level, hsp90 promotes the translation of conserved herpesvirus protein kinases (chpks), including herpes simplex virus type 1 and 2 (hsv-1, hsv-2), varicella-zoster virus (vzv), ebv, kshv. 153 chpks play important roles in multiple processes, including gene expression, 154-156 viral dna replication, [156] [157] [158] capsid nuclear egress, 159, 160 and dna damage responses. 161, 162 the translation of ebv nuclear antigen 1 (ebna1) protein is also manipulated by hsp90. 163, 164 ebna1 is critical for cellular transformation, tumorigenesis, and the maintenance of viral episomes. [165] [166] [167] the ebna1 translation is strictly regulated by hsp90 through the gly-ala repeat domain to keep ebna1 at a relatively low level. 168 it has been demonstrated that hsp90 inhibitors block the translation of ebna1; and mutation of gly-ala repeat domain abrogates the inhibition of ebna1 translation. 163, 164 hsp90 does not interact with ebna1directly, a bridge protein may be involved in this process. inhibiting ebna1 expression strongly suppresses both ebv-induced primary b cell transformation in vitro and lymphoproliferative disease in scid mice in vivo. 163, 164 virus assembly. only a few papers reported the function of hsp90 in dna virus assembly. the activated hsp90 is needed for hbv assembly. 169, 170 hsp90 enhances the affinity of core protein dimers for capsid formation and prevents capsid dissociation. 169 besides, the reactive oxygen species (ros)-promoted hbv capsid assembly also requires an active form of hsp90. 170 virus-induced tumorigenesis. as described before, ebv is the causative regent of several tumors, including burkitt's lymphoma 171 and nasopharyngeal carcinoma (npc). 172 the latent membrane protein 1 (lmp1) is regarded as an oncoprotein that promotes tumor metastasis and invasiveness through inducing the expression of matrix metalloproteinase 9 (mmp9), mimicking the tumor necrosis factor receptor (tnfr) superfamily proteins, and activating the nf-kb, mapk, pi3k/akt and jak/stat signal transduction pathways. 173, 174 hsp90 seems positively promoting cell growth in ebv-positive nasopharyngeal carcinoma cells and ebv-infected t and nk cells. 175, 176 hsp90 inhibitors, at13387 and biib021, potently inhibit cell growth and induce apoptosis by impeding lmp1 function through activating its downstream signaling pathways described above. 175, 176 immunity modulation. we have discussed how hsp90 is hijacked by dna viruses to promote viral replication above. under certain conditions, hsp90 exhibits antiviral activities by promoting cell immunity. in the acute infection stage, hsp90 is induced to express in the cell surface of ebv-infected b cells. it has a strong ability to expand gamma delta t cells (γδ t cells) population. 177 the γδ t cells have potent antiviral ability in the acute phage when a host is infected by hiv, influenza, sendai, coxsackie, vaccinia, vsv, or hsv-1. the γδ t cells work as both early sentinels of the immune system by providing immediate protection and as bridging elements between innate immunity and adaptive immunity. 178 since hsp90 can work as an immune sensor and assist antigen presentation, it may function in the same way in ebv infection. 177, [179] [180] [181] [182] the function of hsp90 family on cell transformation during retrovirus infection several hsps functions as oncoproteins to promote cellular transformation. hsp90 participates in the htlv-1-induced cellular transformation. the tax protein of htlv-1 controls viral replication and induces t lymphocyte transformation. 183 nf-κb pathway is one of the main targets essential for this process; 184 while hsp90 acts as an important partner of tax by binding with and maintaining its stability in nucleus. 153, 185 hsp90 inhibitors (e.g.,17-dmag) or hsp90-depletion by sirnas cause tax degradation in proteasome, inhibition of nf-κb signalling, and activation of the long terminal repeat (ltr) of htlv-1. 153, 185 oral treatment with fig. 4 pim1 signaling promotes ev-a71 replication through hsp90β phosphorylation(unpublished data). a rd cells were treated with scramble/hsp90β sirna for 24h, the effects of hsp90β knockdown were determined by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. b, c rd cells were treated with scramble /hsp90β sirna for 4h, then infected with ev-a71 at moi of 1 for indicated time. the intracellular (b) and extracellular (c) viral rna levels were detected by rt-qpcr assay. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. d, e knockdown of hsp90β by sirna or knockout by cripsr/cas9 mediated gene editing in rd cells, and then cells were infected with ev-a71 at moi of 1 for indicated time. the protein level of ev-a71 was determined by western blot. f rd cells were treated with hsp90 inhibitors at different concentrations and infected with ev-a71 at moi of 0.01 for 48h. the protein level of ev-a71 was determined by western blot. g pim1protein interaction network was predicted using online genemania program. h pim 1 was overexpressed in 293t cells and cell lysate was collected for immunoprecipitation assay. the phosphorylation status of hsp90 was detected by western blot. i pim1 was overexpressed/ knocked down in 293t cells. and cell lysate was collected for native page analysis. j wt rd cells and hsp90β knockout (hsp90β-ko) cells were pretreated with 2 μm pim1 inhibitor sgi-1776 for 2 h, then the cells were infected cells with ev-a71 at moi of 0.01 for 48h. intracellular viral rna level was determined by rt-qpcr. the results are shown as the means, and ±error indicates the standard deviation (sd). data are obtained from triplicate experiments. *p < 0.05 and **p < 0.01 by two-tailed student's t-test. k pim1 was overexpressed in wt/hsp90β -knockout rd cells, and infected with ev-a71 at moi of 1 for 12 h. the protein level of ev-a71 was determined by western blot hsp90 inhibitor 17-dmag significantly suppresses the aggressive infiltration into multiple organs in atl mice. 153, 185 functions of hsp70 family in rna virus infection virus entry. viruses in different families (e.g., picornaviridae, flaviviridae, and reoviridae) take advantage of hsp70 family proteins for their entry into host cells. in the case of picornaviridae, for example, the coxsackievirus a9 (cav-9) uses hsp70 homolog grp78 for its entry. 186 it shows that antibodies against grp78 block 50% of virus binding. integrin αvβ3 is another famous virus receptor. 187 when cells are simultaneously treated with grp78 and integrin αvβ3 antibodies, virus binding is blocked completely. therefore, grp78 functions as a co-receptor of cav-9. besides, grp78 can interact with major histocompatibility complex (mhc) i molecules on the host cell membrane after infection of cav-9. mhc i molecules help virus internalization into mammalian cells. 186 in the course of ev-a71 infection, hsp70 is dramatically upregulated and interacts with ev-a71 on the cell surface. hsp70 antibody significantly inhibits virus binding to the cell surface. 188 besides enteroviruses, many viruses of flaviviridae family also require hsp70 to entry into host cells. by affinity chromatography assay, hsp70 is discovered to form a complex with hsp90 and denv receptor that facilitates viral entry. 110, 189 hsp70 interacts with dnev envelope protein (e protein) and plays a significant role in virus attachment. 190 similarly, antibodies against hsp70 and hsp90 significantly inhibit denv infection. 110, 189 the same mechanism is also observed in jev infection. 191 hsp70 is enriched in the lipid raft and colocalized with the e protein in jev-infected huh7 cells. 192 the depletion of cholesterol disrupts the enrichment and colocalization of the e protein and hsp70 to a raft membrane. eventually, it decreases jev entry without any effects on virus attachment. 192 these results suggest that hsp70 works as a receptor of jev; and lipid rafts serve as an organizing centre to facilitate jev entry. at the late stage of jev entry, hsc70 (isoform d) is upregulated in c6/36 cells upon jev infection. however, it seems that hsc70 is not required for virus attachment to the cell membrane but needed for virus penetration into the host cells. it is suggested that hsc70 holds an intense involvement in clathrinmediated endocytosis at the late stage of viral entry, which helps jev to penetrate into host cells. 193 recently, it is reported that grp78 is also required for jev both in the attachment and entry steps. 194 antibody targeting the n-terminal of grp78 significantly prevents virus attaching to host cells whereas antibody targeting the c-terminus fails to block the attachment. knockdown of grp78 also inhibits jev internalization. the colocalization and interaction between grp78 and jev envelope protein provide solid evidence to show the importance of grp78 in the process of virus attachment and entry. 194 interestingly, grp78 is secreted out of host cells after jev infection, and the secreted grp78 cooperates with jev to promote virus infection. 195 recent studies show that grp78 is a receptor of sars-cov, mers-cov, and sars-cov-2 viruses. 196 zikv infection is positively regulated by hsp70 at multiple stages. 197 hsp70 inhibitors impair virus entry, rna replication, and capsid assembly of different zikv strains in diverse cell lines. 190, 197 rotavirus infection also needs the assistance of membrane-resident hsc70. 198 hsc70-specific monoclonal antibodies inhibit virus internalization and infection without effect on virus attachment. 198 further evidence shows that the whole virus particle and a short domain (or a peptide) in the cterminal region of vp5 is sufficient to bind to hsc70. 199 the atpase domain of hsc70 is proved to be necessary for its interaction with vp5 and induction of virion conformation change for the entry. 200 virus replication. hsp70 family proteins participate viral replication by employing different mechanisms. first, hsp70 family proteins facilitate the formation of virus replication complex and/ or maintain the stability of complex proteins. in some cases, hsp70 family proteins directly interact with viral polymerase to enhance viral replication. for example, during the mumps virus (muv) infection, the expression level of hsp72 is increased. the c-terminal region of hsp72 interacts with the n-terminal region of p protein, which is an essential component of rdrp complex. knockdown of hsp72 results in accumulated ubiquitinated p protein as well as increased cell apoptosis. 201 besides, hsp70 is also reported to regulate l protein, another muv polymerase component. hsp70 cooperates with hsp90 to regulate l protein levels. hsp90 inhibitor, 17-aag, reduces the l protein level through promoting degradation via the c terminus of hsp70interacting protein (chip) -mediated proteasomal pathway. hsp70 inhibitor ver155008 together with 17-aag enhances l protein degradation. therefore, hsp90 and hsp70 together regulate the stability of l protein and ensure the proper virus replication complex (vrc) formation. 202 in the case of canine distemper virus (cdv) infection, the increased hsp70 results in an elevated expression of light nucleocapsid (nc-l) variant, which displays polymerase activity. 203, 204 a more direct evidence is that hsp70 facilitates viral rna production in cell-free transcriptional assays. 204 furthermore, it is demonstrated that hsp70 interacts with and regulates nc polymerase activity dependent on the hsp70 atp activity; because hsp70 antibody significantly inhibits nc polymerase activity and supplementation of purified recombinant hsp70 enhances both the basal and stress-induced nc polymerase activity. 205 the other members of hsp70s also regulate vrc formation. hsp72 physically interacts with several replication proteins of flavivirus including ns5a, ns3, and ns5b (rdrp). for example, hsp72 participates the vrc formation of hcv. 206 downregulating hsp72 leads to a decreased number of vrc in hcv-infected cells, while overexpression of hsp72 raises the number of vrc. 206 hsc70 is associated with vrc by binding on the 3' polyu/uc motif of hcv rna genome. 207 hcv accumulation and virion production are significantly suppressed when cells are treated with hsp70 or hsc70 inhibitors. 208 similar result is reported in the case of rsv infection. ectopic expression of rsv nucleocapsid protein (n protein) and phosphoprotein (p protein) are detected to interact with hsp70 in 293t cells. 209 the n protein is responsible for interacting with the viral rna, and p protein interacts with n protein and with the rdrp l to form the nucleocapsid. in rsvinfected cells, hsp70 redistributes into lipid-raft membranes and colocalizes with virus n protein and lipid raft marker gm1. 210 although hsp70 inhibitors suppress rsv polymerase activity; 210 it only disrupts viral gene expression but do not affect rna polymerization. 211 therefore, more detailed studies are needed to understand the functions of hsp70 in modulating rsv gene expression and replication. in term of ebola virus (ebov) replication, the mechanism of hsp70 involved is much more complicated. by using immunoprecipitation and mass spectrometry assays, it has been identified that the n protein interacts with hsp70, nef, bag2, and the hsp70 co-chaperone dnaja2. 212 the n protein recognizes and binds to the viral rna genome to establish a steady n protein-rna complex structure (rnp). this complex further interacts with viral proteins vp30, vp35 and rdrp l, to finally form vrc. here, hsp70 functions to maintain the stability of n protein and helps to facilitate vrc formation. 212, 213 in addition, hsp70 is also copurified with l polymerase in insect cells. 214 besides, hsc70 interacts with the terminal non-coding regions of the ebov genome. disruption of the interaction by mutating the binding site potently inhibits the minigenome replication of ebov. 215 another mechanism that hsp70 employs to support virus replication is to modulate nuclear import of polymerase or nuclear capsid. some rna viruses also replicate in the nuclear such as cdv and influenza virus. therefore, nuclear transportation becomes a critical step for their replication. upon cdv infection, hsp70 is shown a strong contribution to viral replication by interacting with and promoting the translocation of the nucleocapsid particles from the cytosol to nucleus. 216 similarly, during influenza infection, hsp70 interacts with pb2 or pb1 monomers and pb2/ pb1 heterodimer in hela and hek293t cells, and sequentially translocates into the nucleus with pb2 monomers or pb2/pb1 heterodimers. 217 if hsp70 and pb2/pb1 polymerases are retained in the cytosol, the polymerase activity reduces dramatically. the shuttling of hsp70 between nuclear and cytoplasmic compartments underlies the modulatory effect of hsp70 on influenza virus replication. 217 virus gene expression. besides viral entry and replication, hsp70 also contributes to viral protein translation. positive singlestranded rna viruses (e.g., sars-cov-2, hcv, zika, ev-a71, etc) use the internal ribosome entry site (ires) to initiate the translation of their own proteins but inhibit the host cellular cap-dependent translation through regulation or cleavage of eukaryotic translation initiation /elongation factors (eifs/eefs). in the case of coxsackievirus b3 (cva b3) infection, hsp70 is upregulated to enhance the initiation and elongation of viral translation. 218 in the translation initiation step, hsp70 upregulates ires-acting factor lupus autoantigen protein expression and activates eif4e binding protein 1 (eif4ebp1), a cap-dependent translation suppressor. in the elongation step, hsp70 activates the akt-mammalian target of rapamycin complex 1 (mtorc1) signal cascade, leading to activation of eef2 via kinase p70s6k-and cdc2-mediated phosphorylation and inactivation of eef2 kinase (ef2k). 218 hsc70 enhances the ires activity in ev-a71 infected cells. hsc70 interacts with 2a protease of ev-a71 to enhance eif4g cleavage that impairs host cell cap-dependent translation but enhances viral ires-mediated translation. 219 therefore, hsc70 may serve as an antiviral target against ev-a71 and hcv infections. some viruses do not have ires sequence, and virus replication produces plenty of dsrnas which trigger the activation of protein kinase-rna-activated (pkr)-eif2a signalling cascade that shuts down global translation in cells and releases stresses. 220, 221 to circumvent the pkr-mediated block to viral proliferation, influenza a virus induces the cellular tetratricopeptide repeat (tpr)domains containing jdp protein, p58ipk. influenza virus downregulates pkr in an hsp70-dependent way. [222] [223] [224] [225] [226] in uninfected and unstressed cells, p58ipk activity is clogged with hdj1 by forming a complex. 225, 226 during influenza a infection, the amount of hdj1 in p58ipk-hdj1 complex decreases to an undetectable level. these findings suggest that the activation of p58ipk appears to be a sequel to the hsp70-mediated release of hdj1 from the p58ipk-hdj1 complex, which allows the monomeric p58ipk to inhibit pkr. 225 in the hsp40 chaperone part, we would discuss more details about the regulation of pkr signaling by influenza virus. virion assembly. hsp70 is reported to assist some viruses' assembly. during morphogenesis of the double-stranded rna reovirus, hsp70 contributes to the assembly of trimeric sigma 1 protein, which is responsible for the interaction with host cell receptor. 227 while the n-terminal segment of the sigma 1 protein folds and trimerizes cotranslationally in an hsp70-independent manner, a post-translational fold of the c-terminal globular domain is dependent on hsp70. in this process, hsp70 binds cotranslationally to the region downstream the n-terminal αhelical coiled-coil, which presumably helps to inhibit unwanted interaction and misfolding. trimerization of the c-terminal domain of the sigma 1 protein is coupled to the atp-dependent release of hsp70 from the ribosome. 227 besides, in hela cells infected by poliovirus or coxsackievirus b1, hsp70 is detected to interact with the capsid precursor p1. 228 in the complex, p1 is mainly newly synthesized and has a longer half-life than that of total p1. the hsp70-p1 complex is regarded as an assembly intermediate of picornaviruses. 228 interestingly, some researchers demonstrate that hsp70 inhibits influenza virus replication by blocking nuclear export of viral ribonucleoprotein complex (vrnp), and subsequent viral morphogenesis via disassociating m1 from vrnp. 229, 230 virus release. the evidence of hsps on virus releasing is limited. both hsp70 and hsc70 can interact with ns5a protein; although they play different roles in hcv infection. 231 silencing of hsp70 decreases viral protein expression, but the virus protein level is not affected. 232, 233 instead, interfering hsc70 reduces extracellular virion production. 232, 233 moreover, hsc70 is embedded in the viral capsid. and co-localization between hsc70 and core and e2 structural proteins of hcv has been found in lipid droplets. therefore, hsp70 and hsc70 may regulate hcv infection release at different steps. the function of hsp70 family proteins in dna virus infection virus entry and genome releasing. instead of virus attachment and entry into the host cells, here we talked about virus entry into the cytosol from er. the cytosol entry from er is a key step in sv40 infections. hsc70 is reported to be essential in this step. hsc70 interacts with and is regulated by sgta. 234 further studies show that hsp70 superfamily member hsp105 forms a subunit in the hsc70-sgta complex to facilitate sv40 cytosol entry. 235 in addition, grp78/bip also plays a role in sv40 cytosol entry. grp78 interacts with sv40 capsid protein in a dnajb11-dependent manner to help sv40 disassemble and enter into the cytosol. 236 hsp70 is also needed for the genome release of some dna viruses. such a process is described in adenovirus infection. after the release of the virion from endocytic vesicles into the cytoplasm, hsp70 and hsc70 immediately attach to the hexon protein, one of the major adenovirus coat proteins. 237 hsp70/ hsc70 and its co-chaperone bag3 interact with the penton base protein, the viral capsid constituent responsible for virus internalization. 238, 239 the intact nucleocapsid is transported to nucleus through the typical nls-dependent nuclear import machinery. 240 the nucleocapsid anchors to the nuclear pore through its hexon protein by interacting with components of the pore complex. then viral dna is transferred into the nucleus in a hsp70-dependent manner but leaving hexon outside the nucleus because the purified hexon, instead of viral dna, enter the nucleus in a hsp70-independent manner. 240 a possible explanation is that the intact nucleocapsid is too large to pass through the nuclear pore complex, while the disassembly of nucleocapsid facilitates the entry of viral dna into nucleus. however, more solid evidence is needed for such an explanation. 238 other examples for the contribution of hsc70 in viral genome release in host nucleus are hsv and polyomavirus. in hsv-infected cells, the translocation of hsc70 from the cytosol to nucleus is triggered by the immediate-early viral protein icp0. hsc70 is colocalized with the components of the 26s proteasome and virus ul6 portal protein, which provides the conduit for dna entry and exit from the capsid. ul6 is highly ubiquitinated in the nucleus, indicating that hsc70 may be responsible for the correct folding and degradation of ul6 in the ubiquitin-proteasome pathway, though there was no direct evidence that ul6 is a substrate of hsc70. 241 it is also believed that hsc70 contributes to polyomavirus genome nuclear import through its interaction with all viral capsid proteins vp1, vp2 and vp3 both in vitro and in vivo. hsc70 translocates from the cytosol to the nucleus accompanied by the translocation of capsid proteins upon infection with polyomavirus. 254 gene expression and protein maturation. most viruses manipulate the cellular transcription and translation machinery and shut off host protein synthesis, so that they can take advantage of these machineries and recruit initiation and elongation factors for the expression of viral proteins. some host factors exploited by viruses interact closely with components of hsp70 complex. therefore, the chaperone system is highly important for viral gene expression. several transcription initiation factors are well known to physically interact with the hsp70 co-chaperone bag1 in vitro. bag1 stimulates general transcription activity in an hsp70dependent manner. [242] [243] [244] the stimulation of global transcription is detected in cells upon infections by either human polyomavirus, john cunningham virus (jcv) or hcmv. 245, 246 however, the detailed molecular mechanism of the general transcriptional activation by viruses is to be further investigated. the typical example of hsp70 system in regulating the maturation of viral proteins is shown in hbv large envelope protein (lhbsag). [247] [248] [249] the mature lhbsag has a unique dual transmembrane topology. initially, the c-terminal of lhbsag cotranslationally resides in er, while the n-terminus is resident in the cytosol and later is translocated into er for post-tranreported that the expression of grp78 is stimulatedslation. to ensure the correct topology, hsp70 system strictly regulates the post translocation of n-terminal of lhbsag. during the translation, hsc70 interacts with the n-terminal of lhbsag at amino acids 63 to 107 and suppresses lhbsag translocation into er. 248, 249 hsc70 cochaperones hip and bag1 also regulate the activity of hsc70 in an antagonistic way. 250, 251 overexpressed hip promotes hsc70 activity resulting in more cytosol retention of lhbsag n-terminal; 250 while bag1 overexpression could inhibit hsc70 activity to promote nuclear translocation. 251 in the post-translation process, grp78 binds with lhbsag and hsc70 to facilitate the er translocation of hbv large surface antigen. 247, 248, 252, 253 the function of grp78 is regulated by both positive regulator er-localized dnaj-domain-containing protein 4 (erdj4) and negative regulator bip-associated protein (bap). 253 increased bap destabilizes lhbsag/bip complex. 253 together, hsp70 chaperone system is crucial in modulating the sophisticated topogenesis of hbv envelope protein. 247 virus assembly. a lot of dna viruses normally assemble in the nuclei of infected cells. taking polyomavirus as an example, all capsid proteins are synthesized in the cytosol, whereas subsequent assembly of virions only takes place in the nucleus. during polyomavirus infection, the constitutive form of hsp70 and hsc70 are coimmunoprecipitated with all three viral capsid proteins (vp1, vp2, and vp3). hsp70 is translocated from cytoplasm to the nucleus in the late stage of infection, coincident with localization change of the viral capsid proteins. 254 in vitro studies show that hsp70 functions to keep proper assembly of polyomavirus. 255 the prokaryotic hsp70 chaperone dnak also interacts with recombinant vp1 at the c-terminal domain where it links pentamers in an assembled capsid. 255 when dnak binds to vp1, it inhibits vp1 assembly, which is induced by calcium in vitro. however, combining the hsp70 chaperone system including dnak, dnaj and grpe with vp1 together is sufficient to assemble vp1 into uniform capsids in the presence of atp alone without calcium. 255 the function of hsp70 family in retrovirus infection human t lymphotropic virus type 1 (htlv-1) is a well-investigated example of retrovirus that interacts with hsp70 proteins. during htlv-1 infection, syncytium formation is a key factor for cell-tocell virus transfer. the syncytium formation is subject to close cellto-cell interactions. 256, 257 cell membrane-resident hsc70 is necessary in this process as hsc70-specific monoclonal antibodies eliminates the syncytium formation and htlv-1 infectivity. the same outcome presents when cell is treated with a peptide derived from the htlv-1 glycoprotein gp46, which binds to hsc70. 258, 259 interestingly, although hsc70 enhances the syncytium formation, it has no effect on virus entry. 259 hsp70 also plays an important role in the post-entry steps. during human immunodeficiency virus type 1 (hiv-1) infection, viral protein r (vpr) stimulates interaction between the viral preintegration complex and karyopherin-alpha to facilitate viral nuclear import. hsp70 functionally overlaps with vpr in this process. 260 when vpr is deficient, hsp70 could rescue virus nuclear import by interacting with karyopherin-alpha at the n-terminal that also binds vpr. interestingly, some researchers argue for the antiviral role of hsp70 because hsp70 and vpr share the same substrate. it seems that hsp70 would compete and inhibit vpr function. since hiv-1 needs vpr to manipulate cell cycle and apoptosis, hsp70 neutralizes the function of vpr in hiv-1 infection. 261, 262 recently, hiv is observed to package hsp70 as part of virion core. the virion-incorporated hsp70 atpase activity and correct conformation of hsp70-virion are essential for hiv infection, since inhibition of hsp70 atpase activity interrupts the hsp70-virion core association and diminishes virus infectivity. 263 the effects of hsp70s on host cells upon dna virus infection cellular transformation. dna viruses those do not encode polymerase in their genome are dependent on host dna replication machinery. to replicate in quiescent cells, the virus has to reinitiate cell cycle thereby transforming the host cells. some mechanisms have evolved in enabling viruses to overcome the restriction points of cell cycle. the best-investigated example is sv40. the large and small t antigen (tag) are central for the sv40 transformation ability. at their n terminus, both types of tag contain the j-domain, the signature motif of an hsp70 cochaperone. mutation or deletion of the j-domain disrupts the functional association of tag with hsp70s. the ability of tag to transform mammalian cells is subsequently obliterated. 264 also, tag sequesters the retinoblastoma family proteins (prb, p107, and p130) and liberates members of the e2f family of transcription factors in a hsc70-atp hydrolysis-dependent manner. 265, 266 the free e2f family proteins subsequently trigger the expression of the s-phase genes leading to dna replication. 267 the above observations are interpreted in the following four steps. firstly, the large tag combines with prb-e2f complex. subsequently, the prb-e2f-tag complex associates with hsc70 in the presence of atp as hsc70 atp-bound form presents high substrate association rate to facilitate tag-hsc70 complex formation. the third step is that the j-domain of tag in the tag-hsc70 complex stimulates atp hydrolysis. this process is dependent on the active site of hsc70 and does not occur at the t-antigen binding site. meanwhile, hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. in the last step, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f. after adp dissociation and rebinding of atp to hsc70, e2f and the prb-tag complex is released from the hsc70-substrate complex. [268] [269] [270] [271] a second strategy of tag to transactivate e2f transcription is independent of the disruption of the prb-e2f complex that also involves the jdomain and hsp70 protein. [271] [272] [273] [274] tag functions like bag1 to assiste the assembly of a transcription initiation complex on the respective promoters in the presence of hsc70. alternatively, tag could induce hsc70 to disassemble an inhibitory silencer complex fig. 5 model for the participation of hsc70 in the tag induced disassembling of prband e2f. firstly, tag combines with prb-e2f complex to facilitate t-antigen-hsc70 complex formation. then tag-hsc70 complex stimulates atp hydrolysis, and hsc70 transfers to the high-affinity conformation allowing the trap of the substrate protein prb or e2f. finally, hsc70 induces the conformation of the substrate protein in the complex which assists the dissociation of prb and e2f to initiate cell cycle reprogram or to assist with remodelling the chromatin (fig. 5) . hpv and adenovirus have similar transforming activities by disrupting prb-e2f complexes. although neither e7 (hpv) nor e1a (adenovirus) protein contains a j-domain, both proteins could transform cells in a way similar to that described for sv40 tag. e7 interacts with tumor suppressor htid-1. 275 the c terminus of e7, which mediates the interaction with htid-1, is essential for the physical disruption of the prb-e2f complex though it is not necessary for direct interaction with prb. 276, 277 these observations suggest that the interaction with htid-1 is involved in the disruption of the prb-e2f complex, providing e7 with the jdomain necessary to recruit hsc70 for the complex dissociation in analogy to the function of sv40 tag. alternatively, the binding of e7 to htid-1 could transform cells through inhibition of the assumed tumor suppressor function of htid-1. e1a directly interacts with hsc70 to disrupt the prb-e2f complex. 278 in conclusion, most double-stranded dna viruses depend on hsp70 chaperones for the reprogramming of the host cells to reenter the cell cycle. cell survival and apoptosis. since hsp70 systems are essential modulators for cell survival under stress conditions, the induction of hsp70 protein facilitates virus infection by keeping the cell alive until mature viruses are ready to leave. this is the main reason why the disruption of apoptotic pathway is a quite common phenomenon in viral infections. [279] [280] [281] in the early stage of viral life cycle, viral reproduction is simply vulnerable to cell death. naturally, viruses are evolved in manipulating cellular apoptosis. in ebv-infected cells, nuclear oncoprotein ebna3a helps hsp70 nuclear translocation and hsp70 chaperone complex formation to immortalize b cells through inhibiting apoptosis. 282 in contrast, apoptosis is beneficial for virus spreading when virions are finally assembled. [283] [284] [285] [286] [287] [288] [289] [290] virions are found in apoptotic bodies and subsequently engulfed by phagocytic cells. it has been suggested that virus can infect neighbour cells without being detected by the host immune system. 291, 292 on the other hand, the decrease of hsp70 mrna level may lead to the timed induction of apoptosis at the late stage of adenovirus infections. innate immunity. hsp70 has been previously described to influence innate immunity and inflammatory responses. 293 it has been believed that hepatocyte is devoid of innate immunities. ma et al. reported that the expression of grp78 is stimulated by hbv replication. the elevated grp78 protein in turn activated innate immune response by induction of ifnβ expression. 294 further studies showed that hsp70 greatly contributes to cellular innate immunity in response to either virus or bacterial infections. in the course of bacterial infections, the elevated intracellular levels of hsp70 protect cells from lps-mediated inflammation. through its interaction with tnf receptor associated factor 6 (traf6), hsp70 can inhibit its ubiquitination and thereby block the activation of transcription factor nf-kb. 295, 296 weiss et al. has presented more details on this mechanism. hsp70 binds with ikk, leading to disturbing the function and stability of nf-κb/ikbα/ikk complex and further impairing ikbα phosphorylation. 297 the disturbance of this complex also affects ikbα proteasomal degradation and nuclear translocation of nf-κb complex. 298 the inhibition of nf-κb signalling has great therapeutic significance because it can prevent massive tissue damage medicated by the excessive inflammation response. a more recent study provides another approach for hsp70 to regulate inflammation. pierre et al. described an nf-κb independent pathway that hsp70 could impact nlrp3/asc inflammasome formation through its association with nlrp3. 299 aside from the anti-inflammation function of intracellular hsp70, the secreted extracellular hsp70 binds to dendritic cells and macrophages before being recognized by its binding elements, most of which are innate immune receptors. 293, 300 toll-like receptors (tlrs) detect virus invasion and immediately trigger intracellular innate antiviral response. 301 they belong to type i integral membrane glycoproteins of il-1 receptor (il-1r) superfamily. 302 it has been suggested that tlr2/tlr4 involved in the initiation of innate immunity by extracellular hsp70. hsp70 utilizes both tlr2 and tlr4 to transduce its proinflammatory signal in a cd14-dependent manner to promote proinflammatory cytokine production via myd88/irak/nf-κb axis signalling cascade. 8, 301 another study clearly demonstrated the function of tlr4 and its direct interaction with hsp70. 303 after ligand binding, tlrs dimerize and undergo a conformational change required for recruiting downstream signaling molecules, including the adaptor molecule myeloid differentiation primary-response protein 88 (myd88), il-1r-associated kinases (iraks), tgfβ-activated kinase (tak1), tak1-binding protein 1 (tab1), tab2 and tnf-receptor-associated factor 6 (traf6). [304] [305] [306] [307] many other innate immunity-related signaling pathways would then be activated, for example, phosphorylation of nf-κb via tak1/ikk activation. mapks p38, jnk, and erk pathway are also activated, then subsequently activate creb and ap-1 transcription factors. both ap-1 and nf-κb activate proinflammatory cytokine expression, including tnfα, il-6, il-1β, and a number of other cytokines and chemokines. 308-311 the effects of hsp60s on host cells upon rna virus infection immunity modulation. few studies show the function of hsp60s in the life cycle of rna virus; however, the function of hsp60 in regulating host immunity has been widely studied. [312] [313] [314] [315] [316] the majority of studies show that hsp60 works as an activator of immune response. in the case of jev infection, hsp60 facilitates virus-induced inflammation by promoting il-1β production via increasing nlrp3 inflammasome activity and nfκb phosphorylation. 317 however, under certain conditions, viruses also utilize hsp60 to evade host cell immune response. the genome-wide rna interference (rnai) screen identifies the interaction of hsp60 with pb2 of influenza virus. 318 hsp60 helps pb2 translocation from the cytosol into mitochondria. 38, 39 then the mitochondrial pb2 interacts with and modulates the activity of mitochondrial antiviral signaling protein (mavs) to suppress interferon β (ifnβ) production. 38 therefore, hsp60 determines the effect of pb2 on both mitochondrial stability and the level of ifn-β production. 38, 39 besides, denv infection also elevates hsp60 expression. silencing of hsp60 results in an increase of ifn-α production and decrease of virus reproduction in macrophages. 319 however, the detailed mechanism remains elusive. apoptosis regulation. viruses use different mechanisms to modulate apoptosis. in the case of hcv infection, ros production is regarded as the major contributor of hcc although it also promotes cell apoptosis. 320 study shows that the n-terminal domain of hcv core protein can induce ros production by interacting with hsp60 and inhibiting the normal function of hsp60 in releasing protein stress. 321, 322 while another rna virus, rotavirus sa11, tries its best to delay the early apoptosis through modulating hsp60 stability. 323 hsp60 helps to translocate nsp4 protein of rotavirus into mitochondria from cytosol and induces apoptosis. 323 to yield more viruses, rotavirus infection increases the hsp60 phosphorylation at tyr 228 by activated src kinase that leads to the ubiquitin-mediated proteasomal degradation. 323 even though virus postpones apoptosis via hsp60, the main function of hsp60 is to refold proteins in mitochondria. 323, 324 the function of hsp60 family in dna virus infection virus replication. in the previous sections, we have discussed that hsp90 is important for rt-ε rna complex formation in hbv infection. however, it has been shown that hsp60 directly regulates rt activity before the rt-ε rna complex formation. 325, 326 hbv replication is markedly suppressed when hsp60 is knocked down by specific sirnas. 325 hsp60 transiently interacts with rt to activate rt in an atp-dependent manner. 326 upon rt activation, hsp60 immediately dissociates from the hsp60/rt complex without being encapsidated into viral nucleocapsid. 326 more detailed research shows that at least one of two rt fragments, residues 1-199 of terminal protein (tp) domain and 680-842 of rnase h (rh), is necessary for hsp60 binding. 327 the tp domain is also responsible for the binding of hsp90 in the rt-ε rna priming step. 138 apoptosis regulation. similar to hcv infection, hbv infection also induces strong apoptosis which is thought mainly contributed by hbx protein. 328 studies show that overexpression of hsp60 facilitates hbx-induced apoptosis. 329, 330 the interaction of hbx and hsp60 has been observed and confirmed by different methods including affinity purification, mass spectrometry and co-immunoprecipitation. 329 hsp60 binds on a small domain (residues 88-117) of hbx. 329 furthermore, hsp60 also forms a complex with hbx and hsp70 in the mitochondria. 330 however, the mechanism of how hsp60 enhances the apoptosis remains unclear during hbv infections. immunity modulation. hsp60 is involved in both the innate and adaptive immune response. 314 here, we focus on how hbv harnesses hsp60 to evade host immune response. numerous studies show that hbv employs active means to escape innate immune response and induce immunosuppression. 331 among these strategies, hbv infection increases the population of cd4 + cd25 + t regulatory cells (tregs) which can produce an amount of il-10 and tgf-β. 332 il-10 is also called cytokine synthesis inhibitory factor (csif) displaying anti-inflammatory properties. 333 it influences both the first and the second line of immune defence. 334 hbv infection increases serum shsp60 level and makes use of hsp60 to activate cd4 + cd25 + regulatory t via tlr2/myd88/il10 signaling. 335 the function of hsp60 family in retrovirus infection although the role of hsp60 in dna/rna virus infection process has been widely studied; only limited knowledge has been obtained in retrovirus infection. hsp60 is encapsidated into hiv particles, 336 but we have no idea about its function. the integrase catalyzes the integration of hiv-1 pro-viral dna in the host genome. 337 a small portion of hsp60 colocalizes and interacts with the viral integrase (in). 338 hsp60-hsp10 complex maintains the integrase at an active form and stimulates its activity in an atp-dependent manner. 338 the integration is a critical step for successful infection of hiv-1. the function of hsp40 family in rna virus infection virus replication. hsp40s regulate rna viral replication by modulating polymerase activity, replication complex and nuclear transportation. in jev-infected cells, hsp40/dnaj homolog hdj2 interacts and colocalizes with ns5 protein, an rdrp essential for viral rna genome replication. 339 overexpressed hdj2 promotes jev replication significantly. however, how hdj2 modulates ns5 activity remains elusive. 339 influenza virus also takes advantage of hsp40 to promote replication by assisting vrc to relocate into nucleus. the replication of influenza virus occurs in the nucleus of host cells; therefore, nuclear trafficking of viral ribonucleoprotein (vrnp) complex is required. the vrnp is composed of viral rna (vrna), polymerase heterotrimer (pa, pb1, pb2) and nucleoprotein (np). 340 np has a key function of interacting with importins through its nuclear localization signals. 341, 342 hsp40s have two strategies to help vrnp transport into nucleus. first, hsp40/ dnajb1 interacts with np at the early stage of infection and ensures efficient association between np and importin alpha. 343 this interaction is mediated by the j domain of hsp40 and the nterminal region of np. 343 another strategy is that hsp40/dnaja1 binds with the pb2 and pa polymerase subunits, then cotranslocates into nucleus with pb1-pa complex. 344 besides, hsp40/dnaja1 enhances viral rna synthesis both in vivo and in vitro. 344 different from dnajb1, dnaja1 mainly depends on its c-terminal substrate-binding domain instead of typical j domain to manage viral rna synthesis. 344 in the replication process, hsp70 is reported to enhance polymerase nuclear translocation. 217 thus it is proposed that dnaja1 cooperates with hsp70 and assist rna polymerase nuclear import. 217, 343 virus gene expression. viral rna replication produces plenty of double-stranded rna (dsrna) molecules. host cell detects these dsrna and activates interferon-induced protein kinase (pkr) to restrict viral replication by phosphorylating eukaryotic initiation factor eif2α and preventing protein synthesis. 220, 221, 345 however, in order to escape from the antiviral response of host cells, the influenza virus smartly blocks the activation of pkr/eif-2α. influenza virus ns1 directly binds the n-terminal of pkr and inhibits pkr activation. 225 besides, np protein also interacts with hsp40, leading to dissociation of hsp40 and p58ipk. 346 it is reported that type iii hsp40/p58ipk is an inhibitor of pkr, [222] [223] [224] 226, 347 while hsp40 is the inhibitor of p58ipk. 225, 348 therefore, the dissociation of hsp40 results in activation of p58ipk. subsequently, p58ipk inhibits the activity of pkr/eif-2α. as a result, influenza virus releases the inhibition of protein synthesis. however, in the late stage of infection, influenza virus m2, hsp40 and p58ipk form a stable complex that would lead to pkr activation, er-stress-induced cell death and virion release. 349 protein maturation. flavivirus genome encodes a large polyprotein which is later cleaved into several mature structures and nonstructure proteins. the mature proteins then form vrcs. at the beginning, hsp40 family protein dnajc14 participates in the vrc formation of flavivirus. during yellow fever virus (yfv) infection, dnajc14 is recruited to non-structural protein clustering sites with ns3 and ns5 to form vrc. 350 however, either knockdown or overexpression of dnajc14 inhibits yfv and hcv replication. 350, 351 later, it has been demonstrated that dnajc14 overexpression affects yfv polyprotein processing and alters vrc assembly. overexpression of dnajc14 alters the cleavage sites of ns3/4a and ns4a/2k and gives rise to abnormal ns3 to ns3-4a ratios, suggesting that the chaperone activity of dnajc14 modulates ns3/ 4a/2k cleavage that ensures appropriate expression level of ns3 and ns4a. the inhibition of vrc formation upon ectopic expressing dnajc14 is caused by chaperone dysregulation. 351, 352 immunity modulation. hsp40s sometimes act to help the virus evade host immunity, while sometimes it exhibits antiviral activity by increasing host immune response under certain conditions. it is reported that the expression of dnajb1/hsp40 and hsp70 is induced by polyi:c stimulation. 353 hsp40 cooperates with hsp70 to suppress the mda5/mavs pathway though interacting with mda5 and inhibiting mda5 multimer formation. 353 however, dnaja3 shows its ability to suppress virus replication. during hfdv infection, vp1 is able to suppress the type i interferon signaling via suppression of phosphorylation, dimerization, and nuclear translocation of irf3. 354 however, dnaja3 induces lysosomal degradation of vp1 protein. therefore, dnaja3 indirectly stimulates the immune response of host cells. 354 the function of hsp40 family in dna virus infection virus replication. evidence suggests that hsp40s regulate the initiation of dna virus replication. in the case of hpv replication, it starts with the recognition of protein e2 on the origin (ori) sequence and recruitments of replication initiator e1, which displays atpase and helicase activities. [355] [356] [357] hsp40 (hdj1 and hdj2) and hsp70 enhance e1 binding on the ori independently and additively. hsp40 directly binds with e1 and remains in the e1ori complex, whereas hsp70 transiently interacts with e1 in an atp-dependent manner. 355 subsequent study reveals an additional role of hdj2 in facilitating e1 helicase function by replacing e2 in the e1/e2/ori complex. 358 the stable association of e2 to ori flanking the e1 binding site may act as a dna clamp to prevent dna unwinding. similarly, hsp40 (htid1) also has similar functions to hdj1 and hdj2 with independent chaperone activity. hsp40 interacts with hsv-1 replication initiator protein and helicase protein ul9, thereby promotes their binding to the replication origin. 359, 360 this provides another example of the involvement of j-proteins in the replication process of eukaryotic dna viruses. except for enhancing hbv replication, a possible negative role of hsp40 has also been reported. the core protein is a key component of viral capsid and essential for virion assembly, while hbx is a multifunctional virulence factor implicated in viral replication and hepatocarcinogenesis in human. a yeast 2-hybrid approach is used to identify interactions between the core protein and two hsp40s, hdj1 and htid1. 361, 362 individual expression of each hsp40 in hepatocytes transfected with a replicationcompetent hbv construct shows an inhibitory effect on both viral replication and capsid formation. further studies reveal that both core and hbx proteins are destabilized by co-expression with hdj1 or htid1; because they are targeted for enhanced proteolytic degradation. except inhibiting hbv replication, hdj1 also facilitates proteasome-mediated degradation of hbx. 361 virus induced cellular transformation. as mentioned above, some viruses can induce cell transformation and tumorigenesis. here, we discuss the role of hsp40 proteins on virus-induced cellular transformation. hsp40 may have a negative role in hbv-induced cell transformation. it has been demonstrated that hbx is the major factor for induction of hepatocyte transformation. [363] [364] [365] [366] nevertheless, overexpression of hsp40s (hdj1 and htid1) significantly enhance the proteasome-mediated degradation of hbx. another study suggests that htid1 interacts with e7 oncoprotein and promotes the cellular transformation by hpv16; 275 because the binding sequence of e7 shares high similarity to the tag oncoproteins of sv40. nuclear entry of viral pre-integration complex. hiv can efficiently infect nondividing cells. 367 this requires active transport of the viral pre-integration complex (pic) into the nucleus without breaking down nuclear membrane. some components of pic implicated in regulating nuclear import include the central dna flap and viral proteins in, ma, and vpr of hiv-1 (or vpx of hiv-2). 288,368-372 hsp40/dnajb6 interacts and enhances the nuclear localization of vpx as well as promotes the nuclear import of viral pic. 373 similarly, dnajb6 also promotes vpr nuclear localization during hiv-1 infection; 374, 375 particularly, the long isoform of dnajb6 is extremely important in this process. 375 the expression level of dnajb6 s/l isoform is regulated by the polyadenylation factor cstf64. high level of cstf64 favors dnajb6-s isoform production, whereas a low level of cstf64 enhances dnajb6-l isoform production. 374 regulation of viral gene expression. a notable example of hsp40 involvement in regulating retrovirus gene expression is its importance in enhancing the gene expression during hiv-1 infection. 376 nef protein, an important viral protein associated with pathogenesis and disease progression, stimulates hsp40 expression by enhancing hsp40 promoter activity via hsf1 transcription factor. [377] [378] [379] hsp40 and nef co-translocate into nucleus where they become a part of cdk9-associated transcription complex to enhance long terminal repeat (ltr) mediated gene expression. 376, 380 the binding of hsf1 on hiv-1 ltr promoter induces viral gene expression directly. 380 interestingly, hsp70 seems to act contrary to hsp40, which also presents in the nef-hsp40 complex. different from hsp40, hsp70 suppresses viral replication and gene expression; while hsp40 rescues the hsp70downreguled viral gene expression. hsp70 inhibits cdk9 phosphorylation, an essential event for high-affinity binding of hiv-1 transactivator of transcription-positive transcription elongation factor b complex for transactivating response rna. 381 it is also reported that some other hsp40 family proteins negatively regulate hiv replication. hsp40a1, b1, b6 and c5 (but not c3) are able to limit hiv-1 production, while they have no effect on viral gene expression upon infection by adenovirus, hsv-1 or vaccinia virus. the conserved dnaj domain is suggested to be responsible for the inhibiting hiv-1 reproduction. the hsp70/ hsp40 complex specifically recognizes and inhibits the rev translation or accelerates its degradation, leading to inhibiting viral gene expression. 382 small heat shock proteins are the most upregulated proteins identified in host cells under stress conditions, for example, when cells are exposed to elevated ros level, abnormally high temperature, or pathogen invasions. 383 in most cases, shsps are responsible for recognizing misfolded proteins and transferring them to other atp-dependent chaperones for proper folding, or proteasomes or autophagosomes for degradation. 49, 384 hsp27 is one of the most ubiquitously expressed shsps with the highest level in skeletal, smooth, and cardiac muscles. 59, 385 like all other shsps, hsp27 shares a highly conserved α-crystallin domain, or socalled c-terminal domain. it contains 6-8 β-strands, forming 2 β-sheets as intermolecular interaction sites. 41, 59 because of the importance of hsp27, in this section, we mainly focus on the function of hsp27 in virus infection. hsp27 has been shown particular importance in viral infections. rajaiya et al. suggested that the association of hsp27 with p38 or nfκb/p65 plays key roles in controlling the expression of proinflammatory mediators in virus-infected cells. 64 fukagawa et al. suggested that the phosphorylated hsp27 is upregulated through the pi3k/akt pathway upon ebv infection. 386 we would discuss the special roles of hsp27 in different viral infections in the following sections. the function of hsp27 in rna virus infection hsp27 is upregulated during ev-a71 infection by proteomics analysis. knockout of hsp27 results in the suppression of virus replication and protein expression level, while their restoration appears after hsp27 is restored. also, hsp27 enhances viral ires activity by promoting 2a pro -mediated eif4g cleavage. 57 interestingly, the nuclear translocation of hsp27 from cytosol is reversely correlated with the relocalization of rna chaperone hnrnpa1 from nucleus to the cytosol for initiating viral protein translation upon ev-a71 infection. however, knockout of hsp27 blocks hnrnpa1 cytosol relocolization, indicating a fundamental role of hsp27 in regulating the import/export of nuclear proteins during virus infection (fig. 6) . 383 hsp27 is rapid upregulated at the early stage (4 hours post infection) of coronavirus infection, 387 suggesting an important role in virus early replication and possibly a good target for treating sars-cov-2 infection. swine fever virus (csfv) is a member of the family flaviviridae. hsp27 is found to bind with ns5a, which is a non-structural protein in response to viral replication and assembly. hsp27 depletion enhances the virus replication while the replication is suppressed by ectopic expression of hsp27 through activating nf-κb signaling in pk-15 cells. 388 porcine epidemic diarrhoea virus (pedv) infection causes high fatality in swine. the hsp27 is obviously upregulated in pedv infected marc-145 cells. 389 however, the virus titer is declined by ectopic expression of hsp27. hsp27 could activate nf-κb signalling and enhance the transcription of ifnβ and downstream interferon stimulated genes (isgs). 389 the function of hsp27 in dna virus infection pro-inflammatory cytokines play crucial roles in early antiviral infections. adenovirus infection causes a wide variety of diseases. 390 the internalization of adenovirus leads to the activation of erk1/2, which could in turn trans-activate nf-κb and ap-1 to induce pro-inflammatory cytokine il-8 expression in different experimental systems. [391] [392] [393] a study shows that downregulation of hsp27 leads to an increased release of il-8 from keratinocytes stimulated with uv or tnfα. the increase of il-8 is suppressed by nf-κb inhibitor and correlated with an enhanced iκb-α degradation and phosphorylated iκb-α accumulation in hsp27-depleted cells. in addition, hsp27 is shown to be associated with the iκb kinase (ikk) complex. because the synthesis of prostanoid pge2 and il-8 is regulated by nf-κb, it could be a likely mechanism of hsp27 in modulating inflammatory cytokine production. 70 further studies also show that hsp27 is of particular importance in the cyclooxygenase-2 and il-6 expression via activating p38 mapk/mk2 signalling and the consequential stabilization of cyclooxygenase-2 and il-6 mrnas. 68 aside from its involvement in pro-inflammatory response regulation, researches have also found the antioxidant role of hsp27 in regulating stress caused by ros. 49 it is commonly agreed that hsp27 regulates enzyme activities by upholding glutathione in reduced form, such as glutathione reductase and glucose-6phosphate dehydrogenase. 394 more recent evidence correlates the level of shsps with the intracellular content of iron, a catalyzer of hydroxyl radical generation. hsp27 also exhibits an important role in oxidized protein degradation machinery. 395, 396 however, there is also controversial evidence indicating the involvement of hsp27 in accumulation of oxidized proteins that benefits herpesvirus replication. experimental models are set up using two distantly related herpesviruses rhesus rhadinovirus (rrv) and hsv-1. they are close relative of kaposi's sarcoma-associated herpesvirus (kshv). the oxidized proteins are accumulated during these viral infections. results show the removal of only a part of oxidized proteins in a proteasome-dependent manner, while some others resisting degradation. 397 oxidized proteins resisting proteolysis become sequestered in foci within the nucleus and coincided with hsp27-enriched foci; although they are not associated with virus-induced chaperone enriched domains (vice). furthermore, the accumulation of oxidized proteins is more pronounced in hsp27-depleted cells. 398 one possible explanation is that hsp27 buffers the toxic effects of those defective proteins undergoing proteolysis through aggregation in the nucleus. the roles of hsp27 are most likely not mutually exclusive during virus infection. hsp27 also contributes to virus replication. porcine circovirus type 2 (pcv2) is a single-stranded dna virus that causes the postweaning multisystemic wasting syndrome (pmws) in pigs. the phosphorylated hsp27 is upregulated in the nucleus in pcv2infected pk-15 cells. hsp27 inhibitors such as sb203580 suppress pcv2 replication. the same result appears upon hsp27 knockdown. in contrast, ectopic expression of hsp27 promotes viral replication. 399 moreover, the phosphorylation of hsp27 is also upregulated in ebv-positive cells, as well as the phosphorylated (activated) akt levels. when ebv-positive cells are treated with pi3k inhibitors, the phosphorylated hsp27 level is decreased, suggesting that the phosphorylation of hsp27 is upregulated through the pi3k/akt signalling pathway upon ebv infection. 386 however, studies also show that hsp27 can both positively and negatively regulate the virus replication depending on the virus/ cell types. tong et al. reported that hsp27 works as an antiviral protein against hbv replication through enhancing ifn production in hepatocytes. the hsp27 expression level is increased in both hbv-infected human liver tissues and hbv-producing hepg2.2.15 cells. 400 the function of pdis on virus entry and uncoating the entry of some viruses into eukaryotic cells is governed by redox-regulated processes. one example is newcastle disease virus (ndv), a bird virus in the family of paramyxoviruses. this negative-sense, single-stranded rna paramyxovirus gains entry to its host cell through large conformational changes in its fusogenic f-protein, which involves thiol/disulfide exchange. 401 overexpression of pdi and erdj5 (a pdi family reductase with an extra j domain) leads to an increase of viral membrane fusion, indicating a route whereby virus can take advantage of the pdi family to gain access to host cells. 402 fig. 6 the controversial function of hsp27 during virus infection. hsp27 would enhance 2a protein-mediated eif4g cleavage, which would in turn promote ires function and viral genome replication. it correlates with hnrnpa1 translocation from host cell nuclei to cytosol, and the consequential virus protein translation. hsp27 is also able to suppress apoptosis via caspase 3 inhibition. on the other hand, hsp27 could help activating ikk complex, modulating nf-κb and ap-1 to induce the expression of pro-inflammatory cytokines reduces β1 and β3 integrins allowing denv entry. 403, 404 also, thiol blockers and pdi inhibitors decrease the entry of rotavirus in ma104 cells, indicating the involvement of thiols for infectivity. 405 the entry of hiv is regulated by pdis. the envelope of hiv becomes unhinged by pdi for entry. ryser et al. firstly reported that cleavage of two disulfide bonds in the gp120 surface component of the hiv-1 envelope is required for virion entry into cd4 + cells. in this process, the pdi on cell surface is responsible for this effect. 406 pdi inhibitors sufficiently prevent the reduction and block the cleavage of surface-bound disulfide conjugate, thereby prevent infection at the level of hiv-1 entry. 407 now, there are numerous studies on how envelope binds on host cells. results from different groups have demonstrated that gp120 moves laterally along the membrane surface until it collides with a patch of pdi in a domain of the membrane that distinguishes from a typical lipid raft. pdi reduces two disulfide bonds in gp120, producing conformation changes that likely stabilize the binding of gp120 to cd4 and expose the v3 loop for subsequent binding to the chemokine coreceptor. following this, gp41 undergoes rearrangement into its fusogenic intermediates and entry occurs. 408, 409 during the entry, galectin-9 binds pdi to regulate the redox environment on the cell surface and enhance hiv entry. 410 taken together, the reports from these three groups have profound implications for our understanding of the hiv virion surface structure and viral entry. the other examples are the nonenveloped polyomavirus (py). py particles contain a layer of coat protein vp1. this single protein, arranged as 72 pentamers, forms the shell surrounding the viral genome. 411 after internalization, py penetrates the er membrane to gain access to the cytosol and then the nucleus for viral genome transcription and replication. pdis isomerize or reduce the virus disulfide bonds to generate a membrane transportcompetent intermediate for host cell membrane penetration. 412 for example, sv40 entry involves caveolar/lipid raft-mediated endocytosis, vesicular transport to er and translocation into the cytosol. erp57 isomerizes the interchain disulfides connecting vp1 for virus uncoating. 413 the further study demonstrates that erp57 and pdi operate in concert with erp29 to unfold the vp1 c-terminal arm. pdi and erp72 reduce py, while erp57 principally isomerizes the virus in vitro. mutagenesis study subsequently identified that the residues c 11 and c 15 of vp1 are important for infection, suggesting a role for these residues during isomerization. 414 pdis regulate viral protein translation a little evidence shows that pdis are involved in viral protein translation. some positive single-stranded rna (+ssrna) virus depends on ires-mediated translation for viral protein synthesis. ev-a71 infection is potently inhibited by an active compound oblongifolin m (om), which is isolated from herb garcinia oblongifolia. further studies show that om suppresses the viral ires-mediated translation of polypeptide via suppressing erp57, and ectopic expression of erp57 increases the ires activity and partially rescues the decreased viral replication caused by om treatment. 415 the detailed mechanism how erp57 downregulates ires activity would be further investigated. several studies have demonstrated the implication of redox balance disruption in the establishment of viral infection and the progression of virus-induced diseases. and accumulated ros in turn may modulate the viral replication and cellular response that also contribute to viral pathogenesis. 416, 417 virus-induced oxidative stress has been reported during hiv, 418 influenza virus, 419 hbv, 420 hcv, 421 encephalomyocarditis virus (emcv), 422 respiratory syncytial virus (rsv), 423 and jev 424 infections. considering its thioredoxin-like sites, erp57 has been thought a main player in the mechanisms of cell protection against oxidative stress, regardless of its subcellular location. redox proteomics analysis of hpv positive tissues shows that the expression level of erp57 and gst is positively correlated with tissue redox status, suggesting its potential association with viral-induced oxidative dna and protein damages. 425 er stress is another consequence caused by virus activities. viral infection would lead to exploitation of the er membrane, accumulation of misfolded proteins, and imbalance of calcium concentration. influenza a virus (iav), hbv, jev, denv, and zika virus all hijack host cell process to enhance viral pathogenesis, such as facilitating viral folding and trafficking, affecting receptor interaction, and modulating host immune responses. [426] [427] [428] therefore, as a major factor in er stress response, erp57 would be critical for viral protein glycosylation and maturation, which may in turn affect virus release and infection (fig. 7) . the life cycle of most rna viruses is completed in the cytoplasm of host cells. to complete the life cycle, virus is often able to induce redistribution of many host cell proteins; particularly, those proteins involved in rna metabolism and functional regulation of viral rnas. hnrnps, such as hnrnp a1, 429 hnrnp c1/c2, 430 hnrnp d 431 and hnrnp i, 432 are mainly resident in the nuclear but quite often shuttle to cytoplasm for stabilizing the viral rna and initiating cap-independent translation. viruses employ various means to redistribute hnrnps. for instance, the ebov inhibits the nuclear import of hnrnp c1/c2. vp24 binds to npi-1 subfamily karyopherin-alpha nuclear import proteins at the c-terminal region (amino acids and prevents their interaction with tyrosine-phosphorylated stat1 (pstat1) and hnrnp c1/c2. the inhibition results in cytoplasm retention of pstat1 and hnrnp c1/c2. 430 435 however, the detailed mechanism of how 2a pro and core protein induce the redistribution remains unknown. virus replication. it has been well documented that hnrnps greatly contribute to the virus replication. for example, hnrnp i/ ptb and hnrnp c participate replication of different viruses, e.g., coronavirus, 436 hcv 437 and poliovirus. 438 the role of hnrnp i/ptbs in viral rna synthesis seems to vary among different viruses. ptbs bind ucuaa pentanucleotide repeats at ires and 3′-utr. ptbs modulate coronavirus rna synthesis. 436 however, the function of hnrnp i/ptb is to some degree complicated in hcv rna replication. it selectively interacts with the 3′ end of the hcv genomic rna. the upstream sl2 and sl3 stem-loop structures are essential for hnrnp-i/ptb i binding whereas the most 3′-terminal stem-loop sl1 is not needed. hnrnp i/ptb i and hnrnp c specifically bind the pyrimidine-rich region of in the 3′ntr of hcv rna genome. 437 possibly, hnrnp i/ptb i is recruited for helping to initiate viral negative-strand (−) rna genome replication, to stabilize rna genome, and/or to regulate the encapsidation of genomic rna. 439, 440 up to date, the detailed function of hnrnp i/ptb i binding on 3′utr has not been elucidated. some studies showed that ptbs have an inhibitory effect on the synthesis of hcv rna genome, 441 while aizaki et al. reported that ptbs are required for efficient hcv rna replication. 442 it has been postulated that they suppress the initiation of viral genome rna replication but enhance ires-mediated translation or facilitate the replication-translation switch. it is shown that hur can compete hnrnp i/ptb i binding on 3′ utr of the viral rna to facilitate la binding to the 3′ utr; while la protein is critical for hcv genome replication. [443] [444] [445] although hnrnp c has many similarities with hnrnp i and both bind with the 3′utr of the hcv rna genome to facilitate viral rna replication; 437 more details demonstrate that only hnrnp c binds the 3′-ends of viral rnas with both negative and positive polarities. 446 besides, hnrnp c also binds at the 5′-end on negative-strand rna of poliovirus to facilitate the synthesis of positive-strand rna; 438 while mir-555 decreases the expression of hnrnp c thereby inhibits poliovirus replication. 447, 448 virus rna splicing. the main function of hnrnps is modulating rna process and metabolism, including splicing, stabilization and transportation. it has been documented that hnrnp k helps the rna splicing of iav. iav is a major human pathogen with a genome comprised of eight negative-stranded rna segments. two viral rna segments, ns1 and m, undergo alternative splicing and yield several proteins including ns1, ns2, m1, and m2 proteins. two of the influenza virus rna segments generate spliced products: ns segment codes for non-structural protein ns1 and nuclear export protein nep/ns2; m segment encodes the matrix protein (m1) and ion channel (m2). ns1-bp properly splices the viral m1 mrna segment to yield the m2 mrna without affecting the splicing of m4 mrna or ns mrna segments. in this process, hnrnp k works as a mediator to bridge the interaction of ns1-bp (binding protein) and m mrna. lack of neither ns1-bp nor hnrnp k ensures the proper splicing of m mrna. 449, 450 further studies show that ns1-bp and hnrnp k bind m mrna downstream of the m2 5′ splice site (5′ ss). ns1-bp binds most proximal to the 5′ss, partially overlapping the u1 snrnp binding site, while hnrnp k binds further downstream and promotes u1 snrnp recruitment. mutation of either or both the hnrnp k and ns1-bp-binding sites results in m segment mis-splicing and attenuated iav replication. 451 virus translation. virus rna translation often harnesses host cellular proteins. most hnrnps positively regulate virus translation. positive-sense single-stranded rna viruses normally contain an ires sequence that drives viral protein translation independence of the cellular cap-dependent translation. hnrnps bind with the ires of different viruses to assist their translation. during hcv virus infection, hnrnp i, hnrnp l, hnrnp d, [452] [453] [454] hnrnp a1 and hnrnp k 455 all participate the translation of hcv viral protein by binding with 5′utr sequence. hnrnp i (ptb3), is one of the polypyrimidine tract-binding proteins (ptbs) that has been reported to bind viral rna recurrently. they could bind the ires of picornaviruses, including cardio-aphthovirus group (including emcv, ev-a71) and entero-rhinovirus group (poliovirus and hrv-2). 89, 456 ptbs also bind hcv utr regions, 441 calicivirus rna 457 and coronavirus rnas. 436 for picornavirus and hcv, it is proposed that ptbs could help ires folding into a translation-competent structure. 458 although unlike canonical transient interactions shared by rna chaperones, it is not clear whether ptbs are eliminated after the ires has folded properly. 459 ptb binds the 5' utr of hcv rna genome to initiate translation. in translation assays, ptb antibody efficiently blocks the ires-mediated translation in vitro. 460 three rrm motifs of ptb monomer directly bind with ires of fmdv to stabilize ires structure and enhance eif4g entry to ires. 459 hnrnp l is capable of binding the 3′ border of the ires. [461] [462] [463] hnrnp l binds with single stranded-hcv rna when preannealing singlestranded rna with mir-122. 463 hnrnp d, also referred to as aurich element rna-binding protein 1 (auf1), shuttles between the cytosol and nucleus. it is found that hnrnp d could also interact with the stem-loop ii of hcv 5′ utr, and its overexpression enhances hcv ires-dependent translation. 452 pim1 inhibitors induce hnrnp d relocalization from nucleus to cytosol so that it binds the ires and inhibit ev-a71 protein translation. 122 similarly, hnrnp k interacts with sl1 located in the 5′ -utr of hcv genome where is bound with mir-122 binding site. 464, 465 mir-122 is required for hcv replication. it binds at a conserved sequence in the 5′ -utr and increases the stability of hcv rna. [466] [467] [468] it is also reported that residues 25-91, a hydrophilic region near the n terminus of hcv core protein, binds to proline-rich domains of hnrnp k and negatively regulates the effect on human thymidine kinase transcription. 469 however, its function on viral replication is not well addressed. while hnrnp a1 binds with both 5′-and 3′-utr of hcv rna, they form a complex with ns5b and septin 6 to assist viral protein translation. the c-terminal of hnrnp a1 and n-terminal of septin-6 are required in the translation process. since hnrnp a1 has many homologous such as hnrnp a/b, hnrnp a2/b1, and hnrnp a2; all of them may substitute for hnrnp a1 in regulating ires-mediated translation. 470 the enteroviruses' ires sequence also interacts with hnrnps. hnrnp a1 specifically binds on the 5′-utr of ev-a71 and enhances ires-dependent translation. 471 apigenin, a dietary flavonoid, interacts with hnrnps and interferes with their rna editing activity. 472 the binding of hnrnp a1 with viral rna is significantly blocked when the cells are infected with ev-a71 upon treating with apigenin, leading to marked suppression of iresmediated translation. it is noted that hnrnp a1 redistribution is not affected in this experiment. recent studies show that hnrnp a1 cytoplasmic translocation is strictly regulated by some signalling or stress proteins, such as mink/p38 mapk pathway 473 and hsp27. 57 p38 inhibitors or hsp27 knockout dramatically block hnrnp a1 translocating from nucleus into cytosol, leading to inhibition of virus replication. 57, 473 except for promoting viral protein translation by binding with the viral ires sequence, hnrnps can directly bind with virus proteins to facilitate virus replication. upon cvb3 infection, 3c pro binds and cleaves hnrnp m to facilitate virus replication. 474 however, hnrnp m does not affect ires activity and rna stability. 475 hnrnp k is required for denv replication. 476 the core protein of dnev interacts with hnrnp k to release the inhibitory effects of hnrnp k on transcriptional activator c/ebpb. 477, 478 other studies demonstrate that hnrnp c1/c2 interact with viral rna and ns1 protein of dnev and facilitate virus reproduction. [479] [480] [481] [482] hnrnp d binds on both 5′-and 3′-utr of enteroviruses and inhibits translation without affecting rna decay. 434 on the other hand, virus also applies strategy to release the inhibitory effects of hnrnp d via protease 3cd cleavage. 434 the cytoplasm translocation of hnrnp d is dependent on the expression of 2a pro and viral rna replication. 433, 434 hnrnp d also inhibits nucleocapsid expression by interacting with an au-rich sequence (nt 41 to 60) in the 3′ utr of niv. 483 virus rna export. hnrnp a2/b1 interacts with ns1 of influenza virus, leading to decrease of the viral ns1 rna/protein levels as well as ns1 rna nuclear export. 484 rna polyadenylation. the gag gene of rous sarcoma virus contains a cis-acting negative regulator of splicing (nrs) element. the general function of nrs is usually manifested by binding serine/arginine-rich (sr) protein hnrnp h and u1/u11 snrnps, resulting in inhibition of splicing by acting as a decoy 5′ splice site. evidenced by in vitro polyadenylation analysis, a new function of nrs element is revealed that it is required for 3′ ltr polyadenylation. in this process, hnrnp h binds on nrs element and promotes polyadenylation; however, mutation of the binding sites of u1 and u11 snrnps to the nrs does not affect polyadenylation. 485, 486 an opposite result shows that polyadenylation stimulatory activity of nrs is dependent of u1 and/or u11 snrnp as well as sr proteins; while hnrnp h seems not participating in the splicing control or rous sarcoma virus polyadenylation. 487 the functions of rna chaperones in regulating viral activities of dna viruses virus replication. the transient lytic dna replication of hcmv relies on the cis-acting element orilyt, six viral-encoded core proteins, the proposed dna replication initiator protein ul84, ie2, irs1 and the gene products from the ul112/113 loci. here it is reported that hnrnp k is sufficient to interact with ul84 protein of hcmv thereby promotes viral replication. the interaction is further enhanced by another two virus proteins ul44 and ie2. 488 gene expression. hnrnps regulate dna virus gene expression at both transcriptional and post-transcription levels either positively or negatively. at the transcription level, hnrnp d0b and hnrnp a/b are capable of binding with cis-acting aagtatgca core element of hpv18 late promoter to suppress the late genes l1 and l2, which are components of virus capsid proteins. 425, 489 hnrnp k binds enhancer ii (enii) to promote hbv replication. ectopic expression of hnrnp k augments hbv replication; while hnrnp k knockdown significantly decreases hbv viral load. 490 further study reveals that apobec3b forms a super complex with hnrnp k that alters the enii binding activity via conformational change, therefore suppresses the s gene promoter activity. 491 in the course of hsv infection, the immediate early protein ie63 (icp27), hnrnp k and casein kinase 2 (ck2) together form a big complex. ck2 is capable of phosphorylating hnrnp k and icp27. the phosphorylated icp27 is responsible for its nucleocytoplasmic translocation and interaction with hnrnp k. up to date, the function of the complex formation is not well elucidated. 492, 493 it may affect icp27 to recruit the cellular rna polymerase ii for the transcription of certain late genes. [494] [495] [496] at the post-transcription level, hnrnps regulate the polyadenylation, splicing, and translation during dna virus infections. hpv genome can be divided into an early region and a late region, followed by the proximal early (pae) and the distal late (pal) polyadenylation signals, respectively. 497, 498 the virion production mainly depends on the differentiation-dependent induction of l1 and l2 late genes. it has been shown that hnrnp h downregulates the late gene l2 at an early stage by interacting with the multiple ggg motifs located 174 nucleotides downstream of the early polyadenylation signal pae. the hnrnp h binding promotes polyadenylation at early polyadenylation signal and inhibits the l2 mrna production, since l2 mrna production need read-through into the late region and polyadenylation of the late transcripts at the pal. 499 while at the late infection stage, hnrnp h is captured by l1 to release the inhibitory effect on l2. 499, 500 this process is called late gene autoregulation which enables rapid viral capsid protein production. 500 another example for hnrnps modulating the polyadenylation is that sm polymerase (pol) mrna of ebv early protein is cleaved and polyadenylated inefficiently. 501 under certain conditions, ebv early protein sm may harness hnrnp a1 and hnrnp c to help the processing of polymerase mrna. viral rna splicing is also modulated by hnrnps. hnrnp a1 negatively regulates the expression of hpv late genes by affecting rna splicing. it directly binds with the late regulatory element (lre) in differentiated hpv16-infected cells. 502 hnrnp a1 binds with splicing silencer element to suppress the use of the 3′ splice site located immediately upstream of aug at late 1 (l1) mrna. hnrnp a1 inhibits the splicing of late mrnas through the splicing silencer sequence and prevents the premature expression of l1 gene. [503] [504] [505] on the other hand, there is another 17 nucleotides resident immediately downstream of the splice site which counteracts the effect of hnrnp a1-binding splicing silencers. 506 hnrnp i also interferes with the splicing inhibitory elements locating at the upstream and downstream of major late 5′ splice site sd3632, thereby activates the late gene expressions. 507 at the translation level, hnrnp i and hnrnp k inhibit the translation of hpv late gene l2 via binding on a specific cis-acting element in the 3′ end of l2 mrna; 508 whereas the inhibition of l2 translation can be disrupted by c-src-mediated phosphorylation of hnrnp k at multiple tyrosine residues. 509 cellular transformation. hnrnps also exhibit some other functions during virus infections. auf1 works as a major component of c promoter binding factor 2 (cbf2) to interact with ebna2 and mediate ebna2 targeting to the latency c promoter (cp) of ebv, thereby inducing b-cell immortalization and viral latency in humans. 510 auf1 also binds with the eber1 noncoding rna of ebv. in ebv-positive cells, eber1 is abundant; therefore it may compete with auf1-interacting targets in the host cells. 511 both ebna2 and eber1 are proposed to facilitate cell transformation. immunity modulation. aside from affecting virus life cycle directly, hnrnps are able to regulate viral activities through modulating immune response. during hsv-1 infection, hnrnp a/b form a complex with viral dna, followed by homodimerization and demethylation. these events result in translocating the complex to cytosol and activating natural immunity through type i interferon signalling. moreover, the complex promotes n6methyladenosine modification and cgas-sting-related mrnas translocation upon infection by dna viruses, further enhancing the immune response for virus elimination. 512 the function of rna chaperones in regulating the viral activities of retroviruses as we have mentioned above, hnrnp a1 can bind to rna/proteins and participate intracellular nucleo-cytoplasmic transportation, 513 as well as alternative splicing of mrna in eukaryotes. during retrovirus infection, hnrnps participate in viral rna transcription, 514 splicing, 515-517 translocation 516 and translation. transcription. in hiv-infected cells, viral nef protein is required for high-level viral replication. 518 it is reported that nef, eed, kinase lck and npkc subfamily (pkcδ/θ) form a complex nakc (nef-associated kinase complex) responsible for promoting viral replication. 519 later on, it is found that hnrnp k is also a partner of the complex. it bridges the interaction of nef, eed and the kinases. the hnrnp k-nucleated complex activates erk1/2 and results in suppressing hiv promoter, enabling suboptimal amounts of tat and transcription factors (e.g., nf-kb) for initiation of transcription. 514 post-transcription. hiv-1 takes advantage of alternative splicing to generate doses of messenger rnas to encode the various viral proteins. it is known that over 40 message rnas are created by alternative splicing from a single pre-mrna. 520,521 alteration of splicing patterns dramatically affects the infectivity and pathogenesis of hiv-1. 521, 522 the splicing of hiv mrna is mainly controlled at the early stages of spliceosome assembly on pre-mrna by a stepwise association of the small nuclear ribonucleoprotein particles (snrnps) u1, u2, and u5·u4/u6. the early steps include the recognition of the 5′ splice site and the branch point sequence by u1 and u2 snrnps, respectively. 523 rs splicing factors, which contain a domain rich in arginines and serines (rs domain), assist these steps. u2af, one of the splicing factors which consists of two subunits u2af65 and u2af35, interacts with the polypyrimidine tract and the 3′ splice site, respectively. 524 then the interaction mediates the association of u2 snrnp with the branch point sequence. [525] [526] [527] sr splicing factors are essential for virtually every step of spliceosome assembly, including early recognition of splice sites, recruitment of basic splicing factors to the pre-mrna, and formation of bridging contacts with other rs domain-containing splicing factors. 528 prior to forming spliceosomes, the pre-mrna is packed with hnrnps. 529 it has been documented that pre-mrnas bind with different subsets of hnrnps, indicating sequence specificity at some degrees. a direct role of hnrnps in constitutive splicing has not been observed; although it seems that the binding of hnrnps exhibits an unspecific nature. it is widely believed that hnrnps employ crucial functions of modelling pre-mrna structure and initiating recognition of splice sites. 530 the cis-acting sequence elements of cellular and viral pre-mrnas undergoing alternative splicing regulate the process either positively or negatively. they bind trans-splicing factor machinery together and form splicing complex. the majority of trans-acting factors are either hnrnps or sr family proteins. these proteins also regulate alternative rna splicing either positively or negatively. sr proteins often regulate splicing in a positive way while some hnrnps mainly do the job in a negative way. 528, 530 alternative incomplete splicing of hiv-1 genomic mrna produces more than 40 unique viral mrna species within an hiv-1-infected cell through highly accurate regulation by cis exon splicing silence elements (ess) and trans hnrnps. the production of tat, rev and vpr proteins is highly controlled because they play key roles in hiv-1 multiplication. ess2 is the first identified ess in the hiv-1 genome that locates downstream of 3′ ss a3 within exon 4 and specifically represses tat mrna splicing. 531 ess2 is mapped to a 10 nt core sequence cuagacuaga. 532 ess3, which locates at exon 7, represses splicing at 3′ ss a7. 533 fine structure mutagenesis indicated that ess3 is bipartite; and each sub-elements [agaucc (ess3a) and uuag (ess3b)] inhibits splicing independently. 534 a third ess (auaguuaguccuagg, essv), which locates downstream of 3' ss a2 in exon 3 within the vif coding sequence, represses splicing at 3′ ss a2. 535 to modulate the expression of tat protein, several hnrnps participate the splicing process by interacting with these ess, intron splicing silencer (iss) elements directly or interfering enhancer splicing element (ese) activity. for example, hnrnp a1 and hnrnp k synergistically bind on ess2 and inhibit the utilization of a3 splicing site. 536, 537 the uag triplets in ess2 is required for hnrnp a1 binding, and several hnrnp a1-binding sites are also found at sls3. the c-terminal gly domain of hnrnp a1 is important for the binding. sr proteins sc35 and srp40, the strong activators of site a3, have similar binding sites to hnrnp a1. hence, they may compete with each other to bind ess2 and ese2. 536 another study shows that hnrnp h displays an inhibitory effect on splicing. hnrnp h binds to ess2 and competes with u2af35 for binding to the exon sequence flanking 3′-splice site a3. this binding results in the inhibition of splicing at the 3′-splice site a3. 538 hnrnp a1 also inhibit tat splicing by binding with iss, which is independent of exon splicing silencer (ess2) and blocks the entry of u2 snrnp but not u2af65. [539] [540] [541] the up1 domain of hnrnp a1 is responsible for the iss binding, while the rgg domain of hnrnp a1 is not needed for the alternative splicing activity. 542, 543 in addition to ess2 and iss elements that are regulated by hnrnp a1, blocking the binding of sr protein sc35 on ese is another strategy for the virus to inhibit the tat expression at the early infection stage. hnrnp a/b proteins bind the ese locating at tat exon 2 to repress splicing, whereas sc35 binds the ese to activate splicing. the binding sites of hnrnp a1 and sc35 are overlapping within the juxtaposed ese/ess9. it seems that hnrnp a1 inhibits the splicing of the upstream intron by binding to the ess and directly masking the sc35 binding site. 544, 545 similarly, hnrnp a1 is also reported to compete with asf/sf2 at ese3/(gaa)3. therefore, the ratio of asf/sf2 to hnrnp a1 determines the utilization of ese3/(gaa)3 for activation or repression at site a7. 515, 541, 546 the expression of other hiv proteins is also regulated by hnrnps via modulating splicing. hnrnp a/b proteins inhibit the splicing at 3′ splice site a2 which is used to generate vpr mrna. hnrnp a/b proteins bind with essv with a consensus sequence pyuag. the splicing at splice site a2 is increased when the three pyuag motifs within essv are mutated, leading to increased vpr mrna levels and reduced skipping of the noncoding exon flanked by a2 and d3. 547 others reported that hnrnp d also binds at essv and functions as an inhibitor of splicing. 548 the binding of hnrnp a/b proteins at essv blocks u2af65 binding to the ppt of the repressed 3′ splice site and inhibits the splicing efficiency of 3′ splice site. 549 interestingly, hnrnp h is found to positively regulate the exon 6d splicing of hiv mrna. it interacts with the sequence cgga and enables u1 snrnp assembly onto exon 6d. 550, 551 further study shows that hnrnp h family proteins function as a splicing enhancer through enhancing the atp-dependent spliceosomal complex formation. 552 rna translocation. after transcription and splicing, lots of spliced and unspliced rna molecules of hiv exist in the nucleus. translocation of rna into the cytoplasm is dependent on the rev protein. on one hand, some hnrnps facilitate rna translocation. in hiv-1 genome, two sequences similar to the hnrnp a2 response element (a2re) function as cis-acting rna trafficking sequence that binds to the trans-acting trafficking factor hnrnp a2. the binding mediates a specific rna trafficking pathway characterized extensively in oligodendrocytes. a2re-1 locates within the major homology region of gag gene; while a2re-2 locates at a region overlapped between vpr and tat coding sequence. hnrnp a2 binds to both a2res in vitro to form a complex, which is necessary for rna transportation in oligodendrocytes in vivo. a2re-mediated rna transport requires both microtubule and hnrnp a2. if gag and vpr rnas containing a2re-1 and a2re-2 respectively are differentially labelled, it is observed that they are co-assembled into the same rna trafficking granules and cotransported to the periphery of the cell. although the tat rna contains a2re-2, it is not transported as efficiently as vpr rna. [553] [554] [555] hnrnp d also has a similar function in assisting rna translocation. 556 on the other hand, hnrnp a2b1, hnrnp c and hnrnp u can retain the hiv-1 genomic rna in nucleus. depletion of hnrnp a2b1 results in cytoplasmic redistribution of the virus rna genome in the absence of rev. 557, 558 an n-terminal fragment of the hnrnp u specifically targets the 3′ long terminal repeat (3′ltr) and blocks the cytoplasmic accumulation of mrnas, thereby affecting hiv gene expression. 559 besides hiv, the regulatory protein orf57 of kaposi's sarcomaassociated herpesvirus (kshv) also interacts with hnrnp k. ck2 can phosphorylate orf57 and promote orf57-hnrnp k interaction. the orf57-hnrnpk-ck2 complex may be important for rna export of kshv since orf57 is responsible for the nuclear export of viral mrnas. 560,561 hnrnp a1 interferes with the binding of rex to rexresponse element (xre). the rex protein of htlv-1 mediates the nuclear export of unspliced and incompletely spliced viral mrnas. this process partly depends on the binding of rex to rex-regulatory sequences xre. 562 viral protein translation. hnrnp a1 is induced to redistribute into the cytoplasm in the late infection stage of hiv and enhances the ires-mediated translation. 563 hnrnp d helps the redistribution of hiv mrna into the cytoplasm, and p45 and p42 isoforms increase viral gag protein synthesis while p40 and p37 suppressed this process. 556 hnrnp i interacts with a novel ires within a latently expressed gene (vcyclin) of kshv and enhances vflip expression. 564 the network of hsps and their co-chaperones is essential for cells to maintain protein homeostasis, including nascent protein folding, protein translocation across membranes, and protein complex formation. many stress stimuli can disrupt protein homeostasis such as thermal stress, nutrient starvation, chemical toxicity, oxidative stress, hypoxia, inflammation, and virus infection. [565] [566] [567] [568] stresses can lead to protein misfolding and aggregation that need to be resolved quickly to prevent cell and tissue damage. 568 hsps and their partners may facilitate protein degradation when cells cannot cope with massive damaged proteins. many lines of evidence suggest hsps as major factors for protein surveillance to protect host cells against virus infections. it was observed that hsp70/hsp90 expression increased dramatically in hsv-infected cells. 569 overexpression of hsp70 inhibits the translocation of viral capsid into nucleoli during flavivirus infections. although the interaction has not yet been investigated in natural infection, the in vitro findings have suggested that hsp70 may act as a negative regulator of viral capsid protein to protect host cells against wnv infection by abolishing cytotoxic effects induced by the viral capsid. 570 studies from ours and other groups show that almost all hsp subfamily members (hsp90s, hsc70, hsp70, grp78, erp57, hsp27) are highly responsible to enterovirus infection and play key roles in all stages of virus life cycle. not surprisingly, demonstrated that all most all hsps (hsp90s, hsp70s, hsp60s, hsp40s and hsp27) participate coronavirus infection, 145, 196, 387, 571 suggesting good target for anti-covid-19 drug development. as discussed in above sections, hsps exhibit immunomodulatory effects on both innate and adaptive immune responses. [572] [573] [574] hsps are capable of regulating not only intracellular innate immunity, but extracellular innate and adaptive immunity as well. hsps released from tumor cells can bind to surface receptors on antigen presenting cells (apcs) and elicit tumor-specific killers by means of antigen crosspresentation. 575, 576 for example, hsp27 positively modulates nf-κb phosphorylation, increases ifnβ transcription and downstream antiviral interferon-induced genes (isgs). pedv infection downregulates hsp27 expression to escape host antiviral surveillance. 389 additionally, extracellular hsps can also regulate cytokine production by dendritic cells (dcs), underscoring a connection between innate and adaptive immune responses modulated by hsps. 577 the purified or recombinant hsp70 can stimulate the production of pro-inflammatory cytokines and c-c chemokines in monocytes, macrophages and dcs, and upregulate mhc and costimulatory molecules in dcs. 578 binding of extracellular hsp72 to human monocytes and dendritic cells can induce the production of the pro-inflammatory cytokines including tnf-α, il-1β, il-6 and il-12 and ifn-γ. 579 in the process of above hspinduced cytokine production, hsps act as internal stimulus of the cd14/tlr (tlr2 and tlr4) complex signal transduction pathways that further activates nf-κb and mapks signalling. 580, 581 although the main functions of hsps are to protect cells upon stresses, they are often hijacked by many viruses to achieve successful infections. recent study has demonstrated that denv ns3 protein acts as a viral suppressor of rna silencing by interacting with hsc70, thereby interrupting host antiviral system by rnai pathway and subsequently enhancing denv replication. 582 ev-a71 takes advantage of hsps (hsp90, hsc70, erp57, and hsp27) to enhance 2a pro -mediated eif4g cleavage that is favour viral protein translation and blocks host protein translation. 57, 219, 415 viruses also initiate er stress in host cells after infection. they have to manipulate upr activation leading to cell survival, rather than inflammation induction, autophagy and apoptosis. denv modulates upr activation in a sequential manner to prolong the viral life cycle by allowing cellular adaptation to cope with the infection-induced er stress. upr is transiently induced by perk pathway resulting in phosphorylation of eif2α and subsequently translational attenuation in the early phase of infection. this transient event allows viral protein synthesis and accumulation that finally trigger upr by ire1-xbp1 (x-box binding protein 1) axis in the mid-phase of infection. this results in the increased expression of grp78 to facilitate protein folding and also the increased expression of gadd34 (growth arrest and dna damage 34), which dephosphorylates eif2ɑ, and thus allowing protein translation to be continued. the increased grp78 also prevents cellular apoptosis-mediated by chop (pro-apoptotic protein during stress persistence). finally, the increased viral proteins transiently trigger atf6 arm of upr to provide the active spliced xbp1 for sustaining upr activation in the late phase. 583 jev infection requires hsp70s in particular stages of its life cycle for survival and establishment of infection, including viral entry, replication, 194 and maturation. 195 cell-surface hsp70 directly interacts with jev envelope protein. 192 antibodies against hsp70 or 90 significantly block virus entry. 111 it is also observed that hsp70 colocalizes and directly interacts with jev replicase complex. in addition, hsp70 also interacts with ns3, ns5 and viral dsrna that stabilizes vrc formation during jev infection. 194, 584 er stress and antiviral in mammalian cells, the er stress is sensed and mediated by three er transmembrane receptors: pancreatic er kinase (pkr)-like er kinase (perk), inositol-requiring enzyme 1 (ire1) and activating transcription factor 6 (atf6). in resting cells, these three sensors are maintained in inactive states via interactions with the er resident chaperone grp78. when unfolded or misfolded proteins accumulate in the er lumen, grp78 dissociates from these three transducers, resulting in activation and initiation of the upr. viruses also take advantage of or revolt er stress response by different means for favour its life cycle at specific stage(s) of infection. the er stress response constitutes a cellular process triggered by a variety of conditions disturbing protein folding in the er. eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, i.e., the er stress and upr, aiming to clear unfolded/misfolded, and excessive amount of protein production; thereby restoring er homeostasis. in cases er stress cannot be resolved, upr would be triggered and lead to cell death. er stress and upr could be observed in a large amount of virus infections, as most of viruses require host er machinery for viral protein synthesis and modification. virus infection usually causes er stress. when large amount of misfolded/unfolded proteins or excessively expressed viral proteins accumulate inside er lumen, er stress response is triggered as indicated by fast elevated expression of er chaperone proteins, including grp78/ bip, grp94, calnexin and calreticulin. it is well documented that er stress response is triggered by a number in some cases, er stress plays a role in virus pathogenesis. for instance, jev, bvdv, tulv, severe acute respiratory syndrome coronavirus (sars-cov) and wnv have all been shown to induce apoptosis through upr. [585] [586] [587] [588] [589] oxidative stress is mediated by er stress during hcv infection. 21 certain viruses modulate er stress response or preferentially activate different pathways of upr. hcv infection activates the atf6 pathway while blocks the ire1 pathway. instead, hbv infection stimulates both atf6 and ire1 signalling but has no effects on perk signalling although both virus infect the same kind of hepatocytes. 590-592 hsv-1 develops a virulence factor enabling dephosphorylation of eif2α, one of the downstream effectors of the perk pathway. 593 some consequences of upr are beneficial for viral life cycle. for example, atf6-induced expression of chaperone proteins may help viral protein folding and prevent protein aggregation. the perk-eif2α-activated atf4 may help re-establish cell metabolism and resume protein translation. the ire1-xbp1 pathway may facilitate virus replication by enhancing er protein-folding ability and er membrane biosynthesis. 594 the activated atf6 promotes the replication of asfv and lymphocytic choromeningitis virus (lcmv). 595,596 denv envelope protein directly interacts with grp78, which provides a scaffold for its association with two other chaperones calnexin and calreticulin. this complex significantly improves the proper folding and stability of denv e protein, thereby leading to increase of virus production. 597 other than denv, grp78 is also demonstrated to promote hcmv, jev and rgnnv infections. 195, 598, 599 the ire1-xbp1 pathway is activated by iav to facilitate its replication. 600 however, other upr outcomes are detrimental for virus replication. the perk-eif2α-mediated global translation attenuation is known as an antiviral response to restrict viral replication, such as infection by denv or wnv. 583, 601 the activated pkr phosphorylates eif2α at the ribosomal interface, which in turn causes a general inhibition of protein synthesis and blockage of vsv replication. 602 the ire1-xbp1(s)-mediated er-associated protein degradation (erad) pathway reduces intracellular hbv particles by degrading its envelope proteins. 603 another approach of er stress contributing to virus pathogenesis goes through modulating host cell immune responses. vsv, hcv and sars-cov are able to inhibit the type i ifn signaling pathway by activating the perk signalling, which leads to the phosphorylation-dependent ubiquitination and subsequent degradation of ifnar1, thereby promoting immune evasion and virus pathogenesis. 604, 605 wnv has also been reported to induce er stress and inhibit type i ifn signaling pathway for escaping from the host immune response. 601 us11 protein of hcmv activates upr to facilitate the degradation of class i major histocompatibility complex (mhc1), leading to immune evasion. 606 moreover, er stress is responsible for viral pathogenesis by interconnecting with the inflammatory responses. for example, hcv induces inflammatory responses by activating ire1, which interacts with traf2 to phosphorylate jnk, leading to activation of inflammation mediators. 607 ns4b and ns5a proteins of hcv activate nf-κb via er stress-elicited calcium depletion and ros production. 604 the x protein (hbx) of hbv induces the expression of cyclo-oxygenase 2 (cos2), a key mediator of inflammation, through perk-eif2α-activated atf4. 608 rna chaperons and human diseases caused by viral infections hnrnps impact mrna metabolism including transcript synthesis, processing and degradation as well as translation. 432 the function of hnrnps is determined or modulated by cellular localization. the mechanisms that regulate the nucleo-cytoplasmic shuttling are, therefore, of extreme importance. most hnrnps possess a conventional nuclear localization signal (nls) and are predominantly present in the nucleus during steady state. they are able to translocate in the cytosol upon post-translational stimulation or by the recruitment of other hnrnps. 609 post-translational modifications like methylation, phosphorylation, ubiquitination and sumoylation are reported to affect biological activity and subcellular localization of hnrnps. 610 as stated in the above sections, virus always employs different strategies to redistribute hnrnps in host cells. 430, 431, 433, 434 how dysregulation of hnrnps causing human diseases has gained an increasing interest. the expression level of hnrnps is altered in many types of diseases, including varieties of human cancers and neurodegenerative diseases (e.g., spinal muscular atrophy (sma), amyotrophic lateral sclerosis (als), alzheimer's disease (ad), and fronto-temporal lobe dementia). in this section, we mainly focus on the relationship between hnrnps and diseases caused by virus infections. enteroviruses are common pathogens that cause human diseases worldwide. although most enteroviral infections are subclinical, they may cause a wide spectrum of diseases including mild upper respiratory illness (common cold), febrile rash (hand, foot, and mouth disease and herpangina), aseptic meningitis, heart failture, pleurodynia, encephalitis, acute flaccid paralysis (paralytic poliomyelitis) and neonatal sepsis-like disease. 293, 611 besides, several studies showed that enterovirus sequences could be detected in neuronal cell bodies of the spinal cord of 60-88% amyotrophic lateral sclerosis (als) patients, suggesting that persistent enterovirus infection may have a relationship with als. 295, 297, 299 enterovirus infections are supposed to cause or increase the risk of als; because the replication and translation of poliovirus, ev-a71, and coxsackievirus redistribute numerous hnrnps (such as hnrnp a1) into the cytoplasm, the same localization during als pathogenesis. for example, hnrnp k, hnrnp a1, hnrnp m and hnrnp d shuttle into cytoplasm to assist virus replication or translation. 300, 455, 472, [612] [613] [614] dislocated hnrnp a1 and loss of splicing function have been regarded as a toxic mechanism in als pathogenesis. 301 hsv infection is one of the most common causes of infectious disease in humans. 302 hsv infection often causes watery blisters in the skin or mucous membranes of the mouth, lips, nose, or genitals. 615 as neurotropic and neuroinvasive viruses, hsv-1 and -2 persist in the infected individuals through hiding in the neuron cells from immune surveillance. when the carrier's immunity compromised, hsv is reactivated that causes new sores. more seriously, accumulating evidence shows that hsv infection is associated with the pathogenesis of alzheimer's disease. 105, 616, 617 during hsv infection, hnrnp k positively promotes hsv replication; 618 while hnrnp a2/b1 negatively regulates hsv replication by triggering immunity response. 512 hbv, hcv, ebv, hpv and hiv are oncogenic viruses that account for over 20% of human cancers. hcv or hbv infection leads to a wide spectrum of liver disease ranging from acute hepatitis (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (hcc). 619,620 hdv can only infect people who are already infected with hbv. the coinfection of hdv and hbv increases the risk of liver cirrhosis and liver cancer. 621, 622 except for damaging the liver, 10% of hbv infected patients also have other symptoms outside the liver, such as serumsickness-like syndrome, membranous glomerulonephritis, acute necrotizing vasculitis (polyarteritis nodosa) and papular acrodermatitis of childhood (gianotti-crosti syndrome). 623 multiple steps of hcv reproduction need the presence of hnrnps. the rna replication of hcv needs the presence of hnrnp i, hnrnp c; 437, 442, 446 while virus rna translation requires hnrnp a1, hnrnp d, hnrnp i, hnrnp k and hnrnp l. 452, 460, [462] [463] [464] [465] besides, hnrnp a2, hnrnp l, hnrnp u and hnrnp k are highly expressed in hcc tissue. they promote liver cancer progression. 8, 304, 305, 624 ebv has been implicated in several diseases, including infectious mononucleosis, 625 burkitt's lymphoma, 626 hodgkin's lymphoma, 627 nasopharyngeal carcinoma, 628 multiple sclerosis 629 and lymphomatoid granulomatosis. 630 other diseases associated with ebv infections include gianotti-crosti syndrome, erythema multiforme, acute genital ulcers, oral hairy leukoplakia, 631 and disorders related to α-synuclein aggregation (e.g. multiple system atrophy, dementia with lewy bodies, and parkinson's disease). 632 during ebv infection, hnrnp a1 and hnrnp c assist the polyadenylation of ebv rna. hnrnp d takes part in cellular transformation-induced by ebv. 510 hpvs are a group of dna viruses with more than 150 members that infect cutaneous and mucosal epithelia. the acute infection of hpv causes benign cutaneous lesions such as non-genital and genital warts, or flat cervical condylomas. 633 about 15 hpv subtypes that infect genital tract are capable of inducing malignant tumors, most commonly in the cervix. 308,309 these cancer-associated hpvs are grouped into high-risk types, while those not associated with cervical cancer are regarded as low-risk types. 307 most infections by low-risk type hpvs are asymptomatic, except for infection by hpv6 and hpv11 that cause most cases of genital warts (condyloma acuminatum). 309 hpv-related cancers are the result of long-term persistent infection with high-risk types. hpv16 and hpv18 are the most common carcinogenic types which account for a larger proportion of cervical cancers, squamous cell cancers and adenocarcinomas in all regions. 311 hpvs are also implicated in the development of a variable proportion of vulvar, vaginal, penile, and oropharyngeal cancers, anal cancer, 634 head and neck cancers, 635-637 lung cancer and skin cancers. hnrnp a1 is involved in splicing of hpv rna; while hnrnp h plays a role in the polyadenylation of hpv. hnrnp k and hnrnp i are required for viral protein translation. besides acquired immunodeficiency syndrome (aids), hiv infection is also able to cause human cancers because hiv infection impairs immunity system. people with aids are not only more easily infected with bacteria, viruses, fungi, and parasites; 638 but at high risk for developing various viral-induced cancers as well, including burkitt's lymphoma, kaposi's sarcoma, primary central nervous system lymphoma, and cervical cancer. 639 515, [532] [533] [534] [535] [536] [537] [544] [545] [546] [547] [548] [549] [550] [551] [552] [553] [554] [555] [556] [557] [558] [559] 564 potential therapeutic value by targeting stress proteins interest in targeting stress proteins in various diseases is increasing overtime. in this part, we will discuss the potential therapeutic value by targeting stress proteins. first, targeting stress proteins have a wide spectrum of antiviral ability. for example, hsp90 inhibitors, which are originally developed for anticancer, have been demonstrated to possess antiviral activity in cultured cells against poliovirus, rhinovirus, ev-a71, 107,109 coxsackievirus, 124 rsv, 112, 113 vsv, paramyxoviruses (hpiv2, hpiv3, sv5, sv41), influenza virus, 126 chikv, 114 hcv, 115, 124 and hsv, 131, 144, 145, 148, 149 hbv, 135, 169 ebv, 146, 147, 163, 164 hcmv 151, 152 and htlv. 153, 185 depleting hsp60 by sirna functionally suppresses infections by influenza virus, 38,39 denv, 319 hbv, 325, 329, 331 hcv, 321, 322 rotavirus, 323 and hiv. 338 targeting hnrnp a1 is able to inhibit the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c could be a strategy to combat viral infections by hcv, 438 [461] [462] [463] 475 therefore, stress proteins are particularly attractive as antivirals targets for those lacking therapies and newly emerging viral diseases. second, under disease situations, the cellular need of stress proteins is usually stronger than that in normal conditions which enable specific selectivity of those drugs targeting stress proteins. for example, mutant p53 relies much more on hsp90 function than wild type p53. hsp90 inhibitor gm can easily disturb the association of mutant p53 with hsp90, resulting in mutant p53 degradation while not affecting wild type p53. 310 additionally, stress proteins derived from stressed cells display higher affinity to clients and inhibitors. tumor cell-derived hsp90 exhibits a 100fold higher binding affinity for 17-aag than that from normal cells, since tumor cell-derived hsp90 complexes with activating cochaperones p23 and hop exhibit increased atpase activity and possess higher affinity to hsp90 inhibitors. in contrast, hsp90 in normal cells exists as an uncomplexed species with low atpase activity and low affinity to hsp90 inhibitors. 294 further study demonstrates that the difference is caused by a distinctive portion of hsp90 forming complexes in cancer cells with oncogenic partners, such as bcr-abl-hsp90 complex in mutant b-raf-hsp90 in skmel28 melanoma cells, k562 chronic myeloid leukemia cells, her3-hsp90 and raf1-hsp90 complex in mda-mb-468 breast cancer cells. hsp90 inhibitor pu-h71 selectively binds to specific hsp90-oncoprotein networks in these cancer cells. 570 these characters of cancer cells enable stress proteins to exhibit stronger biological activity and to be discriminated by their inhibition under stress as compared with normal conditions. similarly, inhibitors of stress proteins show high prospect for antiviral. hsp70 inhibitor jc40 inhibits pan-flavivirus (denv2 and denv4) replication in mddcs, with negligible toxicity to host cells. 640 these experiments highlight the feasibility of using hsp inhibitors therapeutically in humans. third, it is not observed drug resistance using hsp inhibitors for antiviral. for instance, hsp90 inhibitors are refractory to develop drug resistance. this is clearly demonstrated in rsv infection. 113 when rsv is repeatedly treated with hsp90 inhibitors, no drug resistance was observed either in extensive passaging of the virus in cultured cells or in mice undergoing long-term treatment with hsp90 inhibitors. similar result is also observed in denv infections. 640 treatment of denv up to 10 passages with hsp70 inhibitor jc40 does not cause any drug resistance. the lack of viral drug resistance to hsp90 and hsp70 inhibitors suggests that such an antiviral approach may be particularly useful for treating chronic viral infections in which drug resistance is most frequently observed. the three features of stress proteins inhibitors make them extremely powerful antiviral agents, suggesting great application potential for treating the diseases caused by virus infections, such as covid-19. challenges and perspectives: target-based drug development targeting hsp90 for antivirus drug development. hsp90 is thought to be the most abundant and evolutionarily conserved heat shock protein. a unique pocket in n-terminal region of hsp90 is required for binding with atp and co-chaperones. 641 hsp90 forms a flexible dimer by interaction of c-terminal domains. the formation and dissociation of compact dimers involving nterminal domains are important for the molecular chaperone activity. 642 the middle domain of hsp90 tends to recruit and facilitate unfolded client proteins to assemble. the c-terminal domain of hsp90 possesses a highly conserved peptide sequence for interacting with co-chaperones. 643 over 20 co-chaperones selectively interact with hsp90 to regulate atpase activity and recruit client proteins to assemble a big complex under certain conditions. [642] [643] [644] [645] it has been shown that more than 300 client proteins require hsp90 and co-chaperones for folding and maturation. 646 the mechanism of selectivity may rely on the direct interaction of co-chaperones with specific clients. therefore, most hsp90 inhibitors achieve their inhibitory effects by suppressing the atpase activity or disrupting the interaction between hsp90 and its co-chaperones. expression of hsp90 and its client proteins are increased during viral infection and in most cancer cells. hsp90 as an effective anticancer drug target has already grabbed attention; and a series of hsp90 inhibitors as potential drugs have been intensively investigated in the laboratories, preclinical and clinical scenarios. the successful use of hsp90 inhibitors in cancer therapy makes it much easier to apply them for treating virus infections. as described above, we have comprehensively summarized the functions of hsp90 and its client proteins in a diversity of virus reproduction processes. the potential of hsp90 inhibitors has been well demonstrated to protect cultured cells against infections by ev-a71, 107,109 poliovirus, rhinovirus, coxsackievirus, 124 paramyxoviruses (hpiv2, hpiv3, sv5, sv41),vsv, rsv, 112,113 influenza virus, 126 chikv, 114 hcv, 115,124 hsv, 131,144,145,148,149 hbv, 135,169 ebv, 146, 147, 163, 164 hcmv, 151, 152 and htlv. 153, 185 notably, administration of hsp90 inhibitors to infected animals exhibits little toxicity while potently suppresses the replication of poliovirus, 107,109 ebv, 163, 164 hbv, 135 chikv 114 and hcv. 647 these experiments highlight the feasibility of using these inhibitors therapeutically in clinic. here we would briefly enumerate the present hsp90 inhibitors and their potential for antiviral therapies. most hsp90 inhibitors hamper hsp90 function by competitively binding to the atp binding site of hsp90, blocking the interaction with co-chaperones, or modulating acetylation. by doing so, we also try to address the possibility of repurposing hsp90 inhibitors as candidate antiviral drugs ( table 2) . inhibitors targeting hsp90 atpase activity. some hsp90 inhibitors competitively bind to the atp pocket in the n-terminus of hsp90, leading to blocking of atp hydrolysis and the closure of nterminus of hsp90 dimer. in addition, another atp binding site has been found in the c-terminus of hsp90. 648 recently, some natural products and their derivatives have been reported to competitively bind to the atp pocket in the c-terminus of hsp90. generally, inhibitors of hsp90 atpase are classified into three types: (i) ansamycins, (ii) non-ansamycins and (iii) those block hsp90 c-terminal atpase activity. ansamycins are antibiotics that possess the benzoquinone as structure core. these antibiotics include geldanamycin (ga), herbimycin a, and the macbecins. they inhibit hsp90 activity and degrade its client proteins. 649 ga competitively binds to the atp pocket in the n-terminus and inhibits the atpase activity. 649 it has shown potent antiviral activity against coronavirus infection in the culture cells, 145 indicating a good potential for treating covid-19. tanespimycin (17-allylamino-17-demethoxygeldanamycin, 17-aag) is an analogue of ga. 650, 651 alvespimycin (17-dimethylaminoethylamino-17demethoxygeldanamycin, 17-dmag) is an analogue of 17-aag, with high solubility in water. in addition, retaspimycin hydrochloride (ipi-504) is a more water-soluble derivative of 17-aag. 652 non-ansamycin inhibitors of hsp90 include luminespib, 653 biib021, 654 ganetespib, 655 onalespib, 656 snx-5422, 657 etc. another type of hsp90 inhibitors binds to the c-terminal atp pocket rather than n-terminal atp pocket. novobiocin blocks the c-terminal atpase activity. 658, 659 in in vitro and in vivo assays, treatment with novobiocin strongly reduces the expression of hsp90-dependent client proteins, such as raf-1 and p60v-src. a natural rotenoid, deguelin, is suggested to bind to the atp pocket in the c-terminus without affecting the atp pocket in the nterminus. 660 treating with deguelin leads to reduced hsp90 clients, such as cdk4, akt and mek1/2. 661 like deguelin, epigallocatechin gallate (ecgc) is reported to bind to the atp pocket in the c-terminus of hsp90 without affecting the nterminal atp pocket. 662 compared with inhibition of the nterminal atp pocket of hsp90, inhibitors targeting the c-terminal atp pocket lead to a stability of hsp70 and a negative instability of client proteins of hsp90. however, the potential of these cterminal inhibitors and their molecular mechanisms remain elusive. the structural features of the c-terminal domain of hsp90 need to be further analysed. such analysis may provide important hints on how to design effective hsp90 inhibitors without hsr induction. inhibitors of hsp90's co-chaperones. as the functions of the hsp90 chaperone machinery are highly dependent on the associated co-chaperones, it is possible to selectively inhibit downstream signaling of hsp90 by disrupting certain protein-protein interactions. inhibitors targeting the interactions may present an alternative approach to prevent the toxicity induced by other hsp90 inhibitors. as a result, great efforts are put into disrupting the interaction between hsp90 and its cochaperones and are rewarded recently in this area. here we briefly present the advance in hsp90-cdc37 complex and hsp90-hop-hsp70 ternary complex. human cdc37 is a well-studied co-chaperone of hsp90. the middle domain and c-terminus of cdc37 are critical for the interaction with hsp90. currently, most of the reported agents for manipulating the hsp90-cdc37 interaction are natural products and their derivatives. these agents include celastrol, aferin a, sulforaphane, kongensin a and platycodin d. they are capable of dissociating the hsp90-cdc37 complex. [663] [664] [665] [666] [667] in addition, a peptide (pep-1, ac-khfgmlrrwdd-nh2) is developed and shows strong inhibitory effects on the formation of hsp90-cdc37 complex. 668 another well-known hsp90/co-chaperone complex is hsp90-hop-hsp70 ternary complex, which facilitates the transfer of unfolded client proteins from hsp70 to hsp90. notably, six active compounds have been identified after screening the ncgc compound library. 669 these compounds possess similar structural cores and have no effect on hsp70 expression. studies on hsp90-co-chaperone inhibitors are limited. great effort should be paid on the efficacy and toxicity for further drug design and clinical studies. hsp90 inhibitors blocking deacetylation of hsp90. the chaperone function of hsp90 is also controlled by posttranslational modification (ptm). the well-studied ptms in hsp90 are acetylation and deacetylation in the m-domain of hsp90. vorinostat (suberoylanilide hydroxamic acid, saha) dissociates her2/erbb2 from hsp90 via acetylation of hsp90 that leads to degradation of her2/erbb2. 670 laq824 induces hsp90 acetylation that reduces hsp90 client protein levels. 671 romidepsin is involved in the dissociation of mutant p53 and raf-1 from hsp90 via acetylation of hsp90. 672 in addition, the nuclear import of iav polymerase needs the deacetylation of hsp90 which is strictly regulated by histone deacetylases 6/8 (hdac6/8). 120 hdac6/8 inhibitors efficiently limit polymerase nuclear import and suppress virus replication. 120 therefore, hsp90 inhibitors that block hsp90 deacetylation would potentially be used to treat influenza a virus infection. their potential usage for combating other viruses (such as sars-cov-2) needs to be extensively explored. novel class of hsp90 inhibitors for hsp90 cleavage. recently, hsp90 cleavage has been observed under various stimuli such as uv irradiation, 673 ascorbate/menadione, 674 hdac inhibitors, 675 proteasome inhibitors 676 etc. therefore, hsp90 cleavage is considered as another mechanism. hsp90 cleavage induced by these inhibitors can be classified into two types: enzymatic cleavage and non-enzymatic cleavage. 674, 676 the enzymatic cleavage produces a 55 kda fragment of hsp90 via caspase 10 activation, while the non-enzymatic cleavage utilizes reactive oxygen species (ros) to chemically degrade hsp90 to an~70 kda fragment. additionally, some substances have been reported to present hsp90 cleavage activity, but the mechanism remains unclear. targeting hsp70s for drug development: one kind of antiviral drugs are currently designed based on different properties of hsps. considering hsp70 and hsc70 are potential targets for hcv, they load the dcs with hsp70 for a prolonged potent antigen-and tumor-specific t cell responses directed against multiple epitopes. 677 hsp70 inhibitors (such as quercetin, ver155008 and jc40) also show great potential for treating flavivirus and enterovirus infections. 202, 208, 640 inhibitors blocking the interaction between grp78 and spike protein of coronavirus would be a useful approach for combating the infection by sars-cov, mers-cov, and sars-cov-2 infections. 196, 387, 571 however, the efficacy and toxicity of these inhibitors are not yet well investigated in clinic studies. targeting hsp60s for drug development: as described above, hsp60s play critical roles in different diseases including autoimmune diseases, human cancers and virus infection-induced diseases. studies show that silencing of hsp60s by sirna significantly decrease influenza virus, 38,39 denv 319 and hbv 325, 331 reproduction by activating immunity response; and is capable of releasing hcv 321,322 , rotavirus, 323 hbv 329 and hiv 338 infectioninduced pathogenesis. therefore, small molecule regents targeting hsp60s are potentially useful as therapeutics in these diseases. 678, 679 currently, the known hsp60 inhibitors are either from natural products or synthetic compounds (table 3) . mechanistically, they can be grouped as two types. type i inhibitors block atp binding and hydrolysis. type ii inhibitors covalently interact with certain cysteine residues of hsp60. however, the detailed binding sites have not been identified. the natural products of hsp60 inhibitors include mizoribine, epolactaene, myrtucommulone a, stephacidin b. mizoribine is an imidazole nucleoside antibiotics isolated from eupenicillium brefeldianum. 680 it directly binds to hsp60 and inhibits the chaperone activity of the hsp60-hsp10 complex. 33 epolactaene is isolated from the fungal strain penicillium sp. bm 1689-p. 681 its derivate tert-butyl ester etb has similar activity as epolactaene. 682 they inhibit hsp60-hsp10's chaperoning activity by covalent and selective bound to hsp60 at residue of cys442, close to the atp binding pocket. 683, 684 interestingly, etb does not inhibit the atpase activity of hsp60, 685 suggesting that the covalent interaction between etb and cys442 may allosterically modulate hsp60-hsp10's chaperoning activity without interfering with its atpase activity. myrtucommulone a (mc) binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. 686 mc is a non-prenylated acylphloroglucinol with multiple reported bioactivities, including antibacterial, 687,688 antioxidant, 689 antiinflammatory, 690, 691 and anti-tumor properties. 692, 693 in addition to these natural products, several other natural products are also reported to interact with hsp60 without direct proof. stephacidin b is isolated from aspergillus ochraceus wc76466 while avrainvillamide is isolated from aspergillus sp. cnc358. 694, 695 interestingly, dimeric stephacidin b is converted into monomeric avrainvillamide in tissue culture media which implies avrainvillamide is the actual active species. 696 pull down assay only shows hsp60 may be a putative target. 696 further validation studies have yet to be performed. besides the natural products identified above as potential hsp60 inhibitors, quite a few synthetic molecules have also been discovered to be able to modulate hsp60 activity. o-carboranylphenoxyacetanilide is firstly identified as an hif-1α inhibitor. 697 later it is found that o-carboranylphenoxyacetanilide selectively and directly binds to hsp60 and inhibits hsp60-hsp10's atpase activity and refolding activity. 685 hsp60 can interact with hif-1α, suggesting that inhibition of hif-1α activity by carboranylphenoxyacetanilide is possibly through targeting hsp60. 685 the other class of synthetic compounds identified to inhibit hsp60 is gold porphyrins. 698 it directly binds to hsp60 and inhibits the refolding activity of the hsp60-hsp10 complex. although several hsp60 inhibitors potently suppress virus reproduction, a lot of work remains to be done to verify if they can serve as drugs for treating diseases caused by virus infections. targeting hnrnps for antivirus drug development. drugs targeting rna chaperones may have a wide spectrum of antivirus capability. hnrnp a1 participates the reproduction of hiv, [539] [540] [541] 563 hpv, 503-505 hcv, 471 ev-a71 472 and htlv-1. 562 inhibition of hnrnp c suppresses the replication of hcv, 438 514, 536, 537 however, the available regents developed targeting hnrnps as antiviral drugs are limited. to date, only three agents targeting hnrnps have been reported. apigenin is a flavonoid abundant in fruits and vegetables. it targets hnrnp a2 and blocks hnrnp a2 dimerization thereafter affects the alternative splicing of key mrnas. 699 apigenin efficiently inhibits the interaction of hnrnp a1 and a2 with ev-a71 rna thereby inhibits ev-a71 rna translation. 472 quercetin is also a flavonoid abundantly present in plants. it binds to the c-terminal region of hnrnp a1, impairing the ability of hnrnp a1 to shuttle between the nucleus and cytoplasm and ultimately resulting in its cytoplasmic retention. 700 in addition, quercetin down-regulates hnrnp a1 expression. 701 the antiviral ability of quercetin has been verified in some viruses such as inhibiting influenza entry, 702 ev-a71, 703 and hcv, 208 jev and denv reproduction. 704 however, in these researches, they explain the antivirus mechanism of quercetin is by inhibiting hsp70 expression 208 or inhibiting virus proteases 703 without mentioning the function of hnrnp a1. another compound vpc-80051 targets rna binding motif of hnrnp a1 and alters hnrnp a1 splicing activity. 705 its antiviral ability remains to be further investigated. targeting er stress pathways for drug development: during the past few years, the enlarging role of er stress-mediated pathways is becoming quite an attractive topic for broad-spectrum antiviral therapy, especially in the field of virus reproduction and pathogenesis, considerable progress has also been made for potential antiviral agents development. among pathways involved in er stress, the three upr pathways are the most thoroughly studied, thus antiviral targets related with these pathways are considered as the first class. extensive investigation for antiviral development has been put int to the perk-eif2α pathway. a small chemical compound named salubrinal, identified by boyce and his colleagues, specifically inhibits the formation of pp1/gadd34 complex as well as suppresses hsv γ134.5-mediated eif2α dephosphorylation. it could also significantly reduce hsv replication. 706 their research suggested that salubrinal was able to attenuate pp1/gadd34-dependent eif2α dephosphorylation, which would in turn inhibit denv replication. 707 a glucose analog 2-deoxy-d-glucose is responsible for the activation and phosphorylation of eif2α, leading to suppression of herpesvirus (kshv) replication. this agent is a potential candidate for anti-herpesvirus, and hopefully is tested in clinical therapies. 708 other than eif2α, tremendous efforts have also been made in developing drugs to target ire1-xbp1 pathway. 3,5-dibromosalicyladehyde is an endoribonuclease that specifically interacts with ire1 and blocks the downstream iav replication. 600 wp1130 is another ire1-xbp1 inhibitor with a broad-spectrum antiviral effects against multiple viruses, namely mnv-1, lcv and so on. 709 er stress-mediated apoptosis signaling is another noteworthy pathway for drug development and selection. rana catesbeiana ribonuclease (rc-rnase) is reported to be efficient in suppression of jev replication via activation of caspase-3, caspase-8 and caspase-9 pathways. 710 dubble-stranded rna activated caspase oligomerizer (draco) is reported to have broad anti-viral effects in clinical therapy. draco selectively activates apoptosis in cells infected with dsdna viruses but has no harmful effect on normal healthy cells. over 15 different kind of viruses can be eradicated by draco, including hiv and dengue virus. 711 drugs targeting other signal transducers are also investigated, for example jnk, jak, bcl-2 and chop. a promising agent vaticanol b can protect host cells from er stress-induced apoptosis. 712, 713 some researchers choose to focus on viral proteins that could trigger er-stress, including non-envelope glycoproteins of sfv, precursor glycoprotein of lcmv, glycoproteins tulv, several nonstructural protein of sars-cov, multiple non-structural proteins of flavivirus family, envelope protein of pedv, e1, e2 and nonstructral protein of hcv, surface protein of mhv, icp0 glycoprotein b of hsv1, structural protein 4 of chikv. viral proteins that are responsible for upr activation and apoptosis induction are given special attentions such as hcv (e1, e2, core), hcmv (pul37x1, pul38) and sv5 v protein. great advances have been made in this area, current progress includes, clemizol for hcv ns4b, vaccine vectors for hsv-1 γ134, and norakin for iav hemagglutinin a. 714, 715 due to their potential side effects on normal cell functions, the usage of inhibitors targeting stress proteins are very cautious in clinic settings; and the gone with wind infection manner of some virus (e.g., sars-cov) limits the opportunity of clinic studies, giving rise to additional challenges for developing stress protein-based drugs against the common and special infectious diseases such as severe acute respiratory symptom (sars) caused by coronavirus. we thank prof. robert weiss at cornell university college of veterinary medicine for critical reading and comments in preparation of this manuscript. the work was partially supported by grants from the science technology and innovation committee of shenzhen municipality [jcyj20170818100531426, jcyj20180507181627057]; grants from national science foundation of china in and out of the er: protein folding, quality control, degradation, and related human diseases a. new puffing pattern induced by temperature shock and dnp in drosophila molecular chaperone functions of heat-shock proteins the growing world of small heat shock proteins: from structure to functions 70 and 90, anti-apoptotic proteins, in clinical cancer therapy molecular chaperones in protein folding and proteostasis the role of heat shock proteins in antigen cross presentation novel signal transduction pathway utilized by extracellular hsp70: role of toll-like receptor (tlr) 2 and tlr4 heat shock protein 70 acts as a potential biomarker for early diagnosis of heart failure hsp27 expression in the human peripheral blood mononuclear cells as an early prognostic biomarker in coronary artery disease patients chaperone machines for protein folding, unfolding and disaggregation the hsp90 family: structure,regulation, function, and implications in health and disease molecular chaperones and protein quality control involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits the hsp90 chaperone machinery heat shock proteins: cellular and molecular mechanisms in the central nervous system structural analysis of substrate binding by the molecular chaperone dnak the human hsp70 family of chaperones: where do we stand? gymnastics of molecular chaperones the kinetic parameters and energy cost of the hsp70 chaperone as a polypeptide unfoldase hepatitis c virus, er stress, and oxidative stress chaperones in hepatitis c virus infection hsp70 chaperone dynamics and molecular mechanism the hsp70 chaperone machinery: j-proteins as drivers of functional specificity two families of chaperonin: physiology and mechanism folding of newly translated proteins in vivo: the role of molecular chaperones mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets group ii chaperonins: new tric(k)s and turns of a protein folding machine structure of holo-chaperonin studied with electron microscopy oligomeric opal0 on top of two layers of cpn60 rings with two stripes each a chaperonin from a thermophilic bacterium, thermus thermophilus. in molecular chaperones review: a structural view of the groe chaperone cycle groel and the groel-groes complex mammalian hsp60 is a major target for an immunosuppressant mizoribine mammalian hsp60 is quickly sorted into the mitochondria under conditions of dehydration mammalian 60-kda stress protein (chaperonin homolog) physicochemical properties of the mammalian molecular chaperone hsp60 down-regulation of hsp60 suppresses the proliferation of glioblastoma cells via the ros/ampk/mtor pathway the pb2 subunit of the influenza virus rna polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon association of the influenza virus rna polymerase subunit pb2 with the host chaperonin cct heat shock protein 60: regulatory role on innate immune cells the small heat shock proteins family: the long forgotten chaperones crystal structure and assembly of a eukaryotic small heat shock protein a first line of stress defense: small heat shock proteins and their function in protein homeostasis the small heat shock protein hsp27: present understanding and future prospects structure and function of small heat shock/α-crystallin proteins: established concepts and emerging ideas mechanism of chaperone function in small heat shock proteins regulation of aging and age-related disease by daf-16 and heatshock factor hsp27 negatively regulates cell death by interacting with cytochrome c some like it hot: the structure and function of small heat-shock proteins small heat shock protein activity is regulated by variable oligomeric substructure small heat shock proteins hsp27 and αb-crystallin: cytoprotective and oncogenic functions heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27 muscle develops a specific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation heat shock protein 27 phosphorylation: kinases, phosphatases, functions and pathology heat shock protein 27: its potential role in vascular disease hsp27 responds to and facilitates enterovirus a71 replication by enhancing viral internal ribosome entry site-mediated translation heterooligomeric complexes formed by human small heat shock proteins hspb1 (hsp27) and hspb6 (hsp20) large potentials of small heat shock proteins blocking tumor cell migration and invasion with biphenyl isoxazole derivative kribb3, a synthetic molecule that inhibits hsp27 phosphorylation quantification of hsp27 and hsp70 molecular chaperone activities human adenovirus type 19 infection of corneal cells induces p38 mapk-dependent interleukin-8 expression mapk and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption heat shock protein 27 mediated signaling in viral infection hsp27 expression regulates cck-induced changes of the actin cytoskeleton in cho-cck-a cells increased hsp27 after androgen ablation facilitates androgenindependent progression in prostate cancer via signal transducers and activators of transcription 3-mediated suppression of apoptosis interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of hsp27 heat shock protein 27 functions in inflammatory gene expression and transforming growth factor-beta-activated kinase-1 (tak1)-mediated signaling hsp27 regulates pro-inflammatory mediator release in keratinocytes by modulating nf-κb signaling increased expression of the m (r) 27,000 heat shock protein (hsp27) in in vitro differentiated normal human keratinocytes 28-kda mammalian heat shock protein, a novel substrate of a growth regulatory protease involved in differentiation of human leukemia cells regulation of hsf 1 function in the heat stress response: implications in aging and disease targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis novel aspects of heat shock factors: dna recognition, chromatin modulation and gene expression proteins of the pdi family: unpredicted non-er locations and functions the protein disulfide isomerase family: key players in health and disease erp57 functions as a subunit of specific complexes formed with the er lectins calreticulin and calnexin protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation cellular functions of endoplasmic reticulum chaperones calreticulin, calnexin, and erp57 rna chaperone stpa loosens interactions of the tertiary structure in the td group i intron in vivo rna chaperones and the rna folding problem probing the folding landscape of the tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway exposing the kinetic traps in rna folding strategies for rna folding and assembly protein enhancement of hammerhead ribozyme catalysis a. chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis domain analysis of the saccharomyces cerevisiae heterogeneous nuclear ribonucleoprotein, nab2p. dissecting the requirements for nab2p-facilitated poly(a) rna export role of rna chaperones in virus replication heat shock proteins in cancer: diagnostic, prognostic, predictive, and treatment implications altered states: selectively drugging the hsp90 cancer chaperone heat shock proteins promote cancer: it's a protection racket heat shock proteins and cancer: intracellular chaperones or extracellular signalling ligands? disruption of rna metabolism in neurological diseases and emerging therapeutic interventions heat shock proteins as potential targets for protective strategies in neurodegeneration severe respiratory viral infections: new evidence and changing paradigms pathogenesis of viral respiratory infection. respiratory disease and infection-a new insight, intechopen lvd viruses causing gastroenteritis: the known, the new and those beyond identification of mw polyomavirus, a novel polyomavirus in human stool discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin a highly prevalent and genetically diversified picornaviridae genus in south asian children immunological features underlying viral hemorrhagic fevers pathogenesis of the viral hemorrhagic fevers the role of viruses in neurodegenerative and neurobehavioral diseases herpes viruses and alzheimer's disease: new evidence in the debate virus infection and human cancer: an overview. recent results cancer res heat shock protein 90: role in enterovirus 71 entry and assembly and potential target for therapy dengue virus entry into liver (hepg2) cells is independent of hsp90 and hsp70 heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells characterization of putative japanese encephalitis virus receptor molecules on microglial cells antiviral activity and rna polymerase degradation following hsp90 inhibition in a range of negative strand viruses hsp90 inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus chikungunya virus nsp3 & nsp4 interacts with hsp-90 to promote virus replication: hsp-90 inhibitors reduce chikv infection and inflammation in vivo heat-shock protein 90 is essential for stabilization of the hepatitis c virus nonstructural protein ns3 destabilization of pdk1 by hsp90 inactivation suppresses hepatitis c virus replication through inhibition of prk2-mediated viral rna polymerase phosphorylation hepatitis c virus rna replication is regulated by fkbp8 and hsp90 identification of hsp90 as a stimulatory host factor involved in influenza virus rna synthesis hsp90 inhibitors reduce influenza virus replication in cell culture mc1568 inhibits hdac6/8 activity and influenza a virus replication in lung epithelial cells: role of hsp90 acetylation autophagy is involved in regulating influenza a virus rna and protein synthesis associated with both modulation of hsp90 induction and mtor/p70s6k signaling pathway pim1 impacts enterovirus a71 replication and represents a potential target in antiviral therapy host cell factor requirement for hepatitis c virus enzyme maturation evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance molecular chaperone hsp90 is a therapeutic target for noroviruses influenza a virus neuraminidase protein interacts with hsp90, to stabilize itself and enhance cell survival viral transport and the cytoskeleton the hepatitis e virus open reading frame 3 product interacts with microtubules and interferes with their dynamics the ins and outs of tubulin acetylation: more than just a post-translational modification? regulation of microtubule assembly and stability by the transactivator of transcription protein of jembrana disease virus heat-shock protein 90 promotes nuclear transport of herpes simplex virus 1 capsid protein by interacting with acetylated tubulin glucocorticoid stimulates hepatitis b viral gene expression in cultured human hepatoma cells geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus hepatitis b virus replication hsp90 is required for the activity of a hepatitis b virus reverse transcriptase requirement of heat shock protein 90 for human hepatitis b virus reverse transcriptase function in vitro reconstitution of a functional duck hepatitis b virus reverse transcriptase: posttranslational activation by hsp90 hbv polymerase interacts independently with nterminal and c-terminal fragments of hsp90beta chaperone activation of the hepadnaviral reverse transcriptase for template rna binding is established by the hsp70 and stimulated by the hsp90 system efficient hsp90-independent in vitro activation by hsc70 and hsp40 of duck hepatitis b virus reverse transcriptase, an assumed hsp90 client protein hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids hsp90 makes the human hbv pol competent for in vitro priming rather than maintaining the human hbv pol/ pregenomic rna complex expression of stable hepatitis b viral polymerase associated with grp94 in e. coli herpes simplex virus type 1 dna polymerase requires the mammalian chaperone hsp90 for proper localization to the nucleus geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro hsp90 inhibitors: a potential treatment for latent ebv infection? nuclear transport of epstein-barr virus dna polymerase is dependent on the bmrf1 polymerase processivity factor and molecular chaperone hsp90 the herpes simplex virus vp16-induced complex: the makings of a regulatory switch herpes simplex virus disrupts nf-kappab regulation by blocking its recruitment on the ikappabalpha promoter and directing the factor on viral genes heat-shock protein 90α is involved in maintaining the stability of vp16 and vp16-mediated transactivation of α genes from herpes simplex virus-1 geldanamycin, a potent and specific inhibitor of hsp90, inhibits gene expression and replication of human cytomegalovirus curcumin inhibits human cytomegalovirus by downregulating heat shock protein 90 a novel hsp90 inhibitor, 17-dmag, induces tax down-regulation and its oral administration to atl-model mice intervenes against the infiltration property of the atl-like lymphocytes and provides extended survival period processing of the herpes simplex virus regulatory protein alpha 22 mediated by the ul13 protein kinase determines the accumulation of a subset of alpha and gamma mrnas and proteins in infected cells icp22 and the ul13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of rna polymerase ii epstein-barr virus-encoded protein kinase (bglf4) is involved in production of infectious virus the human cytomegalovirus ul44 protein is a substrate for the ul97 protein kinase distinct and separate roles for herpesvirus-conserved ul97 kinase in cytomegalovirus dna synthesis and encapsidation the human cytomegalovirus ul97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress viral mimicry of cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress conserved herpesvirus kinases target the dna damage response pathway and tip60 histone acetyltransferase to promote virus replication sumo binding by the epstein-barr virus protein kinase bglf4 is crucial for bglf4 function hsp90 inhibitors block outgrowth of ebv-infected malignant cells in vitro and in vivo through an ebna1-dependent mechanism ganetespib, an hsp90 inhibitor, kills epstein-barr virus (ebv)-infected b and t cells and reduces the percentage of ebv-infected cells in the blood ebp2 plays a key role in epstein-barr virus mitotic segregation and is regulated by aurora family kinases epstein-barr virus provides a survival factor to burkitt's lymphomas structure of the p53 binding domain of hausp/usp7 bound to epstein-barr nuclear antigen 1 implications for ebv-mediated immortalization self-inhibition of synthesis and antigen presentation by epstein-barr virus-encoded ebna1 heat shock protein 90 facilitates formation of the hbv capsid via interacting with the hbv core protein dimers reactive oxygen species promote heat shock protein 90-mediated hbv capsid assembly virus particles in cultured lymphoblasts from burkitt's lymphoma epstein-barr virus in the pathogenesis of npc all three domains of the epstein-barr virus-encoded latent membrane protein lmp-1 are required for transformation of rat-1 fibroblasts epstein-barr virus induces invasion and metastasis factors a novel hsp90 inhibitor at13387 induces senescence in ebvpositive nasopharyngeal carcinoma cells and suppresses tumor formation the heat shock protein 90 inhibitor biib021 suppresses the growth of t and natural killer cell lymphomas heat shock protein 90 expression in epstein-barr virus-infected b cells promotes gammadelta t-cell proliferation in vitro tcrgammadelta cells and viruses heat shock protein 90 (hsp90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway nlr, the nucleotide-binding domain leucine-rich repeat containing gene family the hsp90-sgt1 chaperone complex for nlr immune sensors molecular mechanisms of cellular transformation by htlv-1 tax activation of nf-kappab by htlv-i and implications for cell transformation hsp90 protects the human t-cell leukemia virus type 1 (htlv-1) tax oncoprotein from proteasomal degradation to support nf-κb activation and htlv-1 replication grp78, a coreceptor for coxsackievirus a9, interacts with major histocompatibility complex class i molecules which mediate virus internalization integrin alpha v beta 6 is an rgd-dependent receptor for coxsackievirus a9 heat shock protein 70 as a supplementary receptor facilitates enterovirus 71 infections in vitro surface proteins of c6/36 cells involved in dengue virus 4 binding and entry zika virus dependence on host hsp70 provides a protective strategy against infection and disease heat shock protein 70 on neuro2a cells is a putative receptor for japanese encephalitis virus association of heat-shock protein 70 with lipid rafts is required for japanese encephalitis virus infection in huh7 cells heat shock cognate protein 70 isoform d is required for clathrin-dependent endocytosis of japanese encephalitis virus in c6/36 cells grp78 is an important host factor for japanese encephalitis virus entry and replication in mammalian cells japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity natural products may interfere with sars-cov-2 attachment to the host cell heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells heat shock cognate protein 70 is involved in rotavirus cell entry interaction of rotaviruses with hsc70 during cell entry is mediated by vp5 the peptide-binding and atpase domains of recombinant hsc70 are required to interact with rotavirus and reduce its infectivity heat shock protein 70 regulates degradation of the mumps virus phosphoprotein via the ubiquitin-proteasome pathway heat shock protein 90 ensures efficient mumps virus replication by assisting withfang viral polymerase complex formation constitutive overexpression of the major inducible 70 kda heat shock protein mediates large plaque formation by measles virus enhanced production of morbillivirus gene-specific rnas following induction of the cellular stress response in stable persistent infection the highly inducible member of the 70 kda family of heat shock proteins increases canine distemper virus polymerase activity heat shock protein 72 is associated with the hepatitis c virus replicase complex and enhances viral rna replication tylophorine analogs allosterically regulates heat shock cognate protein 70 and inhibits hepatitis c virus replication the heat shock protein inhibitor quercetin attenuates hepatitis c virus production human respiratory syncytial virus n, p and m protein interactions in hek-293t cells evidence for an association between heat shock protein 70 and the respiratory syncytial virus polymerase complex within lipid-raft membranes during virus infection interactome analysis of the human respiratory syncytial virus rna polymerase complex identifies protein chaperones as important cofactors that promote l-protein stability and rna synthesis elucidation of the cellular interactome of ebola virus nucleoprotein and identification of therapeutic targets an rna polymerase ii-driven ebola virus minigenome system as an advanced tool for antiviral drug screening recombinant rna-dependent rna polymerase complex of ebola virus a small stem-loop structure of the ebola virus trailer is essential for replication and interacts with heat-shock protein a8 cellular stress response induces selective intranuclear trafficking and accumulation of morbillivirus major core protein heat shock protein 70 modulates influenza a virus polymerase activity heat shock protein 70 promotes coxsackievirus b3 translation initiation and elongation via akt-mtorc1 pathway depending on activation of p70s6k and cdc2 hsc70 regulates the ires activity and serves as an antiviral target of enterovirus a71 infection the double-stranded rna-dependent protein kinase is also activated by heparin repression of the pkr protein kinase by the hepatitis c virus ns5a protein: a potential mechanism of interferon resistance the 58,000-dalton cellular inhibitor of the interferon-induced double-stranded rna-activated protein kinase (pkr) is a member of the tetratricopeptide repeat family of proteins purification and partial characterization of a cellular inhibitor of the interferon-induced protein kinase of mr 68,000 from influenza virus-infected cells characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsrna-activated protein kinase the molecular chaperone hsp40 regulates the activity of p58ipk, the cellular inhibitor of pkr the cellular inhibitor of the pkr protein kinase, p58(ipk), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity c-terminal trimerization, but not n-terminal trimerization, of the reovirus cell attachment protein is a posttranslational and hsp70/atp-dependent process association of heat shock protein 70 with enterovirus capsid precursor p1 in infected human cells heat shock protein 70 is related to thermal inhibition of nuclear export of the influenza virus ribonucleoprotein complex heat shock protein 70 inhibits the activity of influenza a virus ribonucleoprotein and blocks the replication of virus in vitro and in vivo polo-like kinase 1 is involved in hepatitis c virus replication by hyperphosphorylating ns5a the ns5a-binding heat shock proteins hsc70 and hsp70 play distinct roles in the hepatitis c viral life cycle structural characterization of the hsp70 interaction domain of the hepatitis c viral protein ns5a sgta-dependent regulation of hsc70 promotes cytosol entry of simian virus 40 from the endoplasmic reticulum a non-enveloped virus hijacks host disaggregation machinery to translocate across the endoplasmic reticulum membrane bip and multiple dnaj molecular chaperones in the endoplasmic reticulum are required for efficient simian virus 40 infection adenovirus capsid proteins interact with hsp70 proteins after penetration in human or rodent cells novel partner proteins of adenovirus penton co-chaperone bag3 and adenovirus penton base protein partnership nuclear import of adenovirus dna in vitro involves the nuclear protein import pathway and hsc70 nuclear sequestration of cellular chaperone and proteasomal machinery during herpes simplex virus type 1 infection transcriptional stimulation by the dna binding protein hap46/bag-1m involves hsp70/hsc70 molecular chaperones transcriptional activation by the human hsp70-associating protein hap50 the hsp70-associating protein hap46 binds to dna and stimulates transcription bag-1, a novel bcl-2-interacting protein, activates expression of human jc virus bag-1m, an isoform of bcl-2-interacting protein bag-1, enhances gene expression driven by cmv promoter chaperone action in the posttranslational topological reorientation of the hepatitis b virus large envelope protein: implications for translocational regulation sequence-specific repression of cotranslational translocation of the hepatitis b virus envelope proteins coincides with binding of heat shock protein hsc70 chaperones involved in hepatitis b virus morphogenesis bag-1m accelerates nucleotide release for human hsc70 and hsp70 and can act concentration-dependent as positive and negative cofactor hsp70 interacting protein hip does not affect glucocorticoid receptor folding by the hsp90-based chaperone machinery except to oppose the effect of bag-1 molecular chaperone grp78/bip interacts with the large surface protein of hepatitis b virus in vitro and in vivo mammalian bip controls posttranslational er translocation of the hepatitis b virus large envelope protein in vivo and in vitro association of hsc70 with polyomavirus capsid proteins chaperone-mediated in vitro assembly of polyomavirus capsids productive infection and cell-free transmission of human t-cell leukemia virus in a nonlymphoid cell line detection of lymphocytes producing a human retrovirus associated with adult t-cell leukemia by syncytia induction assay 71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human t-cell lymphotropic virus type 1 heat shock cognate protein 70 is a cell fusion-enhancing factor but not an entry factor for human t-cell lymphotropic virus type i heat-shock protein 70 can replace viral protein r of hiv-1 during nuclear import of the viral preintegration complex heat-shock proteins reverse the g2 arrest caused by hiv-1 viral protein r heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein r atpgammas disrupts human immunodeficiency virus type 1 virion core integrity the amino-terminal transforming region of simian virus 40 large t and small t antigens functions as a j domain the molecular chaperone activity of simian virus 40 large t antigen is required to disrupt rb-e2f family complexes by an atp-dependent mechanism atp-dependent simian virus 40 tantigen-hsc70 complex formation the regulation of e2f by prb-family proteins mechanism of regulation of hsp70 chaperones by dnaj cochaperones investigation of the interaction between dnak and dnaj by surface plasmon resonance spectroscopy priming polyvalent immunity by dna vaccines expressing chimeric antigens with a stress protein-capturing, viral j-domain loss of p19(arf) eliminates the requirement for the prb-binding motif in simian virus 40 large t antigenmediated transformation novel mechanisms of e2f induction by bk virus large-t antigen: requirement of both the prbbinding and the j domains j domain-independent regulation of the rb family by polyomavirus large t antigen the j domain of simian virus 40 large t antigen is required to functionally inactivate rb family proteins a novel human dnaj protein, htid-1, a homolog of the drosophila tumor suppressor protein tid56, can interact with the human papillomavirus type 16 e7 oncoprotein identification of a novel retinoblastoma gene product binding site on human papillomavirus type 16 e7 protein the human papillomavirus e7 oncoprotein and the cellular transcription factor e2f bind to separate sites on the retinoblastoma tumor suppressor protein differential distribution of the adenovirus e1a proteins and colocalization of e1a with the 70-kilodalton cellular heat shock protein in infected cells to kill or be killed: viral evasion of apoptosis apoptosis in viral pathogenesis viruses and apoptosis epstein-barr virus nuclear antigen (ebna) 3a induces the expression of and interacts with a subset of chaperones and co-chaperones caspase activation is required for permissive replication of aleutian mink disease parvovirus in vitro the mechanisms of direct, virus-induced destruction of neurons the hepatitis c virus core protein interacts with ns5a and activates its caspase-mediated proteolytic cleavage apoptosis in coxsackievirus b3-caused diseases: interaction between the capsid protein vp2 and the proapoptotic protein siva the apoptotic capability of coxsackievirus b3 is influenced by the efficient interaction between the capsid protein vp2 and the proapoptotic host protein siva hiv-1 vpr induces apoptosis through caspase 9 in t cells and peripheral blood mononuclear cells adenovirus encoding hiv-1 vpr activates caspase 9 and induces apoptotic cell death in both p53 positive and negative human tumor cell lines influenza virus ns1 protein induces apoptosis in cultured cells neurovirology and developmental neurobiology programmed cell death in virus infections of the nervous system ts-hsp70 induces protective immunity against trichinella spiralis infection in mouse by activating dendritic cells through tlr2 and tlr4 multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma hsp70 inhibits lipopolysaccharide-induced nf-κb activation by interacting with traf6 and inhibiting its ubiquitination hsp70/dnaja3 chaperone/cochaperone regulates nf-κb activity in immune responses the role of the membrane-initiated heat shock response in cancer enhanced heat shock protein 70 expression alters proteasomal degradation of ikappab kinase in experimental acute respiratory distress syndrome hsp70 is a negative regulator of nlrp3 inflammasome activation functional domains of hsp70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity hsp70 peptidembearing and peptide-negative preparations act as chaperokines toll-like receptor signalling toll-like receptor 4 (tlr4) is essential for hsp70-like protein 1 (hsp70l1) to activate dendritic cells and induce th1 response toll-like receptor signaling and its inducible proteins inflammation in gastric cancer: interplay of the cox-2/prostaglandin e2 and toll-like receptor/myd88 pathways tak1-ecsit-traf6 complex plays a key role in the tlr4 signal to activate nf-kappab cutting edge: traf6 mediates tlr/il-1r signaling-induced nontranscriptional priming of the nlrp3 inflammasome heme oxygenase-1 regulates dendritic cell function through modulation of p38 mapk-creb/atf1 signaling activated apoptotic cells induce dendritic cell maturation via engagement of toll-like receptor 4 (tlr4), dendritic cell-specific intercellular adhesion molecule 3 (icam-3)-grabbing nonintegrin (dc-sign), and beta2 integrins il-1β induction of muc5ac gene expression is mediated by creb and nf-κb and repressed by dexamethasone induction of proinflammatory response in prostate cancer epithelial cells by activated macrophages autoimmune and inflammatory mechanisms in atherosclerosis heat shock proteins as ligands of toll-like receptors the hsp60 immune system network heat shock protein and innate immunity heat shock proteins: mediators of atherosclerotic development hsp60 critically regulates endogenous il-1β production in activated microglia by stimulating nlrp3 inflammasome pathway genome-wide rnai screen identifies human host factors crucial for influenza virus replication rna interference mediated silencing of hsp60 gene in human monocytic myeloma cell line u937 revealed decreased dengue virus multiplication hepatitis c virus: from oxygen free radicals to hepatocellular carcinoma mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis c virus core protein interaction of hepatitis c virus core protein with hsp60 triggers the production of reactive oxygen species and enhances tnf-alpha-mediated apoptosis tyrosine phosphorylation modulates mitochondrial chaperonin hsp60 and delays rotavirus nsp4-mediated apoptotic signaling in host cells cooperation between heat shock proteins in organizing of proteins spatial structure antisense oligodeoxynucleotides targeted against molecular chaperonin hsp60 block human hepatitis b virus replication human hepatitis b virus polymerase interacts with the molecular chaperonin hsp60 binding site analysis of human hbv pol for molecular chaperonin, hsp60 hepatitis b virus x protein: a multifunctional viral regulator interaction of the hepatitis b virus x protein (hbx) with heat shock protein 60 enhances hbx-mediated apoptosis hbx protein of hepatitis b virus (hbv) can form complex with mitochondrial hsp60 and hsp70 innate immune responses in hepatitis b virus (hbv) infection circulating and liver resident cd4 + cd25 + regulatory t cells actively influence the antiviral immune response and disease progression in patients with hepatitis b interferon effects on interleukin-10 secretion mononuclear cell response to interleukin-10 is normal in multiple sclerosis patients il-10-producing regulatory b cells (b10 cells) in autoimmune disease hepatitis b virus replication could enhance regulatory t cell activity by producing soluble heat shock protein 60 from hepatocytes an hsp60 related protein is associated with purified hiv and siv molecular mechanisms in retrovirus dna integration functional interactions of human immunodeficiency virus type 1 integrase with human and yeast hsp60 dnaj homolog hdj2 facilitates japanese encephalitis virus replication structure of the ribonucleoprotein of influenza virus a classical bipartite nuclear localization signal on thogoto and influenza a virus nucleoproteins an unconventional nls is critical for the nuclear import of the influenza a virus nucleoprotein and ribonucleoprotein human heat shock protein 40 (hsp40/dnajb1) promotes influenza a virus replication by assisting nuclear import of viral ribonucleoproteins dnaja1/hsp40 is co-opted by influenza a virus to enhance its viral rna polymerase activity molecular cloning and characterization of the human doublestranded rna-activated protein kinase induced by interferon influenza a virus nucleoprotein exploits hsp40 to inhibit pkr activation p58ipk, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eif2alpha signaling biochemical and genetic evidence for complex formation between the influenza a virus ns1 protein and the interferon-induced pkr protein kinase interaction of hsp40 with influenza virus m2 protein: implications for pkr signaling pathway flavivirus replication complex assembly revealed by dnajc14 functional mapping identification and characterization of the host protein dnajc14 as a broadly active flavivirus replication modulator chaperone-assisted protein folding is critical for yellow fever virus ns3/4a cleavage and replication dnajb1/hsp40 suppresses melanoma differentiation-associated gene 5-mitochondrial antiviral signaling protein function in conjunction with hsp70 cellular dnaja3, a novel vp1-interacting protein, inhibits footand-mouth disease virus replication by inducing lysosomal degradation of vp1 and attenuating its antagonistic role in the beta interferon signaling pathway human hsp70 and hsp40 chaperone proteins facilitate human papillomavirus-11 e1 protein binding to the origin and stimulate cell-free dna replication targeting the e1 replication protein to the papillomavirus origin of replication by complex formation with the e2 transactivator cell-free replication of the human papillomavirus dna with homologous viral e1 and e2 proteins and human cell extracts chaperone proteins abrogate inhibition of the human papillomavirus (hpv) e1 replicative helicase by the hpv e2 protein the human dnaj protein, htid-1, enhances binding of a multimer of the herpes simplex virus type 1 ul9 protein to oris, an origin of viral dna replication activation of the herpes simplex virus type-1 origin-binding protein (ul9) by heat shock proteins turnover of hepatitis b virus x protein is facilitated by hdj1, a human hsp40/dnaj protein negative regulation of hepatitis b virus replication by cellular hsp40/dnaj proteins through destabilization of viral core and x proteins hepatitis b virus x protein in the pathogenesis of hepatitis b virusinduced hepatocellular carcinoma high-level expression of hepatitis b virus hbx gene and hepatocarcinogenesis in transgenic mice x protein of hepatitis b virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis hepatitis b. x antigen and p53 are associated in vitro and in liver tissues from patients with primary hepatocellular carcinoma retroviral infection of non-dividing cells: old and new perspectives hiv-1 infection of nondividing cells through the recognition of integrase by the importin/karyopherin pathway a nuclear localization signal within hiv-1 matrix protein that governs infection of non-dividing cells hiv-1 genome nuclear import is mediated by a central dna flap functional analysis of the simian immunodeficiency virus vpx protein: identification of packaging determinants and a novel nuclear targeting domain simian immunodeficiency virus vpx is imported into the nucleus via importin alphadependent and -independent pathways hsp40 facilitates nuclear import of the human immunodeficiency virus type 2 vpx-mediated preintegration complex interference of dnajb6/mrj isoform switch by morpholino inhibits replication of hiv-1 and rsv large isoform of mammalian relative of dnaj is a major determinant of human susceptibility to hiv-1 infection heat shock protein 40 is necessary for human immunodeficiency virus-1 nef-mediated enhancement of viral gene expression and replication hiv-1 nef and host cell protein kinases nef triggers a transcriptional program in t cells imitating single-signal t cell activation and inducing hiv virulence mediators the plasma membrane as a combat zone in the hiv battlefield cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways reciprocal regulation of human immunodeficiency virus-1 gene expression and replication by heat shock proteins 40 and 70 novel role of hsp40/dnaj in the regulation of hiv-1 replication heat shock proteins as cellular lifeguards structural and functional diversities between members of the human hspb, hsph, hspa, and dnaj chaperone families muscle develops a dpecific form of small heat shock protein complex composed of mkbp/hspb2 and hspb3 during myogenic differentiation epstein-barr virus upregulates phosphorylated heat shock protein 27 kda in carcinoma cells using the phosphoinositide 3-kinase/akt pathway proteomics analysis of differentially expressed proteins in chicken trachea and kidney after infection with the highly virulent and attenuated coronavirus infectious bronchitis virus in vivo cellular hsp27 interacts with classical swine fever virus ns5a protein and negatively regulates viral replication by the nf-κb signaling pathway down-regulating heat shock protein 27 is involved in porcine epidemic diarrhea virus escaping from host antiviral mechanism the heat-shock response adenovirus type 7 induces interleukin-8 production via activation of extracellular regulated kinase 1/2 nf-κb and the map kinases/ap-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein i/ii, a modulin from oral streptococci signaling pathways for glycated human serum albumin-induced il-8 and mcp-1 secretion in human rpe cells mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery heat shock proteins: essential proteins for apoptosis regulation apoptosis versus cell differentiation: role of heat shock proteins hsp90, hsp70 and hsp27 small heat shock proteins hsp27 (hspb1), αb-crystallin (hspb5) and hsp22 (hspb8) as regulators of cell death modification and reorganization of the cytoprotective cellular chaperone hsp27 during herpes simplex virus type 1 infection heat shock protein 27 is involved in pcv2 infection in pk-15 cells hspb1 is an intracellular antiviral factor against hepatitis b virus role of thiol/disulfide exchange in newcastle disease virus entry overexpression of thiol/disulfide isomerases enhances membrane fusion directed by the newcastle disease virus fusion protein endothelial cell surface expression of protein disulfide isomerase activates beta1 and beta3 integrins and facilitates dengue virus infection protein disulfide isomerase mediates dengue virus entry in association with lipid rafts inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent hiv-1 entry the catalytic activity of protein disulfide isomerase is involved in human immunodeficiency virus envelope-mediated membrane fusion after cd4 cell binding protein-disulfide isomerase-mediated reduction of two disulfide bonds of hiv envelope glycoprotein 120 occurs post-cxcr4 binding and is required for fusion galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance t-cell migration and hiv entry structure of murine polyomavirus complexed with an oligosaccharide receptor fragment how viruses use the endoplasmic reticulum for entry, replication, and assembly simian virus 40 depends on er protein folding and quality control factors for entry into host cells a pdi family network acts distinctly and coordinately with erp29 to facilitate polyomavirus infection oblongifolin m, an active compound isolated from a chinese medical herb garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of erp57 cellular self-defense: how cellautonomous immunity protects against pathogens role of free radicals in viral pathogenesis and mutation ex vivo induction of apoptosis in lymphocytes is mediated by oxidative stress: role for lymphocyte loss in hiv infection. free radic role of oxidants in influenza virus-induced gene expression investigation of oxidative stress and antioxidant defense in patients with hepatitis b virus infection and the effect of interferon-alpha plus lamivudine combination therapy on oxidative stress hepatitis c virus core protein inhibits mitochondrial electron transport and increases reactive oxygen species (ros) production oxidative damage to neurons caused by the induction of microglial nadph oxidase in encephalomyocarditis virus infection cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures oxidative stress in hpv-driven viral carcinogenesis: redox proteomics analysis of hpv-16 dysplastic and neoplastic tissues folding and oligomerization of influenza hemagglutinin in the er and the intermediate compartment n-glycosylation of the premembrane protein of japanese encephalitis virus is critical for folding of the envelope protein and assembly of viruslike particles hepatitis c virus glycoprotein folding: disulfide bond formation and association with calnexin an rna chaperone activity of non-specific rna binding proteins in hammerhead ribozyme catalysis the ebola virus vp24 protein prevents hnrnp c1/c2 binding to karyopherin α1 and partially alters its nuclear import hnrnps relocalize to the cytoplasm following infection with vesicular stomatitis virus hnrnp proteins and the biogenesis of mrna direct and indirect effects on viral translation and rna replication are required for auf1 restriction of enterovirus infections in human cells cellular mrna decay protein auf1 negatively regulates enterovirus and human rhinovirus infections heterogeneous nuclear ribonucleoprotein a2 participates in the replication of japanese encephalitis virus through an interaction with viral proteins and rna viral and cellular proteins involved in coronavirus replication hnrnp c and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'ntr of the hcv rna genome mechanistic consequences of hnrnp c binding to both rna termini of poliovirus negative-strand rna intermediates characterization of multimeric complexes formed by the human ptb1 protein on rna heterogeneous nuclear ribonucleoprotein i (hnrnp-i/ptb) selectively binds the conserved 3′ terminus of hepatitis c viral rna role of la autoantigen and polypyrimidine tract-binding protein in hcv replication polypyrimidine-tract-binding protein is a component of the hcv rna replication complex and necessary for rna synthesis hur displaces polypyrimidine tract binding protein to facilitate la binding to the 3′ untranslated region and enhances hepatitis c virus replication the la antigen binds 5′ noncoding region of the hepatitis c virus rna in the context of the initiator aug codon and stimulates internal ribosome entry site-mediated translation hepatitis c virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human la autoantigen hur, a protein implicated in oncogene and growth factor mrna decay, binds to the 3′ ends of hepatitis c virus rna of both polarities delayed kinetics of poliovirus rna synthesis in a human cell line with reduced levels of hnrnp c proteins microrna-555 has potent antiviral properties against poliovirus cellular rna binding proteins ns1-bp and hnrnp k regulate influenza a virus rna splicing ns1-binding protein (ns1-bp): a novel human protein that interacts with the influenza a virus nonstructural ns1 protein is relocalized in the nuclei of infected cells co-regulatory activity of hnrnp k and ns1-bp in influenza and human mrna splicing rna-binding protein hnrnp d modulates internal ribosome entry site-dependent translation of hepatitis c virus rna two distinct binding modes of a protein cofactor with its target rna mechanism for nucleic acid chaperone activity of hiv-1 nucleocapsid protein revealed by single molecule stretching heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 5′ untranslated region and participates in virus replication the polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation feline calicivirus replication: requirement for polypyrimidine tract-binding protein is temperature-dependent internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of unr to two distinct sites on the 5′ untranslated region evidence for an rna chaperone function of polypyrimidine tractbinding protein in picornavirus translation interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis c virus rna genome and its functional requirement in internal initiation of translation hnrnp l is required for the translation mediated by hcv ires heterogeneous nuclear ribonucleoprotein l interacts with the 3 border of the internal ribosomal entry site of hepatitis c virus hnrnp l and nf90 interact with hepatitis c virus 5′-terminal untranslated rna and promote efficient replication a human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein k (hnrnp k) recognizes the 5′ terminal sequence of the hepatitis c virus rna heterogeneous ribonucleoprotein k (hnrnp k) binds mir-122, a mature liver-specific microrna required for hepatitis c virus replication modulation of hepatitis c virus rna abundance by a liver-specific microrna global epidemiology and genotype distribution of the hepatitis c virus infection position-dependent function for a tandem microrna mir-122-binding site located in the hepatitis c virus rna genome hepatitis c virus core protein interacts with heterogeneous nuclear ribonucleoprotein k an rna-binding protein, hnrnp a1, and a scaffold protein, septin 6, facilitate hepatitis c virus replication hnrnp a1 interacts with the 5' untranslated regions of enterovirus 71 and sindbis virus rna and is required for viral replication apigenin inhibits enterovirus-71 infection by disrupting viral rna association with trans-acting factors the role of misshapen nck-related kinase (mink), a novel ste20 family kinase, in the ires-mediated protein translation of human enterovirus 71 n-terminomics tails identifies host cell substrates of poliovirus and coxsackievirus b3 3c proteinases that modulate virus infection heterogeneous nuclear ribonucleoprotein m facilitates enterovirus infection the heterogeneous nuclear ribonucleoprotein k (hnrnp k) is a host factor required for dengue virus and junín virus multiplication the heterogeneous nuclear ribonucleoprotein k (hnrnp k) interacts with dengue virus core protein identification of heterogeneous nuclear ribonucleoprotein k (hnrnp k) as a repressor of c/ebpmediated gene activation role of human heterogeneous nuclear ribonucleoprotein c1/c2 in dengue virus replication vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein 1 and is important for viral replication and release identification of human hnrnp c1/c2 as a dengue virus ns1-interacting protein heterogeneous nuclear ribonucleoprotein k supports vesicular stomatitis virus replication by regulating cell survival and cellular gene expression downregulation of nipah virus n mrna occurs through interaction between its 3′ untranslated region and hnrnp d hnrnp a2/b1 interacts with influenza a viral protein ns1 and inhibits virus replication potentially through suppressing ns1 rna/ protein levels and ns1 mrna nuclear export the negative regulator of splicing element of rous sarcoma virus promotes polyadenylation a cellular protein, hnrnp h, binds to the negative regulator of splicing element from rous sarcoma virus efficient polyadenylation of rous sarcoma virus rna requires the negative regulator of splicing element analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication viral dna replication orientation and hnrnps regulate transcription of the human papillomavirus 18 late promoter host heterogeneous ribonucleoprotein k (hnrnp k) as a potential target to suppress hepatitis b virus replication cytidine deaminase apobec3b interacts with heterogeneous nuclear ribonucleoprotein k and suppresses hepatitis b virus expression the multifunctional herpes simplex virus ie63 protein interacts with heterogeneous ribonucleoprotein k and with casein kinase 2 ck2 protein kinase is stimulated and redistributed by functional herpes simplex virus icp27 protein rna polymerase ii holoenzyme modifications accompany transcription reprogramming in herpes simplex virus type 1-infected cells association of herpes simplex virus type 1 icp8 and icp27 proteins with cellular rna polymerase ii holoenzyme herpes simplex virus 1 icp27 is required for transcription of two viral late (gamma 2) genes in infected cells human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences a downstream polyadenylation element in human papillomavirus type 16 l2 encodes multiple ggg motifs and interacts with hnrnp h specific interaction between hnrnp h and hpv16 l1 proteins: implications for late gene auto-regulation enabling rapid viral capsid protein production the epstein-barr virus (ebv) sm protein enhances pre-mrna processing of the ebv dna polymerase transcript the alternative splicing factor hnrnp a1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element a 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hfip1, cstf-64, hnrnp c1/c2, and polypyrimidine tract binding protein identification of an hnrnp a1-dependent splicing silencer in the human papillomavirus type 16 l1 coding region that prevents premature expression of the late l1 gene inhibition of hpv-16 l1 expression from l1 cdnas correlates with the presence of hnrnp a1 binding sites in the l1 coding region identification of a 17-nucleotide splicing enhancer in hpv-16 l1 that counteracts the effect of multiple hnrnp a1-binding splicing silencers polypyrimidine tract binding protein induces human papillomavirus type 16 late gene expression by interfering with splicing inhibitory elements at the major late 5' splice site, sd3632 translational inhibition in vitro of human papillomavirus type 16 l2 mrna mediated through interaction with heterogenous ribonucleoprotein k and poly(rc)-binding proteins 1 and 2 c-src-mediated phosphorylation of hnrnp k drives translational activation of specifically silenced mrnas regulation of the epstein-barr virus c promoter by auf1 and the cyclic amp/protein kinase a signaling pathway auf1/hnrnp d is a novel protein partner of the eber1 noncoding rna of epstein-barr virus nuclear hnrnpa2b1 initiates and amplifies the innate immune response to dna viruses hnrnp a1 selectively interacts through its gly-rich domain with different rna-binding proteins hiv nef enhances tat-mediated viral transcription through a hnrnp-k-nucleated signaling complex cooperative-binding and splicing-repressive properties of hnrnp a1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly actin-associated hnrnp proteins as transacting factors in the control of mrna transport and localization genomic structure of an attenuated quasi species of hiv-1 from a blood transfusion donor and recipients novel (n)pkc kinases phosphorylate nef for increased hiv transcription, replication and perinuclear targeting cloning and functional analysis of multiply spliced mrna species of human immunodeficiency virus type 1 alternative splicing of human immunodeficiency virus type 1 mrna modulates viral protein expression, replication, and infectivity a naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production exon recognition in vertebrate splicing intron recognition comes of age identification, purification, and biochemical characterization of u2 small nuclear ribonucleoprotein auxiliary factor interaction of u2af65 rs region with pre-mrna of branch point and promotion base pairing with u2 snrna a potential role for u2af-sap 155 interactions in recruiting u2 snrnp to the branch site sorting out the complexity of sr protein functions hnrnp complexes: composition, structure, and function alternative pre-mrna splicing: the logic of combinatorial control balanced splicing at the tat-specific hiv-1 3'ss a3 is critical for hiv-1 replication splicing efficiency of human immunodeficiency virus type 1 tat rna is determined by both a suboptimal 3' splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2 the tat/rev intron of human immunodeficiency virus type 1 is inefficiently spliced because of suboptimal signals in the 3′ splice site behind the scenes of hiv-1 replication: alternative splicing as the dependency factor on the quiet an exonic splicing silencer downstream of the 3' splice site a2 is required for efficient human immunodeficiency virus type 1 replication biochemical and nmr study on the competition between proteins sc35, srp40, and heterogeneous nuclear ribonucleoprotein a1 at the hiv tat exon 2 splicing site rna biology identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new hiv-1 splicing regulator, protein hnrnp k a second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of tat mrna and binds protein hnrnp h control of hiv-1 env rna splicing and transport: investigating the role of hnrnp a1 in exon splicing silencer (ess3a) function the hnrnp a1 protein regulates hiv-1 tat splicing via a novel intron silencer element hnrnp a1 controls hiv-1 mrna splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure solution structure of the hiv-1 intron splicing silencer and its interactions with the up1 domain of heterogeneous nuclear ribonucleoprotein (hnrnp) a1 a truncated hnrnp a1 isoform, lacking the rgg-box rna binding domain, can efficiently regulate hiv-1 splicing and replication sc35 and heterogeneous nuclear ribonucleoprotein a/b proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate hiv-1 tat exon 2 splicing hnrnp a1 recruited to an exon in vivo can function as an exon splicing silencer a janus splicing regulatory element modulates hiv-1 tat and rev mrna production by coordination of hnrnp a1 cooperative binding rna splicing at human immunodeficiency virus type 1 3' splice site a2 is regulated by binding of hnrnp a/b proteins to an exonic splicing silencer element differential hnrnp d isoform incorporation may confer plasticity to the essv-mediated repressive state across hiv-1 exon 3 human immunodeficiency virus type 1 hnrnp a/b-dependent exonic splicing silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts sr proteins and hnrnp h regulate the splicing of the hiv tev-specific exon 6d the secondary structure of the human immunodeficiency virus type 1 transcript modulates viral splicing and infectivity members of the heterogeneous nuclear ribonucleoprotein h family activate splicing of an hiv-1 splicing substrate by promoting formation of atp-dependent spliceosomal complexes rna trafficking signals in human immunodeficiency virus type 1 trafficking of hiv-1 rna is mediated by heterogeneous nuclear ribonucleoprotein a2 expression and impacts on viral assembly a late role for the association of hnrnp a2 with the hiv-1 hnrnp a2 response elements in genomic rna, gag, and vpr localization differential effects of hnrnp d/auf1 isoforms on hiv-1 gene expression depletion of hnrnp a2/b1 overrides the nuclear retention of the hiv-1 genomic rna mapping of determinants required for the function of the hiv-1 env nuclear retention sequence inhibition of hiv-1 gene expression by a fragment of hnrnp u nuclear mrna export: insights from virology protein kinase ck2 phosphorylation regulates the interaction of kaposi's sarcoma-associated herpesvirus regulatory protein orf57 with its multifunctional partner hnrnp k heterogeneous nuclear ribonucleoprotein a1 interferes with the binding of the human t cell leukemia virus type 1 rex regulatory protein to its response element human immunodeficiency virus type 1 (hiv-1) induces the cytoplasmic retention of heterogeneous nuclear ribonucleoprotein a1 by disrupting nuclear import: implications for hiv-1 gene expression a polypyrimidine tract facilitates the expression of kaposi's sarcoma-associated herpesvirus vflip through an internal ribosome entry site heat shock protein hsp72 plays an essential role in her2-induced mammary tumorigenesis. oncogene the role of heat shock protein 70 in mediating agedependent mortality in sepsis peptides and aptamers targeting hsp70: a novel approach for anticancer chemotherapy activation of hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation monitering heat shock protein 70 and heat shock protein 90 during herpes simplex virus type 1 infection hsp70 functions as a negative regulator of west nile virus capsid protein through direct interaction mitochondrial hsp70, hsp40, and hsp60 bind to the 3′ untranslated region of the murine hepatitis virus genome immunomodulatory activity of extracellular hsp70 mediated via paired receptors siglec-5 and siglec-14 extracellular cell stress proteins as biomarkers of human disease molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation cross-presentation of the oncofetal tumor antigen 5t4 from irradiated prostate cancer cells-a key role for heat-shock protein 70 and receptor cd91 membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen stress for maintaining memory: hsp70 as a mobile messenger for innate and adaptive immunity novel heat shock protein hsp70l1 activates dendritic cells and acts as a th1 polarizing adjuvant chaperokine activity of heat shock proteins hsp70 as endogenous stimulus of the toll/interleukin-1 receptor signal pathway chlamydial heat shock protein 60 activates macrophages and endothelial cells through toll-like receptor 4 and md2 in a myd88-dependent pathway association of hadha with human rna silencing machinery dengue virus modulates the unfolded protein response in a time-dependent manner heat shock protein 70 is associated with replicase complex of japanese encephalitis virus and positively regulates viral genome replication japanese encephalitis virus activates autophagy through xbp1 and atf6 er stress sensors in neuronal cells bovine viral diarrhea virus infection induces autophagy in mdbk cells the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis the emerging roles of viroporins in er stress response and autophagy induction during virus infection stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy hepatitis c virus suppresses the ire1-xbp1 pathway of the unfolded protein response glucose-regulated protein 78 is an intracellular antiviral factor against hepatitis b virus hepatitis b virus x protein (hbx) activates atf6 and ire1-xbp1 pathways of unfolded protein response herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase perk and mediates eif-2alpha dephosphorylation by the gamma(1)34.5 protein membrane biogenesis and the unfolded protein response the atf6 branch of unfolded protein response and apoptosis are activated to promote african swine fever virus infection role of the host cell's unfolded protein response in arenavirus infection interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone bip/grp78, which is required for virion assembly betanodavirus up-regulates chaperone grp78 via er stress: roles of grp78 in viral replication and host mitochondria-mediated cell death influenza a viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (ire1) stress pathway west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2α kinases perk and pkr activation of erad pathway by human hepatitis b virus modulates viral and subviral particle production hepatitis c virus ns4b induces unfolded protein response and endoplasmic reticulum overload response-dependent nf-κb activation the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor human cytomegalovirus protein us11 provokes an unfolded protein response that may facilitate the degradation of class i major histocompatibility complex products hcv causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response endoplasmic reticulum stress induced by hepatitis b virus x protein enhances cyclo-oxygenase 2 expression via activating transcription factor 4 functional diversity of the hnrnps: past, present and perspectives heterogeneous nuclear ribonucleoproteins (hnrnps) in cellular processes: focus on hnrnp e1's multifunctional regulatory roles acute and chronic disease caused by enteroviruses hnrnp a1 alters the structure of a conserved enterovirus ires domain to stimulate viral translation heterogeneous nuclear ribonuclear protein k interacts with the enterovirus 71 59 untranslated region and participates in virus replication molecular mechanism of action of hnrnp k and rtn3 in the replication of enterovirus 71. chin sherris medical microbiology, 6e anti-herpetic medications and reduced risk of dementia in patients with herpes simplex virus infections-a nationwide, population-based cohort study in taiwan antivirals reduce the formation of key alzheimer's disease molecules in cell cultures acutely infected with herpes simplex virus type 1 the heterogeneous nuclear ribonucleoprotein k is important for herpes simplex virus-1 propagation hepatitis b virus epidemiology and natural history of hcv infection pathobiology of chronic hepatitis virus infection and hepatocellular carcinoma (hcc). tal hepatitis delta virus polyarteritis nodosa and extrahepatic manifestations of hbv infection: the case against autoimmune intervention in pathogenesis autoantibody recognition of an n-terminal epitope of hnrnp l marks the risk for developing hbv-related hepatocellular carcinoma benign lymphadenopathies epstein-barr virus-associated lymphoproliferative disorders: experimental and clinical developments epstein-barr virus-associated hodgkin's lymphoma human papillomavirus and epstein-barr virus in nasopharyngeal carcinoma in a low-incidence population epstein-barr virus infection and multiple sclerosis: a review lymphomatoid granulomatosis: a practical review for pathologists dealing with this rare pulmonary lymphoproliferative process epstein-barr virus and skin manifestations in childhood monoclonal antibodies against epstein-barr virus cross-react with alpha-synuclein in human brain human papillomavirus (hpv) type distribution and serological response to hpv type 6 virus-like particles in patients with genital warts the global health burden of infection-associated cancers in the year 2002 trends in human papillomavirus-associated cancers-united states epidemiology and natural history of human papillomavirus infections in the female genital tract case-control study of human papillomavirus and oropharyngeal cancer review of human immunodeficiency virus type 1-related opportunistic infections in sub-saharan africa the treatment of patients with hiv defining hsp70 subnetworks in dengue virus replication reveals key vulnerability in flavivirus infection the hsp90 mosaic: a picture emerges the chaperone hsp90: changing partners for demanding clients structure of tpr domain-peptide complexes: critical elements in the assembly of the hsp70-hsp90 multichaperone machine hsp90 and co-chaperones twist the functions of diverse client proteins the 'active life' of hsp90 complexes structure and functional relationships of hsp90 cellular growth kinetics distinguish a cyclophilin inhibitor from an hsp90 inhibitor as a selective inhibitor of hepatitis c virus novobiocin and additional inhibitors of the hsp90 cterminal nucleotide-binding pocket the amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an atp/adp switch domain that regulates hsp90 conformation 17-allylamino-17-demethoxygeldanamycin induces downregulation of critical hsp90 protein clients and results in cell cycle arrest and apoptosis of human urinary bladder cancer cells inhibition of signal transduction by the hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis antitumor efficacy of ipi-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib nvp-auy922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis biib021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein hsp90 ganetespib, a unique triazolone-containing hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy the heat shock protein 90 inhibitor, at13387, displays a long duration of action in vitro and in vivo in non-small cell lung cancer snx2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against her kinase-dependent cancers novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins the heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized atpbinding domain in the carboxyl terminus of the chaperone design, synthesis, and biological evaluation of novel deguelinbased heat shock protein 90 (hsp90) inhibitors targeting proliferation and angiogenesis structural basis for depletion of heat shock protein 90 client proteins by deguelin epigallocatechin-3-gallate is a novel hsp90 inhibitor novel hsp90 inhibitor platycodin d disrupts hsp90/cdc37 complex and enhances the anticancer effect of mtor inhibitor sulforaphane inhibits pancreatic cancer through disrupting hsp90-p50cdc37 complex and direct interactions with amino acids residues of hsp90 withaferin a targets heat shock protein 90 in pancreatic cancer cells a novel hsp90 inhibitor to disrupt hsp90/cdc37 complex against pancreatic cancer cells natural product kongensin a is a non-canonical hsp90 inhibitor that blocks rip3-dependent necroptosis discovery and identification of cdc37-derived peptides targeting the hsp90-cdc37 protein-protein interaction targeting the hsp90-cdc37-client protein interaction to disrupt hsp90 chaperone machinery activity of suberoylanilide hydroxamic acid against human breast cancer cells with amplification of her-2 chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor laq824 modulation of p53, erbb1, erbb2, and raf-1 expression in lung cancer cells by depsipeptide fr901228 caspase-10-mediated heat shock protein 90β cleavage promotes uvb irradiation-induced cell apoptosis hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death suberoylanilide hydroxamic acid induces ros-mediated cleavage of hsp90 in leukemia cells proteasome inhibitor-induced cleavage of hsp90 is mediated by ros generation and caspase 10-activation in human leukemic cells dendritic cells transfected with heat-shock protein 70 messenger rna for patients with hepatitis c virus-related hepatocellular carcinoma: a phase 1 dose escalation clinical trial hsp60 chaperonopathies and chaperonotherapy: targets and agents hsp60 as a drug target studies on bredinin. i. isolation,characterization and biological properties hiroyuki epolactaene, a novel neuritogenic compound in human neuroblastoma cells, produced by a marine fungus structure-activity relationships of epolactaene derivatives: structural requirements for inhibition of hsp60 chaperone activity epolactaene binds human hsp60 cys442 resulting in the inhibition of chaperone activity crystal structure of the human mitochondrial chaperonin symmetrical football complex identification of hsp60 as a primary target of o-carboranylphenoxyacetanilide, an hif-1α inhibitor mitochondrial chaperonin hsp60 is the apoptosis-related target for myrtucommulone isolation and antibacterial activity of acylphloroglucinols from myrtus communis oligomeric acylphloroglucinols from myrtle (myrtus communis) antioxidant activity of oligomeric acylphloroglucinols from myrtus communis l myrtucommulone from myrtus communis, exhibits potent antiinflammatory effectiveness in vivo identification of molecular targets of the oligomeric nonprenylated acylphloroglucinols from and myrtle (myrtus communis) and their implication as anti-inflammatory compounds myrtucommulone from myrtus communis induces apoptosis in cancer cells via the mitochondrial pathway involving caspase-9 dual induction of mitochondrial apoptosis and senescence in chronic myelogenous leukemia by myrtucommulone a stephacidin a and b: two structurally novel, selective inhibitors of the testosterone-dependent prostate lncap cells avrainvillamide, a cytotoxic marine natural product, and derivatives thereof evidence for the rapid conversion of stephacidin b into the electrophilic monomer avrainvillamide in cell culture boron-containing phenoxyacetanilide derivatives as hypoxiainducible factor (hif)-1α inhibitors anticancer gold(iii) porphyrins target mitochondrial chaperone hsp60 molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets chemical proteomics identifies heterogeneous nuclear ribonucleoprotein (hnrnp) a1 as the molecular target of quercetin in its anti-cancer effects in pc-3 cells quercetin targets hnrnpa1 to overcome enzalutamide resistance in prostate cancer cells quercetin as an antiviral agent inhibits influenza a virus (iav) entry. viruses inhibition of enterovirus 71 replication and viral 3c protease by quercetin antiviral activity of baicalein and quercetin against the japanese encephalitis virus computer-aided discovery of small molecules targeting the rna splicing activity of hnrnp a1 in castration-resistant prostate cancer a selective inhibitor of eif2alpha dephosphorylation protects cells from er stress dengue virus serotype infection specifies the activation of the unfolded protein response activation of the unfolded protein response by 2-deoxy-dglucose inhibits kaposi's sarcoma-associated herpesvirus replication and gene expression antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response rana catesbeiana ribonuclease inhibits japanese encephalitis virus (jev) replication and enhances apoptosis of jev-infected bhk-21 cells broad-spectrum antiviral therapeutics vaticanol b, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress the hepatitis c virus (hcv) ns4b rna binding inhibitor clemizole is highly synergistic with hcv protease inhibitors spontaneous and engineered compensatory hsv mutants that counteract the host antiviral pkr response open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source [81671995]; the general research grant of hong kong [11100215] ; and strategic funds from city university of hong kong. competing interests: the authors declare no competing interests.commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-305085-bv7udg9k authors: lawrence, robert m. title: chapter 13 transmission of infectious diseases through breast milk and breastfeeding date: 2011-12-31 journal: breastfeeding doi: 10.1016/b978-1-4377-0788-5.10013-6 sha: doc_id: 305085 cord_uid: bv7udg9k nan a large body of evidence clearly demonstrates the protective effects of breastfeeding and documents the transmission of specific infections to infants through breast milk. the fear and anxiety that arise with the occurrence of any infectious disease are even greater in the situation of the breastfeeding mother-infant dyad. uncertainty and lack of knowledge often lead to proscribing against breastfeeding out of fear, which then deprives the infant of the potential protective, nutritional, and emotional benefits of breastfeeding exactly at the time when they are most needed (see the discussion of immunologic benefits of human milk in chapter 5). decisions concerning breastfeeding in a mother with an infectious illness should balance the potential benefits of breastfeeding versus the known or estimated risk for the infant acquiring a clinically significant infection via breastfeeding and the potential severity of the infection. documenting transmission of infection from mother to infant by breastfeeding requires not only the exclusion of other possible mechanisms of transmission but also the demonstration of the infectious agent in the breast milk and a subsequent clinically significant infection in an infant that was caused by a plausible infectious process. the first step is to establish the occurrence of a specific infection (clinically or immunologically evident) in a mother and demonstrate the persistence of the infectious agent such that it could be transmitted to the infant. isolation or identification of the infectious agent from the colostrum, breast milk, or an infectious lesion of the breast is important but not necessarily proof of transmission to an infant. epidemiologic evidence of transmission must be considered, including identifying characteristics of the organism that relate an isolate from an infant to the maternal isolate. infectious organisms can reach the breast milk either by secretion in the fluid or cellular components of breast milk or by contamination of the milk at the time of or after expression. a reasonable mechanism of infection via breast milk should be evident and proved through either animal or human studies. demonstration of a subclinical or clinically evident infection in an infant should follow these outlined steps. exclusion of other possible mechanisms of transmission (exposure to mother or other persons/ animals via airborne, droplet, arthropod, or vector modes of transmission or through direct contact with other infectious fluids) would complete the confirmation of transmission of infection via breastfeeding. it is essential to exclude prenatal or perinatal transmission of infection to a fetus/infant, but doing this can often be difficult. clinical case reports or studies confirming the isolation of an infectious agent from the milk are important. to determine a reasonable estimate of the risk for infection via breast milk, larger epidemiologic studies are needed that compare infection rates in breastfed infants versus formula-fed infants, robert m. lawrence addressing the issues just identified. timing of breastfeeding is important relative to the timing of maternal infection and to the presence of a pathogen in colostrum or breast milk. the duration of breastfeeding is another important variable to consider in the estimate of risk because shedding of a pathogen in breast milk may be intermittent. these considerations are only some of the variables to be taken into account, in general, to assess the risk for transmission of an infectious agent from mother to infant via breast milk or breastfeeding. efforts to prove transmission of infection in a particular maternal-infant dyad can be just as difficult and must consider many of the same factors. this chapter focuses on a discussion of specific, clinically relevant, infectious agents and diseases, with reasonable estimates of the risk for infection to infants from breastfeeding. the basic tenet concerning breastfeeding and infection is that breastfeeding is rarely contraindicated in maternal infection. 243 the few exceptions relate to specific infectious agents with strong evidence of transmission and to the association of an infant' s illness with significant morbidity and mortality. the risk or benefit of breastfeeding relative to immunization of a mother or infant is discussed for certain microorganisms. appendix d addresses drugs in breast milk and includes table d-1, on antiinfective agents, and chapter 5 reviews how breastfeeding may protect against infection. chapter 21 addresses specific concerns relating to banked breast milk and includes standards developed by the human milk banking association of north america to guide the appropriate handling of banked human milk relative to possible infectious agents. isolation precautions have undergone some revisions in terminology and conceptualization. 143 understanding that the transmission of microorganisms can occur with a known infection and with unrecognized sources of infection, recommendations have been made for standard precautions to be applied to all patients to protect health care workers from potentially infectious body fluids. additionally, precautions based on the predominant modes of transmission have been recommended to protect against infection through the airborne route, direct contact, or contact with droplets. although these precautions are intended to be used in clinical situations to protect health care workers, they may be applied in certain situations to the mother-infant dyad to prevent transmission of infectious agents from one to the other or to other hospitalized mothers and infants. these precautions are useful most often when a mother and infant are still hospitalized. the use of such precautions within the home is not meant to limit breastfeeding. they are intended to allow breastfeeding in the majority of cases and to facilitate the continuation of breastfeeding with some additional safeguards in certain situations, after short temporary periods of stopping breastfeeding, and when to safely use expressed breast milk (see appendix f). standard precautions include preventing contact with blood, all body fluids, secretions and excretions, nonintact skin, and mucous membranes by (1) careful handwashing before and after every patient contact; (2) use of gloves when touching body fluids, nonintact skin, or mucous membranes or any items contaminated with body fluids (linens, equipment, devices, etc.); (3) use of nonsterile gowns to prevent contact of clothing with body fluids; (4) use of masks, eye protection, or face shields when splashing with body fluids is possible; and (5) appropriate disposal of these materials. standard precautions should be applied to all patients regardless of actual or perceived risks. the centers for disease control and prevention (cdc) does not consider breast milk a body fluid with infectious risks and thus these policies do not apply to breast milk. (see section on misadministration of breast milk later in this chapter as a possible exception to this concept.) in considering breastfeeding infant-mother dyads and standard precautions, body fluids other than breast milk should be avoided, and only in specified situations should breast milk also be avoided. in general, clothing or a gown for the mother and bandages, if necessary, should prevent direct contact with nonintact skin or secretions. avoiding infant contact with maternal mucous membranes requires mothers to be aware of and understand the risks and to make a conscious effort to avoid this type of contact. the use of gloves, gowns, and masks on infants for protection is neither practical nor appropriate. the recommendations concerning the appropriateness of breastfeeding and breast milk are addressed for specific infectious agents throughout this chapter. human immunodeficiency virus (hiv) infection is an example of one infection that can be prevented by the use of standard precautions, including avoiding breast milk and breastfeeding. the recommendations concerning breastfeeding and hiv and the various variables and considerations involved are discussed later. airborne precautions are intended to prevent transmission via droplet nuclei (dried respiratory particles smaller than 5 mcm that contain microorganisms and can remain suspended in the air for long periods) or dust particles containing microorganisms. airborne precautions include the use of a private room with negative-air-pressure ventilation and masks at all times. in the case of pulmonary tuberculosis (tb), respiratory protective devices (requiring personal fitting and seal testing before use) should be worn. airborne precautions are recommended with measles, varicella or disseminated zoster, and tb. breastfeeding in the presence of these maternal infections is prohibited for the infectious period. this is to protect against airborne transmission of the infection from the mother and to allow the infant to be fed the mother' s expressed breast milk by another individual. the exception to allowing breast milk would be local involvement of the breast by varicella-zoster lesions or mycobacterium tuberculosis, such that the milk becomes contaminated by the infectious agent. transmission via droplets occurs when an individual produces droplets that travel only a short distance in the air and then contact a new host's eyes, nose, mouth, or skin. the common mechanisms for producing droplets include coughing, sneezing, talking (singing or yelling), suctioning, intubation, nasogastric tube placement, and bronchoscopy. in addition to standard precautions applied to all patients, droplet precautions include the use of a private room (preferred) and a mask if within 3 feet (0.9 m) of the patient. droplet precautions are recommended for adenovirus, diphtheria, respiratory infections, haemophilus influenzae, neisseria meningitidis or invasive infection, influenza, mumps, mycoplasma, parvovirus, pertussis, plague (pneumonic), rubella, and streptococcal pharyngitis, pneumonia, or scarlet fever. the institution of droplet precautions with a breastfeeding mother who has these infections should be specified for each particular infection. this may require some period of separation for the infant and mother (for duration of the illness, for short-term or complete treatment of the mother, for the infectious period) with use of expressed breast milk for nutrition in the interim. prophylactic treatment of the infant, maternal use of a mask during breastfeeding or close contact combined with meticulous handwashing, and the mother's avoidance of touching her mucous membranes may be adequate and reasonable for certain infections. contact precautions are meant to prevent transmission of infection via direct contact (contact between the body surfaces of one individual with another) and indirect contact (contact of a susceptible host with an object contaminated with microorganisms from another individual). contact precautions include cohorting or a private room, gloves and gowns at all times, and handwashing after removal of gown and gloves. contact precautions are recommended for a long list of infections, such as diarrhea in diapered or incontinent patients with clostridium difficile infection, escherichia coli o157:h7, shigella, rotavirus, hepatitis a, respiratory illness with parainfluenza virus or respiratory syncytial virus (rsv), multidrug-resistant (mdr) bacteria (e.g., enterococci, staphylococci, gramnegative organisms), enteroviral infections, cutaneous diphtheria, impetigo, herpes simplex virus (hsv) infection, herpes zoster (disseminated or in immunocompromised individuals), pediculosis, scabies, staphylococcus aureus skin infection, viral hemorrhagic fevers (e.g., ebola, lassa), conjunctivitis and abscesses, cellulitis, or decubitus that cannot be contained by dressings. 94 for a breastfeeding infant-mother dyad, implementation of precautions for each of these infections in a mother requires meticulous attention to gowning and handwashing by the mother and a specialized plan for each situation. each of these transmission-based precautions can be used together for organisms or illnesses that can be transmitted by more than one route. they should always be used in conjunction with standard precautions, which are recommended for all patients. the red book: report of the committee on infectious diseases by the american academy of pediatrics (aap) 96 remains an excellent resource for infection control guidelines and recommendations to prevent transmission in specific situations and infections. routine culturing of breast milk or culturing breast milk to screen for infectious agents is not recommended except when the milk is intended as donor milk to another mother' s child directly or through human milk banks. see chapter 21 for specific bacterial count standards for raw donor milk and for pasteurization of donor milk. breastfeeding and the expression of or pumping of breast milk (referred to as expressed breast milk) for later use are not sterile activities. in general expressed breast milk should not contain large numbers of microorganisms (less than 10 4 for raw milk and less than 10 6 for milk to be pasteurized), nor should it contain potential pathogens such as s. aureus, β-hemolytic streptococci, pseudomonas species, proteus species, or streptococcus faecalis or faecium. few studies have examined "routine" culturing of milk and the significance of specific bacterial colony counts relative to illness in infants. the studies have been primarily concerned with premature or low-birth-weight (lbw) infants who remain hospitalized and are commonly fed via enteral tubes. a study from canada tested 7610 samples of milk for use in 98 preterm infants. 242 the study did not identify any adverse events in the infants attributed to organisms growing in the milk samples, and routine bacteriological testing of expressed breast milk was not recommended. a study from chicago examined gram-negative bacilli in the milk used in premature infants. 48 samples were tested before feeding and from the nasogastric tubes during feeding. milk samples from before feeding were less likely to contain gram-negative bacilli (36%) than milk samples from the nasogastric tubing (60%). feeding intolerance was observed when there were more than 10 3 colony-forming units per milliliter (cfu/ml), and episodes of sepsis were identified when the bacterial counts in the milk were greater than or equal to 10 6 cfu/ml. this study recommended the routine bacteriologic testing of expressed breast milk. another study from arkansas focused on contamination of feeding tubes during administration of expressed breast milk or formula. 277 ten infants in the neonatal intensive care unit (nicu) were exposed to greater than 10 5 gram-negative bacteria in their feeding tubes. the three infants who were fed expressed breast milk with contamination at greater than 10 5 organisms remained well, but the seven formula-fed infants with high levels of bacterial contamination in the feeding tubes developed necrotizing enterocolitis. the gram-negative bacteria with high level contamination in the feeding tubes were either enterobacter or klebsiella in all cases. many nicus consider 10 5 to 10 6 cfu/ml as the significant bacterial count for gram-negative bacilli in breast milk that places premature and lbw infants at greater risk for infection. even less data are available concerning specific bacterial colony counts for gram-positive organisms and the risk to the infant. generally less than 10 3 gram-positive organisms per ml of milk is considered acceptable, with only case reports and no controlled trials to support this cutoff. when the presence of an infectious illness in an infant and/or the breastfeeding mother' s breast when breast milk is seriously considered as a possible mechanism of transmission to the infant, culturing breast milk to identify the organism may be warranted and useful. more important than hurrying to culture breast milk is the careful instruction of mothers on the proper technique for collecting expressed breast milk, storing it, and cleaning the collection unit. the reinforcement of proper technique from time to time, especially when a question of contamination arises, is equally important. many small reports comment on the contamination of breast milk with different collection methods. relative comparisons suggest decreasing contamination of expressed breast milk when collected by the following methods; drip milk, hand pumped milk, manual expression, modern electric pumped milk. one group from malaysia published results showing no difference in contamination between milk collected by electric pump versus manual expression when collected in the hospital. expressed breast milk collected at home by breast pump had higher rates of contamination with staphylococci and gram-negative bacteria. 46 discussion continues about the need to discard the first few milliliters of milk to lower bacteria numbers in expressed breast milk without any evidence to suggest if this is truly necessary. 62, 337 no evidence shows that cleansing the breast with anything other than tap water decreases the bacterial counts in cultured expressed breast milk. 414 if an infant is directly breastfeeding, collecting milk for culture by manual expression and trying to obtain a "midstream" sample (as is done with "midstream" urine collection for culture) is appropriate. if an infant is being fed expressed breast milk, collecting and culturing the milk at different points during collection (utilizing the same technique the mother uses [manual expression, hand pump, or electric pump]) and administration is appropriate. this might include a sample from immediately after collection, another of stored expressed breast milk, and a sample of milk from the most recent infant feeding at the time the decision to culture is made. please see box 13-1 for the basic steps in culturing expressed breast milk. interpretation of such culture results can be difficult and should involve a pediatric infectious disease expert, a microbiologist, and hospital epidemiologist. additional organism identification is often required, utilizing antibiogram patterns or molecular fingerprinting by various techniques to correlate a bacterial isolate from breast milk with an isolate causing disease in infant or mother. misadministration of breast milk, also known as misappropriation, breast milk exposure, and accidental ingestion of breast milk, and other terms, is a medical-legal issue when it occurs in a hospital. this scenario occurs when one infant receives breast milk from another mother by mistake. this occurrence can be very distressing to the families (recipient patient, recipient parent, and donor mother) and medical staff involved. the actual risk for transmission of an infectious agent to an infant via a single ingestion of expressed breast milk (the most common occurrence) from another mother is exceedingly low. in this scenario, the cdc recommends treating this as an accidental exposure to a body fluid, which could be infectious. 84 bacterial, fungal, or parasitic infection from the one exposure is highly unlikely. the concern is about viral pathogens, known to be blood-borne pathogens, which have been identified in breast milk and include but are not limited to hepatitis b virus (hbv), hepatitis c virus (hcv), cytomegalovirus (cmv), west nile virus, human t-cell lymphotropic virus (htlv), and hiv. most hospitals have protocols for managing the situation from both the infection control/prevention and the medical-legal perspectives. these protocols advise informing both families about what occurred, discussing the theoretical risks of harm from the exposure, and reviewing test results and/ or recommending testing to determine the infectious status of each mother relative to the above mentioned viruses. hcv is not a contraindication to breastfeeding and west nile virus infection in lactating women is rare. 74, 177 neither infection has a documented effective form of prevention or acute treatment. testing either mother (donor or of recipient infant) for these agents is not warranted. prenatal testing for hiv is more commonplace throughout the world. the incidence of hiv among women of childbearing age is low, although it varies significantly by geographic location, and the hospital or locale-specific incidence would be important to know to estimate risk. most women and medical staff are aware that hiv can be transmitted by breastfeeding; therefore breast milk from hiv-positive women is rarely if ever stored in hospitals. the risk for transmission of hiv via breastfeeding is due to the volume of feedings over months (estimated at 400 to 500 feedings in the first 2 months of life) compared with the small "dose of exposure" from one or two "accidental feedings." transmission of hiv from a single breast milk exposure has never been documented. immunologic components in breast milk, along with time and cold of storage, inactivate the hiv in expressed breast milk. for these reasons, the risk for transmission of hiv via expressed breast milk consumed by another child is thought to be extremely low. htlv-i/ii infection in childbearing women is uncommon except in certain geographic regions (japan, africa, the caribbean, and south america). transmission of htlv via breast milk does occur and, like hiv, appears to be related to the volume and duration of breastfeeding. limiting the duration of breastfeeding is effective in decreasing transmission. 407, 409, 446 freezing and thawing expressed breast milk decreases the infectivity of htlv-i. 11 in areas of low prevalence, a positive test in a mother should be suspected to be a false positive test, and retesting with both antibody and polymerase chain reaction (pcr) testing should be performed. for these reasons the transmission of htlv-i/ii via accidental expressed breast milk exposure is thought to be extremely low. although the majority of women are cmv positive by childbearing age and cmv transmission occurs via breastfeeding, the risk for cmv in a full-term infant is low. premature or lbw infants are at greater risk for developing disease with cmv infection. freezing expressed breast milk (at −20° c) for 3 to 5 days significantly decreases the infectivity of cmv. here again the risk for cmv transmission from a single accidental exposure to cmv-positive expressed breast milk is extremely low. with a discussion of theoretical risk should be a discussion of possible preventive interventions, such as vaccination or antimicrobial postexposure prophylaxis. if donor mothers are positive for hbv, it is appropriate to give recipient infants hepatitis b virus immunoglobulin (hbig) and hbv vaccines if they have not already received them. if a box 13-1. culturing breast milk 1. wash hands as per routine. 2. wash breast with warm tap water and a clean washcloth. 3. manually express breast milk ("midstream" collection is not required) or attach breast pump flange (previously cleaned as per routine) for collection and collect milk. 4. place a 3 to 5 ml sample of expressed breast milk in a sterile container with a nonleakable top. 5. deliver to the labatory in less than 1 hour or refrigerate at 4° c until delivery. before sending samples to the viral lab or for nucleic acid/ po lymerase chain reaction (pcr) testing, confirm that the laboratory will accept and process the sample as requested and that the appropriate collection container and prelaboratory management of the specimen are utilized. 6 324 it may also be appropriate to consult a pediatric infectious disease specialist. additional important components of the hospital-based protocols for managing accidental expressed breast milk exposure include ongoing psychosocial support for the families and staff, documentation of medical discussions with the families, investigative steps, consents and interventions, and the demonstration of ongoing infection control efforts to prevent additional events of misadministration of breast milk. microorganisms produce a whole spectrum of clinical illnesses affecting mothers and infants. many situations carry the risk for transmission of the involved organism from a mother to the infant, or vice versa; in general, however, infants are at greater risk because of such factors as inoculum size and immature immune response. as always, an infection must be accurately diagnosed in a timely manner. empiric therapy and initial infection control precautions should begin promptly based on the clinical symptoms and the most likely etiologic agents. when dealing with a maternal infection, clarifying the possible modes of transmission and estimating the relative risk for transmission to the infant are essential first steps to decision-making about isolating a mother from her infant and the appropriateness of continuing breastfeeding or providing expressed breast milk. breastfeeding infrequently is contraindicated in specific maternal infections. 243 often the question of isolation and interruption of breastfeeding arises when symptoms of fever, pain, inflammation, or other manifestations of illness first develop in a mother and the diagnosis is still in doubt. a clinical judgment must be made based on the site of infection, probable organisms involved, possible or actual mechanisms of transmission of these organisms to the infant, estimated virulence of the organism, and likely susceptibility of the infant. additionally, by the time the illness is clearly recognized or diagnosed in a mother, the infant has already been exposed. given the dynamic nature of the immunologic benefits of breast milk, continuation of breastfeeding at the time of diagnosis or illness in a mother can provide the infant protection rather than continued exposure in most illnesses. stopping breastfeeding is rarely necessary. many situations associated with maternal fever do not require separation of mother and infant, such as engorgement of the breasts, atelectasis, localized nonsuppurative phlebitis, or urinary tract infections. appendix f lists a number of clinical syndromes, conditions, and organisms that require infection control precautions in hospitals. this appendix also includes short lists of possible etiologic agents for these conditions and appropriate precautions and recommendations concerning breastfeeding for different scenarios or organisms. this chapter considers specific infectious agents that are common, clinically significant, or of particular interest. bacillus anthracis, a gram-positive, spore-forming rod, causes zoonotic disease worldwide. human infection typically occurs due to contact with animals or their products. three forms of human disease occur: cutaneous anthrax (the most common), inhalation anthrax, and gastrointestinal (gi) disease (rare). person-to-person transmission can occur as a result of discharge from cutaneous lesions, but no evidence of human-to-human transmission of inhalational anthrax is available. no evidence of transmission of anthrax via breast milk exists. standard contact isolation is appropriate for hospitalized patients or patients with draining skin lesions. the issue of anthrax as a biologic weapon has exaggerated its importance as a cause of human disease. the primary concerns regarding anthrax and breastfeeding are antimicrobial therapy or prophylaxis in breastfeeding mothers and the possibility that infant and mother were exposed by intentional aerosolization of anthrax spores. the cdc published recommendations for treatment and prophylaxis in infants, children, and breastfeeding mothers. 72 the recommendations include the use of ciprofloxacin, doxycycline, amoxicillin, and several other agents without discontinuing breastfeeding. little available is information on ciprofloxacin and doxycycline in breast milk for prolonged periods of therapy or prophylaxis (60 days) and possible effects on infants' teeth and bone/cartilage growth during that time period. depending on the clinical situation and sensitivity testing of the identified anthrax strain, other agents can be substituted to complete the 60-day course. the cdc has approved the use of ciprofloxacin and doxycycline for breastfeeding women for short courses of therapy (less than several weeks). simultaneous exposure of infant and mother could occur from primary aerosolization or from spores "contaminating" the local environment. in either case decontamination of the mother-infant dyad' s environment should be considered. breastfeeding can continue during a mother' s therapy for anthrax as long as she is physically well. open cutaneous lesions should be carefully covered and, depending on the situation, simultaneous prophylaxis for the infant may be appropriate. considerable justifiable concern has been expressed because of the reports of sudden infant death from botulism. infant botulism is distinguished from food-borne botulism from improperly preserved food containing the toxin and from wound botulism from spores entering the wound. infant botulism occurs when the spores of clostridium botulinum germinate and multiply in the gut and produce the botulinal toxin in the gi tract. 17 the toxin binds presynaptically at the neuromuscular junction, preventing acetylcholine release. the clinical picture is a descending, symmetric flaccid paralysis. not every individual who has c. botulinum identified in the stool experiences a clinical illness. the age of infants seems to relate to their susceptibility to illness. the illness is mainly in children younger than 12 months of age; the youngest patient described in the literature was 6 days old. 17 most children become ill between 6 weeks and 6 months of age. the onset of illness seems to occur earlier in formula-fed infants compared with breastfed infants. when a previously healthy infant younger than 6 months of age develops constipation, then weakness and difficulty sucking, swallowing, crying, or breathing, botulism is a likely diagnosis. the organisms should be looked for in the stools, and electromyography may or may not be helpful. in a group reviewed by arnon et al, 19 33 of 50 patients hospitalized in california were still being nursed at onset of the illness. a beneficial effect of human milk was observed in the difference in the mean age at onset, with breastfed infants being twice as old as formula-fed infants with the disease. the breastfed infants' symptoms were milder. breastfed infants receiving iron supplements developed the disease earlier than those who were breastfed but unsupplemented. of the cases of sudden infant death from botulism, no infants were breastfed within 10 weeks of death. all were receiving iron-fortified formulas. in most cases, no specific food source of c. botulinum can be identified, but honey is the food most often implicated, and corn syrup has been implicated in infants older than 2 months of age. honey may contain botulism spores, which can germinate in the infant gut. however, botulin toxin has not been identified in honey. it has been recommended that honey not be given to infants younger than 12 months of age. this includes putting honey on a mother' s nipples to initiate an infant' s interest in suckling. arnon 18 reviewed the first 10 years of infant botulism monitoring worldwide. the disease has been reported from 41 of the 50 states in the united states and from eight countries on four continents. the relationship to breastfeeding and human milk is unclear. in general the acid stools (ph 5.1 to 5.4) of human milk fed infants encourage bifidobacterium species. few facultative anaerobic bacteria, or clostridia, existing as spores, are present in breastfed infants. in contrast, formula-fed infants have stool phs ranging from 5.9 to 8.0, with few bifidobacteria, primarily gram-negative bacteria, especially coliforms and bacteroides species. c. botulinum growth and toxin production decrease with declining ph and usually stops below ph 4.6. breast milk also contains additional protective immunologic components, which purportedly have activity against botulinum toxin. 269 the relationship between the introduction of solid foods or weaning in both formula-fed and breastfed infants and the onset of botulism remains unclear. for a breastfed infant, the introduction of solid food may cause a major change in the gut with a rapid rise in the growth of enterobacteria and enterococci followed by progressive colonization by bacteroides species, clostridia, and anaerobic streptococci. feeding solids to formula-fed infants minimally changes the gut flora as these organisms already predominate. although more hospitalized infants have been breastfed, sudden-death victims are younger and have been formula fed, which supports the concept of immunologic protection in the gut of a breastfed infant. much work remains to understand this disease. clinically, constipation, weakness, and hypotonicity in a previously healthy child constitute botulism until ruled out, especially with recent dietary changes. at this time, no reason exists to suspect breastfeeding as a risk for infant botulism, and some evidence suggests a possible protective effect from breastfeeding. breastfeeding should continue if botulism is suspected in mother or infant. brucella melitensis has been isolated in the milk of animals. foods and animals represent the primary sources of infection in humans. brucellosis demonstrates a broad spectrum of illness in humans, from subclinical to subacute to chronic illness with nonspecific signs of weakness, fever, malaise, body aches, fatigue, sweats, arthralgia, and lymphadenitis. in areas where the disease is enzootic, childhood illness has been described more frequently. the clinical manifestations in children are similar to those in adults. 259 infection can occur during pregnancy, leading to abortion (infrequently), and can produce transplacental spread, causing neonatal infection (rarely). the transmission of b. melitensis through breast milk has been implicated in neonatal infection. 259, 260 there have been eight cases of brucellosis in infants that were possibly associated with breastfeeding, but brucella was not isolated from the breast milk in any of those cases.* one case of brucellosis in an infant caused by breast milk transmission, with b. melitensis isolated from the breast milk, before antibiotic treatment was given to the mother has been documented. 415 additionally, brucella melitensis has been cultured from women with breast lumps and abscesses. 295 only one of six women described in this report was lactating at the time of diagnosis, and no information about the infant was given. brucellosis mastitis or abscess should be considered in women presenting with appropriate symptoms and occupational exposure to animals, contact with domestic animals in their environment, or exposure to animal milk or milk products (especially unpasteurized products). the breast inflammation tends to be granulomatous in nature (without caseation) and is often associated with axillary adenopathy; occasionally systemic illness in the woman is evident. treatment of brucellosis mastitis or abscess should be treated with surgery or fine needle aspiration as indicated and 4 to 6 weeks of combination antibiotic therapy with two or three medications. temporary interruption of breastfeeding with breast pumping and discarding the milk to continue stimulation of milk production is appropriate. breastfeeding should then continue after an initial period of 48 to 96 hours of therapy in the mother. acceptable medications for treating the mother while continuing breastfeeding include gentamicin, streptomycin, tetracycline, doxycycline, trimethoprim-sulfamethoxazole, and rifampin (see appendix d). chlamydial infection is the most frequent sexually transmitted disease (std) in the united states and is a frequent cause of conjunctivitis and pneumonitis in an infant from perinatal infection. the major determinant of whether chlamydial infection occurs in a newborn is the prevalence rate of chlamydial infection of the cervix. 364 chlamydial immunoglobulin a (iga) has been found in colostrum and breast milk in a small number of postpartum women who were seropositive for chlamydia. no information is available on the role of milk antibodies in protection against infection in infants. 389 it is not believed that chlamydia is transmitted via breast milk. use of erythromycin or tetracycline to treat mothers and oral erythromycin and ophthalmic preparations of tetracyclines, erythromycin, or sulfonamides to treat suspected infection in infants are appropriate during continued breastfeeding. separating infants from mothers with chlamydial infections or stopping breastfeeding is not indicated. simultaneous treatment of mothers and infants may be appropriate in some situations. corynebacterium diphtheriae causes several forms of clinical disease, including membranous nasopharyngitis, obstructive laryngotracheitis, and cutaneous infection. complications can include airway obstruction from membrane formation and toxinmediated central nervous system (cns) disease or myocarditis. the overall incidence of diphtheria has declined even though immunization does not prevent infection but does prevent severe disease from toxin production. fewer than five cases are reported annually in the united states. transmission occurs via droplets or direct contact with contaminated secretions from the nose, throat, eye, or skin. infection occurs in individuals whether they have been immunized or not, but infection in those not immunized is more severe and prolonged. as long as the skin of the breast is not involved, no risk for transmission exists via breast milk. no toxin-mediated disease from toxin transmitted through breast milk has been reported in an infant. breastfeeding, along with chemoprophylaxis and immunization of affected infants, is appropriate in the absence of cutaneous breast involvement (see appendix f). maternal infection with neisseria gonorrhoeae can produce a large spectrum of illness ranging from uncomplicated vulvovaginitis, proctitis, pharyngitis, conjunctivitis, or more severe and invasive disease, including pelvic inflammatory disease, meningitis, endocarditis, or disseminated gonococcal infection. the risk for transmission from mother to infant occurs mainly during delivery in the passage through the infected birth canal and occasionally from postpartum contact with the mother (or her partner). risk for transmission from breast milk is negligible, and n. gonorrhoeae does not seem to cause local infection of the breasts. infection in neonates is most often ophthalmia neonatorum and less often a scalp abscess or disseminated infection. mothers with presumed or documented gonorrhea should be reevaluated for other stds, especially chlamydia trachomatis and syphilis, because some therapies for gonorrhea are not adequate for either of these infections. with the definitive identification of gonorrhea in a mother, empiric therapy should begin immediately, and the mother should be separated from the infant until completion of 24 hours of adequate therapy. treatment of the mother with ceftriaxone, cefixime, penicillin, or erythromycin is without significant risk to the infant. single-dose treatment with spectinomycin, ciprofloxacin, ofloxacin, or azithromycin has not been adequately studied but presumably would be safe for the infant given the 24-hour separation and a delay in breastfeeding without giving the infant the expressed breast milk (pump and discard). doxycycline use in a nursing mother is not routinely recommended. careful preventive therapy for ophthalmia neonatorum should be provided, and close observation of the infant should continue for 2 to 7 days, the usual incubation period. empiric or definitive therapy against n. gonorrhoeae may be necessary depending on an infant' s clinical status and should be chosen on the basis of the maternal isolate' s sensitivity pattern. the mother should not handle other infants until after 24 hours of adequate therapy, and the infant should be separated from the rest of the nursery population, with or without breastfeeding. haemophilus influenzae type b can cause severe invasive disease such as meningitis, sinusitis, pneumonia, epiglottitis, septic arthritis, pericarditis, and bacteremia. shock can also occur. because the increased utilization of the h. influenzae type b conjugate vaccines, invasive disease caused by haemophilus has decreased dramatically, more than 95%, in the united states. most invasive disease occurs in children 3 months to 3 years of age. older children and adults rarely experience severe disease but do serve as sources of infection for young children. children younger than 3 months of age seem to be protected because of passively acquired antibodies from the mothers, and some additional benefits may be received from breast milk. transmission occurs through contact with respiratory secretions, and droplet precautions are protective. no evidence suggests transmission through breast milk or breastfeeding. evidence supports that breast milk limits the colonization of h. influenzae in the throat. 185 in the rare case of maternal infection, an inadequately immunized infant in a household is an indication to provide rifampin prophylaxis and close observation for all household contacts, including the breastfeeding infant. expressed breast milk can be given to an infant during the 24-hour separation after the mother' s initiation of antimicrobial therapy, or if the mother' s illness prevents breastfeeding, it can be reinitiated when the mother is able (see appendix f). although uncommon in the united states, leprosy occurs throughout the world. this chronic disease presents with a spectrum of symptoms depending on the tissues involved (typically the skin, peripheral nerves, and mucous membranes of the upper respiratory tract) and the cellular immune response to the causative organism, mycobacterium leprae. transmission occurs through long-term contact with individuals with untreated or multibacillary (large numbers of organisms in the tissues) disease. leprosy is not a contraindication to breastfeeding, according to jeliffe and jeliffe. 202 the importance of breastfeeding and urgency of treatment are recognized by experts who treat infants and mothers early and simultaneously. no mother-infant contact is permitted except to breastfeed. dapsone, rifampin, and clofazimine are typically and safely used for infant and mother regardless of the method of feeding (see appendix d). listeriosis is a relatively uncommon infection that can have a broad range of manifestations. in immunocompetent individuals, including pregnant women, the infection can vary from being asymptomatic to presenting as an influenza-like illness, occasionally with gi symptoms or back pain. severe disease occurs more frequently in immunodeficient individuals or infants infected in the perinatal period (pneumonia, sepsis, meningitis, granulomatosis infantisepticum). although listeriosis during pregnancy may manifest as mild disease in a mother and is often difficult to recognize and diagnose, it is typically associated with stillbirth, abortion, and premature delivery. it is thought that transmission occurs through the transplacental hematogenous route, infecting the amniotic fluid, although ascending infection from the genital tract may occur. 122 early and effective treatment of a woman can prevent fetal infection and sequelae. 206, 257 neonatal infection occurs as either early-or late-onset infection from transplacental spread late in pregnancy, ascending infection during labor and delivery, infection during passage through the birth canal, or, rarely, during postnatal exposure. no evidence in the literature suggests that listeria is transmitted through breast milk. treatment of the mother with ampicillin, penicillin, or trimethoprim-sulfamethoxazole is not a contraindication to breastfeeding as long as the mother is well enough. expressed colostrum or breast milk also can be given if the infant is able to feed orally. the management of lactation and feeding in neonatal listeriosis is conducted supportively, as it is in any situation in which an infant is extremely ill, beginning feeding with expressed breast milk or directly breastfeeding as soon as reasonable. n. meningitidis most often causes severe invasive infections, including meningococcemia or meningitis often associated with fever and a rash and progressing to purpura, disseminated intravascular coagulation, shock, coma, and death. transmission occurs via respiratory droplets. spread can occur from an infected, ill individual or from an asymptomatic carrier. droplet precautions are recommended until 24 hours after initiation of effective therapy. despite the frequent occurrence of bacteremia, no evidence indicates breast involvement or transmission through breast milk. the risk for maternal infection to an infant after birth is from droplet exposure and exists whether the infant is breastfeeding or bottle feeding. in either case the exposed infant should receive chemoprophylaxis with rifampin, 10 mg/kg/dose every 12 hours for 2 days (5 mg/kg/dose for infants younger than 1 month of age), or ceftriaxone, 125 mg intramuscularly (im) once, for children younger than 15 years of age. close observation of the infant should continue for 7 days, and breastfeeding during and after prophylaxis is appropriate. the severity of maternal illness may prevent breastfeeding, but it can continue if the mother is able, after the mother and infant have been receiving antibiotics for 24 hours. a period of separation from the index case for the first 24 hours of effective therapy is recommended; expressed breast milk can be given during this period. respiratory illness caused by bordetella pertussis evolves in three stages: catarrhal (nasal discharge, congestion, increasing cough), paroxysmal (severe paroxysms of cough sometimes ending in an inspiratory whoop, i.e., whooping cough), and convalescent (gradual improvement in symptoms). transmission is via respiratory droplets. the greatest risk for transmission occurs in the catarrhal phase, often before the diagnosis of pertussis. the nasopharyngeal culture usually becomes negative after 5 days of antibiotic therapy. chemoprophylaxis for all household contacts is routinely recommended. no evidence indicates transmission through breast milk, with similar risk to breastfed and bottle-fed infants. in the case of maternal infection with pertussis, chemoprophylaxis for all household contacts, regardless of age or immunization status, is indicated. in addition to chemoprophylaxis of the infant, close observation and subsequent immunization (in infants older than 6 weeks of age) are appropriate. despite chemoprophylaxis, droplet precautions and separation of mother and infant during the first 5 days of effective maternal antibiotic therapy are recommended. expressed breast milk can be provided to the infant during this period. staphylococcal infection in neonates can be caused by either s. aureus or coagulase-negative staphylococci (most often s. epidermidis) and can manifest in a wide range of illnesses. localized infection can be impetigo, pustulosis in neonates, cellulitis, or wound infection, and invasive or suppurative disease includes sepsis, pneumonia, osteomyelitis, arthritis, and endocarditis. s. aureus requires only a small inoculum (10 to 250 organisms) to produce colonization in newborns, most often of the nasal mucosa and umbilicus. 193 by the fifth day of life, 40% to 90% of the infants in the nursery will be colonized with s. aureus. 126 the organism is easily transmitted to others from mother, infant, family, or health care personnel through direct contact. outbreaks in nurseries were common in the past. mothers, infants, health care workers, and even contaminated, unpasteurized, banked breast milk were sources of infection. 298, 326 careful use of antibiotics, changes in nursery layout and procedures, standard precautions, and cohorting as needed decreased the spread of s. aureus in nurseries. now the occurrence of methicillin-resistant s. aureus (mrsa) is again a common problem, requiring cohorting, occasionally epidemiologic investigation, and careful infection control intervention. there are numerous reports of mrsa outbreaks in nicus.* the significance of colonization with staphylococcus and the factors leading to development of disease in individual patients are not clear. the morbidity and mortality related to s. aureus infection in neonates is well described. 192, 195, 219 management of such outbreaks has been reviewed. 147, 250 little has been written about the role of breastfeeding in colonization with s. aureus in nicus, wellbaby nurseries, or at home. mrsa is an important pathogen worldwide. community-acquired mrsa is different from hospital-acquired mrsa. community-acquired mrsa is usually defined as occurring in an individual without the common predisposing variables associated with hospital-acquired mrsa, lacking a mdr phenotype (common with hospital-acquired mrsa), frequently carrying multiple exotoxin virulence factors (such as panton-valentine leukocidin toxin), as well as carrying the smaller type iv staphylococcal cassette cartridge for the meca gene on a chromosome (hospital-acquired mrsa carries types i-iii staphylococcal cassette cartridge) and as being molecularly distinct from the common nosocomial strains of hospital-acquired mrsa. community-acquired mrsa is most commonly associated with skin and soft tissue infections and necrotizing pneumonia and less frequently associated with endocarditis, bacteremia, necrotizing fasciitis, myositis, osteomyelitis, or parapneumonic effusions. community-acquired mrsa is so common, it is now being observed in hospital outbreaks. 24, 144, 164, 358 community-acquired mrsa transmission to infants via breast milk has been reported. 34, 144, 210, 253, 286 premature or small-forgestational-age infants are more susceptible to and at increased risk for significant morbidity and mortality due to mrsa due in part to prolonged hospitalization, multiple courses of antibiotics, invasive procedures, and intravenous (iv) lines, their relative immune deficiency due to prematurity and illness, and altered gi tract due to different flora and decreased gastric acidity. therefore colonization with mrsa may pose a greater risk to infants in nicus in the long run. full-term infants develop pustulosis, cellulitis, and soft tissue infections, but rarely has invasive disease been reported. 82, 132, 298 fortunov et al 132 from texas reported 126 infections in term or late-preterm previously well infants including 43 with pustulosis, 68 with celluliltis or abscesses, and 15 invasive infections. family history of soft tissue skin infections and male sex were the only variables associated with risk for infection; cesarean delivery, breastfeeding, and circumcision were not. 132 nguyen et al 298 reported mrsa infections in a well-infant nursery from california. the eleven cases were all in full-term boys with pustularvesicular lesions in the groin. the infections were associated with longer length of stay, lidocaine injection use in infants, maternal age older than 30 years, and circumcision. breastfeeding was not an associated risk factor for mrsa infection. 298 the question of the role of circumcision in mrsa outbreaks was addressed by van howe and robson. 426 they reported that circumcised boys are at greater risk for staphylococcal colonization and infection. 426 others report that s. aureus carriage in infants (and subsequent infection) is most likely affected by multiple variables including infant factors (antibiotics, surgical procedures [circumcision being the most common], duration of hospital stay as a newborn), maternal factors (previous colonization, previous antibiotic usage, mode of delivery, length of stay), and environmental factors (mrsa in the family or hospital, nursery stay versus rooming-in, hand hygiene).* gerber et al 147 from the chicago area published a consensus statement for the management of mrsa outbreaks in the nicu. the recommendations, which were strongly supported by experimental, clinical, and epidemiologic data, included using a waterless, alcohol-based hand hygiene product, monitoring and enforcing hand hygiene, placing mrsa-positive infants in contact precautions with cohorting if possible, using gloves and gowns for direct contact and masks for aerosolgenerating procedures, cohorting nurses for care of mrsa-positive infants when possible, periodic screening of infants for mrsa using nares or nasopharyngeal cultures, clarifying the mrsa status of infants being transferred into the nicu, limiting overcrowding, and maintaining ongoing instruction and monitoring of health care workers in their compliance with infection control and hand hygiene procedures. evaluation of the outbreak could include screening of health care workers and environmental surfaces to corroborate epidemiologic data and laboratory molecular analysis of the mrsa strains if indicated epidemiologically. the use of mupirocin or other decolonizing procedures should be determined on an individual basis for each nicu. s. aureus is the most common cause of mastitis in lactating women. 317, 394, 395, 436 recurrence or persistence of symptoms of mastitis is a well described occurrence and an important issue in the management of mastitis. communityacquired mrsa has been associated with mastitis as well. 342 pasteurization, s. aureus was not detected in any of the 6820 samples of expressed breast milk. colonization of one infant with mrsa was identified, but no mrsa infections were identified in any of the hospitalized infants in the nicu during the 18 months of the study. 26 novak et al 300 identified mrsa in 57 of 500 samples (11%) of expressed fresh-frozen milk from 500 different donors from five brazilian milk banks. only 3 of the 57 samples were positive with high-level bacterial counts of mrsa: greater than 10,000 cfu/ml. these were the only samples that would not have been acceptable by bacteriological criteria according to brazilian or american criteria for raw milk use. they did not investigate other epidemiologic data to identify possible variables associated with low or high level contamination of expressed breast milk with mrsa. 300 management of an infant and/or mother with mrsa infection relative to breastfeeding or use of breast milk should be based on the severity of disease and whether the infant is premature, lbw, very-lowbirth-weight (vlbw), previously ill, or full term. full-term infants who themselves or their mothers develop mild to moderate infections (impetigo, pustulosis, cellulitis/abscess, mastitis/breast abscess, or soft tissue infection) can continue breast feeding after a short period of interruption (24 to 48 hours). during this time, pumping to maintain the milk supply should be supported, an initial evaluation for other evidence of infection should be done in the maternalinfant dyad, the infected child and/or mother should be placed on "commonly" effective therapy for the mrsa infection, and ongoing observation for clinical disease should continue. the mother and infant can "room-in" together in the hospital, if necessary, with standard and contact precautions. culturing the breast milk is not necessary. empiric therapy for the infant may be chosen based on medical concerns for the infant and the known sensitivity testing of the mrsa isolate. appropriate antibiotic choices include short-term use of azithromycin (erythromycin use during infancy [less than 6 weeks of age], or breastfeeding associated with an increased risk for hypertrophic pyloric stenosis), sulfamethoxazoletrimethoprim (in the absence of g6pd deficiency and older than 30 days of age), clindamycin, and perhaps linezolid for mild to moderate infections. infants in nicus (premature, lbw, vlbw, and/ or previously ill), who themselves or their mothers have a mrsa infection, should have the breast milk cultured and suspend breastfeeding or receiving breast milk from their mother until the breast milk is shown to be culture negative for mrsa. the infant should be treated as indicated for their infection or empirically treated if symptomatic (with pending culture results) and closely observed for development of new signs or symptoms of infection. pumping to maintain the milk supply and the use of banked breast milk are appropriate. the infant should be placed on contact precautions, in addition to the routine standard precautions. the infant can be cohorted with other mrsa-positive infants with nursing care cohorted as well. for the mother with mrsa infection, she should be instructed concerning hand hygiene, the careful collection, handling, and storage of breast milk, contact precautions to be used with her infant, and the avoidance of contact with any other infants. the mother can receive several possible antibiotics for mrsa that are compatible with breastfeeding when used for a short period. if the mother remains clinically well, including without evidence of mastitis, but her breast milk is positive for mrsa greater than 10 4 cfu/ml, empiric therapy to diminish or eradicate colonization would be appropriate. various regimens have been proposed to "eradicate" mrsa colonization, but none have been proven to be highly efficacious. these regimens usually include systemic antibiotics with one or two medications (rifampin added as the second medication), nasal mupirocin to the nares twice daily for 1 to 2 weeks with routine hygiene, with or without the usage of hexachlorophene (or similar topical agent or cleanser) for bathing during the 1 to 2 week treatment period. there is no clear information concerning the efficacy of using similar colonization eradication regimens for other household members or pets in preventing recolonization of the mother or infant. before reintroducing the use of the mother' s breast milk to the infant at least two to three negative breast milk cultures should be obtained after completion of therapy. routine screening of breast milk provided by mothers for their infants in nicus for the presence of mrsa is not indicated in the absence of mrsa illness in the maternal-infant dyad, an mrsa outbreak in nicus, or a high frequency of mrsa infection in a specific nicu. one case of staphylococcal scalded skin syndrome was reported by katzman and wald 208 in an infant breastfed by a mother with a lesion on her areola that did not respond to ampicillin therapy for 14 days. subsequently the infant developed conjunctivitis with s. aureus, which produced an exfoliative toxin, and a confluent erythematous rash without mucous membrane involvement or nikolsky sign. no attempt to identify the exfoliative toxin in the breast milk was made, and the breast milk was not cultured for s. aureus. the child responded to iv therapy with nafcillin. this emphasizes the importance of evaluating mother and infant at the time of a suspected infection and the need for continued observation of the infant for evidence of a pyogenic infection or toxin-mediated disease, especially with maternal mastitis or breast lesions. this case also raises the issue of when and how infants and their mothers become colonized with s. aureus and what factors lead to infection and illness in each. the concern is that staphylococcus can be easily transmitted through skin to skin contact, colonization readily occurs, and potentially serious illness can occur later, long after colonization. in the case of staphylococcal scalded skin syndrome or toxic shock syndrome (tss), the primary site of infection can be insignificant (e.g., conjunctivitis, infection of a circumcision, or simple pustulosis), but a clinically significant amount of toxin can be produced and lead to serious disease. toxic shock syndrome can result from s. aureus or streptococcus pyogenes infection and probably from a variety of antigens produced by other organisms. tss-1 has been identified as a "superantigen" that affects the t lymphocytes and other components of the immune response, producing an unregulated and excessive immune response and resulting in an overwhelming systemic clinical response. tss has been reported in association with vaginal delivery, cesarean delivery, mastitis, and other local infections in mothers. mortality rate in the mother may be as high as 5%. the case definition of staphylococcal tss includes meeting all four major criteria: fever greater than 38.9° c, rash (diffuse macular erythroderma), hypotension, and desquamation (associated with subepidermal separation seen on skin biopsy). the definition also includes involvement of three or more organ systems (gi, muscular, mucous membrane, renal, hepatic, hematologic, or central nervous system); negative titers for rocky mountain spotted fever, leptospirosis, and rubeola; and lack of isolation of s. pyogenes from any source or s. aureus from the cerebrospinal fluid (csf). 368 a similar case definition has been proposed for streptococcal tss. 451 aggressive empiric antibiotic therapy against staphylococci and streptococci and careful supportive therapy are essential to decreasing illness and death. oxacillin, nafcillin, first-generation cephalosporins, clindamycin, erythromycin, and vancomycin are acceptable antibiotics, even for a breastfeeding mother. the severity of illness in the mother may preclude breastfeeding, but it can be reinitiated when the mother is improving and wants to restart. standard precautions, but allowing breastfeeding, are recommended. staphylococcal enterotoxin f has been identified in breast milk specimens collected on days 5, 8, and 11 from a mother who developed tss at 22 hours postpartum. 428 s. aureus that produced staphylococcal enterotoxin f was isolated from the mother' s vagina but not from breast milk. infant and mother lacked significant antibody against staphylococcal enterotoxin f in their sera. the infant remained healthy after 60 days of follow-up. staphylococcal enterotoxin f is pepsin inactivated at ph 4.5 and therefore is probably destroyed in the stomach environment, presenting little or no risk to the breastfeeding infant. 35 breastfeeding can continue if the mother is able. coagulase-negative staphylococcal infection (the predominant isolate is staphylococcus epidermidis) produces minimal disease in healthy, full-term infants but is a significant problem in hospitalized or premature infants. factors associated with increased risk for this infection include prematurity, high colonization rates in specific nurseries, invasive therapies (e.g., iv lines, chest tubes, intubation), and antibiotic use. illness produced by coagulasenegative staphylococci can be invasive and severe in high-risk neonates, but rarely in mothers. there are reports of necrotizing enterocolitis associated with coagulase-negative staphylococcus. at 2 weeks of age, for infants still in the nursery, s. epidermidis is a frequent colonizing organism at multiple sites, with colonization rates as high as 75% to 100%. serious infections with coagulase-negative staphylococci (e.g., abscesses, iv line infection, bacteremia/sepsis, endocarditis, osteomyelitis) require effective iv therapy. many strains are resistant to penicillin and the semisynthetic penicillins, so sensitivity testing is essential. empiric or definitive therapy may require treatment with vancomycin, gentamicin, rifampin, teicoplanin, linezolid, or combinations of these for synergistic activity. transmission of infection in association with breastfeeding appears to be no more common than with bottle feeding. as with s. aureus infection control includes contact and standard precautions. occasionally, during presumed outbreaks, careful epidemiologic surveillance may be required, including cohorting, limiting overcrowding and understaffing, surveillance cultures of infants and nursery personnel, reemphasis of meticulous infection control techniques for all individuals entering the nursery, and, rarely, removal of colonized personnel from direct infant contact. s. epidermidis has been identified as part of fecal microbiota of breastfed infants. 203 s. epidermidis has also been identified in the breast milk of women with clinical evidence of mastitis. 107 nevertheless, s. epidermidis is rarely associated with infection in full-term infants. conceivably breast milk for premature infants could be a source of s. epidermidis colonization in the nicus. the other factors associated with hospitalization in a nicu noted previously presumably play a significant role in both colonization and infection in premature infants. the benefits of early full human milk feeding potentially outweigh the risk for colonization with s. epidermidis via breast milk. 348 ongoing education and assistance should be provided to mothers about the careful collection, storage, and delivery of human breast milk for their premature infants. 353 s. pyogenes (β-hemolytic group a streptococcus [gas]) is a common cause of skin and throat infections in children, producing pharyngitis, cellulitis, and impetigo. illnesses produced by gas can be classified in three categories: (1) impetigo, cellulitis, or pharyngitis without invasion or complication; (2) severe invasive infection with bacteremia, necrotizing fasciitis, myositis, or systemic illness (e.g., streptococcal tss); and (3) autoimmune-mediated phenomena, including acute rheumatic fever and acute glomerulonephritis. gas can also cause puerperal sepsis, endometritis, and neonatal omphalitis. significant morbidity and mortality rates are associated with invasive gas infection; mortality rate is 20% to 50%, with almost half the survivors requiring extensive tissue débridement or amputation. 347 infants are not at risk for the autoimmune sequelae of gas (rheumatic fever or poststreptococcal glomerulonephritis). transmission is through direct contact (rarely indirect contact) and droplet spread. outbreaks of gas in the nursery are rare, unlike with staphylococcal infections. either mother or infant can be initially colonized with gas and transmit it to the other. in the situation of maternal illness (extensive cellulitis, necrotizing fasciitis, myositis, pneumonia, tss, mastitis), it is appropriate to separate mother and infant until effective therapy (penicillin, ampicillin, cephalosporins, erythromycin) has been given for at least 24 hours. breastfeeding should also be suspended and may resume after 24 hours of therapy for the mother. group b streptococcus (gbs, streptococcus agalactiae) is a significant cause of perinatal bacterial infection. in parturient women, infection can lead to asymptomatic bacteriuria, urinary tract infection (often associated with premature birth), endometritis, or amnionitis. in infants, infection usually occurs between birth and 3 months of age (1 to 4 cases per 1000 live births). it is routinely classified by the time of onset of illness in the infant: early onset (0 to 7 days, majority less than 24 hours) and late onset (7 to 90 days, generally less than 4 weeks). infants may develop sepsis, pneumonia, meningitis, osteomyelitis, arthritis, or cellulitis. early-onset gbs disease is often fulminant, presenting as sepsis or pneumonia with respiratory failure; three quarters of neonatal disease is early onset. type iii is the most common serotype causing disease. transmission is believed to occur in utero and during delivery. colonization rates of mothers and infants vary between 5% and 35%. postpartum transmission is thought to be uncommon, although it has been documented. risk factors for early-onset gbs disease include delivery before 37 weeks' gestation, rupture of membranes for longer than 18 hours before delivery, intrapartum fever, heavy maternal colonization with gbs, or low concentrations of anti-gbs capsular antibody in maternal sera. 95 the common occurrence of severe gbs disease before 24 hours of age in neonates has lead to prevention strategies. revised guidelines developed by the aap committees on infectious diseases and on the fetus and newborn 95 have tried to combine various variables for increased risk for gbs infection (prenatal colonization with gbs, obstetric and neonatal risk factors for early-onset disease) and provide intrapartum prophylaxis to those at high risk ( figure 13 -1) the utilization of these guidelines and intrapartum prophylaxis across the united states has decreased the incidence of early-onset disease by approximately 80%. in 2005, the incidence of early-onset disease was 0.35 cases per 1000 live births. 95 late-onset gbs disease is thought to be the result of transmission during delivery or in the postnatal period from maternal, hospital, or community sources. dillon et al 112 demonstrated that 10 of 21 infants with late-onset disease were colonized at birth, but the source of colonization was unidentified in the others. gardner et al 141 showed that only 4.3% of 46 children who were culture negative for gbs at discharge from the hospital had acquired gbs by 2 months of age. anthony et al 15 noted that many infants are colonized with gbs, but the actual attack rate for gbs disease is low and difficult to predict. acquisition of gbs through breast milk or breastfeeding is uncommon. cases of late-onset gbs disease associated with gbs in the maternal milk have been reported. 58, 214, 313, 366, 438 some of the mothers had bilateral mastitis, at least one had delayed evidence of unilateral mastitis, and the others were asymptomatic. it was not clear when colonization of the infants occurred or when infection or disease began in the infants. the authors discussed the possibility that the infants were originally colonized during delivery, subsequently colonized the mothers' breasts during breastfeeding, and then became reinfected at a later time. butter and demoor 56 showed that infants initially colonized on their heads at birth had gbs cultured from their throat, nose, or umbilicus 8 days later. whenever they cultured gbs from the nipples of mothers, the authors also found it in the nose or throat of the infants. byrne et al 58 presented a review of gbs disease associated with breastfeeding and made recommendations to decrease the risk for transmission of gbs to infants via breastfeeding or breast milk. some of their recommendations included confirming appropriate collection and processing procedures for gbs cultures 370 in medical facilities to decrease false-negative cultures, reviewing proper hygiene for pumping, collection, and storage of expressed breast milk with mothers, reviewing the signs and symptoms of mastitis with mothers, and utilizing banked human milk as needed instead of mother' s milk. when a breastfed infant develops late-onset gbs disease, it is appropriate to culture the milk. (see discussion of culturing breast milk earlier in this chapter.) consider treatment of the mother to prevent reinfection if the milk is culture positive for gbs (greater than 10 4 cfu/ml), with or without clinical evidence of mastitis in the mother. withholding the mother' s milk until it is confirmed to be culture negative for a pathogen is appropriate and should be accompanied by providing ongoing support and instruction to the mother concerning pumping and maintaining her milk supply. serial culturing of expressed breast milk after treatment of the mother for gbs disease or colonization would be appropriate to insure the ongoing absence of a pathogen in the expressed breast milk. there are reports of reinfection of the infant from breast milk. 23, 225 eradication of gbs mucosal colonization in the infant or the mother may be difficult. some authors have recommended using rifampin prophylactically in both the mother and infant at the end of treatment to eradicate mucosal colonization. 23 (see chapter 16 for management of mastitis in the mother.) a mother or infant colonized or infected with gbs should be managed with standard precautions 94 while in the hospital. ongoing close evaluation of the infant for infection or illness and empiric therapy for gbs in the infant are appropriate until the child has remained well and cultures are subsequently negative at 72 hours. occasionally, epidemiologic investigation in the hospital will utilize culturing medical staff and family members to detect a source of late-onset gbs disease in the nursery. this can be useful when more than one case of late-onset disease is detected with the same serotype. cohorting in such a situation may be appropriate. selective prophylactic therapy for colonized infants to eradicate colonization may be considered, but unlike gas or staphylococcus infection, gbs infection in nurseries has not been reported to cause outbreaks. no data support screening all breastfeeding mothers and their expressed breast 4 cbc including wbc count with differential and blood culture. 5 applies only to penicillin, ampicillin, or cefazolin and assumes recommended dosing regimens. 6 a healthy-appearing infant who was ≥ 38 weeks' gestation at delivery and whose mother received ≥ 4 hours of iap before delivery may be discharged home after 24 hours if other discharge criteria have been met and a person able to comply fully with instructions for home observation will be present. if any one of these conditions is not met, the infant should be observed in the hospital for at least 48 hours and until criteria for discharge are achieved. milk for gbs as a reasonable method for protecting against spread of gbs infection via expressed breast milk. selective culturing of expressed breast milk may be appropriate in certain situations. the face of tuberculosis (tb) is changing throughout the world. in the united states the incidence of tb rose during 1986 through 1993 and has been declining since then. 60 increased rates of tb were noted in adults between 25 and 45 years of age, and because these are the primary childbearing years, the risk for transmission to children increased. tb during pregnancy has always been a significant concern for patients and physicians alike. 340 it is now clear that the course and prognosis of tb in pregnancy are less affected by the pregnancy and more determined by the location and extent of disease, as defined primarily by chest radiograph, and by the susceptibility of the individual patient. untreated tb in pregnancy is associated with maternal and infant mortality rates of 30% to 40%. 365 effective therapy is crucial to the clinical outcome in both pregnant and nonpregnant women. tb during pregnancy rarely results in congenital tb. any individual in a high-risk group for tb should be screened with a tuberculin skin test (tst). no contraindication or altered responsiveness to the tst exists during pregnancy or breastfeeding. interpretation of the tst should follow the most recent guidelines, using different sizes of induration in different-risk populations as cutoffs for a positive test, as proposed by the cdc. 68 figure 13 -2 outlines the evaluation and treatment of a pregnant woman with a positive tst. 398 treatment of active tb should begin as soon as the diagnosis is made, regardless of the fetus' gestational age, because the risk for disease to mother and fetus clearly outweighs the risks of treatment. isoniazid, rifampin, and ethambutol have been used safely in all three trimesters. isoniazid and pyridoxine therapy during breastfeeding is safe, although the risk for hepatotoxicity in the mother may be a concern during the first 2 months postpartum. 391 congenital tb is extremely rare if one considers that 7 to 8 million cases of tb occur each year worldwide and that less than 300 cases of congenital tb have been reported in the literature. as with other infectious diseases presenting in the perinatal period, distinguishing congenital infection from perinatal or postnatal tb in infants can be difficult. postnatal tb infection in infancy typically presents with severe disease and extrapulmonary extension (meningitis, lymphadenopathy, and bone, liver, spleen involvement). airborne transmission of tb to infants is the major mode of postnatal infection because of close and prolonged exposure in enclosed spaces, especially in their own household, to any adult with infectious pulmonary tb. potential infectious sources could be the mother or any adult caregiver, such as babysitters, day care workers, relatives, friends, neighbors, and even health care workers. the suspicion of tb infection or disease in a household with possible exposure of an infant is a highly anxiety-provoking situation ( figure 13 -3). although protection of an infant from infection is foremost in everyone' s mind, separation of the infant from the mother should be avoided when reasonable. every situation is unique, and the best approach will vary according to the specifics of the case and accepted principles of tb management. the first step in caring for the potentially exposed infant is to determine accurately the true tb status of the suspected case (mother or household contact). this prompt evaluation should include a complete history (previous tb infection or disease, previous or ongoing tb treatment, tst status, symptoms suggestive of active tb, results of most recent chest radiograph, sputum smears, or cultures), physical examination, a tst if indicated, a new chest radiograph, and mycobacterial cultures and smears of any suspected sites of infection. all household contacts should be evaluated promptly, including history and tst with further evaluation as indicated. 68 continued risk to the infant can occur from infectious household contacts who have not been effectively evaluated and treated. an infant should be separated temporarily from the suspected source if symptoms suggest active disease or a recent tst documents conversion, and separation should continue until the results of the chest radiograph are seen. because of considerable variability in the course of illness and the concomitant infectious period, debate continues without adequate data about the appropriate period of separation. 278 this should be individualized given the specific situation. hiv testing and assessment of the risk for mdr tb should be done in every case of active tb. sensitivity testing should be done on every mycobacterium tuberculosis isolate. table 13 -1 summarizes the management of the newborn infant whose mother (or other household contact) has tb. initiation of prophylactic isoniazid therapy in the infant has been demonstrated to be effective in preventing tb infection and disease in the infant. therefore continued separation of infant and mother is unnecessary after therapy in both mother and child has begun. 114 the real risk to an infant requiring separation is from airborne transmission. separation of the infant from a mother with active pulmonary tb is appropriate, regardless of the method of feeding. however, in many parts of the world, after therapy in the mother and prophylaxis with isoniazid in the infant has begun, the infant and mother are not separated. with or without separation, the mother and infant should continue to be closely observed throughout the course of maternal therapy to ensure good compliance with medication by both mother and infant and to identify, early on, any symptoms in the infant suggestive of tb. tuberculous mastitis occurs rarely in the united states but does occur in other parts of the world* and can lead to infection in infants, frequently involving the tonsils. a mother usually has a single breast mass and associated axillary lymph node swelling and infrequently develops a draining sinus. tb of the breast can also present as a painless mass or edema. involvement of the breast can occur with or without evidence of disease at other sites. evaluation of extent of disease is appropriate, including lesion cultures by needle aspiration, biopsy, or wedge resection and milk cultures. therapy should be with multiple anti-tb medications, but surgery should supplement this, as needed, to remove extensive necrotic tissue or a persistently draining sinus. 16 neither breastfeeding nor breast milk feeding should be done until the lesion is healed, usually 2 weeks or more. continued anti-tb therapy for 6 months in the mother and isoniazid for the infant for 3 to 6 months is indicated. in the absence of tuberculous breast infection in the mother, transmission of tb through breast milk has not been documented. thus even though temporary separation of infant and mother may occur pending complete evaluation and initiation of adequate therapy in the mother and prophylactic isoniazid therapy (10 mg/kg/day as a single daily dose) in the infant, breast milk can be expressed and given to the infant during the short separation. breastfeeding can safely continue whether the mother, infant, or both are receiving anti-tb therapy. anti-tb medications (isoniazid, rifampin, pyrazinamide, aminoglycosides, ethambutol, ethionamide, p-aminosalicylic acid) have been safely used in infancy, and therefore the presence of these medications in smaller amounts in breast milk is not a contraindication to breastfeeding. although conflicting, reports indicate that breastfeeding by tst-positive mothers does influence infants' responses to bacille calmette-guérin notes: 1 further workup should always include evaluation of tb status of all other household (or close) contacts by tuberculin skin testing (tst), review of symptoms, physical examination, and chest x-ray (cxr). sputum smears and cultures should be done as indicated. 2 separation should occur until interpretation of cxr film confirms absence of active disease, or, with active disease, separation should continue until individual is no longer considered infectious: three negative consecutive sputum smears, adequate ongoing empiric therapy, and decreased fever, cough, and sputum production. separation means in a different house or location, not simply separate rooms in a household. duration of separation should be individualized for each case in consultation with tb specialist. 3 this assumes no evidence of breast involvement, suspected tb mastitis, or lesion (except in status 5, when breast involvement is considered). risk to infant is via aerosolized bacteria in sputum from the lung. expressed breast milk can be given even if separation of mother and infant is advised. 4 tst positive, no symptoms or physical findings suggestive of tb, negative cxr film. 5 prophylactic therapy: isoniazid 10 mg/kg/day, maximum 300 mg for 6 months; pyridoxine 25 to 50 mg/day for 6 months. empiric therapy: standard three-or four-drug regimens for 2 months, and treatment should continue for total of 6 months with isoniazid and rifampin when organism is shown to be sensitive. suspected multidrug-resistant (mdr) tb requires consultation with tb specialist to select optimum empiric regimen and for ongoing monitoring of therapy and clinical response. vaccine, the tst, and perhaps the m. tuberculosis bacillus. despite efforts to identify either a soluble substance or specific cell fractions (gamma/delta t cells) in colostrum and breast milk that affect infants' immune responsiveness, no unified theory explains the various reported changes and no evidence has identified a consistent, clinically significant effect. 39, 213, 319, 367 viral infections arboviruses arboviruses were originally a large collection of viruses grouped together because of the common mode of transmission through arthropods. they have now been reclassified into several different families: bunyaviridae, togaviridae, flaviviridae, reoviridae, and others. they include more than 30 human pathogens. these organisms primarily produce either cns infections (encephalitis, meningoencephalitis) or undifferentiated illnesses associated with fever and rash, severe hemorrhagic manifestations, and involvement of other organs (hepatitis, myalgia, polyarthritis). infection with this array of viruses may also be asymptomatic and subclinical, although how often this occurs is uncertain. some of the notable human pathogens include bunyaviridae (california serogroup viruses), hantavirus, hantaan virus, phlebovirus (rift valley fever), nairovirus (crimean-congo hemorrhagic fever), alphavirus (western, eastern, and venezuelan equine encephalomyelitis viruses, chikungunya virus), flavivirus (st. louis encephalitis virus, japanese encephalitis virus, dengue viruses, yellow fever virus, tick-borne encephalitis viruses), and orbivirus (colorado tick fever). other than for crimean-congo hemorrhagic fever and for reported cases of colorado tick fever associated with transfusion, direct person-to-person spread has rarely been described. recent outbreaks of chikungunya virus infection in reunion island and in india described infection in young infants probably secondary to vertical spread from mother to infant transplacentally. 146, 339, 422 a few cases of early fetal deaths were associated with infection in pregnant women. the cases of vertical transmission occurred with near-term infection in the mothers, and the infants developed illness within 3 to 7 days of delivery. 146, 339 no evidence for transmission via breast milk or breastfeeding is available. little evidence indicates that these organisms can be transmitted through breast milk. the exceptions to this include evidence of transmission of two flaviviruses via breast milk, west nile virus, and yellow fever vaccine virus. standard precautions are generally sufficient. with any of these infections in a breastfeeding mother, the severity of the illness may determine the mother' s ability to continue breastfeeding. providing the infant with expressed breast milk is acceptable. (see the discussion of west nile virus and yellow fever vaccine virus later in this chapter.) in general, treatment for these illnesses is supportive. however, ribavirin appears to decrease the severity of and mortality from hantavirus pulmonary syndrome, hemorrhagic fever with renal failure, and crimean-congo hemorrhagic fever. ribavirin has been described as teratogenic in various animal species and is contraindicated in pregnant women. no information is available concerning ribavirin in breast milk, with little information available on the use of iv or oral ribavirin in infants. arenaviruses are single-stranded ribonucleic acid (rna) viruses that infect rodents and are acquired by humans through the rodents. the six major human pathogens in this group are (1) lymphocytic 6 sensitivity testing should be done on any positive culture. 7 isoniazid 10 mg/kg/day for 3 to 9 months depending on mother's or contact's status; repeat tst at 3 months and obtain normal cxr in infant before stopping isoniazid. before beginning therapy, workup of infant for congenital or active tb may be appropriate. this workup should be determined by clinical status of infant and suspected potential risk, and may include tst after 4 weeks of age, with cxr, complete blood count, and erythrocyte sedimentation rate, liver function tests, cerebrospinal fluid analysis, gastric aspirates, sonography/computed tomography of liver/spleen, and chest if congenital tb is suspected. 8 breastfeeding is proscribed when separation of mother and infant is indicated because of risk for aerosolized transmission of bacteria. expressed breast milk given to infant via bottle is acceptable in absence of mastitis or breast lesions. 9 consult with tb specialist about mdr tb. empiric therapy will be chosen based on the most recent culture sensitivities of index patient or perhaps suspected source case, if known, as well as medication toxicities and other factors. 10 tb mastitis usually involves a single breast with associated axillary lymph node swelling and, infrequently, a draining sinus tract. it can also present as a painless mass or edema of breast. 11 with suspected mastitis or breast lesion caused by tb, even breast milk is contraindicated until lesion or mastitis heals, usually 2 weeks or more. 12 patient has a documented, recent tst conversion but has not been completely evaluated. evaluation should begin and cxr done and evaluated in less than 24 hours to minimize separation of this person from infant. further workup should proceed as indicated by symptoms, physical findings, and cxr results. choriomeningitis virus, (2) lassa fever virus, (3) junin virus (argentine hemorrhagic fever), (4) machupo virus (bolivian hemorrhagic fever), (5) guanarito virus (venezuelan hemorrhagic fever), and (6) sabia virus. the geographic distribution of these viruses and the illness they cause are determined by the living range of the host rodent (reservoir). the exact mechanism of transmission to humans is unknown and hotly debated. 25, 69, 131 direct contact and aerosolization of rodent excretions and secretions are probable mechanisms. lymphocytic choriomeningitis virus is well recognized in europe, the americas, and other areas. perinatal maternal infection can lead to severe disease in the newborn, but no evidence suggests transmission through breast milk. 28, 224 standard precautions with breastfeeding are appropriate. lassa fever (west africa) and argentine hemorrhagic fever (argentine pampas) are usually more severe illnesses with dramatic bleeding and involvement of other organs, including the brain. these fevers more frequently lead to shock and death than do the forms of hemorrhagic fever caused by the other viruses in this group. person-to-person spread of lassa fever is believed to be common, and transmission within households does occur. 212 this may relate to prolonged viremia and excretion of the virus in the urine of humans for up to 30 days. 330 the possibility of persistent virus in human urine, semen, and blood after infection exists for each of the arenaviruses. the possibility of airborne transmission is undecided. current recommendations by the cdc 69 are to use contact precautions for the duration of the illness in situations of suspected viral hemorrhagic fever. no substantial information describes the infectivity of various body fluids, including breast milk, for these different viral hemorrhagic fevers. considering the severity of the illness in mothers and the risk to the infants, it is reasonable to avoid breastfeeding in these situations if alternative forms of infant nutrition can be provided. as more information becomes available, reassessment of these recommendations is advisable. a vaccine is in clinical trials in endemic areas for junin virus and argentine hemorrhagic fever. preliminary studies suggest it is effective, but data are still being accumulated concerning the vaccine' s use in children and pregnant or breastfeeding women. cytomegalovirus (cmv) is one of the human herpesviruses. congenital infection of infants, postnatal infection of premature infants, and infection of immunodeficient individuals represent the most serious forms of this infection in children. the time at which the virus infects the fetus or infant and the presence or absence of antibodies against cmv from the mother are important determinants of the severity of infection and the likelihood of significant sequelae (congenital infection syndrome, deafness, chorioretinitis, abnormal neurodevelopment, learning disabilities). 234 about 1% of all infants are born excreting cmv at birth, and approximately 5% of these congenitally infected infants will demonstrate evidence of infection at birth (approximately five symptomatic cases per 10,000 live births). approximately 15% of infants born after primary infection in a pregnant woman will manifest at least one sequela of prenatal infection. 96 various studies have detected that 3% to 28% of pregnant women have cmv in cervical cultures and that 4% to 5% of pregnant women have cmv in their urine. 120, 172 perinatal infection certainly occurs through contact with virus in these fluids but usually is not associated with clinical illness in fullterm infants. the lack of illness is thought to result from transplacental passive transfer of protective antibodies from the mother. postnatal infection later in infancy occurs via breastfeeding or contact with infected fluids (e.g., saliva, urine) but, again, rarely causes clinical illness in full-term infants. seroepidemiologic studies have documented transmission of infection in infancy, with higher rates of transmission occurring in daycare centers, especially when the prevalence of cmv in the urine and saliva is high. cmv has been identified in the milk of cmv-seropositive women at varying rates (10% to 85%) using viral cultures or cmv deoxyribonucleic acid (dna) pcr. 172, 301, 397, 430 cmv is more often identified in the breast milk of seropositive mothers than in vaginal fluids, urine, and saliva. the cmv isolation rate from colostrum is lower than that from mature milk. 172, 396 the reason for the large degree of variability in identification of cmv in breast milk in these studies probably relates to the intermittent nature of reactivation and excretion of the virus in addition to the variability, frequency, and duration of sampling of breast milk in the different studies. some authors have hypothesized that the difference in isolation rates between breast milk and other fluids is caused by viral reactivation in cells (leukocytes or monocytes) in the breast leading to "selective" excretion in breast milk. 301 vochem et al 430 reported that the rate of virolactia was greatest at 3 to 4 weeks postpartum, and yeager et al 455 reported significant virolactia between 2 and 12 weeks postpartum. antibodies (e.g., secretory iga) to cmv are present in breast milk, along with various cytokines and other proteins (e.g., lactoferrin). these may influence virus binding to cells, but they do not prevent transmission of infection.* several studies have documented increased rates of postnatal cmv infection in breastfed infants (50% to 69%) compared with bottle-fed infants (12% to 27%) observed through the first year of life 120, 281, 397, 430 in these same studies, full-term infants who acquired cmv infection postnatally were only rarely mildly symptomatic at the time of seroconversion or documented viral excretion. also, no evidence of late sequelae from cmv was found in these infants. postnatal exposure of susceptible infants to cmv, including premature infants without passively acquired maternal antibodies against cmv, infants born to cmv-seronegative mothers, and immunodeficient infants, can cause significant clinical illness (pneumonitis, hepatitis, thrombocytopenia).* in one study of premature infants followed up to 12 months, vochem et al 430 found cmv transmission in 17 of 29 infants (59%) exposed to cmv virolactia and breastfed compared with no infants infected of 27 exposed to breast milk without cmv. no infant was given cmv-seropositive donor milk or blood. five of the 12 infants who developed cmv infection after 2 months of age had mild signs of illness, including transient neutropenia, and only one infant had a short increase in episodes of apnea and a period of thrombocytopenia. five other premature infants with cmv infection before 2 months of age had acute illness, including sepsis-like symptoms, apnea with bradycardia, hepatitis, leukopenia, and prolonged thrombocytopenia. 430 vollmer et al 431 followed premature infants with early postnatal cmv infection acquired through breast milk for 2 to 4.5 years to assess neurodevelopment and hearing function. none of the children had sensorineural hearing loss. there was no difference between the 22 cmv-infected children and 22 matched premature control cmv-negative infants in terms of neurologic, speech and language, or motor development. 431 neuberger et al 296 examined the symptoms and neonatal outcome of cmv infection transmitted via human milk in premature infants in a case-control fashion; 40 cmv-infected premature infants were compared with 40 cmv-negative matched premature infants. neutropenia, thrombocytopenia, and cholestasis were associated with cmv infection in these infants. no other serious effects or illnesses were found directly associated with the infection including intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity, necrotizing enterocolitis, bronchopulmonary dysplasia, duration of mechanical ventilation or oxygen therapy, duration of hospital stay or weight, gestational age, or head circumference at the time of discharge. exposure of cmv-seronegative or premature infants to cmv-positive milk (donor or natural mother' s) should be avoided. 379 various methods of inactivating cmv in breast milk have been reported, including holder pasteurization, freezing (−20° c for 3 days), and brief high temperature (72° c for 10 seconds). 120, 135, 155, 393, 455 one small, prospective study suggests that freezing breast milk at −20°c for 72 hours protects premature infants from cmv infection via breast milk. sharland et al 379 reported on 18 premature infants (less than 32 weeks) who were uninfected at birth and exposed to breast milk from their cmv seropositive mothers. only one of 18 (5%) infants became positive for cmv at 62 days of life, and this infant was clinically asymptomatic. this transmission rate is considerably lower than others reported in the literature. cmvseronegative and leukocyte-depleted blood products were used routinely. banked breast milk was pasteurized and stored at −20° c for various time periods and maternal expressed breast milk was frozen at −20° c before use whenever possible. the infants received breast milk for a median of 34 days (range 11 to 74 days) and they were observed for a median of 67 days (range 30 to 192 days). breast milk samples pre-or postfreezing were not analyzed by pcr or culture for the presence of cytomegalovirus. 379 buxmann et al 57 demonstrated no transmission of cmv in 23 premature infants receiving thawed frozen breast milk until 33 weeks (gestational age + postnatal age) (less than or equal to 31 weeks gestational age) born to 19 mothers who were cmv-igg negative. cmv infection was found in five premature infants of 35 infants born to 29 mothers who were cmv-igg positive and who provided breast milk for their infants. three of the five children remained asymptomatic. one child development a respirator-dependent pneumonia and the second developed an upper respiratory tract infection and thrombocytopenia in association with their cmv infections. 57 yasuda et al 454 reported on 43 preterm infants (median gestational age 31 weeks) demonstrating a peak in cmv dna copies, detected by a real-time pcr assay, in breast milk at 4 to 6 weeks postpartum. thirty of the 43 infants received cmv dna-positive breast milk. three of the 30 had cmv dna detected in their sera, but none of the three had symptoms suggestive of cmv infection. much of the breast milk had been stored at −20° c before feeding, which the authors propose is the probable reason for less transmission in this cohort. 454 lee et al 248 reported on the use of maternal milk frozen at −20°c for a minimum of 24 hours before feeding to premature infants in a nicu; 23 infants had cmv-seropositive mothers and 39 infants had cmv-seronegative mothers. two infants developed cmv infection, which was symptomatic. they were both fed frozen thawed milk from cmv-seropositive mothers. 248 others have reported individual cases of cmv infection in premature infants despite freezing and thawing breast milk. 268, 314 simple freezing and thawing of breast milk does not completely prevent transmission of cmv to premature infants. the efficacy of freezing and thawing breast milk for varying lengths of time to prevent cmv infection in premature infants has not been studied prospectively in a randomized controlled trial. eleven of 36 neonatal units in sweden (27 of which have their own milk banks) freeze maternal milk to reduce the risk for cmv transmission to premature infants. 314 a prominent group of neonatologists and pediatric infectious disease experts in california who recognize the significant benefits of providing human milk to premature and lbw infants recommend screening mothers of premature infants for cmv igg at delivery and, when an infant' s mother is cmv igg positive at delivery, using either pasteurized banked human milk or frozen then thawed maternal breast milk for premature infants until they reach the age of 32 weeks. 445 in consideration of the low rates of cmv virolactia in colostrum 169, 397 and the predominant occurrence of virolactia between 2 and 12 weeks (peak at 3 to 4 weeks) postpartum, 430,455 they reasonably propose beginning colostrum and breast milk feedings for all infants until the maternal cmv serologic screening is complete. they appropriately recommend close observation and follow-up of premature infants older than 3 weeks of age for signs, symptoms, and laboratory changes of cmv infection until discharge from the hospital. 445 cmv-seropositive mothers can safely breastfeed their full-term infants because, despite a higher rate of cmv infection than in formula-fed infants observed through the first year of life, infection in this situation is not associated with significant clinical illness or sequelae. dengue viruses (serotypes dengue 1 to 4) are flaviviruses associated primarily with febrile illnesses and rash; dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. the mosquito aedes aegypti is the main vector of transmission of dengue virus in countries lying between latitudes 35 degrees north and 35 degrees south. more than 2.5 billion people live in areas where transmission occurs; dengue virus infects over 100 million individuals a year and casuses approximately 24,000 deaths a year. 159, 163 although dengue hemorrhagic fever and dengue shock syndrome occur frequently in children younger than 1 year of age, they are infrequently described in infants younger than 3 months of age. 167 there are also differences in the clinical and laboratory findings of dengue virus infection in children compared with adults. 222 boussemart et al 49 reported on two cases of perinatal/prenatal transmission of dengue and discussed eight additional cases in neonates from the literature. prenatal or intrapartum transmission of the same type of dengue as the mother was confirmed by serology, culture, or pcr. phongsamart et al 333 described three additional cases of dengue virus infection late in pregnancy and apparent transmission to two of the three infants with passive acquisition of antibody in the third infant. sirinavin et al 386 reported on 17 cases in the literature of vertical dengue infection, all presenting at less than 2 weeks of age, but no observations or discussion of breast milk or breastfeeding as a potential source of infection were published. watanaveeradej et al 439 presented an additional three cases of dengue infection in infants documenting normal growth and development at follow-up at 12 months of age. it has been postulated that more severe disease associated with dengue disease occurs when an individual has specific igg against the same serotype as the infecting strain in a set concentration, leading to antibody-dependent enhancement of infection. the presence of preexisting dengue serotype specific igg in an infant implies either previous primary infection with the same serotype, passive acquisition of igg from the mother (who had a previous primary infection with the same serotype), or perhaps acquisition of specific igg from breast milk. watanaveeradej et al 439 documented transplacentally transferred antibodies against all four serotypes of dengue virus in 97% of 2000 cord sera at delivery. follow-up of 100 infants documented the loss of antibodies to dengue virus over time with losses of 3%, 19%, 72%, 99%, and 100% at 2, 4, 6, 9, and 12 months of age, respectively. no evidence is available in the literature about more severe disease in breastfed infants compared with formula-fed infants. no interhuman transmission of dengue virus in the absence of the mosquito vector and no evidence of transmission via breast milk are known. only one report of a factor in the lipid portion of breast milk, which inhibits the dengue virus, is available, and no evidence for antibody activity against dengue virus in human breast milk is known. 127 breastfeeding during maternal or infant dengue disease should continue as determined by the mother' s or infant' s severity of illness. epstein-barr virus (ebv) is a common infection in children, adolescents, and young adults. it is usually asymptomatic but most notably causes infectious mononucleosis and has been associated with chronic fatigue syndrome, burkitt lymphoma, and nasopharyngeal carcinoma. because ebv is one of the human herpesviruses, concern has been raised about lifelong latent infection and the potential risk for infection to a fetus and neonate from the mother. primary ebv infection during pregnancy is unusual because few pregnant women are susceptible. 149, 189 although abortion, premature birth, and congenital infection from ebv are suspected, no distinct group of anomalies is linked to ebv infection in fetus or neonate. also, no virologic evidence of ebv as the cause of abnormalities was found in association with suspected ebv infection. culturing of ebv from various fluids or sites is difficult. the virus is detected by its capacity to transform b lymphocytes into persistent lymphoblastoid cell lines. pcr and dna hybridization studies have detected ebv in the cervix and in breast milk. one study, which identified ebv dna in breast milk cells in more than 40% of women donating milk to a breast milk bank, demonstrated that only 17% had antibody to ebv (only igg, no igm). 204 another study examining serologic specimens from breastfed and bottle-fed infants showed similar seroprevalence of ebv at 12 to 23 months of age (36/66 [54.5%] and 24/43 [55.8%]) in the breastfed and bottle-fed children, respectively. 236 the question of the timing of ebv infection and the subsequent immune response and clinical disease produced requires continued study. differences exist among the clinical syndromes that manifest at different ages. infants and young children are asymptomatic, have illness not recognized as related to ebv, or have mild episodes of illness, including fever, lymphadenopathy, rhinitis and cough, hepatosplenomegaly, or rash. adolescents or young adults who experience primary ebv infection more often demonstrate infectious mononucleosis syndrome or are asymptomatic. chronic fatigue syndrome is more common in adolescents and young adults. burkitt lymphoma, observed primarily in africa, and nasopharyngeal carcinoma, seen in southeast asia, where primary ebv infection usually occurs in young children, are tumors associated with early ebv infection. 246 these tumors are related to "chronic" ebv infection and tend to occur in individuals with persistently high antibody titers to ebv viral capsid antigen and early antigen. the questions of why these tumors occur with much greater frequency in these geographic areas and what cofactors (including altered immune response to infection associated with coinfections, immune escape by ebv leading to malignancy, or increased resistance to apoptosis secondary to ebv gene mutations) may contribute to their development remain unanswered. 21, 289 it also remains unknown to what degree breast milk could be a source of early ebv infection compared with other sources of ebv infection in an infant' s environment. similar to the situation of postnatal transmission of cmv in immunocompetent infants, clinically significant illness rarely is associated with primary ebv infection in infants. more data concerning the pathogenesis of ebv-associated tumors should be obtained before proscribing against breastfeeding is warranted, especially in areas where these tumors are common but the protective benefits of breastfeeding are high. in areas where burkitt lymphoma and nasopharyngeal carcinoma are uncommon, ebv infection in mother or infant is certainly not a contraindication to breastfeeding. marburg and ebola viruses cause severe and highly fatal hemorrhagic fevers. the illness often presents with nonspecific symptoms (conjunctivitis, frontal headache, malaise, myalgia, bradycardia) and progresses with worsening hemorrhage to shock and subsequent death in 50% to 90% of patients. personto-person transmission through direct contact, droplet spread, or airborne spread is the common mode of transmission. however, the animal reservoir or source of these viruses in nature for human infection has not been identified. attack rates in families are 5% to 16%. 330 no postexposure interventions have proved useful in preventing spread, and no treatment other than supportive is currently available. a recent report documented the presence of ebola virus in numerous body fluids including in breast milk. one acute breast milk sample on day 7 after the onset of illness and a "convalescent" breast milk sample on day 15 from the same woman were positive for ebola virus by both culture and pcr testing. 30 in the same study, saliva remained virus positive for a mean of 16 days after disease onset, urine was positive for a mean of 28 days, and semen for a mean of 43 days after the onset of disease. no information is available concerning the risk for transmission of these viruses in breast milk or additional risks or benefits from breastfeeding. contact precautions are recommended for marburg virus infections and contact and airborne precautions for ebola virus infection. given the high attack and mortality rates, these precautions should be carefully instituted and breastfeeding not allowed. if any other suitable source of nutrition can be found for an infant, expressed breast milk should also be proscribed for the infant of a mother with either of these infections for at least 3 weeks postrecovery. the diagnosis of hepatitis in a pregnant woman or nursing mother causes significant anxiety. the first issue is determining the etiology of the hepatitis, which then allows for an informed discussion of risk to the fetus/infant. the differential diagnosis of acute hepatitis includes (1) common causes of hepatitis, such as hepatitis a, b, c, and d; (2) , igm anti-hbcag, anti-hcv) as the initial diagnostic tests. simultaneous consideration of other etiologies of acute liver dysfunction is appropriate depending on a patient' s history. if the initial diagnostic tests are all negative, subsequent additional testing for anti-hepatitis d virus (hdv), hcv rna, hepatis g virus (hgv) rna, anti-hepatis e virus (hev), or hev rna may be necessary. if initial testing reveals positive hbsag, testing for anti-hdv, hbeag, and hbv dna is appropriate. these additional tests are useful in defining the prognosis for a mother and the risk for infection to an infant. during the diagnostic evaluation, it is appropriate to discuss with the mother or parents the theoretic risk for transmitting infectious agents that cause hepatitis via breastfeeding. the discussion should include an evaluation of the positive and negative effects of suspending or continuing breastfeeding until the exact etiologic diagnosis is determined. the relative risk for transmission of infection to an infant can be estimated and specific preventive measures provided for the infant (table 13-2) . hepatitis a virus (hav) is usually an acute selflimited infection. the illness is typically mild, and generally subclinical in infants. occasionally, hav infection is prolonged or relapsing, extending 3 to 6 months, and rarely it is fulminant, but hav infection does not lead to chronic infection. the incidence of prematurity after maternal hav infection is increased, but no evidence to date indicates obvious birth defects or a congenital syndrome. 372, 464 hav infection in premature infants may lead to prolonged viral shedding. 349 transmission is most often person to person (fecal-oral), and transmission in food-borne or water-borne epidemics has been described. transmission via blood products and vertical transmission (mother to infant) are rare. 440 transmission in daycare settings has been clearly described. infection with hav in newborns is uncommon and does not seem to be a significant problem. the usual period of viral shedding and presumed contagiousness lasts 1 to 3 weeks. acute maternal hav infection in the last trimester or in the postpartum period could lead to infection in an infant. symptomatic infection can be prevented by immunoglobulin (ig) administration, and 80% to 90% of disease can be prevented by ig administration immune serum globulin within 2 weeks of exposure. hav vaccine can be administered simultaneously with ig without affecting the seroconversion rate to produce rapid and prolonged hav serum antibody levels. transmission of hav via breast milk has been implicated in one case report, but no data exist on the frequency of isolating hav from breast milk. 440 because hav infection in infancy is rare and usually subclinical without chronic disease and because exposure has already occurred by the time the etiologic diagnosis of hepatitis in a mother is made, no reason exists to interrupt breastfeeding with maternal hav infection. the infant should receive ig and hav vaccine, administered simultaneously. hepatitis b virus (hbv) infection leads to a broad spectrum of illness, including asymptomatic seroconversion, nonspecific symptoms (fever, malaise, fatigue), clinical hepatitis with or without jaundice, extrahepatic manifestations (arthritis, rash, renal involvement), fulminant hepatitis, and chronic hbv infection. chronic hbv infection occurs in up to 90% of infants infected via perinatal and vertical transmission and in 30% of children infected between 1 to 5 years of age. given the increased risk for significant sequelae from chronic infection (chronic active hepatitis, chronic persistent hepatitis, cirrhosis, primary hepatocellular carcinoma), prevention of hbv infection in infancy is crucial. transmission of hbv is usually through blood or body fluids (stool, semen, saliva, urine, cervical secretions). 94 vertical transmission either transplacentally or perinatally during delivery has been well described throughout the world. vertical transmission rates in areas where hbv is endemic (taiwan and japan) are high, whereas transmission to infants from hbv carrier mothers in other areas where hbv carrier rates are low is uncommon. 399 transmission of hbv to infants occurs in up to 50% of infants when the mothers are acutely infected immediately before, during, or soon after pregnancy. 462 hbsag is found in breast milk, but transmission by this route is not well documented. beasley 31 and beasley et al 32 demonstrated that although breast milk transmission is possible, seroconversion rates are no different between breastfed and nonbreastfed infants in a long-term follow-up study of 147 hbsag-positive mothers. hill et al 176 followed 101 breastfed infants and 268 formula-fed infants born to women who were chronically hbsag positive. all infants received hepatitis b immunoglobulin at birth and a full series of hepatitis b vaccine. none of the breastfed infants and nine of the formulafed infants were positive for hbsag after completion of the hbv vaccine series. breastfeeding had occurred for a mean of 4.9 months (range 2 weeks to 1 year). transmission, when it does happen, probably occurs during labor and delivery. another report from china followed 230 infants born to hbsag-positive women. the infants received appropriate dosing and timing of hbig and hbv vaccine. at 1 year of age, anti-hbs antibody was present in 90.9% of the breastfed infants and 90.3% of the bottle-fed infants. 437 risk factors associated with immunoprophylaxis failure against vertical transmission of hbv include hbeag-seropositive mothers and elevated hbv dna "viral loads" in the mothers. 392 in 2009 the aap committee on infectious diseases stated that "that breastfeeding of the infant by a hbsag-positive mother poses no additional risk for acquisition of hbv infection by the infant with appropriate administration of hepatitis b vaccine and hbig." 96 screening of all pregnant women for hbv infection is an essential first step to preventing vertical transmission. universal hbv vaccination at birth and during infancy, with administration of hepatitis b immunoglobulin (hbig) immediately after birth to infants of hbsag-positive mothers, prevents hbv transmission in more than 95% of cases. breastfeeding by hbsag-positive women is not contraindicated, but immediate administration of hbig and hbv vaccine should occur. two subsequent doses of vaccine should be given at appropriate intervals and dosages for the specific hbv vaccine product. this decreases the small theoretic risk for hbv transmission from breastfeeding to almost zero. when acute peripartum or postpartum hepatitis occurs in a mother and hbv infection is a possibility, with its associated increased risk for transmission to the infant, a discussion with the mother or parents should identify the potential risks and benefits of continuing breastfeeding until the etiology of the hepatitis can be determined. if an appropriate alternative source of nutrition is available for the infant, breast milk should be withheld until the etiology of the hepatitis is identified. hbig and hbv vaccine can be administered to the infant who has not already been immunized or has no documented immunity against hbv. 400 if acute hbv infection is documented in a mother, breastfeeding can continue after immunization has begun. acute infection with hcv can be indistinguishable from hepatitis a or b infection; however, it is typically asymptomatic or mild. hcv infection is the major cause of blood-borne non-a, non-b hepatitis (nanbh). chronic hcv infection is reported to occur 70% to 85% of the time regardless of age at time of infection. sequelae of chronic hcv infection are similar to those associated with chronic hbv infection. bortolotti et al 47 the two commonly identified mechanisms of transmission of hcv are transfusions of blood or blood products and iv drug use. however, other routes of transmission exist because hcv infection occurs even in the absence of obvious direct contact with significant amounts of blood. other body fluids contaminated with blood probably serve as sources of infection. transmission through sexual contact occurs infrequently and probably requires additional contributing factors, such as coinfection with other sexually transmitted agents or high viral loads in serum and other body fluids. studies of transmission in households without other risk factors have demonstrated either low rates of transmission or no transmission. the reported rates of vertical transmission vary widely. in mothers with unknown hiv status or known hiv infection, the rates of vertical transmission were 4% to 100%, whereas the rates varied between 0% and 42% in known hiv-negative mothers. 113 these same studies suggest that maternal coinfection with hiv, hcv genotype, active maternal liver disease, and the serum titer of maternal hcv rna may be associated with increased rates of vertical transmission. 263, 307, 461 the correlation between hcv viremia, the hcv viral load in a mother, and vertical transmission of hcv is well documented. 288, 355, 406, 456 the clinical significance and risk for liver disease after vertical transmission of hcv are still unknown. the timing of hcv infection in vertical transmission is also unknown. in utero transmission has been suggested by some studies, 125 whereas intrapartum or postpartum transmission was proposed by ohto et al 308 when they documented the absence of hcv rna in the cord blood of neonates who later became hcv rna positive at 1 to 2 months of age. more recently, gibb et al 150 reported two pieces of data supporting the likelihood of intrapartum transmission as the predominant time of vertical transmission: (a) low sensitivity of pcr for hcv rna testing in the first month of life with a marked increase in sensitivity after that for diagnosing hcv infection in infants and (b) a lower transmission risk for elective cesarean delivery (without prolonged rupture of membranes) compared with vaginal or emergency cesarean delivery. 150 another group, mcmenamin et al, 275 analyzed vertical transmission in 559 mother-infant pairs. the overall vertical transmission rate was 4.1% (18/441), with another 118 infants not tested or lost to follow-up. comparison of the vertical transmission rate was no different for vaginal delivery or emergency cesarean in labor versus planned cesarean (4.2% vs. 3.0%). this held true even when mothers had hepatitis c rna detected antenatally (7.2% vs. 5.3%). the authors did not support planned cesarean delivery to decrease vertical transmission of hepatitis c infection. no prospective, controlled trials of cesarean versus vaginal delivery and the occurrence of vertical hepatitis c transmission are available. the risk for hcv transmission via breast milk is uncertain. anti-hcv antibody and hcv rna has been demonstrated in colostrum and breast milk, although the levels of hcv rna in milk did not correlate with the titers of hcv rna in serum. 36, 162, 256, 355 nevertheless, transmission of hcv via breastfeeding (and not in utero, intrapartum, or from other postpartum sources) has not been proven in the small number infants studied. transmission rates in breastfed and nonbreastfed infants appear to be similar, but various important factors have not been controlled, such as hcv rna titers in mothers, examination of the milk for hcv rna, exclusive breastfeeding versus exclusive formula feeding versus partial breastfeeding, and duration of breastfeeding.* zanetti et al 461 including 23 infants whose mothers were seropositive for hcv rna. eight infants in that study were infected with hcv, their mothers had both hiv and hcv, and three of these eight infants were infected with both hiv and hcv. the hcv rna levels were significantly higher in the mothers coinfected with hiv compared with those mothers with hcv alone. overall, the risk for hcv infection via breastfeeding is low, the risk for hcv infection appears to be more frequent in association with hiv infection and higher levels of hcv rna in maternal serum, no effective preventive therapies (ig or vaccine) exist, and the risk for chronic hcv infection and subsequent sequelae with any infection is high. it is therefore appropriate to discuss the theoretic risk for breastfeeding in hcv-positive mothers with the mother or parents and to consider proscribing breast milk when appropriate alternative sources of nutrition are available for the infants. hiv infection is a separate contraindication to breastfeeding. additional study is necessary to determine the exact role of breastfeeding in the transmission of hcv, including the quantitative measurement of hcv rna in colostrum and breast milk, the relative risk for hcv transmission in exclusively or partially breastfed infants versus the risk in formulafed infants, and the effect of duration of breastfeeding on transmission. the current position of the cdc is that no data indicate that hcv virus is transmitted through breast milk. 83 therefore breastfeeding by a hcvpositive, hiv-negative mother is not contraindicated. infants born to hcv rna-positive mothers require follow-up through 18 to 24 months of age to determine infants' hcv status, regardless of the mode of infant feeding. infants should be tested for alanine aminotransferase and hcv rna at 3 months and 12 to 15 months of age. alanine aminotransferase and anti-hcv antibody should be tested at 18 to 24 months of age to confirm an infant' s status: uninfected, ongoing hepatitis c infection, or past hcv infection. hepatitis d virus (hdv) is a defective rna virus that causes hepatitis only in persons also infected with hbv. the infection occurs as either an acute coinfection of hbv and hdv or a superinfection of hbv carriers. this "double" infection results in more frequent fulminant hepatitis and chronic hepatitis, which can progress to cirrhosis. the virus uses its own hbv rna (circular, negative-strand rna) with an antigen, hdag, surrounded by the surface antigen of hbv, hbsag. hdv is transmitted in the same way as hbv, especially through the exchange of blood and body fluids. hdv infection is uncommon where the prevalence of hbv is low. in areas where hbv is endemic, the prevalence of hdv is highly variable. hdv is common in tropical africa and south america as well as in greece and italy but is uncommon in the far east and in alaskan inuit despite the endemic occurrence of hbv in these areas. 390 transmission of hdv has been reported to occur from household contacts and, rarely, through vertical transmission. no data are available on transmission of hdv by breastfeeding. hdv infection can be prevented by blocking infection with hbv; therefore hbig and hbv vaccine are the best protection. in addition to hbig and hbv vaccine administration to the infant of a mother infected with both hbv and hdv, discussion with the mother or parents should include the theoretic risk for hbv and hdv transmission through breastfeeding. as with hbv, once hbig and hbv vaccine have been given to the infant, the risk for hbv or hdv infection from breastfeeding is negligible. therefore breastfeeding after an informed discussion with the parents is acceptable. hepatitis e virus (hev) is a cause of sporadic and epidemic, enterically transmitted nanbh, which is typically self-limited and without chronic sequelae. hev is notable for causing high mortality rate in pregnant women. transmission is primarily via the fecal-oral route, commonly via contaminated water or food. high infection rates have been reported in adolescents and young adults (ages 15 to 40 years). tomar 416 reported that 70% of cases of hev infections in the pediatric population in india manifest as acute hepatitis. maternal-neonatal transmission was documented when the mother developed hepatitis e infection in the third trimester. although hev was demonstrated in breast milk, no transmission via breast milk was confirmed in the report. five cases of transfusion-associated hepatitis e were reported. 416 epidemics are usually related to contamination of water. person-to-person spread is minimal, even in households and day care settings. although ig may be protective, no controlled trials have been done. animal studies suggest that a recombinant subunit vaccine may be feasible. 344 hev infection in infancy is rare, and no data exist on transmission of hev by breastfeeding. no evidence of clinically significant postnatal hev infection in infants or of chronic sequelae in association with hev infection and no documented hev transmission through breast milk is available. currently no contraindication exists to breastfeeding with maternal hev infection. ig has not been shown to be effective in preventing infection, and no vaccine is available for hev. hepatitis g virus (hgv) has recently been confirmed as a cause of nanbh distinct from hepatitis viruses a through e. several closely related genomes of hgv, currently named gbv-a, -b, and -c, appear to be related to hcv, the pestiviruses, and the flaviviruses. epidemiologically, hgv is most often associated with transfusion of blood, although studies have identified nontransfusion-related cases. hgv genomic rna has been detected in some patients with acute and chronic hepatitis and a small number of patients with fulminant hepatitis. gbv-c/hgv has also been found in some patients with inflammatory bile duct lesions, but the pathogenicity of this virus is unconfirmed. hgv rna has been detected in 1% to 3% of healthy blood donors in the united states. 8 feucht et al 128 described maternal-to-infant transmission of hgv in three of nine children. two of the three mothers were coinfected with hiv and the third with hcv. none of these infants developed signs of liver disease. neither the timing nor the mode of transmission was clarified. lin et al 255 reported no hgv transmission in three mother-infant pairs after cesarean delivery and discussed transplacental spread via blood as the most likely mode of hgv infection in vertical transmission. wejstal et al 442 reported on perinatal transmission of hgv to 12 of 16 infants born to hgv viremic mothers, identified by pcr. hgv did not appear to cause hepatitis in the children. 442 fischler et al 130 followed eight children born to hgv-positive mothers and found only one to be infected with hgv. that child remained clinically well, while his twin, also born by cesarean delivery and breastfed, remained hgv negative for 3 years of observation. five of the other six children were breastfed for variable periods without evidence of hgv infection. ohto et al 309 examined hgv mother-to-infant transmission. of 2979 pregnant japanese women who were screened, 32 were identified as positive for gbv-c/hgv rna by pcr; 26 of 34 infants born to the 32 hgv positive women were shown to be hgv rna positive. reportedly, none of the infants demonstrated a clinical picture of hepatitis, although two infants had persistent mild elevations (less than two times normal) of alanine aminotransferase. the viral load in mothers, who transmitted hgv to their infants, was significantly higher than in nontransmitting mothers. infants born by elective cesarean delivery had a lower rate of infection (3 in 7) compared with infants born by emergency cesarean delivery (2 of 2) or born vaginally (21 of 25) . in this study, hgv infection in breastfed infants was four times more common than in formula-fed infants, but this difference was not statistically significant because only four infants were formula fed. the authors report no correlation between infection rate and duration of breastfeeding was seen. testing of the infants was not done frequently and early enough routinely through the first year of life to determine the timing of infection in these infants. 309 schröter et al 371 reported transmission of hgv to 3 of 15 infants born to hgv rna positive mothers at 1 week of age. none of 15 breast milk samples were positive for gbv-c/hgv rna, and all of the children who were initially negative for hgv rna in serum remained negative at follow-up between 1 to 28 months of age. 371 the foregoing data suggest that transmission is more likely to be vertical, before, or at delivery rather than via breastfeeding. the pathogenicity and the possibility of chronic disease due to hgv infection remain uncertain at this time. insufficient data are available to make a recommendation concerning breastfeeding by hgv-infected mothers. herpes simplex virus types 1 and 2 (hsv-1, hsv-2) can cause prenatal, perinatal, and postnatal infections in fetuses and infants. prenatal infection can lead to abortion, prematurity, or a recognized congenital syndrome. perinatal infection is the most common form of infection (1 in 2000 to 5000 live births, 700 to 1500 cases per year in the united states) and is often fatal or severely debilitating. the factors that facilitate intrapartum infection and predict the severity of disease have been extensively investigated. postnatal infection is uncommon but can occur from a variety of sources, including oral or genital lesions and secretions in mothers or fathers, hospital workers and home caregivers, and breast lesions in breastfeeding mothers. a number of case reports have documented severe hsv-1 or hsv-2 infections in infants associated with hsvpositive breast lesions in the mothers. 116, 161, 338, 403 cases of infants with hsv gingivostomatitis inoculating the mothers' breasts have also been reported. in the absence of breast lesions breastfeeding in hsv-seropositive or culture-positive women is reasonable when accompanied by careful handwashing, covering the lesions, and avoiding fondling or kissing with oral lesions until all lesions are crusted. breastfeeding during maternal therapy with oral or iv acyclovir can continue safely as well. inadequate information exists concerning valacyclovir, famciclovir, ganciclovir, and foscarnet in breast milk to make a recommendation at this time. breastfeeding by women with active herpetic lesions on their breasts should be proscribed until the lesions are dried. treatment of the mothers' breast lesions with topical, oral, and/or iv antiviral preparations may hasten recovery and decrease the length of viral shedding. human herpesvirus 6 (hhv-6) is a cause of exanthema subitum (roseola, roseola infantum) and is associated with febrile seizures. hhv-6 appears to be most similar to cmv based on genetic analysis. no obvious congenital syndrome of hhv-6 infection has been identified, although prenatal infection has been reported. 118 seroepidemiologic studies show that most adults have already been infected by hhv-6. therefore primary infection during pregnancy is unlikely, but reactivation of latent hhv-6 infection may be more common. no case of symptomatic hhv-6 prenatal infection has been reported. the significance of reactivation of hhv-6 in a pregnant woman and the production of infection and disease in the fetus and infant remains to be determined. primary infection in children occurs most often between 6 and 12 months of age, when maternally acquired passive antibodies against hhv-6 are waning. febrile illnesses in infants younger than 3 months of age have been described with hhv-6 infection, but infection before 3 months or after 3 years is uncommon. various studies involving serology and restriction enzyme analysis of hhv-6 isolates from mother/infant pairs support the idea that postnatal transmission and perhaps perinatal transmission from the mothers are common sources of infection. one study was unable to detect hhv-6 in breast milk by pcr analysis in 120 samples, although positive control samples seeded with hhv-6-infected cells did test positive. 119 given the limited occurrence of clinically significant disease and the absence of sequelae of hhv-6 infection in infants and children, the almost universal acquisition of infection in early childhood (with or without breastfeeding) and the absence of evidence that breast milk is a source of hhv-6 infection, breastfeeding can continue in women known to be seropositive for hhv-6. human herpesvirus 7 (hhv-7) is closely related to hhv-6 biologically. primary infection with hhv-7 occurs primarily in childhood, usually later in life than hhv-6 infection. the median age of infection is 26 months, with 75% of children becoming hhv-7 positive by 5 years of age. 63 seroprevalence of hhv-7 antibody has been reported to be 80% to 98% in adults, and passive antibody is present in almost all newborns. 306, 408 like hhv-6, hhv-7 infection can be associated with acute febrile illness, febrile seizures, and irritability, but in general it is a milder illness than with hhv-6 with fewer hospitalizations. virus excretion of hhv-7 occurs in saliva, and pcr testing of blood cells and saliva are frequently positive in individuals with past infection. 463 congenital infection of hhv-6 was detected via dna pcr testing in 57 of 5638 of cord blood samples (1%), but hhv-7 was not detected in any of 2129 cord blood specimens. 165 hhv-7 dna was detected by pcr in 3 of 29 breast milk mononuclear cell samples from 24 women who were serum positive for hhv-7 antibody. 137 in the same study, small differences were seen in the hhv-7 seropositive rates between breastfed infants and bottle-fed infants at 12 months of age (21.7% versus 20%), at 18 months of age (60% versus 48.1%), and at 24 months of age (77.3% versus 58.3%, respectively,). none of these differences were statistically significant. given that, in general, hhv-6 infection occurs earlier than hhv-7 infection in most infants and that hhv-6 is rarely found in breast milk, it seems unlikely that hhv-7 in breast milk is a common source of infection in infants and children. the infrequent occurrence of significant illness with hhv-7 infection, with the absence of sequelae except in patients who had transplantation surgery at older ages and the common occurrence of infection in childhood argue, that no reason to proscribe against breastfeeding for hhv-7 positive women exists. human papillomavirus (hpv) is a dna virus with at least 100 different types. these viruses cause warts, genital dysplasia, cervical carcinoma (types 6 and 11), and laryngeal papillomatosis. transmission occurs through direct contact and sexual contact. laryngeal papillomas are thought to result from acquiring the virus in passage through the birth canal. infection in pregnant women or during pregnancy does not lead to an increase in abortions or the risk for prematurity, and no evidence indicates intrauterine infection. hpv is one of the most common viruses in adults and one of the most commonly sexually transmitted infections. diagnosis is usually by histologic examination or dna detection. spontaneous resolution does occur, but therapy for persistent lesions or growths in anatomically problematic locations is appropriate. therapy can be with podophyllum preparations, trichloroacetic acid, cryotherapy, electrocautery, and laser surgery. interferon is being tested in the treatment of laryngeal papillomas, with mixed results. 109 prevention against transmission means limiting direct or sexual contact, but this may not be sufficient because lesions may not be evident and transmission may still occur. rintala et al 346 examined the occurrence of hpv dna in the oral and genital mucosa of infants during the first 3 years of life. hpv dna was identified in 12% to 21% of the oral scrape samples and in 4% to 15% of the genital scrape samples by pcr. oral hpv infection was acquired by 42% of children, cleared by 11%, and persisted in 10% of children; 37% of the children were never infected. they did not report on breast milk or breastfeeding in that study. the question of the source of the infection remains undetermined. the breast is a rare site of involvement. 110 hpv types 16 and 18 can immortalize normal breast epithelium in vitro. 441 hpv dna has been detected in breast milk in 10 of 223 (4.5%) of milk samples from 223 mothers, collected 3 days postpartum. 361 no attempt was made to correlate the presence of hpv dna in breast milk with the hpv status of an infant or to assess the "viral load" of hpv in breast milk or its presence over the course of lactation. a second study found dna of cutaneous and mucosal hpv types in 2 of 25 human milk samples and 1 of 10 colostrum samples. 64 no reports of hpv lesions of the breast or nipple and documented transmission to an infant secondary to breastfeeding are available. no increased risk for acquiring hpv from breast milk is apparent, and breastfeeding is acceptable. even in the rare occurrence of an hpv lesion of the nipple or breast, no data suggest that breastfeeding or the use of expressed breast milk is contraindicated. measles is another highly communicable childhood illness that can be more severe in neonates and adults. measles is an exanthematous febrile illness following a prodrome of malaise, coryza, conjunctivitis, cough, and often koplik spots in the mouth. the rash usually appears 10 to 14 days after exposure. complications can include pneumonitis, encephalitis, and bacterial superinfection. with the availability of vaccination, measles in pregnancy is rare (0.4 in 10,000 pregnancies), 148 although respiratory complications (primary viral pneumonitis, secondary bacterial pneumonia), hepatitis, or other secondary bacterial infections often lead to more severe disease in these situations. prenatal infection with measles may cause premature delivery without disrupting normal uterine development. no specific group of congenital malformations have been described in association with in utero measles infection, although teratogenic effects of measles infection in pregnant women may rarely manifest in the infants. perinatal measles includes transplacental infection when measles occurs in an infant in the first 10 days of life. infection from extrauterine exposure usually develops after 14 days of life. the severity of illness after suspected transplacental spread of virus to an infant varies from mild to severe and does not seem to vary with the antepartum or postpartum onset of rash in the mother. it is uncertain what role maternal antibodies play in the severity of an infant's disease. more severe disease seems to be associated with severe respiratory illness and bacterial infection. postnatal exposure leading to measles after 14 days of life is generally mild, probably because of passively acquired antibodies from the mother. severe measles in children younger than 1 year of age may occur because of declining passively acquired antibodies and complications of respiratory illness and rare cases of encephalitis. measles virus has not been identified in breast milk, whereas measles-specific antibodies have been documented. 1 infants exposed to mothers with documented measles while breastfeeding should be given immunoglobulin (ig) and isolated from the mother until 72 hours after the onset of rash, which is often only a short period after diagnosis of measles in the mother. the breast milk can be pumped and given to the infant because secretory iga begins to be secreted in breast milk within 48 hours of onset of the exanthem in the mother. table 13 -3 summarizes management of the hospitalized mother and infant with measles exposure or infection. 148 mumps is an acute transient benign illness with inflammation of the parotid gland and other salivary glands and often involves the pancreas, testicles, and meninges. mumps occurs infrequently in pregnant women (1 to 10 cases in 10,000 pregnancies) and is generally benign. mumps virus has been isolated from saliva, respiratory secretions, blood, testicular tissue, urine, csf in cases of meningeal involvement, and breast milk. the period of infectivity is believed to be between 7 days before and 9 days after the onset of parotitis, with the usual incubation period being 14 to 18 days. prenatal infection with the mumps virus causes an increase in the number of abortions when infection occurs in the first trimester. a small increase in the number of premature births was noted in one prospective study of maternal mumps infection. 383 no conclusive evidence suggests congenital malformations are associated with prenatal infection, not even with endocardial fibroelastosis, as originally reported in the 1960s. perinatal mumps (transplacentally or postnatally acquired) has rarely if ever been documented. natural mumps virus has been demonstrated to infect the placenta and infect the fetus, and live attenuated vaccine virus has been isolated from the placenta but not from fetal tissue in women vaccinated 10 days before induced abortion. antibodies to mumps do cross the placenta. postnatal mumps in the first year of life is typically benign. no epidemiologic data suggest that mumps infection is more or less common or severe in breastfed infants compared with formula-fed infants. although mumps virus has been identified in breast milk and mastitis is a rare complication of mumps in mature women, no evidence indicates that breast involvement occurs more frequently in lactating women. if mumps occurs in the mother breastfeeding can continue because exposure has already occurred throughout the 7 days before the development of symptoms in the mother and secretory iga in the milk may help to mitigate the symptoms in the infant. human parvovirus b19 causes a broad range of clinical manifestations, including asymptomatic infection (most frequent manifestation in all ages), erythema infectiosum (fifth disease), arthralgia and arthritis, red blood cell (rbc) aplasia (less often decreased white blood cells or platelets), chronic infection in immunodeficient individuals, and rarely myocarditis, vasculitis, or hemophagocytic syndrome. vertical transmission can lead to severe anemia and immune-mediated hydrops fetalis, which can be treated, if accurately diagnosed, by intrauterine transfusion. inflammation of the liver or cns can be seen in the infant, along with vasculitis. if the child is clinically well at birth, hidden or persistent abnormalities are rarely identified. no evidence indicates that parvovirus b19 causes an identified pattern of birth defects. postnatal transmission usually occurs person to person via contact with respiratory secretions, saliva, and rarely blood or urine. seroprevalence in children at 5 years of age is less than 5%, with the peak age of infection occurring during the schoolage years (5% to 40% of children infected). the majority of infections are asymptomatic or undiagnosed seroconversions. 417 severe disease, such as prolonged aplastic anemia, occurs in individuals with hemoglobinopathies or abnormal rbc maturation. attack rates have been estimated to be 17% to 30% in casual contacts but up to 50% among household contacts. in one study of 235 susceptible pregnant women, the annual seroconversion rate was 1.4%. 223 no reports of transmission to an infant through breastfeeding are available. excretion in breast milk has not been studied because of limitations in culturing techniques. rat parvovirus has been demonstrated in rat milk. the very low seroconversion rate in young children and the absence of chronic or frequent severe disease suggest that the risk for parvovirus infection via breast milk is not significant. the possibility of antibodies against parvovirus or other protective constituents in breast milk has not been studied. breastfeeding by a mother with parvovirus infection is acceptable. poliovirus infections (types 1, 2, and 3) cause a range of illness, with 90% to 95% subclinical, 4% to 8% abortive, and 1% to 2% manifest as paralytic poliomyelitis. a review by bates 29 from 1955 of 58 cases of poliomyelitis in infants younger than 1 month of age demonstrated paralysis or death in more than 70% and only one child without evidence of even transient paralysis. more than half the cases were ascribed to transmission from the mothers, although no mention was made of breastfeeding. breastfeeding rates at the time were approximately 25%. prenatal infection with polioviruses does cause an increased incidence of abortion. prematurity and stillbirth apparently occur more frequently in mothers who developed paralytic disease versus inapparent infection. 188 although individual reports of congenital malformations in association with maternal poliomyelitis exist, no epidemiologic data suggest that polioviruses are teratogenic. also, no evidence indicates that live attenuated vaccine poliovirus given during pregnancy is associated with congenital malformations. 89, 170 perinatal infection has been noted in several case reports of infants infected in utero several days before birth who had severe disease manifesting with neurologic manifestations (paralysis) but without fever, irritability, or vomiting. additional case reports of infection acquired postnatally demonstrate illness more consistent with poliomyelitis of childhood. these cases were more severe and involved paralysis, which may represent reporting bias. 89 no data are available concerning the presence of poliovirus in breast milk, although antibodies to poliovirus types 1, 2, and 3 have been documented. 270 in this era of increasing worldwide poliovirus vaccination, the likelihood of prenatal or perinatal poliovirus infection is decreasing. maternal susceptibility to poliovirus should be determined before conception and poliovirus vaccine offered to susceptible women. an analysis of the last great epidemic in italy in 1958 was done using a population-based case-control study. 336 in 114,000 births, 942 infants were reported with paralytic poliomyelitis. a group of matched control subjects was selected from infants admitted to the hospital at the same time. using the dichotomous variable of never breastfed and partially breastfed, 75 never-breastfed infants were among the cases and 88 among the control group. the authors determined an odds ratio of 4.2, with 95% confidence interval of 1.4 to 14, demonstrating that the risk for paralytic poliomyelitis was higher in infants never breastfed and lowest among those exclusively breastfed. because by the time the diagnosis of poliomyelitis is made in a breastfeeding mother, the exposure of the infant to poliovirus from maternal secretions has already occurred, and because the breast milk already contains antibodies that may be protective, no reason exists to interrupt breastfeeding. breastfeeding also does not interfere with successful immunization against poliomyelitis with oral or inactivated poliovirus vaccine. 71 the occurrence of human t-cell leukemia virus type i (htlv-i) is endemic in parts of southwestern japan, 66, 105, 207, 450 the caribbean, south america, 156 and sub-saharan africa. htlv-i is associated with adult t-cell leukemia/lymphoma and a chronic condition with progressive neuropathy. the progressive neuropathy is called htlv-i associated myelopathy or tropical spastic paraparesis. 136 other illnesses have been reported in association with htlv-i infection including dermatitis, uveitis, arthritis, sjögren syndrome in adults, and infective dermatitis and persistent lymphadenitis in children. transmission of htlv-i occurs most often through sexual contact, via blood or blood products, and via breast milk. infrequent transmission does occur in utero or at delivery and with casual or household contact. 291 seroprevalence generally increases with age and varies widely in different regions and in populations of different backgrounds. in some areas of japan, seropositivity can be as high as 12% to 16%, but in south america, africa, and some caribbean countries the rates are 2% to 6%. in latin america seropositive rates can be as high as 10% to 25% among female sex workers or attendees to std clinics. 156 in blood donors in europe, the seroprevalence of htlv-i has been reported at 0.001% to 0.03%. the seroprevalence in pregnant women in endemic areas of japan is as high as 4% to 5% and in nonendemic areas as low as 0.1% to 1.0%. htlv-1 is not a major disease in the united states. in studies from europe the seroprevalence in pregnant women has been noted to be up to 0.6%. these pregnant women were primarily of african or caribbean descent. 138 htlv-i antigen has been identified in breast milk of htlv-i positive mothers. 220 another report shows that basal mammary epithelial cells can be infected with htlv-i and can transfer infection to peripheral blood monocytes. 254 human milk from htlv-i positive mothers caused infection in marmosets. 221, 453 htlv-i infection clearly occurs via breastfeeding and a number of reports document an increased rate of transmission of htlv-i to breastfed infants compared with formula-fed infants.* ando et al 12, 13 in two separate reports demonstrated a parallel decline in antibodies against htlv-i in both formula-fed and breastfed infants to a nadir at approximately 1 year of age and a subsequent increase in antibodies from 1 to 2 years of age. the percentage of children seropositive at 1 year of age in the breastfed and formula-fed groups, respectively, was 3.0% and 0.6%, at 1.5 years of age it was 15.2% and 3.9%, and at 2 years of age it was 41.9% and 4.6%. a smaller group of children followed through 11 to 12 years of age demonstrated no newly infected children after 2 years of age and *references 9, 10, 12, 13, 178-180, 407. no loss of antibody in any child who was seropositive at 2 years of age. 12, 13 transmission of htlv-i infection via breastfeeding is also clearly associated with the duration of breastfeeding. 407, 409, 446, 447 it has been postulated that the persistence of passively acquired antibodies against htlv-i offers some protection through 6 months of life (table 13-4) . other factors relating to htlv-i transmission via breast milk have been proposed. yoshinaga et al 460 presented data on the htlv-i antigen producing capacity of peripheral blood and breast milk cells and showed an increased mother-to-child transmission rate when the mother' s blood and breast milk produced large numbers of antigen-producing cells in culture. 460 hisada et al 183 reported on 150 mothers and infants in jamaica, demonstrating that a higher maternal provirus level and a higher htlv-i antibody titer were independently associated with htlv-i transmission to the infant. ureta-vidal et al 421 reported an increased seropositivity rate in children of mothers with a high proviral load and elevated maternal htlv-i antibody titers. various interventions have been proposed to decrease htlv-i transmission via breastfeeding. complete avoidance of breastfeeding was shown to be an effective intervention by hino et al 180, 181 in large population of japanese in nagasaki. avoiding breastfeeding led to an 80% decrease in transmission. breastfeeding for a shorter duration is another effective alternative. ando et al 11 showed that freezing and thawing breast milk decreased the infectivity of htlv-i. sawada et al 363 demonstrated in a rabbit model that htlv-i immunoglobulin protected against htlv-i transmission via milk. it is reasonable to postulate that any measure that would decrease the maternal provirus load or increase the anti-htlv-i antibodies available to infants might decrease the risk for transmission. the overall prevalence of htlv-i infection during childhood is unknown because the majority of individuals do not manifest illness until much later in life. the timing of htlv-i infection in a breastfeeding population has been difficult to assess because of passively acquired antibodies from the mother and issues related to testing. furnia et al 139 in areas where the prevalence of htlv-i infection (in the united states, canada, or europe) is rare, the likelihood that a single test for antibody against htlv-i would be a false positive test is high compared with the number of true positive tests. repeat testing is warranted in many situations. 66 quantification of the antibody titer and the proviral load is appropriate in a situation when mother-to-child transmission is a concern. a greater risk for progression to disease in later life has not been shown for htlv-i infection through breast milk, but early-life infections are associated with the greatest risk for adult t-cell leukemia. 402 the mother and family should be informed about all these issues. if the risk for lack of breast milk is not too great and formula is readily available and culturally acceptable, then the proscription of breastfeeding, or at least a recommendation to limit the duration of breastfeeding to 6 months or less, is appropriate to limit the risk for htlv-i transmission to the infant. freezing and thawing breast milk before giving it to an infant might be another reasonable intervention to decrease the risk for transmission, although no controlled trials document the efficacy of such an intervention. neither ig nor antiviral agents against htlv-i are available at this time. human t-cell leukemia virus type ii (htlv-ii) is endemic in specific geographic locations, including africa, the americas, the caribbean, and japan. transmission is primarily through intravenous drug use, contaminated blood products, and breastfeeding. sexual transmission occurs but its overall contribution to the prevalence of htlv-ii in different populations remains uncertain. many studies have examined the presence of htlv-i and ii in blood products. pcr testing and selective antibody tests suggest that about half of the htlv seropositivity in blood donors is caused by htlv-ii. htlv-ii has been associated with two chronic neurologic disorders similar to those caused by htlv-i, tropical or spastic ataxia. 258 a connection between htlv-ii and glomerulonephritis, myelopathy, arthritis, t-hairy cell leukemia, and large granulocytic leukemia has been reported. mother-to-child transmission has been demonstrated in both breastfed and formula-fed infants. it appears that the rate of transmission is greater in breastfed infants.* htlv-ii has been detected in breast milk. 174 nyambi et al 304 reported that htlv-ii transmission did correlate with the duration of breastfeeding. the estimated rate of transmission was 20%. the time to seroconversion (after the initial loss of passively acquired maternal antibodies) for infected infants seemed to range between 1 and 3 years of age. 304 at this time avoidance of breastfeeding and limiting the duration of breastfeeding are the only two possible interventions with evidence of effectiveness for preventing htlv-ii mother-to-child transmission. 207 with the current understanding of retroviruses, it is appropriate in cases of documented htlv-ii maternal infection to recommend avoiding or limiting the duration of breastfeeding and provide alternative nutrition when financially practical and culturally acceptable. mothers should have confirmatory testing for htlv-ii and measurement of the proviral load. infants should be serially tested for antibodies to htlv-ii and have confirmatory testing if seropositive after 12 to 18 months of age. further investigation into the mechanisms of transmission via breast milk and possible interventions to prevent transmission should occur as they have for hiv-1 and htlv-i. human immunodeficiency virus type 1 (hiv-1) is transmitted through human milk. refraining from breastfeeding is a crucial aspect of preventing perinatal hiv infection in the united states and many other countries. the dilemma is the use of replacement feeding versus breastfeeding in countries where breastfeeding provides infants with significant protection from illness and death due to malnutrition or other infections. the question of the contribution of breastfeeding in mother-to-child hiv-1 transmission is not a trivial one when one considers the following: 3. the who estimates that 2.7 million people were newly infected with hiv-1 in 2007, with children younger than 14 years old making up 370,000 of that 2.7 million. (this number has declined due to increasing access to interventions to prevent mother-to-infant transmission. availability of antiretroviral therapy for prevention of mother-to-child hiv transmission in developing countries in 2007 was estimated to reach 33% of the mothers who needed it.) 419 4. breastfeeding contributes an estimated 10% to 20% increase in the overall mother-to-child transmission rates, over and above intrauterine and intrapartum transmission, when no specific interventions to prevent transmission via breastfeeding are utilized. 5. despite a dramatic increase in the number of people receiving antiretroviral therapy in developing countries (3 million), this represented only 31% of the individuals who needed treatment. 419 the evidence of hiv transmission via breastfeeding is irrefutable. multiple publications summarize the current evidence for hiv transmission via breastfeeding in the literature. 232, 341, 420 since 1985, case reports have documented hiv transmission via breast milk to children around the world. 182, 198, 249, 465 primary hiv infection in breastfeeding mothers, with the concomitant high viral load, is associated with a particularly high rate of hiv transmission via breast milk. palasanthiran et al 322 estimated that risk at 27%.large observational studies have demonstrated higher rates of hiv transmission in breastfed infants of mothers with chronic hiv infection compared with formula-fed infants. 43, 108, 124 a systematic analysis of published reports estimated the additional risk for perinatal hiv transmission due to breastfeeding to be 14% (95% confidence interval 7% to 22%). 117 more recently published cohort studies similarly attributed additional risk for hiv transmission due to breastfeeding at 4% to 22% over and above the risk from prenatal and intrapartum transmission. 38, 104, 121 laboratory reports demonstrate the presence of cell-free virus and cell-associated virus in breast milk as well as various immunologic factors that could block or limit infection.* a dose-response relationship has been observed, correlating the hiv viral load in human milk as well as a mother' s plasma viral load with an increased transmission risk for the breastfed infant. 335, 345, 351, 373 many of the potential risk factors associated with human milk transmission of hiv is higher the longer the duration of breastfeeding. 108, 251, 282, 290, 424 maternal characteristics related to transmission of hiv via human milk include younger maternal age, higher parity, lower cd4+ counts, higher plasma viral loads, and breast abnormalities (mastitis, abscess, or nipple lesions). characteristics of human milk that relate to a higher risk for transmission include higher viral load in the milk, lower concentrations of antiviral substances (lactoferrin, lysozyme), and lower concentrations of virus-specific cytotoxic t-lymphocytes, levels of various interleukins (il-7, il-15), 434, 435 secretory iga, and igm. mixed breastfeeding is also associated with a higher risk for hiv transmission compared with exclusive breastfeeding. 99, 100, 410 the measurable benefits of breast milk versus the relative risk for hiv transmission to the infant due to exclusive breastfeeding (with optimization of other factors to decrease hiv transmission) have been reported in a couple of studies. 97, 229 the measurable benefits of receiving breast milk versus the relative increased risk for hiv transmission will need to be determined in a prospective fashion in different locales. 247 a number of potential interventions to prevent breastfeeding transmission of hiv-1 can be utilized (box 13-3) . the simplest and most effective is the compete avoidance of human milk. this is a practical solution in places like the united states and other countries where replacement feeding as well as other strictly medical interventions are feasible and reasonable, and the risk for not providing breast milk to the infant is negligible. in resourcepoor situations, where the risk for other infections is high without the benefits of breast milk, exclusive breastfeeding is appropriate, with any other reasonable and culturally acceptable interventions to decrease hiv transmission via breast milk. potentially effective interventions include exclusive breastfeeding, early weaning versus breastfeeding for longer durations, education, and support to decrease the likelihood of mastitis or nipple lesions. 191 other possible interventions include treating a mother with antiretroviral therapy for her own health (cd4 counts less than 350) or prophylactically to decrease the human milk viral load, treating an infant prophylactically for a prolonged period of time (6 weeks to 6 months) to protect against transmission via breastfeeding, treating the milk itself to decrease the viral load (by pasteurization or other methods), 316, 318 treating acute conditions in mothers and infants (e.g., mastitis, breast lesions, infant candidiasis), and enhancing an infant' s own defenses via vitamins, immunization, or antiretroviral therapy. some of these may not be feasible in certain settings such as pasteurization or maternal antiretroviral therapy. others may not be culturally acceptable, such as treating expressed breast milk before giving it to an infant or even exclusive breastfeeding. significant data demonstrate the advantage of breastfeeding, even for hiv-infected or hivexposed infants. the complete avoidance of breastfeeding in certain situations may lead to increased risk for illness and death due to other reasons besides hiv transmission. 106 a study from kenya showed improved hiv-1-free survival rates in a formula-fed group of children born to hiv-positive mothers, but the breastfed and formula groups had similar mortality rates (24.4% versus 20.0%, respectively) and similar incidences of diarrhea and pneumonia in the first 2 years of life. 272 no difference in the two groups was seen in the prevalence of malnutrition, but the breastfed infants had better nutritional status in the first 6 months of life. arpadi et al 20 recommend additional nutritional interventions to complement breastfeeding in this population after 6 months of age. two reports from zambia document the benefit of exclusive breastfeeding for decreasing late hiv transmission and the lower mortality at 12 months in infants who had continued breastfeeding rather than had discontinued breastfeeding at 4 months of age. 229, 385 in malawi, hiv-infected and hiv-exposed infants who were breastfed (exclusive breastfeeding for 2 months and mixed feeding after that) had lower mortality at 24 months than those who were not breastfed. 405 a report from botswana examined breastfeeding plus infant zidovudine prophylaxis for 6 months versus formula feeding plus infant zidovudine for 1 month; this study showed a decreased risk for vertical transmission with formula feeding, but also increased cumulative mortality for the hiv-infected infants at 7 months of age who were in the formula-fed group. 411 a study from south africa examining the use of vitamin a also demonstrated less morbidity in hiv-infected children who were breastfed than not breastfed. 102 other abstract reports have shown increased morbidity in hiv-infected children due to diarrhea, gastroenteritis, and hospitalization after weaning from breastfeeding. 205, 226, 315, 413 exclusive breastfeeding in most areas of the world is essential to infant health and survival, even in the situation of maternal hiv infection. 97, 99, 100, 229 the duration of exclusive breastfeeding is crucial to decreasing the risk for hiv infection in infants versus the risk for malnutrition and other infections with early weaning. in the mashi study in botswana, thior et al 411 evaluated infants randomized to breastfeeding plus infant zidovudine for 6 months or formula feeding plus 1 month of infant zidovudine. the cumulative infant mortality was significantly higher at 7 months for the formula-fed group but at 18 months it was similar between the two groups. the breastfed infants were more likely to become hiv infected despite the 6 months of zidovudine prophylaxis. 411 becquet et al 33 analyzed data from cote d'ivoire for 2001 to 2005; 47% of the hivexposed infants were breastfed for a median of 4 months, and 53% were formula fed and observed for 2 years. no significant difference in the rate of hiv infection was seen in the two groups, and no significant difference between the two groups was seen for morbid events (diarrhea, acute respiratory infections or malnutrition) or hospitalization or death. the authors attributed these good outcomes to effective nutritional counseling and care, access to clean water, and the provision of a safe and continuous supply of breast milk substitute. 33 coovadia et al 97 studied exclusive breastfeeding in the first 6 months of life as an intervention in south africa. of the exclusively breastfed infants, 14.1% at 6 weeks of age and 19.5% at 6 months of age were hiv infected. breastfed infants who also were fed solids or formula milk were more likely to acquire infection than exclusively breastfed infants. the cumulative mortality at 3 months of age was markedly lower for exclusively breastfed infants (6.1%) versus 15.1% in the infants receiving mixed feedings. kuhn et al 230 examined the effects of early, abrupt weaning on hiv-free survival of 958 children in zambia. infants were randomly assigned to two different counseling programs that advised either abrupt weaning at 4 months or prolonged breastfeeding. in the weaning intervention group, 69% of mothers stopped breastfeeding by 5 months compared with a median duration of breastfeeding of 16 months in the control group. the study found no significant difference in hiv-free survival at • women and their health care providers need to be aware of the potential risk for transmission of hiv infection to infants during pregnancy and in the peripartum period and through breast milk. • documented, routine hiv education and routine testing with the consent of women seeking prenatal care are strongly recommended so that each woman knows her hiv status and the methods available both to prevent the acquisition and transmission of hiv and to determine whether breastfeeding is appropriate. • at delivery, education about hiv and testing with the consent of women whose hiv status during pregnancy is unknown are strongly recommended. knowledge of a woman' s hiv status assists in counseling on breastfeeding and helps each woman understand the benefits to herself and her infant of knowing her serostatus and the behaviors that would decrease the likelihood of acquisition and transmission of hiv. • women who are known to have hiv infections must be counseled not to breastfeed or provide their milk for the nutrition of their own or other' s infants. • in general, women who are known to be hiv seronegative should be encouraged to breastfeed. however, women who are hiv seronegative but at particularly high risk for seroconversion (e.g., injection drug users and sexual partners of known hiv-positive persons or active drug users) should be educated about hiv with an individualized recommendation concerning the appropriateness of breastfeeding. in addition, during the perinatal period, information should be provided on the potential risk for transmitting hiv through breast milk and about methods to reduce the risk for acquiring hiv infection. • each woman whose hiv status is unknown should be informed of the potential for hiv-infected women to transmit hiv during the peripartum period and through breast milk and the potential benefits to her and her infant of knowing her hiv status and how hiv is acquired and transmitted. the health care provider needs to make an individualized recommendation to assist the woman in deciding whether to breastfeed. 24 months in the two groups (83.9% versus 80.7%). children already infected by 4 months of age had a higher mortality if they were assigned to the early weaning group (73.6% versus 54.8%). additional analysis showed that in mothers with less severe hiv disease early weaning was clearly harmful to the infant. 231 arpadi et al 20 studied the growth of hiv-exposed, uninfected children who were exclusively breastfed for 4 months with rapid weaning to replacement foods or exclusively breastfed until 6 months and then continued breastfeeding with complementary foods. weight-for-age z scores dropped markedly in both groups from 4 to 15 months of age but less so in the continued breastfeeding group. length-for-age z score also dropped dramatically, but was not influenced by continued breastfeeding. even in this hiv-exposed, uninfected group of children, additional nutritional interventions are essential to complement breastfeeding beyond 6 months of age. 20 in recent years the discussion around preventing hiv transmission via breastfeeding and in the number of studies examining the important issues have increased. 98, 233, 283 the fact that intrapartum and perinatal transmission of hiv from mothers to infants has decreased markedly due to the increased utilization of antiretroviral therapy during pregnancy, delivery, and postnatally for prevention emphasizes the importance of now working harder to decrease breast milk transmission of hiv. in considering different possible interventions to decrease mother-infant hiv transmission, it is crucial to reemphasize the goals of optimizing maternal health and survival and optimizing infant health and survival at the same time. a laboratory report shows that mothers receiving highly active antiretroviral therapy (haart) while breastfeeding do have decreased whole breast milk hiv-1 viral loads (23/26 mothers had less than 50 copies/ml) compared with mothers who did not receive haart (9/25 with less than 50 copies/ml). however, the whole milk hiv-1 dna load (measured as "undetectable" at less than 10 copies/10 6 cells) was not significantly different in the haart (13 of 26 mothers)] and non-haart (15 of 23) groups. 378 hiv-1 dna is incorporated into cells found in breast milk. another group showed significantly lower hiv rna levels in the breast milk of women treated with nevirapine, zidovudine, and lamivudine compared with women not receiving antiretroviral therapy. 152 the use of maternal haart seems to decrease hiv-1 transmission via breastfeeding. one group working in mozambique, malawi, and tanzania working with mother-infants pairs receiving haart as prevention during pregnancy compared one cohort (809 mother-infant pairs) who received supplementary formula and water filters for the first 6 months of life with a second cohort (251 motherinfant pairs) breastfeeding exclusively and the mothers receiving haart for the first 6 months. the cumulative incidence rate of hiv infection at 6 months of age was 2.7% for the formula-fed infants and 2.2% for breastfed infants. through 6 months of age no apparent additional risk for late postnatal transmission of hiv was observed. 323 the petra study team working in tanzania, south africa, and uganda examined the efficacy of three shortcourse regimens of zidovudine and lamivudine in preventing early and late hiv transmission in this predominantly breastfeeding population. 332 there were four regimens: a, zidovudine and lamivudine starting at 36 weeks' gestation plus intrapartum medication and 7-days' postpartum treatment; b, same as a without the prepartum component; c, intrapartum zidovudine and lamivudine only; d, placebo. at week 6 the hiv transmission rates were 5.7% in group a, 8.9% in group b, 14.2% in group c, and 15.3% in group d. at 18 months the hiv infection rates were 15% in group a, 18% in group b, 20% in group c, and 22% in group d. although a measurable decrease in transmission at 6 weeks of age was observed, limited protection was seen at 18 months of age. an observational study from tanzania compared maternal haart for 6 months with exclusive breastfeeding and abrupt weaning at 5 to 6 months of age with a historical control of the same feeding schedule without the postnatal maternal haart. in the treatment group the cumulative hiv transmission was 4.1% at 6 weeks, 5.0% at 6 months, and 6.0% at 18 months of age. the cumulative hiv infection or death rate was 8.6% at 6 months and 13.6% at 18 months of age. the cumulative risk for hiv transmission was 1.1% between 6 and 18 months. the hiv transmission in this treatment group was half the transmission rate in the historical control group. 218 another study in sub-saharan africa with 6 months of maternal haart and exclusive breastfeeding for 6 months demonstrated 94% hiv-free survival at 12 months of age; the maternal and infant mortality rates for the treated mother-infant pairs were significantly lower than the country' s maternal and infant mortality rates. 264 antiretroviral therapy prophylaxis for infants is another investigated intervention to decrease hiv transmission via breastfeeding. in a study from cote d'ivoire comparing different groups over time, infants received zidovudine (zdv) alone as zdv prophylaxis, a single dose of nevirapine (nvp), and 7 days of zidovudine (zdv) as nvp/ zdv prophylaxis, or a single dose of nevirapine plus zidovudine and lamivudine (3tc) for 7 days as nvp/zdv/3tc prophylaxis. formula feeding (ff) was compared with exclusive shortened breastfeeding (esb) upto 4 months of age and prolonged breastfeeding (pb). the cumulative transmission rates at 18 months were 22.3% in 238 infants in the zdv + pb group, 15.9% in 169 infants in the nvp/zdv + esb group, 9.4%, in the 195 infants in the nvp/zdv +ff group, 6.8% in the 198 infants in the nvp/zdv/3tc + esb group, and 5.6% in the 126 infants in the nvp/zdv/3tc + ff group. 252 kumwenda et al 235 working in malawi demonstrated decreased hiv transmission with breastfeeding and two different infant prophylaxis regimens. at 9 months of age, they observed a 10.6% occurrence of hiv transmission for infants receiving a single dose of nevirapine plus 1 week of zidovudine compared with 5.2% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of daily nevirapine, and 6.4% in the group receiving a single dose of nevirapine plus 1 week of zidovudine plus 14 weeks of nevirapine and zidovudine. 235 in the mitra study in tanzania in which the median time of breastfeeding was 18 weeks, the hiv transmission rate at 6 months in the infants who received zidovudine plus 3tc for 1 week plus 3tc alone for breastfeeding through 6 months of age was less than 50% of the transmission rate for those infants receiving only 1 week of zidovudine plus 3tc. 217 a summary of three trials in ethiopa, india, and uganda compared a single dose of nevirapine at birth for infants with 6 weeks of daily nevirapine in predominantly breastfed infants whose mothers were counselled regarding feeding per the who/ unicef guidelines. at 6 months 87 of 986 infants in the single-dose group and 62 of 901 in the extended-dose group were hiv infected, which was not statistically significant. the authors suggested that a longer course of infant antiretroviral prophylaxis might be more effective. 388 the potential effect of breastfeeding on the hivpositive mother needs to be adequately assessed in relation to the mother's health status. from uganda and zimbabwe mbizvo et al 271 reported no difference in the number of hospital admissions or mortality between hiv-positive and hiv-negative women during pregnancy. in the 2 years after delivery the hiv-positive women had higher hospital admission (approximately two times increased risk) and death rates (relative risk greater than 10) than hiv-negative women. 271 chilongozi et al 90 reported on 2292 hiv-positive mothers from four sub-saharan sites followed for 112 months. serious adverse events occurred in 166 women (7.2%); 42 deaths occurred in the hiv-positive women, and no deaths occurred in 331 hiv-negative women. 90 several studies have examined breastfeeding relative to mothers' health and reported conflicting results. the first study from kenya demonstrated a significantly higher mortality rate in breastfeeding mothers compared with a formula-feeding group in the 2 years after delivery. the hypothesized explanation offered by the authors for this difference was increased metabolic demands, greater weight loss, and nutritional depletion. 294 a second study from south africa showed an overall lower mortality rate in the two groups with no significant difference in mortality rate in the 10 months of observation. 101 kuhn et al 227 reported no difference in mortality at 12 months after delivery between 653 women randomly assigned to a short breastfeeding group (326 women, median breastfeeding duration 4 months, 21% still breastfeeding at 12 months) and a long breastfeeding group (327 women, 90% breastfeeding at 5 months, 72% breastfeeding at 12 months, median 15 months). the hiv-related mortality rates were high (4.9%), but not associated with prolonged lactation. 227 walson et al 433 followed 535 hiv-positive women for 1 to 2 years in kenya. the mortality risk was 1.9% at 1 year and 4.8% at 2 years of follow-up. although less than 10% of women reported a hospitalization during the 2 years, they experienced various common infections (pneumonia, diarrhea, tb, malaria, stds, urinary tract infections, mastitis). breastfeeding was a significant cofactor for diarrhea and mastitis but not for pneumonia, tb, or hospitalization. 433 in summary, breastfeeding of infants by hiv-positive mothers does lead to an increased risk for hiv infection in the infants. much remains to be understood about the mechanisms of hiv transmission via breast milk and the action and efficacy of different interventions to prevent such transmission. the complete avoidance of breastfeeding is a crucial component for the prevention of perinatal hiv infection in the united states and many other countries. in resource-poor settings, where breastfeeding is the norm and where it provides vital nutritional and infection protective benefits, the who, unicef, and the joint united nations programme on hiv/ aids (unaids) recommend education, counseling, and support for hiv-infected mothers so they can make an informed choice concerning infant feeding. mothers choosing to breastfeed should receive additional education, support, and medical care to minimize the risk for hiv transmission and to optimize their own health status during and after breastfeeding. mothers choosing to use replacement feedings should receive parallel education, support, and medical care for themselves and their infants to minimize the effect of the lack of breastfeeding. good evidence now shows that antiretroviral prophylactic regimens for mothers or infants while continuing breastfeeding does decrease postnatal hiv transmission. early weaning is associated with increased morbidity and mortality. further carefully controlled research is indicated to adequately assess the risks and benefits to infants and mothers of prolonged breastfeeding with antiretroviral prophylaxis for either or both mothers and infants. along with this, hiv testing rates must be improved at the same time as increased availability and access to antenatal care, hiv prevention services, and hiv medical care for everyone must be increased. the availability and free access to antiretroviral medications must also improve. the decision about infant feeding for hivpositive mothers remains a difficult one, but this is slowly changing with increasing options. the goals remain 100% hiv transmission prevention, optimal maternal health and survival, and long-term infant health and survival. human immunodeficiency virus type 2 (hiv-2) is an rna virus in the nononcogenic, cytopathic lentivirus genus of retroviruses. it is genetically closer to simian immunodeficiency virus than to hiv-1. the clinical disease associated with hiv-2 has similar symptoms to hiv-1 infection but progresses at a slower rate to severe immunosuppression. hiv-2 is endemic in western africa and parts of the caribbean and found infrequently in europe and north and south america. 190, 305 it is transmitted via sexual contact, blood, or blood products and from mother to child. routine testing for hiv-2 is recommended in blood banks. antibody tests used for hiv-1 are only 50% to 90% sensitive for detecting hiv-2. 65 specific testing for hiv-2 is appropriate whenever clinically or epidemiologically indicated. vertical transmission occurs infrequently. ekpini et al 121 followed a large cohort of west african mothers and infants: 138 hiv-1 positive women, 132 hiv-2 positive women, 69 women seropositive for both hiv-1 and 2, and 274 hiv seronegative women. a few cases of perinatal hiv-2 transmission occurred, but no case of late postnatal transmission was observed. 121 it is probable that hiv-2 transmission via breast milk is less common than with hiv-1, but insufficient data support that the risk for transmission is zero. mothers who test positive for hiv-2 should be tested for hiv-1, and guidelines for breastfeeding should follow those for hiv-1 until additional information is available. rabies virus produces a severe infection with progressive cns symptoms (anxiety, seizures, altered mental status) that ultimately proceeds to death; few reports of survival exist. rabies occurs worldwide except in australia, antarctica, and several island groups. in 1992 more than 36,000 cases of rabies were reported to the who, a number that is probably a marked underestimate of the actual cases. 67 between 1990 and 2003, 37 cases of human rabies were reported in the united states. 70, 78 postexposure prophylaxis is given to thousands of patients each year. rabies virus is endemic in various animal populations, including raccoons, skunks, foxes, and bats. because of aggressive immunization programs, rabies in domesticated dogs and cats in the united states is uncommon. the virus is found in the saliva and tears and nervous tissue of infected animals. transmission occurs by bites, licking, or simply contact of oral secretions with mucous membranes or nonintact skin. many cases of rabies in humans now lack a history of some obvious contact with a rabid animal. this may be a result of the long incubation period (generally 4 to 6 weeks, but can be up to 1 year, with reports of incubation periods of several years), a lack of symptoms early in an infectious animal, or airborne transmission from bats in enclosed environments (caves, laboratories, houses). person-to-person transmission via bites has not been documented, although it has occurred in corneal transplants. 44 rabies viremia has not been observed in the spread of the virus. no evidence exists indicating transmission through breast milk. in the case of maternal infection with rabies, many scenarios can occur before the onset of progressive, severe cns symptoms. the progression and severity of maternal illness can preclude breastfeeding, but separation of an infant from the mother is appropriate regardless of the mother' s status and method of infant feeding (especially to avoid contact with saliva and tears). breastfeeding should not continue when the mother has symptoms of rabies, and the infant should receive postexposure immunization and close observation. an infant may received expressed breast milk, but the expression must occur without possible contamination with saliva or tears from the mother. depending on the scenario, the nature of a mother' s illness, the possible exposure of an infant to the same source as the mother, and the exposure of a child to the mother, postexposure immunization of an infant may be appropriate. a more common scenario is a mother' s apparent exposure to rabies (without exposure for the infant), necessitating postexposure immunization of the mother with rabies vaccine. in the majority of cases, in the absence of maternal illness, breastfeeding can reasonably continue during the mother' s five-dose immunization series in 28 days. in a rare situation in which apparent exposure of mother and infant to rabies occurs together, postexposure treatment of both mother and infant should be instituted, and breastfeeding can continue. respiratory syncytial virus (rsv) is a common cause of respiratory illness in children and is relatively common in adults, usually producing milder upper respiratory tract infection in adults. no evidence indicates that rsv causes intrauterine infection, adversely affects the fetus, or causes abortion or prematurity. rsv does produce infection in neonates, causing asymptomatic infection, afebrile upper respiratory tract infection, bronchiolitis, pneumonia, and apnea. mortality rate can be high in neonates, especially in premature infants and ill full-term infants, particularly those with preexisting respiratory disease (hyaline membrane disease, bronchopulmonary dysplasia) or cardiac disease associated with pulmonary hypertension. rsv is believed to be transmitted via droplets or direct contact of the conjunctiva, nasal mucosa, or oropharynx with infected respiratory secretions. documentation of rsv infection is rarely made in adults, and spread from a mother or other household contacts probably occurs before a diagnosis can be made. therefore risk for rsv transmission from breast milk is probably insignificant compared with transmission via direct or droplet contact in families. in nurseries, however, it is appropriate to make a timely diagnosis of rsv infection in neonates to isolate infants from the others and prevent spread in the nursery. ribavirin is not recommended for routine use. it is infrequently used in patients with potentially life-threatening rsv infection. rsv infection should be suspected in any infant with rhinorrhea, nasal congestion, or unexplained apnea, especially in october through march in temperate climates. prophylaxis against rsv with rsv-specific immunoglobulin iv (rsv-igiv) during this season for infants at highest risk for severe disease is appropriate. debate surrounds the topic of the effect of passively acquired antibodies (in infants from mothers before birth) against rsv on the occurrence and severity of illness in neonates and infants. it appears that a higher level of neutralizing antibody against rsv in neonates decreases the risk for severe rsv disease. 153, 239 some controversy remains concerning the measurable benefit of breastfeeding for preventing serious rsv disease. 3, 54, 115 some studies have shown benefit and others no effect. controlling for possible confounding factors (e.g., smoking, crowded living conditions) in these studies has been difficult. at this point, no reason exists to stop breastfeeding with maternal rsv infection; a potential exists for benefit from nonspecific factors in breast milk against the rsv. infants with rsv infection should breastfeed unless their respiratory status precludes it. rotavirus infections usually result in diarrhea, accompanied by emesis and low-grade fever. in severe infections the clinical course can include dehydration, electrolyte abnormalities, and acidosis and can contribute to malnutrition in developing countries. generally, every child will have at least one episode of rotavirus infection by 5 years of age. 157 in developed countries, rotavirus is often associated with diarrhea requiring hospitalization in children younger than 2 years of age, but rarely associated with death. worldwide rotavirus is the leading cause of diarrhea-related deaths in children younger than 5 years old. estimates suggest that in children younger than 5 years old rotavirus infection leads to more than 100 million occurrences of diarrhea, 2 million hospital admissions, and 500,000 deaths each year. 157 fecal-oral transmission is the most common route, but fomites and respiratory spread may also occur. spread of infection occurs most often in homes with young children or in daycare centers and institutions. in hospitalized infants or mothers with rotavirus infection, contact precautions are indicated for the duration of the illness. no evidence indicates prenatal infection from rotavirus, but perinatal or postnatal infection from contact with the mother or others can occur. no case of transmission of rotavirus via breast milk has been documented. breast milk does contain antibodies to rotavirus for up to 2 years. human milk mucin has been demonstrated to inhibit rotavirus replication and prevent experimental gastroenteritis. 457 the mechanisms of rotavirus immunity are not well understood. they are thought to be multifactorial with cell-mediated immunity limiting severity and the course of infection, while humoral immunity protects against subsequent infections. innate and adaptive responses at the level of the mucosa are probably the most important. 134 exclusive breastfeeding may decrease the likelihood of severe rotavirus-related diarrhea by as much as 90%. 93, 377 although breastfeeding does not prevent infection with rotavirus, it seems to decrease the severity of rotavirus-induced illness in children younger than 2 years old. 93, 123, 184 at least one study suggested that this may represent simply the postponement of severe rotavirus infection until an older age. 93 one study suggested that protection against rotavirus rapidly declines upon discontinuation of breastfeeding. 356 this delay in rotavirus infection until the child is older may be beneficial in that the older child may be able to tolerate the infection or illness with a lower likelihood of becoming dehydrated or malnourished. continuing breastfeeding during an episode of rotavirus illness with or without vomiting is appropriate and often helpful to the infant. no reason to suspend breastfeeding by a mother infected with rotavirus is apparent. two rotavirus vaccines (rotateq and rotarix) have been licensed for use in more than 90 countries, but less than 20 countries have routine immunization programs. additional types of rotavirus vaccines are undergoing study in various countries, specifically examining the efficacy of the vaccines in low and medium income countries. 444 some of the explanations for the slow implementation of an effective vaccine globally include differences in protection with specific vaccines in high income countries compared with low or medium income countries, the unfortunate association with intussusception in the united states, the delayed recognition of the significant rotavirus-related morbidity and mortality, and the cost of the new vaccines. the question of variable efficacy of the specific rotavirus vaccines in developed and developing countries remains an important one. several trials are examining this issue and attempting to address factors such as maternal transplacentally transferred antibodies, breastfeeding practices (especially immediately before immunization with a live oral rotavirus vaccine), stomach acid, micronutrient malnutrition, interfering gut flora, and differences in the epidemiology of rotavirus in different locations. 327 evidence indicates that maternal immunization with rotavirus vaccine can increase both transplacental acquisition of antibodies and secretory iga in breast milk. 334 additionally, oral rotavirus vaccines have been able to stimulate a good serologic response in both formula-fed and breastfed infants, although the antigen titers may need to be modified to create an optimal response in all infants. 86 the actual protective effect of these vaccines in different situations and strategies will require measurement in ongoing prospective studies. congenital rubella infection has been well described, and the contributing variables to infection and severe disease have been elucidated. the primary intervention to prevent congenital rubella has been to establish the existence of maternal immunity to rubella before conception, including immunization with rubella vaccine and reimmunization if indicated. perinatal infection is not clinically significant. postnatal infection occurs infrequently in children younger than 1 year of age because of passively acquired maternal antibodies. the predominant age of infection is 5 to 14 years old, and more than half of those with infections are asymptomatic. postnatal rubella is a self-limited, mild viral infection associated with an evanescent rash, shotty adenopathy, and low-grade transient fever. it most often occurs in the late winter and spring. infants with congenital infection shed the virus for prolonged periods from various sites and may serve as a source of infection throughout the year. contact isolation is appropriate for suspected and proved congenital infection for at least 1 year, including exclusion from day care and avoidance of pregnant women, whereas postnatal rubella infection requires droplet precautions for 7 days after the onset of rash. rubella virus has been isolated from breast milk after natural infection (congenital or postnatal) and after immunization with live attenuated vaccine virus. both iga antibodies and immunoreactive cells against rubella have been identified in breast milk. breastfed infants can acquire vaccine virus infection via milk but are asymptomatic. because postpartum infection with this virus (natural or vaccine) is not associated with clinically significant illness, no reason exists to prevent breastfeeding after congenital infection, postpartum infection with this virus, or maternal immunization with rubella vaccine. severe acute respiratory syndrome (sars) is a term that could be applied to any acute serious respiratory illness caused by or associated with a variety of infections agents; since 2003, however, it has been linked with sars-associated coronavirus (sars-cov). in the global outbreak of 2002 to 2003, more than 8400 probable cases of sars and more than 800 deaths occurred. more than the actual number of affected individuals or its associated mortality rate (approximately 10% overall, and closer to 50% in persons older than 65 years of age), it was what we did not know about this new unusual illness, and the tremendous publicity surrounding it, that made sars such a sensation. we now know the cause of this illness, known as the sars-cov. sars-cov was shown not to be closely related to the previously characterized coronavirus groups. 265, 350 despite intense international collaboration to study the illness and the virus, many things are not known, such as the degree of infectiousness, the actual period of transmissibility, all the modes of transmission, how many people have an asymptomatic infection compared with those with symptoms or severe illnesses, how to make a rapid diagnosis of confirmed cases, and where it originated. at least 21 cases of probable sars in children have been described in the literature. 42, 187, 380, 387 in general, the illness in children is a mild, nonspecific respiratory illness, but in adolescents and adults it is more likely to progress to severe respiratory distress. it has been reported that children are less likely to transmit sars than adults. 187 the overall clinical course, the radiologic evolution, and the histologic findings of these illness are consistent with the host' s immune response playing a significant role in disease production. five infants were born to mothers with confirmed sars. the infants were born prematurely (26 to 37 weeks), presumably due to maternal illness. although two of the five infants had serious abdominal illnesses (other coronaviruses have been associated with reported outbreaks of necrotizing enterocolitis), the presence of sars-cov could not be demonstrated in any of these infants. 380 no evidence of vertical transmission of sars is available. the mode of feeding for any of the reported cases of young children with sars or the infants born to mothers with sars was not mentioned. as with other respiratory viruses predominantly transmitted by droplets, transmission via breast milk is an insignificant mode of transmission, if it occurs at all. the benefits of breastfeeding being what they are, mothers with sars should continue breastfeeding if they are able, or expressed breast milk can be given to an infant until the mother is able to breastfeed. in this era of worry about biologic terrorism, smallpox is an important concern. the concern for infants (breastfed or formula-fed) is direct contact with mothers or household members with smallpox. smallpox is highly contagious in the household setting due to person-to-person spread via droplet nuclei or aerosolization from the oropharynx and direct contact with the rash. additional potential exposures for infants include the release of a smallpox aerosol into the environment by terrorists, contact with a smallpox-contaminated space or the clothes of household members exposed to an aerosol, and infection via contact with a mother' s or a household member' s smallpox vaccination site. these risks are the same for breastfed and formulafed infants. no evidence for transmission of the smallpox virus via breast milk exists. a contact is defined as a person who has been in the same household or had face-to-face contact with a patient with smallpox after the onset of fever. patients do not transmit infection until after progression from the fever stage to the development of the rash. an exposed contact does not need to be isolated from others during the postcontact observation period (usually 17 days) until the person develops fever. temperature should be monitored daily in the exposed contact. personal contact and breastfeeding between mother and infant can continue until the onset of fever, when immediate isolation (at home) should begin. providing expressed breast milk for the infant of a mother with smallpox should be avoided because of the extensive nature of the smallpox rash and the possibility of contamination (from the rash) of the milk during the expression process. no literature documents transmission of the smallpox virus via expressed breast milk. the other issue for breastfeeding infants is the question of maternal vaccination with smallpox in a preexposure event vaccination program. children older than 1 year of age can be safely and reasonably vaccinated with smallpox in the face of a probable smallpox exposure. smallpox vaccination of infants younger than 1 year of age is contraindicated. breastfeeding is listed as a contraindication to vaccination in the preevent vaccination program. it is unknown whether vaccine virus or antibodies are present in breast milk. the risk for infection due to contact or aerosolization of virus from a mother' s smallpox vaccination site is the same for breastfed and formula-fed infants. the advisory committee on immunization practices also does not recommend preevent smallpox vaccination of children younger than 18 years old. 443 a report documents tertiary contact vaccinia in a breastfeeding infant. 140 a united states military person received a primary smallpox vaccination and developed a local reaction at the inoculation site. despite reportedly observing appropriate precautions, the individual' s wife developed vesicles on both areolae (secondary contact vaccinia). subsequently, the breastfeeding infant developed lesions on her philtrum, cheek, and tongue. both the mother and infant remained well and the infections resolved without therapy. culture and pcr testing confirmed vaccinia in both the mother' s and the infant' s lesions. the breast milk was not tested. 140 in a review from 1931 to 1981, sepkowitz 375 reported on 27 cases of secondary vaccinia in households. the cdc reported 30 suspected cases of secondary/tertiary vaccinia with 18 of those cases confirmed by culture or pcr. the 30 cases were related to 578,286 vaccinated military personnel. this is an incidence of 5.2 cases per 100,000 vac cinees and 7.4 cases per 100,000 primary vaccinees. 79 in a separate report on the civilian preevent smallpox vaccination program, 37,802 individuals were vaccinated between january and june 2003, and no cases of contact vaccinia were reported. 77 the risk for contact vaccinia is low. the risk is from close or intimate contact. in the above-mentioned case, the risk for the infant was contact with the mother' s breasts, the inadvertent site of her contact vaccinia. breastfed and formulafed infants are equally at risk from close contact in the household of a smallpox vaccinee or a case of secondary vaccinia, and separation from the individual is appropriate in both situations. if the breast of the nursing mother is not involved, expressed breast milk can be given to the infant. tt virus (ttv) is a recently identified virus found in a patient (tt) with posttransfusion hepatitis not associated with the other hepatitis-related viruses a through g. ttv has been described as an unenveloped, circular, single-stranded dna virus. 311 this virus is prevalent in healthy individuals, including healthy blood donors, and has been identified in patients with hepatitis. ttv dna has been detected in infants of ttv-positive and ttvnegative mothers. ohto et al 310 reported no ttv dna was detected in cord blood from 38 infants, and it was detected in only 1 of 14 samples taken at 1 month of age. they noted an increasing prevalence from 6 months (22%) to 2 years (33%), which they ascribed to acquisition via nonparenteral routes. in comparisons of the ttv dna in ttvpositive mothers and their ttv-positive infants, 6 of 13 showed high level nucleotide sequence similarity, and 7 of 13 differed by greater than 10%. 310 schröter et al 371 reported on ttv dna in breast milk examined retrospectively. notably, ttv dna was detected in 22 of 23 serum samples of infants at 1 week of age, who were born to 22 women viremic for ttv dna. twenty-four women who were negative for ttv dna gave birth to 24 children who were initially negative for ttv dna and remained negative throughout the observation period (mean 7.5 months, range 1 to 28 months). ttv dna was detected in 77% of breast milk samples from ttv viremic women and in none of the breast milk samples from ttv-negative women. no clinical or laboratory evidence of hepatitis was found in the 22 children who were observed to be ttv dna positive during the period of the study. 371 other authors have reported ttv in breast milk detected by pcr. they describe the absence of ttv dna in infants at 5 days and 3 months of age, and 4 of 10 infants were positive for ttv dna at 6 months of age, suggesting the late acquisition of infection via breastfeeding. 197 tt virus is transmitted in utero and is found in breast milk. no evidence of clinical hepatitis in infants related to ttv infection and no evidence for a late chronic hepatitis exist. given the current available information, no reason to proscribe breastfeeding by ttv-positive mothers is compelling. certainly more needs to be understood concerning the chronic nature of this infection and the possible pathogenesis of liver disease. no documented evidence indicates that women with breast cancer have rna of tumor virus in their milk. no correlation between rna-directed dna polymerase activity has been found in women with a family history of breast cancer. rna-directed dna polymerase activity, a reserve transcriptase, is a normal feature of the lactating breast. 91, 129, 352 epidemiologic data conflict with the suggestion that the tumor agent is transmitted through the breast milk. the incidence of breast cancer is low among groups who had nursed their infants, including lower economic groups, foreign-born groups, and those in sparsely populated areas. 262 the frequency of breast cancer in mothers and sisters of a woman with breast cancer is two to three times that expected by chance. this could be genetic or environmental. cancer actually is equally common on both sides of the family of an affected woman. if breast milk were the cause, it should be transmitted from mother to daughter. when mother-daughter incidence of cancer was studied, no relationship was found to breastfeeding. sarkar et al 360 reported that human milk, when incubated with mouse mammary tumor virus, caused degradation of the particular morphology and decreased infectivity and reverse transcriptase activity of the virions. they suggest that the significance of this destructive effect of human milk on mouse mammary tumor virus may account for the difficulty in isolating the putative human mammary tumor agent. sanner 359 showed that the inhibitory enzymes in milk can be removed by special sedimentation technique. he ascribes the discrepancies in isolating virus particles in human milk to these factors, which inhibit rna-directed dna po lymerase. the fear of cancer in breastfed female offspring of a woman with breast cancer does not justify avoiding breastfeeding. breastfed women have the same breast cancer experience as nonbreastfed women, and no increase is seen in benign tumors. daughters of breast cancer patients have an increased risk for developing benign and malignant tumors because of their heredity, not because of their breastfeeding history. 280, 287 unilateral breastfeeding (limited to the right breast) is a custom of tanka women of the fishing villages of hong kong. ing et al 194 investigated the question, "does the unsuckled breast have an altered risk for cancer?" they studied breast cancer data from 1958 to 1975. breast cancer occurred equally in the left and the right breasts. comparison of patients who had nursed unilaterally with nulliparous patients and with patients who had borne children but not breastfed indicated a highly significantly increased risk for cancer in the unsuckled breast. the authors conclude that in postmenopausal women who have breastfed unilaterally, the risk for cancer is significantly higher in the unsuckled breast. they thought that breastfeeding may help protect the suckled breast against cancer. 194 others 274 have suggested that tanka women are ethnically a separate people and that left-sided breast cancer may be related to their genetic pool and not to their breastfeeding habits. no mention has been made of other possible influences, such as the impact of their role as "fishermen" or any inherent trauma to the left breast. 274 in 1926, lane-claypon 240 stated that a breast that had never lactated was more liable to become cancerous. nulliparity and absence of breastfeeding had been considered important risk factors for breast cancer. macmahon et al 262 reported in 1970 that age at first full-term pregnancy was the compelling factor, and the younger the mother, the less the risk. in a collective review of the etiologic factors in cancer of the breast in humans, papaioannou 325 concludes, "genetic factors, viruses, hormones, psychogenic stress, diet, and other possible factors, probably in that order of importance, contribute to some extent to the development of cancer of the breast." 325 wing 449 concluded in her 1977 review on human milk and health that "in view of the complete absence of any studies showing a relationship between breastfeeding and increased risk of breast cancer, the presence of virus-like particles in breast milk should not be a contraindication to breastfeeding." henderson et al 173 gradually, studies have appeared challenging the dogma. brinton et al, 52 mctiernan and thomas, 276 and layde et al 245 showed the clearly protective effects of breastfeeding. another example is a study conducted to clarify whether lactation has a protective role against breast cancer in an asian people, regardless of confounding effects of age at first pregnancy, parity, and closely related factors. 458 in a hospital-based case-control study of 521 women without breast cancer, statistical adjustment for potential confounders and a likelihood ratio test for linear trend were done by unconditional logistic regression. total months of lactation regardless of parity was the discriminator. regardless of age of first pregnancy and parity, lactation had an independent protective effect against breast cancer in japanese women. 458 although breast cancer incidence is influenced by genetics, stress, hormones, and pregnancy, breastfeeding clearly has a protective effect. "there is a reduction in the risk of breast cancer among premenopausal women who have lactated. no reduction in the risk of breast cancer occurred among postmenopausal women with a history of lactation," according to newcombe et al, 299 reporting a multicenter study in 1993. varicella-zoster virus infection (varicella/chickenpox, zoster/shingles) is one of the most communicable diseases of humans, in a class with measles and smallpox. transmission is thought to occur via respiratory droplets and virus from vesicles. varicella in pregnancy is a rare event, although disease can be more severe with varicella pneumonia, and can be fatal. congenital varicella-zoster virus infection occurs infrequently, causing abortion, prematurity, and congenital malformations. a syndrome of malformations has been carefully described with congenital varicella-zoster virus infection, typically involving limb deformity, skin scarring, and nerve damage, including to the eye and brain. 148 perinatal infection can lead to severe infection in infants if maternal rash develops 5 days or less before delivery and within 2 days after delivery. illness in infants usually develops before 10 days of age and is believed to be more severe because of the lack of adequate transfer of antibody from the mother during this period and transplacental spread of virus to the fetus and infant during viremia in the mother. varicella in a mother occurring before 5 days before delivery allows sufficient formation and transplacental transfer of antibodies to the infant to ameliorate disease even if the infant is infected with varicella-zoster virus. mothers who develop varicella rash more than 2 days after delivery are less likely to transfer virus to the infant transplacentally; they pose a risk to the infants from postnatal exposure, which can be diminished by the administration of varicellazoster ig to the infant. postnatal transmission is believed to occur through aerosolized virus from skin lesions or the respiratory tract entering the susceptible infant's respiratory tract. airborne precautions are therefore appropriate in the hospital setting. infants infected with varicella-zoster virus in utero or in the perinatal period (younger than 1 month of age) are more likely to develop zoster (reactivation of latent varicella-zoster virus) during childhood or as young adults. postnatal varicella from nonmaternal exposure can occur but is generally mild when it develops after 3 weeks of age or when a mother has passed on antibodies against varicella-zoster virus via the placenta. severe postnatal varicella does occur in premature infants or infants of varicella-susceptible mothers. when a mother' s immune status relative to varicella-zoster virus is uncertain and measurement of antibodies to varicella-zoster virus in mother or infant cannot be performed promptly (less than 72 hours), administration of vzig 81 or ivig to the infant exposed to varicella or zoster in the postnatal period is indicated. ideally a mother' s varicella status should be known before pregnancy, when varicella virus vaccine could be given if indicated. varicella-zoster virus virus has not been cultured from milk, but varicella-zoster virus dna has been identified in breast milk. 459 antibody against varicella-zoster virus has also been found in breast milk. 270 breast milk from mothers who had received the varicella vaccine in the postpartum period was tested for varicella-zoster virus dna. varicella dna was not detected in any of the 217 breast milk samples from the 12 women, all of whom seroconverted after vaccination. 45 one case of suspected transfer of varicella-zoster virus to an infant via breastfeeding has been reported, but virus may have been transmitted by respiratory droplet or exposure to rash before the mother began antiviral therapy. 459 isolation of an infant from the mother with varicella and interruption of breastfeeding should occur only while the mother remains clinically infectious, regardless of the method of feeding. as soon as the infant has received varicella-zoster ig, expressed breast milk can be given to an infant if no skin lesions involve the breasts. persons with varicella rash are considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted, usually in 6 to 10 days. immunocompetent mothers who develop zoster can continue to breastfeed if the lesions do not involve the breast and can be covered because antibodies against varicella-zoster virus are provided to the infant via the placenta and breast milk and will diminish the severity of disease, even if not preventing it. conservative management in this scenario would include giving an infant varicella-zoster ig as well (see table 13 -5). 331 it is estimated that 150 to 300 asymptomatic cases of west nile infection occur for every 20 febrile illnesses and for every one case of meningoencephalitis associated with west nile virus. west nile fever is usually a mild illness of 3 to 6 days' duration. the symptoms are relatively nonspecific, including malaise, nausea, vomiting, headache, myalgia, lymphadenopathy, and rash. west nile disease is characterized by severe neurologic symptoms (e.g., meningitis, encephalitis, or acute flaccid paralysis, and occasionally optic neuritis, cranial nerve abnormalities, and seizures). children are infrequently sick with west nile virus infection and infants younger than 1 year of age have rarely been reported. 331 the case-fatality rate for 2003 in the united states was approximately 2.5%, but has been reported as high as 4% to 18% in hospitalized patients. the case-fatality rate for persons older than 70 years of age is considered to be higher, 15% to 29% among hospitalized patients in outbreaks in romania and israel. 331 the primary mechanism of transmission is via a mosquito bite. mosquitoes from the genus culex are primary vectors. the bird-mosquito-bird cycle serves to maintain and amplify the virus in the environment. humans and horses are incidental hosts. the pathogenesis of the infection is believed to occur via replication of the virus in the skin and lymph nodes, leading to a primary viremia that seeds secondary sites before a second viremia causes the infection of the cns and other affected organs. 59, 111 transmission has been reported in rare instances during pregnancy 7,73 via organ transplant 199 and percutaneously in laboratory workers. 75 a study of west nile virus infection in pregnancy documented four miscarriages, two elective abortions, and 72 live births. cord blood samples were tested in 55 infants and 54 of 55 were negative for anti-west nile virus igm. three infants had west nile virus infection, which could have been acquired congenitally. three of 7 infants who had congenital malformations might have been caused by maternal west nile virus infection based on timing in pregnancy, but no evidence of west nile virus etiology is conclusive. 312 west nile virus transmission occurs via blood and blood product transfusion, 186 and the incidence has been estimated to be as high as 21 per 10,000 donations during epidemics in specific cities. 40 no evidence of direct person-to-person transmission without the mosquito vector has been found. one case of possible west nile virus transmission via breastfeeding has been documented. 74 the mother acquired the virus via packed rbc transfusions after delivery. the second unit of blood she received was associated with other blood products from the same donation causing west nile infection in another transfusion recipient. eight days later the mother had a severe headache and was hospitalized with fever and a csf pleocytosis on day 12 after delivery. the mother' s csf was positive for west nile virus-specific igm antibody. the infant had been breastfed from birth through the second day of hospitalization of the mother. samples of breast milk were west nile virus-specific igg and igm positive on day 16 after delivery and west nile virus-specific igm positive on day 24. the same milk was west nile virus rna positive by pcr testing on day 16, but not on day 24 after delivery. the infant tested positive for west nile virus-specific igm in serum at day 25 of age, but remained well without fever. no clear-cut exposure to mosquitoes for the infant were reported. the cord blood and placenta were not available to be tested. igm antibodies can be found in low concentrations in breast milk, but this is not common or as efficient as the transfer of iga, secretory iga, or igg into breast milk. a review of west nile virus illness during the breastfeeding identified six occurrences of breastfeeding during maternal west nile virus illness. 177 five of the six infants had no illness or detectable antibodies to west nile virus in their blood. one infant developed a rash and was otherwise well after maternal west nile virus illness, but was not tested for west nile virus infection. two infants were identified who developed west nile virus illness while breastfeeding, but no preceding west nile virus infection was demonstrated in their mothers. two other breastfeeding infants developed west nile virus-specific antibodies after their mothers acquired west nile virus illness in the last week of pregnancy, but congenital infection could not be ruled out. live virus was not cultured from 45 samples of breast milk from mothers infected with west nile virus during pregnancy, but west nile virus rna was detected in two samples and 14 samples had igm antibodies to west nile virus. 177 the above data suggest that west nile virus infection through breastfeeding is rare. to date evidence of significant disease due to west nile virus infection in young breastfeeding children is lacking. at this time, no reason exists to proscribe breastfeeding in the case of maternal west nile virus infection if a mother is well enough to breastfeed. as with many other maternal viral illnesses, by the time the diagnosis is made in a mother, the infant may have already been exposed during maternal viremia and possible virolactia. the infant can and should continue to receive breast milk for the potential specific and nonspecific antiviral immunologic benefits. yellow fever virus is a flavivirus which is transmitted to humans by infected aedes and haemogogus mosquitos in tropical areas of south america and africa. large outbreaks occur when mosquitos in a populated area become infected from biting viremic humans infected with yellow fever virus. transmission from the mosquitos to other humans occurs after an incubation period in the mosquito of 8 days. direct person-to-person spread has not been reported. illness due to yellow fever virus usually begins after an incubation period of 3 to 6 days, with acute onset of headache, fever, chills, and myalgia. photophobia, back pain, anorexia, vomiting, and restlessness are other common symptoms. the individual is usually viremic for the first 4 days of illness until the fever and other symptoms diminish. liver dysfunction and even failure can develop as can myocardial dysfunction. cns infection is uncommon but symptoms can include seizures and coma. medical care should include intensive supportive care and fluid management. one case of congenital infection after immunization of a pregnant woman with the attenuated vaccine strain has been reported. one of 41 infants whose mothers had inadvertently received the yellow fever virus vaccine during pregnancy developed igm and elevated neutralizing antibodies against the yellow fever virus without any evidence of illness or abnormalities. 418 a more recent study 404 from brazil examined inadvertent yellow fever virus immunization during pregnancy during a mass vaccination campaign in 2000; 480 pregnant women received the yellow fever virus at a mean of 5.7 weeks' gestation, the majority of whom did not know their pregnancy status at the time. seroconversion occurred in 98.2% of the women after at least 6 weeks after vaccination. mild postvaccination illness (headache, fever, or myalgia) was reported by 19.6% of the 480 women. the frequency of malformations, miscarriages, stillbirths, and premature deliveries was similar to that found in the general population. at the 12-month follow-up point, 7% of the infants still demonstrated neutralizing antibodies against yellow fever virus, but after 12 months only one child was still seropositive. 404 transmission of the yellow fever vaccine virus through breastfeeding was recently reported from brazil. 85 the mother was immunized during a yellow fever epidemic in a nonendemic area in brazil; 15 days after delivering a healthy female infant (39 weeks' gestational age) the mother received the 17dd yellow fever vaccine, and 5 days later the mother reported headache, malaise, and low-grade fever that persisted for 2 days. the mother continued breastfeeding and did not seek medical care for herself. at 23 days of age the infant became irritable, developed fever, and refused to nurse. the infant developed seizures and subsequent evaluation of the infant demonstrated an abnormal csf and ct of the brain showed bilateral areas of diffuse low density suggestive of inflammation and consistent with encephalitis. yellow fever-specific igm antibodies were identified in the infant' s serum and csf. reverse-transcriptase polymerase chain reaction (rt-pcr) testing of the csf also demonstrated yellow fever virus rna identical to the 17dd yellow fever vaccine virus. breast milk and maternal serum were not tested for yellow fever virus. 85 yellow fever virus, wild or vaccine type, has not been identified in human breast milk, although another flavivirus, west nile virus, has been detected in milk from a few lactating women with west nile virus infection. 177 (see the section on west nile virus.) yellow fever vaccine-associated neurologic disease occurs at different rates in different age-groups, including 0.5 to 4.0 cases per 1000 infants younger than 6 months of age. 285 the 17d-derived yellow fever vaccines are contraindicated in infants younger than 6 months of age. since 2002, the advisory committee on immunization practices has recommended, based on theoretical risk, that yellow fever vaccine be avoided in nursing mothers, except when exposure in highrisk yellow fever endemic areas is likely to occur. 76 no case of transmission of yellow fever virus from an infected mother to her infant via breastfeeding or breast milk has been reported. published information on the severity of yellow fever virus infection in infants younger than 1 year of age, potential protection from passively acquired antibodies, or protection from breast milk is limited. no information on a differential risk in breastfed versus formula-fed infants is available. given the well documented method of transmission of yellow fever virus via mosquitos, and the lack of evidence of transmission via breast milk, it makes more sense to protect all infants against mosquito bites than to proscribe breastfeeding, even in the mother infected with yellow fever virus. continued breastfeeding or use of expressed breast milk will depend on a mother's health status and ability to maintain the milk supply while acutely ill. if another source of feeding is readily available then temporarily discarding expressed breast milk for at least 4 days of acute illness in the mother is a reasonable precaution. lyme disease, as with other human illnesses caused by spirochetes, especially syphilis, is characterized by a protean course and distinct phases (stages) of disease. lyme borreliosis was described in europe in the early twentieth century. since the 1970s, tremendous recognition, description, and investigation of lyme disease have occurred in the united states and europe. public concern surrounding this illness is dramatic. lyme disease is a multisystem disease characterized by involvement of the skin, heart, joints, and nervous system (peripheral and central). stages of disease are identified as early localized (erythema migrans, often accompanied by arthralgia, neck stiffness, fever, malaise, and headache), early disseminated (multiple erythema migrans lesions, cranial nerve palsies, meningitis, conjunctivitis, arthralgia, myalgia, headache, fatigue, and, rarely, myocarditis), and late disease (recurrent arthritis, encephalopathy, and neuropathy). the varied manifestations of disease may relate to the degree of spirochetemia, the extent of dissemination to specific tissues, and the host' s immunologic response. the diagnosis of lyme disease is often difficult in part because of the broad spectrum of presentations, inapparent exposure to the tick, and the lack of adequately standardized serologic tests. culture of the spirochete, borrelia burgdorferi, is not readily available. enzyme-linked immunosorbent assay (elisa), immunofluorescent assay, and immunoblot assay are the usual tests. pcr detection of spirochetal dna requires additional testing in clinical situations to clarify and standardize its utility. gardner 142 reviewed infection during pregnancy, summarizing a total of 46 adverse outcomes from 161 cases reported in the literature. the adverse outcomes included miscarriage and stillbirth (11% of cases), perinatal death (3%), congenital anomalies (15%), and both early-and late-onset progressive infection in the infants. silver 384 reviewed 11 published reports and concluded that lyme disease during pregnancy is uncommon, even in endemic areas. although the spirochete can be transmitted transplacentally, a significant immune response in the fetus is often lacking, and the association of lyme infection with congenital abnormalities is weak. 401, 448 little published information exists on whether b. burgdorferi can be transmitted via breast milk. one report showed the detection of b. burgdorferi dna by pcr in the breast milk of two lactating women with untreated erythema migrans, but no evidence of lyme disease or transmission of the spirochete in the one infant followed for 1 year. 369 no attempt to culture the spirochete was made, so it is not possible to determine if the detectable dna was from viable spirochetes or noninfectious fragments. in that same study of 56 women with untreated erythema migrans who had detectable b. burgdorferi dna in the urine, 32 still had detectable dna in the urine 15 to 30 days after starting treatment, but none had it 6 months after initiating therapy. ziska et al 466 reported on the management of nine cases of lyme disease in women associated with pregnancy; seven of the nine women were symptomatic at conception and six received antibiotics throughout pregnancy. follow-up of the infants showed no transmission of lyme disease, even in the seven infants who had been breastfed. 466 the lack of adequate information on transmission of b. burgdorferi via breast milk cannot be taken as proof that it is not occurring. if one extrapolates from data on syphilis and the treponema pallidum spirochete, it would be prudent to discuss the lack of information on the transmission of b. burgdorferi via breast milk with the mother or parents and to consider withholding breast milk at least until therapy for lyme disease has begun or been completed. if the infection occurred during pregnancy and treatment has already been completed, an infant can breastfeed. if infection occurs postpartum or the diagnosis is made postpartum, infant exposure may have already occurred. again, discussion with the mother or parents about withholding versus continuing breastfeeding is appropriate. after prenatal or postnatal exposure, an infant should be closely observed and empiric therapy considered if the infant develops a rash or symptoms suggestive of lyme borreliosis. treatment of mother and infant with ceftriaxone, penicillin, or amoxicillin is acceptable during breastfeeding relative to the infant' s exposure to these medications. doxycycline should not be administered for more than 14 days while continuing breastfeeding because of possible dental staining in the neonate. continued surveillance for viable organisms in breast milk and evidence of transmission through breastfeeding is recommended. a large body of information is available on various "lyme vaccines" used in dogs, but these vaccines are only partially protective and must be repeated yearly. preliminary information suggests that a vaccine for use in humans safely produces good serologic responses, but protective efficacy has not been demonstrated, and no information exists on its use during pregnancy or breastfeeding. syphilis is the classic example of a spirochetal infection that causes multisystem disease in various stages. both acquired syphilis and congenital syphilis are well-described entities. acquired syphilis is almost always transmitted through direct sexual contact with open lesions of the skin or mucous membranes of individuals infected with the spirochete, treponema pallidum. congenital syphilis occurs by infection across the placenta (placentitis) at any time during the pregnancy or by contact with the spirochete during passage through the birth canal. any stage of disease (primary, secondary, tertiary) in a mother can lead to infection of the fetus, but transmission in association with secondary syphilis approaches 100%. infection with primary syphilis during pregnancy, without treatment, leads to spontaneous abortion, stillbirth, or perinatal death in 40% of cases. similar to acquired syphilis, congenital syphilis manifests with moist lesions or secretions from rhinitis (snuffles), condylomata lata, or bullous lesions. these lesions and secretions contain numerous spirochetes and are therefore highly infectious. postnatal infection of an infant can occur through contact with open, moist lesions of the skin or mucous membranes of the mother or other infected individuals. if the mother or infant has potentially infectious lesions, isolation from each other and from other infants and mothers is recommended. if lesions are on the breasts or nipples, breastfeeding or using expressed milk is contraindicated until treatment is complete and the lesions have cleared. spirochetes are rarely identified in open lesions after more than 24 hours of appropriate treatment. penicillin remains the best therapy. evaluation of an infant with suspected syphilis should be based on the mother' s clinical and serologic status, history of adequate therapy in the mother, and the infant' s clinical status. histologic examination of the placenta and umbilical cord, serologic testing of the infant' s blood and csf, complete analysis of the csf, long bone and chest radiographs, liver function tests, and a complete blood cell count are all appropriate given the specific clinical situation. treatment of the infant should follow recommended protocols for suspected, probable, or proven syphilitic infection. 96 no evidence indicates transmission of syphilis via breast milk in the absence of a breast or nipple lesion. when a mother has no suspicious breast lesions, breastfeeding is acceptable as long as appropriate therapy for suspected or proven syphilis is begun in the mother and infant. giardiasis is a localized infection limited to the intestinal tract, causing diarrhea and malabsorption. immunocompetent individuals show no evidence of invasive infection, and no evidence exists that documents fetal infection from maternal infection during pregnancy. giardiasis is rare in children younger than 6 months of age, although neonatal infection from fecal contamination at birth has been described. 22 human milk has an in vivo protective effect against giardia lamblia infection, as documented by work from central africa, where the end of breastfeeding heralds the onset of giardia infection. 145 this has been reaffirmed in undeveloped countries around the world. the protective effect of breast milk has been identified in the milk of noninfected donors. 151 the antiparasitic effect does not result from specific antibodies but rather from lipase enzymatic activity. the lipase acts in the presence of bile salts to destroy the trophozoites as they emerge from their cysts in the gi tract. hernell et al 175 demonstrated that free fatty acids have a marked giardiacidal effect, which supports the conclusion that lipase activity releasing fatty acids is responsible for killing g. lamblia. g. lamblia have also been reported to appear in the mother' s milk, and the parasite has been transmitted to newborns via that route. the exact relationship of breastfeeding to transmission of g. lamblia and the effect on infants continue to be studied, even though symptomatic infection in breastfed infants is rare. 151 one report from the middle east suggests that even partial breastfeeding is protective against infection with intestinal parasites, including cryptosporidium and giardia lamblia. 41 breastfeeding by mothers with giardiasis is problematic mainly because of the medications used for therapy. metronidazole' s safety in infants has not been established, and little information is available on quinacrine hydrochloride and furazolidone in breast milk. paromomycin, a nonabsorbable aminoglycoside, is a reasonable alternative recommended for treatment of pregnant women. breastfeeding by a mother with symptomatic giardiasis is acceptable when consideration is given to the presence of the therapeutic agents in the breast milk. hookworm infection, most often caused by ancylostoma duodenale and necator americanus, is common in children younger than the age of 4 years, and there is at least one report on infantile hookworm disease from china. 374 this publication from the chinese literature reports hundreds of cases of infantile hookworm disease that include the common symptoms of bloody stools, melena, anorexia, listlessness, and edema. anemia, eosinophilia and even leukemoid reactions occur as part of the clinical pictures in young children. they also note at least 20 cases of hookworm diseases in newborn infants younger than 1 month of age. in the discussion of infantile hookworm infection, they note four routes of infection: direct contact with contaminated soil, "sand-stuffed" diapers, contaminated "washed/wet" diapers, and vertical equal to transmammary transmission or transplacental transmission. they postulated that infection of infants before 40 to 50 days of age would most likely be due to transplacental transmission and infection before environmental contact would most likely be due to transmammary transmission. ample evidence is available in veterinary medicine of transmammary spread of helminths. 302, 382 at least two reports suggest the possibility of transmammary transmission of hookworms in humans. setasuban et al 376 described the prevalence of necator americanus in 128 nursing mothers as 61% and identified n. americanus in breast milk in one case. nwosu 303 documented positive stool samples for hookworms in 33 of 316 neonates (10%) at 4 to 5 weeks of age in southern nigeria. the majority of neonatal infections were due to ancylostoma duodenale although necator americanus is more prevalent in that area of nigeria. examination of colostral milk did not demonstrate any hookworm larvae. 303 additional epidemiologic work is necessary to determine the potential significance of transmammary spread of helminths in humans, and more careful examination of breast milk as a source of hookworm infection is required before reasonable recommendation are possible. malaria is recognized as a major health problem in many countries. the effect of malaria infection on pregnant and lactating women and thus on the developing fetus, neonate, and growing infant can be significant. the four species of malaria, plasmodium vivax, p. ovale, p. malariae, and p. falciparum, vary in the specific aspects of the disease they produce. p. vivax exists throughout the world, but p. falciparum predominates in the tropics and is most problematic in its chloroquine-resistant form. malaria in the united states is most often seen in individuals traveling from areas where malaria is endemic. the parasite can exist in the blood for weeks, and infection with p. vivax and p. malariae can lead to relapses years later. transmission occurs through the bite of the anopheline mosquito and can occur via transfusion of blood products and transplacentally. congenital malaria is rare but seems to occur more often with p. vivax and p. falciparum. it usually presents in the first 7 days of life (range 1 day to 2 months). it may resemble neonatal sepsis, with fever, anemia, and splenomegaly occurring in the most neonates and hyperbilirubinemia and hepatomegaly in less than half. malaria in infants younger than 3 months of age generally manifests with less severe disease and death than in older children. possible explanations include the effect of less exposure to mosquitoes, passive antibody acquired from the mother, and the high level of fetal hemoglobin in infants at this age. 22 the variations in the infection rates in children younger 3 months of age during the wet and dry seasons support the idea that postnatal infection is more common than congenital infection. no evidence indicates that malaria is transmitted through breast milk. the greatest risk to infants is exposure to the anopheline mosquito infected with malaria. the main issues relative to malaria and breastfeeding are how to protect both mothers and infants effectively from mosquitoes and what drugs for treating malaria in mothers are appropriate during lactation. protection from mosquito bites includes screened-in living areas, mosquito nets while sleeping, protective clothing with or without repellents on the clothes, and community efforts to eradicate the mosquitoes. chloroquine, quinine, and tetracycline are acceptable during breastfeeding. sulfonamides should be avoided in the first month of an infant' s life, but pyrimethamine-sulfadoxine (fansidar) can be used later. mefloquine is not approved for infants or pregnant women. however, the milk/plasma ratio for mefloquine is less than 0.25, there is a large volume of distribution of the drug, high protein binding of the drug limits its presence in breast milk, and the relative importance of breastfeeding in areas where malaria is prevalent shifts the risk/benefit ratio in favor of treatment with mefloquine. the single dose recommended for treatment or the onceweekly dose for prevention allows for continued breastfeeding with discarding of the milk for short periods after a dose (1 to 6 hours). maternal plasma levels of primaquine range from 53 to 107 ng/ml, but no information is available on levels in human milk. primaquine is used in children, and once daily dosing in the mother would allow discarding milk with peak levels of drug. therefore breastfeeding during maternal malaria even with treatment is appropriate with specific medications. strongyloides stercoralis is a nematode (roundworm). most infections are asymptomatic, but clinically significant infection in humans can include larval skin invasion, tissue migration, intestinal invasion with abdominal pain and gi symptoms, and a loeffler-like syndrome due to migration to the lungs. immune-compromised individuals can develop dissemination of larvae systemically, causing various clinical symptoms. humans are the principal hosts, but other mammals can serve as reservoirs. infection via the skin by filariform larvae is the most common form of transmission; ingestion is an uncommon occurrence. transmammary transmission of strongyloides species has been described in dogs, ewes, and rats. 211, 302, 382 only one report of transmammary passage of strongyloides larvae in humans is available. in 76 infants younger than 200 days of age, 34% demonstrated the presence of strongyloides fuelleborni on stool examination. the clinical significance of this was not elucidated. strongyloides larvae was identified in only one sample of milk from 25 nursing mothers. 53 in the absence of an understanding of the clinical significance of strongyloides in the stools of young infants, given the lack of exclusion of the most common mechanism of transmission (through the skin) in the single report and the apparent infrequent evidence of these larvae in human milk, it is difficult to make any recommendations concerning breastfeeding and strongyloides. toxoplasmosis is one of the most common infections of humans throughout the world. the infective organism, toxoplasma gondii, is ubiquitous in nature. the prevalence of positive serologic test titers increases with age, indicating past exposure and infection. the cat is the definitive host, although infection occurs in most species of warmblooded animals. postnatal infection with toxoplasmosis is usually asymptomatic. symptomatic infection typically manifests with nonspecific symptoms, including fever, malaise, myalgia, sore throat, lymphadenopathy, rash, hepatosplenomegaly, and occasionally a mononucleosis-like illness. the illness usually resolves without treatment or significant complications. congenital infection or infection in an immunodeficient individual can be persistent and severe, causing significant morbidity and even death. although most infants with congenital infection are asymptomatic at birth, visual abnormalities, learning disabilities, and mental retardation can occur months or years later. the syndrome of congenital toxoplasmosis is clearly defined, with the most severe manifestations involving the cns, including hydrocephalus, cerebral calcifications, microcephaly, chorioretinitis, seizures, or simply isolated ocular involvement. the risk for fetal infection is related to the timing of primary maternal infection, although transmission can occur with preexisting maternal toxoplasmosis. 241 in the last months of pregnancy the protozoan is more readily transmitted to the fetus, but the infection is more likely to be subclinical. early in pregnancy the transmission to a fetus occurs less frequently but does result in severe disease. treatment of documented congenital infection is currently recommended, although duration and optimal regimen have not been determined, and reversal of preexisting sequelae generally does not occur. 343 prevention of infection in susceptible pregnant women is possible by avoiding exposure to cat feces or the organism in the soil. pregnant or lactating women should not change cat litter boxes, but if they must, it should be done daily and while wearing gloves. the oocyst is not infective for the first 24 to 48 hours after passage. mothers can avoid ingestion of the organism by fully cooking meats and carefully washing fruits, vegetables, and food preparation surfaces. 94 in various animal models, t. gondii has been transmitted through the milk to the suckling young. the organism has been isolated from colostrum as well. the newborn animals became asymptomatically infected when nursed by an infected mother whose colostrum contained t. gondii. only one report has identified t. gondii in human milk, and some question surrounds the reliability of that report. 241 transmission during breastfeeding in humans has not been demonstrated. breast milk may contain appropriate antibodies against t. gondii. given the benign nature of postnatal infection, the absence of documented transmission in human breast milk, and the potential antibodies in breast milk, no reason exists to proscribe breastfeeding by a mother known to be infected with toxoplasmosis. trichomonas vaginalis is a flagellated protozoan that can produce vaginitis (see chapter 16 for a discussion of vaginitis) but frequently causes asymptomatic infection in both men and women. the parasite is found in 10% to 25% of women in the childbearing years. it is transmitted predominantly by sexual intercourse, but it can be transmitted to the neonate by passage through the birth canal. this parasite often coexists with other stds, especially gonorrhea. infection during pregnancy or while taking oral contraceptives is more difficult to treat. some evidence suggests that infection with and growth of the parasite are enhanced by estrogens or their effect on the vaginal epithelium. no evidence indicates adverse effects on the fetus in association with maternal infection during pregnancy. occasionally female newborns have vaginal discharge during the first weeks of life caused by t. vaginalis. this is thought to be influenced by the effect of maternal estrogen on the infant' s vaginal epithelium and acquisition of the organism during passage through the birth canal. the organism does not seem to cause significant disease in a healthy infant. no documentation exists on transmission of t. vaginalis via breast milk. the difficulty encountered with maternal infection during lactation stems from metronidazole (flagyl), the drug of choice, being contraindicated for infants. case reports describe treatment of neonates with metronidazole without adverse effect. although topical agents containing povidone-iodine (betadine) or sodium lauryl sulfate (trichotine) can be effective when given as douches, creams, or suppositories, metronidazole remains the treatment of choice. the aap advises using metronidazole only with a physician' s discretion and considers its effect on a nursing infant unknown but possibly a concern. the potential concerns are metronidazole' s disulfiram-like effect in association with alcohol, tumorigenicity in animal studies, and leukopenia and neurologic side effects described in adults. on the other hand, metronidazole is given to children beyond the neonatal period to treat serious infections with various other parasites, such as entamoeba histolytica. the current recommendation for lactating women is to try local treatment first, and if these fail, then to try metronidazole. a 2-g single-dose treatment produces peak levels after 1 hour, and discarding expressed breast milk for the next 12 to 24 hours is recommended. if this treatment also fails, a 1-g twice-daily regimen for 7 days or a 2-g single daily dose for 3 to 5 days is recommended, with discarding of breast milk close to the dose and timing of feedings distant from the dose. infants who exclusively breastfeed are presumed at greater risk from exposure to metronidazole than those who are only partially breastfed. candida consists of multiple species. the most common species affecting humans include c. albicans as the dominant agent and c. tropicalis, c. krusei, and c. parapsilosis, as well as many other uncommon species. in general, candida exists as a commensal organism colonizing the oropharynx, gi tract, vagina, and skin without causing disease until some change disrupts the balance between the organism and the host. mild mucocutaneous infection is the most common illness, which can lead to vulvovaginitis, mastitis, or, uncommonly, oral mucositis in a mother, and thrush (oral candidiasis) and candidal diaper rash in an infant. invasive candidal infection occurs infrequently, usually when a person has other illness, impaired resistance to infection (hiv, diabetes mellitus, neutropenia; decreased cell-mediated immunity in premature infants or lbw or vlbw infants), or disrupted normal mucosal and skin barriers and has received antibiotics or corticosteroids. invasive disease can occur through local spread, and may occur more often in the genitourinary tract (urethra, bladder, ureters, kidneys), but usually develops in association with candidemia. the bladder and kidney are more frequently involved, but when dissemination occurs via candidemia, a careful search for other sites of infection should be made (e.g., retina, liver, spleen, lung, meninges). 279 transmission usually occurs from healthy individuals colonized with candida through direct contact with them or through contact with their oral or vaginal secretions. intrauterine infection can occur through ascending infection through the birth canal but is rare. no distinct syndrome of congenital candidal infection exists. most often an infant is infected in passing through the birth canal and remains colonized. postnatal transmission can occur through direct contact with caregivers. the mother and infant serve as an immediate source of recolonization for each other, especially during the direct contact of breastfeeding. for this reason, an infant and breastfeeding mother should be treated simultaneously when treating thrush, vulvovaginitis, diaper candidiasis, or mastitis. colonization with this organism usually occurs in the absence of any clinical evidence of infection. simultaneous treatment should occur even in the absence of any clinical evidence of candida infection or colonization in the apparently uninvolved individual of the breastfeeding dyad. no well-controlled clinical trials define the most appropriate or most effective method(s) of treatment for candidal infection in breastfeeding mother-infant pairs. the list of possible treatment products is extensive and includes many anecdotal and empirical regimens. in the face of this absence of data, brent 51 conducted a survey of members of the academy of breastfeeding medicine concerning the respondents' approach to diagnosis and treatment of thrush in the breastfeeding dyad. most of the respondents relied on the history and physical examination of the infant, but only a third rated the examination of the mother as very important in making a diagnosis. only 7% reported using laboratory testing to make the diagnosis. twentyone percent of the respondents reported using only oral nystatin for the infant when the mother was asymptomatic. almost half treated the infant and the mother with topical nystatin, and 13% used oral nystatin for the infant and oral fluconazole for the mother when the mother had breast pain. less than 5% used oral fluconazole for both infant and mother, and other therapies were used by about 15% of the respondents. for recurrence of persistence of the thrush, more respondents reported treating the mother or both the infant and mother with fluconazole, and almost a quarter reported using other therapies. considerable discussion of mammary candidosis/candidiasis, the clinical diagnosis of candidal involvement of the breast, the significance of pain with breastfeeding, and the presence or absence of candida albicans in milk samples is ongoing. 14, 133, 166 this topic will continue to be debated because additional prospective studies are necessary to clarify specific issues. data are inadequate to make specific recommendations about various clinical situations regarding candida and breastfeeding. clinical practice will vary with experience, especially for the more problematic clinical situations. some general guidelines follow. (see chapter 16 for a discussion of mastitis.) the treatment of mucocutaneous candidiasis should probably begin with a topical agent, such as nystatin, clotrimazole, miconazole, econazole, butaconazole, terconazole, or ciclopirox. treatment should continue for at least 2 weeks, even with obvious improvement in 1 or 2 days. failures most often result from inadequate therapy involving the frequency of application, careful washing and drying before application, or, in the case of diaper candidiasis, decreasing the contact of the skin with moisture. nystatin oral suspension is less effective for the treatment of oral candidiasis in infants, now compared with the past, supposedly due to increasing resistance. 154 gentian violet (diluted to 0.25% to 1.0%) applied to the breast or painted onto an infant' s mouth is being recommended more frequently. other topical preparations have been recommended for the mother' s breast including mupirocin, grapefruit seed extract, or mixtures of mupirocin, betamethasone ointments, and miconazole powder. controlled clinical trials for efficacy and toxicity are not available. when good adherence to the proposed regimen with topical agents fails, or when infant or mother are severely affected by pain and decreased breastfeeding, systemic therapy is appropriate. fluconazole and ketoconazole are the most commonly used systemic agents for oral or diaper candidiasis and vulvovaginitis or mastitis. fluconazole has a better side effect profile than ketoconazole, and more data are available concerning its safe use in children younger than 6 months of age and even neonates and premature infants. 87, 154, 209 fluconazole is not currently approved for use in infants younger than 6 months of age. for severe invasive infections in infants, amphotericin b with or without oral flucytosine, iv fluconazole, voriconazole or caspofungin are reasonable choices in different situations. use of itraconazole in infants has not been adequately studied to date. maternal use of fluconazole during breastfeeding is not contraindicated because only a small amount of medicine compared with the usual infant dose reaches the infant through breast milk. amphotericin or caspofungin therapy in mothers is also not contraindicated because these are both poorly absorbed from the gi tract. whenever a mother is treated for candidal mastitis or vulvovaginitis, the infant should be treated simultaneously, at least with nystatin oral suspension as the first choice of medication. any predisposing risk factors for candidal infection in mothers and infants should be reduced or eliminated to improve the chance of rapid, successful treatment and to decrease the likelihood of chronic or recurrent disease. for mothers, such interventions might include decreasing sugar consumption, stopping antibiotic use as soon as possible, and consuming some form of probiotic bacteria, such as acidophilus (in yogurt, milk, or pill form), to reestablish a normal colonizing bacterial flora. for infants, breastfeeding can enhance the growth of specific colonizing bacterial flora such as lactobacillus, which can successfully limit fungal growth. breastfeeding should continue with appropriate support and problem-solving with a professional who is knowledgeable about breastfeeding. hiv-1, hiv-2, htlv-i, and htlv-ii are the only infectious diseases that are considered absolute contraindications to breastfeeding in developed countries. when the primary route of transmission is via direct contact or respiratory droplets/particles, temporary separation of mother and infant may be appropriate (whether the infant is breastfed or formula fed), but expressed breast milk should be given to the infant for the organism-specific immunologic benefits in the mother' s milk. in most instances, by the time a specific diagnosis of infection is made for a mother, the infant has already been exposed to the organism and providing expressed breast milk to the infant should continue. (refer to appendix f for specific exceptions, such as lassa fever.) regarding antimicrobial therapy for mothers and continued breastfeeding, the majority of the medications commonly used in adults can be used to treat the same infection in infants. the additional amount of medication received by infants via breast milk is usually insignificant. in almost all instances, an appropriate antimicrobial agent for treating mothers that is also compatible with breastfeeding can be chosen. unless the risk to infants for transmission of an infectious agent via breast milk that leads to a clinically significant illness in the infants is documented, breastfeeding should continue. measles antibodies in the breast milk of nursing mothers spectrum of breast tuberculosis respiratory syncytial virus infection among young children with acute respiratory tract infection in iraq probable breast milk borne brucellosis in a young infant breast milk transmission of cytomegalovirus (cmv) infection congenital and perinatal cytomegalovirus infections intrauterine west nile virus: ocular and systemic findings the cloning and clinical implications of hgv and hgbv-c bottle feeding can prevent transmission of htlv-i from mothers to their babies transmission of adult t-cell leukemia retrovirus (htlv-i) from mother to child: comparison of bottle-with breastfed babies effect of freezethawing breast milk on vertical htlv-i transmission from seropositive mothers to children long-term follow up study of vertical htlv-i infection in children breastfed by seropositive mothers long-term followup study of htlv-i infection in bottle-fed children born to seropositive mothers the yeast connection: is candida linked to breastfeeding associated pain? epidemiology of group b streptococcus: maternal and nosocomial sources for acquisition tuberculosis and pregnancy and tuberculous mastitis infant botulism infant botulism: anticipating the second decade protective role of human milk against sudden death from infant botulism growth faltering due to breastfeeding cessation in uninfected children born to hiv-infected mothers in zambia other viral infections of the fetus and newborn protozoan and helminth infections (including pneumocystis carinii) recurrent group b streptococcal disease in infants: who should receive rifampin? methicillin-resistant staphylococcus aureus sccmec type iv: nosocomial transmission and colonisation of healthcare workers in a neonatal intensive care unit stringent precautions are advisable when caring for patients with viral hemorrhagic fevers prevalence of methicillin-resistant staphylococcus aureus in expressed breast milk in a neonatal intensive care unit transmision de brucelosis por lactancia materna: presentacion de dos casos congenital lymphocytic choriomeningitis virus infection in twins poliomyelitis in pregnancy, fetus and newborn assessment of the risk of ebola virus transmission from bodily fluids and fomites transmission of hepatitis by breastfeeding evidence against breast feeding as a mechanism for vertical transmission of hepatitis b two-year morbidity-mortality and alternatives to prolonged breast feeding among children born to hiv infected mothers in cote d'ivorie transmission of methicillin-resistant staphylococcus aureus to preterm infants through breast milk a new staphylococcal enterotoxin, enterotoxin f, associated with tss staphylococcus aureus isolate mother-to-infant transmission of hepatitis c outbreak of methicillin-resistant staphylococcus aureus colonization and infection in a neonatal intensive care unit epidemiologically linked to a healthcare worker with chronic otitis estimating the timing of mother-to-child transmission of human immunodeficiency virus in a breast-feeding population in kinshasa mycobacteriareactive t cells are present in human colostrum from tuberculin-positive, but not tuberculin-negative nursing mothers estimated risk of transmission of the west nile virus through blood transfusion in the us partial breastfeeding protects bedouin infants from infection morbidity: prospective cohort study children hospitalized with severe acute respiratory syndrome-related illness in toronto a prospective study of infants born to women seropositive for human immunodeficiency virus type 1. hiv infection in newborns french collaborative study group clinical virology postpartum varicella vaccination: is the vaccine virus excreted in breast milk? contamination of breast milk obtained by manual expression and breast pumps in mothers of very low birth weight hepatitis c virus infection and related liver disease in children of mothers with antibodies to the virus clinical and laboratory observations, gram-negative bacilli in human milk feedings: quantitation and clinical consequences for premature infants prenatal transmission of dengue: two new cases community associated methicillin-resistant staphylococcus aureus in hospital nursery and maternity units thrush in the breastfeeding dyad: results of a survey on diagnosis and treatment reproductive factors in the aetiology of breast cancer transmammary passage of strongyloides sp. larvae in the human host alaska rsv study group: risk factors for severe respiratory syncytial virus infection among alaska native children detection of human immunodeficiency virus type 1 (hiv-1) proviral dna in breast milk and colostrum of seropositive mothers streptococcus agalactiae as a cause of meningitis in the newborn and bacteraemia in adults incidence and clinical outcome of cytomegalovirus transmission via breast milk in preterm infants </=31 weeks neonatal group b streptococcal infection related to breast milk epidemiology of tuberculosis in the united states probable transmission of brucellosis by breast milk does discarding the first few milliliters of breast milk improve the bacteriological quality of bank breast milk? primary human herpes virus 7 infection: a comparison of human herpes virus 7 and human herpes virus 6 infections in children analysis of the presence of cutaneous and mucosal papillomavirus types in ductal lavage fluid, milk and colostrum to evaluate its role in breast carcinogenesis centers for disease control and prevention: testing for antibodies to hiv-2 in the united states public health service working group: recommendations for counseling persons infected with human t-lymphotropic virus, types i and ii centers for disease control and prevention: screening for tuberculosis and tuberculosis infection in high-risk populations update: management of patients with suspected viral hemorrhagic fever-united states poliomyelitis prevention in the united states: introduction of a sequential vaccination schedule of inactivated poliovirus vaccine followed by oral poliovirus vaccine-united states prevention: recommendations for antimicrobial prophylaxis for children and breastfeeding mothers and treatment of children with anthrax prevention: possible west nile virus transmission to an infant through breastfeeding-michigan prevention: yellow fever vaccine; recommendations of the advisory committee on immunization practices (acip) update: cardiac and other adverse events following civilian smallpox vaccination-united states centers for disease control and prevention: secondary and tertiary transfer of vaccinia virus among u.s. military personnel-united states and worldwide centers for disease control and prevention: a new product (varizig) for post exposure prophylaxis of vericells available under an investigational new drug application expanded access protocol centers for disease control and prevention: communityassociated methicillin-resistant staphylococcus aureus infection among healthy newborns: chicago and los angeles county centers for disease control and prevention: what to do if an infant or child is mistakenly fed another woman' s expressed breast milk prevention: transmission of yellow fever vaccine through breast feeding: brazil take of rhesushuman reassortment tetravalent rotavirus vaccine in breastfed infants candida infections in the neonate prevalence of methicillin-sensitive and methicillin-resistant staphylococcus aureus in pregnant women coxsackieviruses, and enteroviruses morbidity and mortality among a cohort of human immunodeficiency virus type 1 infected and uninfected pregnant women and their infants from malawi, zambia, and tanzania electron microscopic detection of simian-type virus particles in human milk staphylococcus aureus outbreaks among newborns: new frontiers in an old dilemma breastfeeding and the risk of life-threatening rotavirus diarrhea: prevention or postponement? committee on infectious diseases: american academy of pediatrics: red book report of the committee on infectious disease committee on infectious diseases: american academy of pediatrics: red book report of the committee on infectious disease committee on infectious disease: american academy of pediatrics: red book report of the committee on infectious disease mother-tochild transmission of hiv-1 infection during exclusive breastfeeding in the first 6 months of life: an intervention cohort breastfeeding, hiv transmission and infant survival: balancing pros and cons influence of infantfeeding patterns on early mother-to-child transmission of hiv-1 in durban, south africa: a prospective cohort study. south african vitamin a study group method of feeding and transmission of hiv-1 from mothers to children by 15 months of age: prospective cohort study from durban are hivinfected women who breastfeed at increased risk of mortality? morbidity in children born to women infected with human immunodeficiency virus in south africa: does mode of feeding matter? pilot epidemiologic study of transmission of cytomegalovirus from mother to preterm infant by breastfeeding mother-to-child transmission of human immunodeficiency virus type: report from the nairobi study human milk and hiv infection: epidemiologic and laboratory data hiv, breastfeeding and under 5 mortality: modelling the impact of policy decisions for or against breastfeeding staphylococcus epidermidis strains isolated from breast milk of women suffering infectious mastitis: potential virulence traits and resistance to antibiotics hiv-1 transmission through breast-milk: appraisal of risk according to duration of feeding task force on recurrent respiratory papillomatosis presence of papillomavirus in condylomatous lesions of the mamillae and in invasive carcinoma of the breast variations in biological features of west nile viruses group b streptococcal carriage and disease: a 6 year prospective study hepatitis in the obstetric patient prophylactic isoniazid protection of infants in a tuberculosis hospital breast-feeding protects against respiratory syncytial virus infections neonatal herpes simplex infection possibly acquired via maternal breast milk risk of human immunodeficiency virus type 1 transmission through breastfeeding serologic evidence for congenital transmission of human herpesvirus 6 examination of human breast milk for evidence of human herpesvirus 6 by pcr cytomegalovirus infection of breast milk and transmission in infancy late postnatal mother-to-child transmission of hiv-1 in abidjan listeriosis during pregnancy and in neonates rotavirus infections in young nicaraguan children european collaborative study: children born to women with hiv-1 infection: natural history and risk of transmission european paediatric hepatitis c virus network: effects of mode of delivery and infant feeding on the risk for motherto-child transmission of hepatitis c virus flora of the umbilical stump: 2479 cultures a lipid inhibitor of dengue virus in human colostrum and milk, with a note on the absence of anti-dengue secretory antibody vertical transmission of hepatitis g nonspecific antiviral substances in human milk against arbovirus and murine leukemia virus genetic evidence for mother-to-infant transmission of hepatitis g virus stringent precautions are not advisable when caring for patients with viral hemorrhagic fevers evalution and treatment of community acquired staphylococcus aureus infections in term and late-preterm previously healthy neonates diagnostic value of signs and symptoms of mammary candidosis among lactating women immunity and correlates of protection for rotavirus vaccines rate of inactivation of cytomegalovirus in raw banked milk during storage at 20 degrees c and pasteurisation htlv-i transmission from mother to child detection of human herpesvirus 7 (hhv-7) dna in breast milk by polym erase chain reaction and prevalence of hhv-7 antibody in breast-fed and bottle-fed children a new endemic focus of human t-lymphotropic virus type ii carriers among orinoco natives in colombia estimating the time of htlv-i infection following mother-to-child transmission in a breast-feeding population in jamaica tertiary contact vaccinia in a breastfeeding infant community acquisition of group b streptococcus by infants of colonized mothers infectious diseases of the fetus and newborn infant hospital infection control practices advisory committee: guidelines for isolation precautions in hospitals transmission of community-associated methicillian-resistant staphylococcus aureus from breast milk in the neonatal intensive care unit giardiasis and breastfeeding in urban africa multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of la reunion management of outbreaks of methicillin-resistant staphylococcus aureus infection in the neonatal intensive care unit: a consensus statement chickenpox, measles and mumps seroepidemiology in various population groups of the greater montreal area mother-to-child transmission of hepatitis c virus: evidence for preventable peripartum transmission cholate-dependent killing of giardia lamblia by human milk triple antiretroviral prophylaxis administered during pregnancy and after delivery significantly reduces breast milk viral load: a study within the drug resource enhancement against aids and malnutrition program risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level comparison of fluconazole and nystatin oral suspensions for treatment of oral candidiasis in infants rapid high temperature treatment of human milk htlv-i: a new problem for latin america rotavirus vaccines opportunities and challenges detection of human immunodeficiency virus type 1 (hiv-1) dna and p24 antigen in breast milk of hiv-1-infected ugandan women and vertical transmission denuge and denuge hemorrhagic fever tubercular mastitis child to mother transmission of herpes simples virus-1 infection at an unusal site vertical transmission of hepatitis c virus dengue: an update epidemiology and diagnosis of hospital acquired conjunctivitis among neonatal intensive care unit patients congenital infections with human herpes virus 6 (hhv6) and human herpes virus 7 (hhv7) the absence of candida albicans in milk samples of women with clinical symptoms of ductal candidiasis dengue hemorrhagic fever in infants: research opportunities ignored epidemiology of transmission of cytomegalovirus from mother to preterm infant by breastfeeding cytomegalovirus transmission to preterm infants during lactation congenital malformations and oral poliovirus vaccination during pregnancy mammary tuberculosis: analysis of thirty-eight patient cytomegalovirus in human milk an epidemiologic study of breast cancer detection of htlv-ii in breast milk of htlv-ii infected mothers killing of giardia lamblia by human milk lipases: an effect mediated by lipolysis of milk lipids risk of hepatitis b transmission in breast-fed infants of chronic hepatitis b carriers transmission of west nile virus through human breast milk seems to be rare milk-borne transmission of htlv-i as a major route in the endemic cycle primary prevention of htlv-i in japan primary prevention of htlv-1 in japan apparent vertical transmission of human immunodeficiency virus type 1 by breast-feeding in zambia virus markers associated with vertical transmission of human t lymphotropic virus type 1 in jamaica rotavirus antibodies in the mother and her breastfed infant incidence of haemophilus influenzae in the throats of healthy infants with different feeding methods transfusion transmission of west nile virus: a merging of historical and contemporary perspectives clinical presentations and outcomes of severe acute respiratory syndrome in children poliomyelitis in pregnancy: a twenty-year report from long-term serologic follow-up of patients for epstein-barr virus after recovery from infectious mononucleosis the global distribution of human immunodeficiency virus type 2 (hiv-2) infection interventions for preventing late postnatal mother-to-child transmission of hiv methicillin-resistant staphylococcus aureus colonization and its association with infection among infants hospitalized in neonatal intensive car units staphylococcus aureus in the infant upper respiratory tract. i. observations on hospital-born babies unilateral breast feeding and breast cancer staphylococcus aureus in australasian neonatal nurseries identification of human t-cell lymphotropic virus type iia infection in the kayapo, an indigenous population of brazil mother-to-infant transmission of tt virus in japan italian register for hiv infection in children: hiv-1 infection and breast milk transmission of west nile virus from an organ donor to four transplant recipients tuberculosis mastitis methicillin-resistant staphylococcus aureus infections among healthy full-term newborns human milk in the modern world staphylococcus epidermidis: a differential trait of the fecal microbiota of breastfed infants epstein-barr virus shedding in breast milk post weaning gastroenteritis and mortality in hiv-1 uninfected african infants receiving antiretroviral prophylaxis to prevent mtct of hiv-1 successful antepartum treatment of listeriosis low risk for motherto-child transmission of human t lymphotropic virus type ii in non-breastfed infants staphylococcal scalded skin syndrome in a breastfed infant fluconazole prophylaxis against fungal colonization and infection in preterm infants transmission of staphylococus aureus between healthy lactating mothers and their infants by breastfeeding transmammary transmission of strongyloides ratti case-control study of mastomys natalensis and humans in lassa virusinfected households in sierra leone transfer of tuberculin immunity from mother to infant recurrent group b streptococcal disease in an infant associated with the ingestion of infected mother' s milk mammary tuberculosis: report on 52 cases eradication of methicillin-resistant staphylococcus aureus from a neonatal intensive care unit by active surveillance and aggressive infection control measures prevention of mother-to-child transmission of hiv-1 through breast feeding by treating infants prophylactically with lamivudine in dar es salaam prevention of mother-to-child transmission of hiv through breastfeeding by treating mothers with triple antiretroviral therapy in dar es salaam, tanzania: the mitra plus study clinical outcomes in methicillin-resistant staphylococcus aureus colonized neonates in the neonatal intensive care unit demonstration of adult t-cell leukemia virus antigen in milk from three seropositive mothers oral infection of a common marmoset with human t-cell leukemia virus type i (htlv-i by fresh human milk of htlv-i carrier mothers the differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection human parvovirus b19 infection in women of childbearing age and within families lymphocytic choriomeningitis in the newborn late-onset and recurrent neonatal group b streptococcal disease associated with breast-milk transmission diarrhoea in uninfected infants of hiv-infected mothers who stop breastfeeding at 6 months: the ban study experience accessed 12/19 prolonged breastfeeding and mortality up to two years postpartum among hiv positive women in zambia hiv specific secretory iga in breast milk of hiv-positive mothers is not associated with protection against hiv transmission among breast-fed infants high uptake of exclusive breastfeeding and reduced postnatal hiv transmission; prospective results from the zambia exclusive breastfeeding study accessed 12/19 effect of early, abrupt weaning on hiv-free survival of children in zambia differential effects of early weaning for hiv-free survival of children born to hiv-infected mothers by severity of maternal disease breastfeeding and aids in the developing world hiv prevention is not enough: child survival in the context of prevention of mother to child hi transmission congenital and postnatally acquired cytomegalovirus infections: long-term follow-up extended antiretroviral prophylaxis to reduce breast milk hiv-1 transmission breast milk is not a significant source for early epstein-barr virus or human herpes virus 6 infection in infants: a seroepidemiologic study in 2 endemic areas of human t-cell lymph tropic virus type i in japan evidence for mother-to-child transmission of human t-lymphotropic virus type ii mother-to-child transmission of human t-lymphotropic virus type ii (htlv-ii) role of maternal antibody in pneumonia and bronchiolitis due to respiratory syncytial virus a further report on cancer of the breast repeated congenital infection with toxoplasma gondii is ingestion of milk associated bacteria by premature infants fed raw human milk controlled by routine bacteriologic screening? maternal and child health technical information bulletin cytomegalovirus in human breast milk: risk to the premature infant the independent associations of parity, age at first full term pregnancy, and duration of breastfeeding with risk for breast cancer epstein-barr virus estimating infant mortality from hiv and other causes in breastfeeding and bottle-feeding populations postnatal cytomegalovirus infection from frozen breast milk in preterm low birth weight infants postntal transmission of hiv from mother to child eradication of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit: which measures for which success? international multicentre pooled analysis of late postnatal mother-to-child transmission of hiv-1 infection. ghent international working group on mother-to-child transmission of hiv 18 month effectiveness of short-course antiretroviral regimens combined with alternatives to breastfeeding to prevent hiv mother-tochild transmission breast milk transmission of a panton-valentine leukocidin-producing staphylococcus aureus strain causing infantile pneumonia mammary epithelial cells support and transfer productive human t cell lymphotropic virus infections mechanism of vertical transmission of hepatitis g absence of infection in breast fed infants born to hepatitis c virus-infected mothers intrauterine listeria infection: prenatal diagnosis by biophysical assessment and amniocentesis ejpidemiologic features of htlv-ii: serologic and molecular evidence neonatal brucellosis probable transmission of brucellosis from breast milk to a newborn a multicenter therapeutic study of 1100 children with brucellosis lactation and cancer of the breast: a summary of an international study human immunodeficiency virus infection as risk factor for mother-to-child hepatitis c virus transmission: persistence of anti-hepatitis c virus in children is associated with the mother' s antihepatitis c virus immunoblotting pattern increased infant human immunodeficiency virus type one free survival at one year of age in sub-saharan africa with maternal use of highly active antiretroviral therapy during breast feeding the genome sequence of the sars-associated coronavirus diagnostic approach cytomegalovirus infection of extremely low-birth weight infants via breast milk freezethawing of breast milk does not prevent cytomegalovirus transmission to a preterm infant human milk siga binds to botulinum type b 16 s toxin and limits toxin adherence on t84 cells antimicrobial factors and microbial contaminants in human milk: recent studies morbidity and mortality patterns in hiv-1 seropositive/seronegative women in kampal and harare during pregnancy and in the subsequent two years morbidity and mortality in breastfed and formula-fed infants of hiv-1-infected women: a randomized clinical trial predominance of left-sided breast tumors obstetric management of hepatitis c-positive mothers: analysis of vertical transmission in 559 mother-infant pairs evidence for a protective effect of lactation on risk of breast cancer in young women: results from a case-control study prospective study of the incidence and complications of bacterial contamination of enternal feeding in neonates effect of treatment on the contagiousness of patients with active pulmonary tuberculosis infectious diseases of the fetus and newborn infant does breast feeding increase the child' s risk of breast cancer? role of breast milk in acquisition of cytomegalovirus infection hiv transmission through breastfeeding: a study in malawi prevention of breast milk tranmission of hiv: the time is now mother-to-infant transmission of hepatitis c virus: rate of infection and assessment of viral load and igm anti-hcv as risk factors yellow fever vaccine nosocomial transmission of methicillin-resistant staphylococus aureus from a mother to her preterm quadruplet infants breastfeeding family history and breast disease transmission of hepatitis c virus from mothers to infants: its frequency and risk factors revisited immune escape by epstein-barr virus associated malignancies the duration of breastfeeding by hiv-1 infected mothers in developing countries: balancing benefits and risks search for possible routes of vertical and horizontal transmission of adult t-cell leukemia virus outbreak of invasive disease caused by methicillin-resistant staphylococcus aureus in neonates and prevalence in the neonatal intensive care unit human immunodeficiency virus type 1-infected cells in breast milk: association with immunosuppression and and vitamin a deficiency effect of breastfeeding on mortality among hiv-1 infected women: a randomised trial breast brucellosis in taif, saudi arabia: cluster of six cases with emphasis on fna evaluation case control study of symptoms and neonatal outcome of human milktransmitted cytomegalovirus infection in premature infants human milk glycosaminoglycans inhibit hiv glycoprotein gp 120 binding to its host cell cd4 receptor risk factors for neonatal methicillin-resistant staphylococcus aureus infection in a well-infant nursery lactation and a reduced risk of premenopausal breast cancer contamination of expressed human breast milk with an epidemic multiresistant staphylococus aureus clone human cytomegalovirus infection of breast milk strongyloides papillosus: prenatal and transmammary infection in ewes human neonatal infections with hookworms in an endemic area of southern nigeria. a possible transmammary route mother-to-child transmission of human t-cell lymphotoropic virus types i and ii (htlv-i/ii) in gabon: a prospective follow-up of 4 years transfer of human herpes virus 6 and 7 antibodies from mother to their offspring vertical transmission of hepatitis c virus transmission of hepatitis c virus from mothers to infants for the vertical transmission of hepatitis viruses collaborative study group, motherto-infant transmission of gb virus type c/hgv for the vertical transmission of hepatitis viruses collaborative study group: tt virus infection during childhood tt virus: virological and genomic characteristics and disease associations birth outcomes following west nile virus infection of pregnant women in the united states neonatal group b streptococcal disease associated with infected breast milk transmission of cytomegalovirus to extremely preterm infants through breast milk early breastfeeding cessation among hiv-exposed negative infants and risk of serious gastroenteritis: findings from a perinatal prevention trial in kampala, uganda inactivation of human immunodeficiency virus type i in human milk: effects of intrinsic factors in human milk and of pasteurization lactation mastitis: bacterial cultivation of breast milk, symptoms, treatment, and outcome human immunodeficiency virus and other viruses in human milk: placing the issues in broader prospective effect of breast-feeding on immune response to bcg vaccination brucellosis in a mother and her young infant: probable transmission by breast milk brucellar arthritis of knee in a child breast-feeding during primary maternal human immunodeficiency virus infection and risk of transmission from mother to infant treatment acceleration program and the experience of the dream program in prevention of mother-to-child transmission of hiv updated u.s. public health service guidelines for the management of occupational exposures to hiv and recommendations for post exposure prophylaxis etiologic factors in cancer of the breast in humans: collective review methicillin-resistant staphylococcus aureus in milk oral rotavirus vaccines: how well will they work were they are needed most? determinants of acquisition and carriage of staphylococcus aureus in infancy early acquisition of cytomegalovirus infection clinical virology west nile virus: a primer for the clinician petra study team: efficancy of three short course regimens of zidovudine and lamivudine in preventing early and late transmission of hiv-1 from mother to child in tanzania, south africa, and uganda (petra study): a randomized, double-blind, placebo-controlled trial dengue virus infection in late pregnancy and transmission to the infants effect of maternal rotavirus immunization on milk and serum antibody titers cell-free virus in breast milk of hiv-1-seropositive women role of breastfeeding in paralytic poliomyelitis bacterial contamination of human milk: container type and method of expression maternal herpetic breast infection: another hazard of neonatal herpes simplex mother-to-child transmission of chikungunya virus infection care of the pregnant woman with tuberculosis and her newborn infant: a pediatrician' s perspective read jsand the committee on pediatric aids: human milk, breastfeeding, and transmission of human immunodeficiency virus type 1 in the united states postpartum mastitis and community-acquired methicillin-resistant staphylococcus aureus infectious diseases of the fetus and newborn infant hepatitis e virus breastmilk infectivity in human immunodeficiency virus type 1-infected mothers high risk types of human papillomavirus (hpv) dna in oral and genital mucosa of infants during their first 3 years of life: experience from the finnish hpv family study streptococcal toxic shock syndrome, including necrotizing fasciitis and myositis late-onset septicemia in a norwegian national cohort of extremely premature infants receiving very early full human milk feeding hepatitis a outbreak in a neonatal intensive care unit: risk factors for transmission and evidence of prolonged viral excretion among preterm infants characterization of a novel corona virus associated with severe acute respiratory syndrome longitudinal analysis of human immunodeficiency virus type 1 rna in breast milk and of its relationship to infant infection and maternal disease attempts to detect rna tumour virus in human milk is breast milk collected at home suitable for raw consumption by neonates in brizilian public neonatal intensive care units? prevalence of hiv-1 dna and p24 antigen in breast milk and correlation with maternal factors follow-up of transmission of hepatitis c to babies of human immunodeficiency virus-negative women: the role of breastfeeding in transmission occurrence of acute diarrhea in atopic and nonatopic infants: role of prolonged breast-feeding an outbreak of methicillin-resistant staphylococcus aureus in a neonatal intensive care unit hospital transmission of community acquired methicillin-resistant staphylococcus aureus among postpartum women removal of inhibitors against rna-directed dna polymerase activity in human milk effect of human milk on mouse mammary tumor virus human papillomavirus dna detected in breast milk control of a cluster of a community-associated methicillin-resistant stahpylococcus aureus in neonatology immunoglobulin prophylaxis against milkborne transmission of human t cell leukemia virus type 1 in rabbits chlamydial infections pregnancy and pulmonary tuberculosis possible breast milk transmission of group b streptococcal infection evidence for transmission of lymphocyte responses to tuberculin by breast-feeding, lancet i:529 toxic shock syndrome detection of borrelia burgdorferi dna by pcr in the urine and breast milk of patients with lyme borreliosis prevention of perinatal group b streptococcal disease. revised guidelines from cdc detection of tt virus dna and gb virus type c/hepatitis g virus rna in serum and breast milk: determination of mother-to-child transmission effects of hepatitis a and b in pregnancy on the mother and fetus human immunodeficiency virus load in breast milk, mastitis and mother-to-child transmission of human immunodeficiency virus type 1 how contagious is vaccinia? transmammary transmission of necator americanus larva in the human host a study of infectious intestinal disease in england: risk factors associated with group a rotavirus in children highly active antiretroviral therapy started during pregnancy or postpartum suppresses hiv-1 rna, but not dna, in breast milk prevention of postnatal cytomegalovirus infection in preterm infants infants born to mothers with severe acute respiratory syndrome tuberculosis of the breast masquerading as carcinoma: a study of 100 patients transmammary transmission of strongyloides stercolis in dogs low birth weight and maternal virus diseases: a prospective study of rubella, measles, mumps, chickenpox, and hepatitis lyme disease during pregnancy no benefit of early cessation of breastfeeding at 4 months on hiv-free survival of infants born to hiv-infected mothers in zambia: the zambia exclusive breastfeeding study vertical dengue infection: case reports and reviews a young infant with severe acute respiratory syndrome six week extended-dose neviapine (swen) study team: extended-dose nevirapine to 6 weeks of age for infants to prevent hiv transmission via breastfeeding in ethiopia, india, and uganda: an analysis of three randomized controlled trials chlamydial secretory iga antibodies in human milk hepatitis d virus factors associated with immunoprophylaxis failure against vertical transmission of hepatitis b virus phagocytosis-associated oxidative metabolism in human milk macrophages management of mastitis in breastfeeding women communityacquired methicillin-resistant staphylococcus aureus among patients with puerperal mastitis requiring hospitalization working parents: the impact of day care and breast-feeding on cytomegalovirus infection in offspring breast milk and the risk of cytomegalovirus infection tuberculosis, an old disease but a new threat to mother, fetus, and neonate vertical transmission of hepatitis b antigen in taiwan yeast-recombinant hepatitis b vaccine: efficacy with hepatitis b immune globulin in prevention of perinatal hepatitis b virus transmission maternal lyme disease and congenital heart disease: a case-control study in an endemic area significance of postnatal mother-to-child transmission of htlv-i on the development of adult t-cell leukemia/lymphoma disseminated neonatal herpes simplex virus type i from a maternal breast lesion the effects of yellow fever immunization (17dd) inadvertently used in early pregnancy during a mass campaign in brazil the impact of breastfeeding on the health of hiv positive mothers and their children in sub-saharan africa prospective study of mother-to-infant transmission of hepatitis c virus inhibitory effect of maternal antibody on mother-to-child transmission of human t-lymphotropic virus type i seroepidemiological study of human herpes virus 6 and 7 in children of different ages and detection of these two viruses throat swabs by polymerase chain reaction short-term breast-feeding may reduce the risk of vertical transmission of htlv-i. the tsushima atl study group infant feeding and risk of mother-to-child transmission of hiv-1 in sao paulo state, brazil. sao paulo collaborative study for vertical transmission of hiv-1 breast feeding plus infant zidovudine prophylaxis for 6 months vs. formula feeding plus infant zidovudine for 1 month to reduce mother-to-child hiv transmission in botswana: a randomized trial: the mashi study isolation of the aids virus from cell-free breast milk of three healthy virus carriers rates of diarrhoea associated with early weaning among infants in kisumu, kenya. in program and abstracts of the 14th conference on retroviruses and opportunistic infections contamination in expressed breast milk following breast cleansing brucellar meningitis in an infant-evidence for human breast milk transmission hepatitis e in india human parvovirus b19 congential yellow fever virus infection after immunization in pregnancy unaids/who: report on the global hiv/aids epidemic a review of hiv transmission through breastfeeding mother-to-child transmission of human t-cell leukemia/ lymphoma virus type i: implication of high antiviral antibody titer and high proviral load in carrier mothers clinical profile of chikungunya in infants infective and antiinfective properties of breast milk from hiv-1 infected women postnatal transmission of the human immunodeficiency virus type 1 from mother to infant: a prospective cohort study in kigali, rwanda mother-tochild transmission of human t-lymphotropic virus type ii the possible role of circumcision in newborn outbreaks of community associated methicillin-resistant staphylococcus aureus brucellosis chez un nourrisson de 3 mois recovery of staphylococcal enterotoxin f from the breast milk of a woman with toxic-shock syndrome evidence for sexual and mother-to-child transmission of human t lymphotrophic virus type ii among guaymi indians transmission of cytomegalovirus to preterm infants through breast milk postnatally acquired cytomegalovirus infection via breast milk: effects on hearing and development in preterm infants pregnancy and lactation in relation to breast cancer risk morbidity among hiv-1-infected mothers in kenya: prevalence and correlates of illness during 2-year postpartum follow-up high concentrations of interleukin 15 in breast milk are associated with protection against postnatal hiv transmission low and undectable breast milk interleukin-7 concentrations are associated with reduced risk of postnatal hiv transmission lactation mastitis: a descriptive study of the experience breastfeeding does not pose any additional risk of immunoprophylaxis failure on infants of hbv carrier mothers recurrent neonatal group b streptococcal disease associated with infected breast milk transplacentally transferred maternal-infant antibodies to dengue virus vertical transmission of hepatitis a resulting in an outbreak in a neonatal intensive care unit immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 e6 or e7 perinatal transmission of hepatitis g virus (gb virus type c) and hepatitis c virus infections: a comparison advisory committee on immunization practices; healthcare infection control practices advisory committee: recommendations for using smallpox vaccine in a pre-event vaccination program global rotavirus surveillance: determining the need and measuring the impact of rotavirus vaccines recommendations for minimizing cmv exposure in breast milk fed very low birth weight (vlbw) preterm infants. available at www.breastfeeding. org/articals/cmvpremi.pdf maternal-infant transmission of htlv-i: frequency and time course of seroconversion. abstract w-53 mother-to-child transmission of human t-cell lymphotropic virus type i associated with prolonged breast-feeding maternal lyme disease and congenital malformations: a cord blood serosurvey in endemic and control areas human versus cow' s milk in infant nutrition and health human t-lymphocyte retroviruses working group on severe streptococcal infections: defining gas streptococcal toxic shock syndrome: rationale and consensus definition acquired cytomegalovirus infection of breast milk in infancy oral transmission of human t-cell leukemia virus type 1 into a common marmoset as an experimental model for milk-borne transmission evaluation of cytomegalovirus infections transmitted via breast milk in preterm infants with a real-time polymerase chain reaction assay sequelae of maternally derived cytomegalovirus infections in premature infants mother-to-infant transmission of hepatitis c virus human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis independent protective effect of lactation against breast cancer: a case-control study in japan a maternal factor for mother-to-child transmission: viral antigen-producing capacities in culture of peripheral blood and breast milk cells mother-to-infant transmission of hepatitis c virus infectious diseases of the fetus and newborn infant sensitive method for detection of human herpes viruses 6 and 7 in saliva collected in field studies survey of 34 pregnant women with hepatitis a and their neonates postnatal transmission of aids-associated retrovirus from mother to infant disseminated lyme disease and pregnancy. 9th annual international scientific conference on lyme disease and other tick-borne disorders a. siblings at home have active chickenpox when neonate and mother are ready for discharge from hospital.no no 1. mother: if she has a history of chickenpox, she may return home. without a history, she should be tested for varicellazoster virus antibody titer.* if test is positive, she may return home. if test is positive, she may return home. if test is negative, varicella-zoster ig † is administered and she is discharged home. 2. neonate: may be discharged home with mother if mother has history of varicella or is varicella-zoster virus-antibody positive. if mother is susceptible, administer varicella-zoster ig to infant and discharge home or place in protective isolation. b. mother has no history of chickenpox; exposed during period 6-20 days antepartum. ‡ ‡if exposure occurred less than 6 days antepartum, mother would not be potentially infectious until at least 72 hours postpartum. §considered noninfectious when no new vesicles have appeared for 72 hours and all lesions have crusted. ¶dosage of acyclovir for pregnant woman is 30 mg/kg/day; for seriously ill infant with varicella, 750 to 1500 mg/m 2 /day. elisa, enzyme-linked immunosorbent assay; fama, fluorescent antibody to membrane antigen; la, latex agglutination. key: cord-000083-3p81yr4n authors: nan title: poster exhibition date: 2009-01-31 journal: hepatol int doi: 10.1007/s12072-009-9123-4 sha: doc_id: 83 cord_uid: 3p81yr4n nan background: biliary atresia (ba) is one of the most common causes of neonatal cholestasis and the most frequent hepatic cause of death in early childhood. the incidence rate of ba is higher in asian countries, occurring in approximately 1 of 8,000 (asian countries ) to 1 of 18,000 (european countries) live births. early identification and prompt intervention is very important. to im prove the early diagnosis, we used proteomic technology to screen serum biomarker for ba. methods: two-dimensional electrophoresis (2-de) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (maldi-tof-ms) were employed to screen serum biomarkers specific to ba sera from idiopathic neonatal hepatitis. after pretreatment including albumin and immunoglobulin (igg ) depletion, sera were subjected to 2-de and there after image analysis. the differentially expressed protein spots were identified by maldi-tof-ms. result: from optimized 2-de gel images, thirty-four spots were differentially expressed and identified by maldi-tof-ms to be eight proteins. overall, kininogen 1 variant was under expressed and alpha-1-b-glycoprotein, leucine-rich alpha-2-glycoprotein 1, 'sp40,40', a1bg protein, vitamin d-binding protein/group specific component , apolipoproteina-, aqgv 3103 were over expressed in ba group com pared to idiopathic neonatal hepatitis. conclusion: 2-de based serum proteome analysis can be useful in detecting protein expression alteration and new discovered biomarkers might be an aid in the diagnosis of ba, though further validation is needed. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 ims, bhu, varanasi, india background: a variety of autoreactive antibodies are detected in patients with chronic liver disease. this prospective, nonrandomized study was undertaken to evaluate the nature & prevalence of various autoantibodies in patients with chronic liver disease of diverse etiologies. methods: study population included 53 patients (75% males), who met defined criteria for chronic liver disease. detailed clinical, laboratory and sonographic evaluation was done. sera were tested for asma, anti-lkm type1, ama, apa, ana, by standard methods. p<0.05 was considered significant. results: among various etiologies for chronic liver disease, hepatitis b was most common (28%), followed by alcohol (19%), autoimmune hepatitis in 15%, hepatitis c (6%) and miscellaneous (2%). 30% of patients were labeled as cryptogenic after detailed investigations. ana (>1/80) was positive in 100% of definite aih, 33% of hcv related cld but at titer of >1/40, 66.6% of hcv related cld & 60% of probable aih were found positive. asma (>1/40) was positive in 6% of hbv related cld, 10% of alcohol related cld, 33% of definite aih, 40% of probable aih, 33% of hcv related cld but asma in titer of >1/80 was positive only in 33% oh definite aih. apa was detected in 12.5% of cryptogenic cld, 13.3% of hbv related cld & 20% of alcohol & probable aih related cld each. ama was detected in 1% of cryptogenic, hbv, aih (definite) & hcv related cld each, and 2% of alcohol related cld & 100% of pbc. conclusions: apart from aih there is high prevalence of ana & sma in hcv related cld while other antibodies has low prevalence in non-aih related clds. this study also suggests that prevalence of various autoantibodies should be borne in mind while considering the diagnosis of cld especially of mixed etiology. conclusions: oa infusion did not lower ammonia levels or improve survival. results: the mortality (50.0%) of patients in lamivudine group with meld score from 30 to 40 was lower than that (86.1%) of control group ( 2 =23.319, p=0.000). univariate analysis showed that mortality was significantly related to age (p=0.005), meld score (p=0.009), treatment method (p=0.000), pretreatment hbv dna load (p=0.000), the decline of hbv dna load during therapy (p=0.006) and encephalopathy (p=0.007). in multivariate analysis, in patients with meld scores 30-40, treatment method (p=0.004), pretreatment hbv dna load (p=0.009), decline of hbv dna load during therapy (p=0.014) and encephalopathy (p=0.019) were independent predictors of mortality; for meld scores above 40, only meld score (p=0.015) was independent predictive. conclusions: lamivudine treatment significantly decreases the 3 month's mortality of patients with meld score 30-40, and a low viral load pre-treatment and quick decline of hbv dna load are good predictors for the survival of lamivudine treatment. background/aims: early identification of patients with fulminant hepatic failure (fhf) who need a liver transplantation is very important. to construct a prediction model for early diagnosis and prognosis of fhf, we studied dynamics of metabolic profiles using a d-galactosamine/lipopolysaccharide (galn/lps)-treated mouse model. methods: balb/c mice were used to construct fhf model and sacrificed for blood collection at 4, 5, and 6 hour after treatment, respectively. levels of plasma metabolites were quantified using gas chromatography/time-of-flight mass spectrometry and data were processed using partial least squares discriminant analysis (pls-da). results: distinct clustering differences were observed 5 and 6 h after treatment between survival and dead groups. at 5 h, plasma levels of some metabolites differed significantly between survival, dead and control groups. ketogenesis and the tca cycle were inhibited in both survival and dead groups, but in dead group, the urea cycle was also inhibited and glycolysis was elevated. pls-da indicated that principal component weighting was greatest for plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate. the y-predicted scatter plot in pls model assigned samples to survival or dead groups using an apriori cutoff of 0.10 with 100% sensitivity and specificity. similar results were observed in 11 fhf patients with different outcomes. pe012 association between polymorphisms in the interleukin-10 gene promoter and hepatitis b-related acute liver failure conclusions: the pls model based on metabonomics analysis can be used to predict outcomes well, and plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate may constitute a set of markers for early diagnosis and prognosis of fhf. il-10 promoter are associated with the susceptibility to hepatitis b-related alf in the chinese population. il-10 a-592c may be a regulatory polymorphism that affects gene regulation. hepatocyte cell death in aclf: mechanism and significance -an immunohistochemical study. p. sakhuja 1 , a. rastogi 2 , s. s hissar 1 , a. singh 1 , a. kumar 2 , r. gondal 1 , s.k. sarin 1 1 gb pant hospital, 2 institute of liver and biliary sciences, new delhi, india background: acute on chronic liver failure (aclf) is defined as acute hepatic insult complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. caspases play an essential role in apoptosis. cox-2 is an inducible immediate early gene responsible for the release of prostaglandins during inflammatory response. we studied the immunohistochemical expression of cox-2 and caspase-1 in liver tissue to assess their role in pathophysiology and in predicting outcome of aclf. method: a retrospective analysis of 50 liver biopsies with clinical diagnosis of aclf was undertaken. patients were divided into two groups a and d based on clinical outcome (alive/died respectively). immunohistochemical analysis for cox-2 and caspase was performed on 39 and 36 cases respectively and scored from 0-8 as per intensity and distribution. score 6-8 indicated high intensity with focal to diffuse distribution, and was considered significant. results: etiology of acute liver failure was viral or alcoholic. increased expression of caspase was observed in 10/21 cases in group d and none of the cases in group a(n=15) (p=0.001). increased expression of cox-2 was observed in 4/21 cases in group d and none of the cases in group a (n=18) (p=0.052). conclusion: increased immunoreactivity of caspase in liver biopsies of patients of aclf may indicate worse prognosis and its important role in the pathophysiology of aclf. immunostaining for caspase is useful for assessment of prognosis and possibility of anti-apoptotic and anti-fibrotic therapies in future. conclusion: hhgf expression vector (pcmv-hhgf) has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hhgf in vivo. j.h. kim 1 , k.w. kim 1 1 asan medical center, seoul, korea background: splenic artery embolization (sae) is performed to increase hepatic arterial flow or to decrease portal venous flow in recipients of liver transplantation (lt). thus, the purpose of this study was to estimate sae effect on the basis of changes in caliber of related vessels and splenic volume on pre-sae and serial post-sae ct scans in lt recipients. methods: between 2003 and 2007, among 73 lt recipients who underwent sae and serial follow-up ct, 43 with no compounding factor that may obscure sae effect were included in this study. they underwent ct before and after (1week, 1month, and 1year) sae. a radiologist retrospectively measured diameters of ca, cha, sa, sv and splenic volume on serial ct scans. their diameters and splenic volume on each ct were compared with those on the prior and pre-sae ct. the difference was compared using repeated-measures anova tests. results: cas decreased between 1week and 1month after sae (p<.05), but were stable before 1week and after 1 month. chas increased within 1week (p<.05) but decreased between 1week and 1month (p<.05) and remained stable after 1month. compared with pre-sae ct, chas were larger for 1month after sae. sas continuously decreased for 1year (p<.05). svs decreased for 1 month (p<.05) and remained stable after 1 month. compared with pre-sae ct, sas and svs were smaller from 1week after sae and on. splenic volume continuously decreased for 1year except a period between 1week and 1month. conclusion: the increase of hepatic arterial flow persists for 1month after sae, but returns to baseline thereafter. the decrease of portal flow may lasts for at least 1year after sae. poster exhibition -cholangioca and other liver neoplasm poster session, hall 5b background: now, rfa has becoming an important practice of hcc therapy. in this study, we evaluated whether rfa therapy for metastatic liver tumor has a beneficial effect on patients' survival. methods: forty six patients were treated by rfa for metastatic liver tumor from july 2001 through february 2008 in our hospital, of the 46, 33 patients were metastasis either form colon or stomach cancer. these 33 patients were analyzed in this investigation. cumulative survival rate from initial rfa therapy was calculated by kaplan-meier method. predictive factors for survival were identified using cox proportional hazard regression model. results: the mean age of the 33 patients were 64. 6 (range, 40-79) . the mean size of the tumor is 28mm (range,8-70mm) and the numbers of tumor foci are 2.7 nodules range,1 18. the survival rates of patients treated by rfa were 49.5% at 3 years and 31.8% at 5 years in colon cancer, 15.6% at 3 years and 15.6% at 5 years in gastric cancer. in this series of 33 patients, primary cancer: colon (p=0.002 odds ratio 0.132 95%ci 0.037-0.473), younger patients ( 64) (p=0.041 odds ratio 0.312 95%ci 0.102-0.955) and multiagent chemotherapy (p=0.012 odds ratio 0.223 95% ci 0.069-0.723) were significantly correlated with better survival. conclusion: the survival of patients treated by rfa for metastatic colon cancers had better survival than those of gastric cancers. in addition, good indication of rfa is for metastatic colon cancers, younger patients and has to be treated by multiagent chemotherapies. utility of contrast enhanced ultrasonography with sonazoid in radiofrequency ablation (rfa) for liver metastasis e. goto 1 , s. shiina 1 , r. tateishi 1 , r. masuzaki 1 , k. enooku 1 , t. sato 1 , j. imamura 1 , t. goto 1 , y. sugioka 2 , h. ikeda 1,2 , h. yoshida 1 , m. omata 1 1 department of gastroenterology, university of tokyo, 2 department of clinical laboratory, tokyo, japan background & aims: contrast enhanced ultrasonography (ceus) with sonazoid is effective for liver metastasis because enhance defect in kupffer imaging is well delineated. the aim of this study is to investigate the detection ability of ceus and the utility of sonazoid in rfa for metastasis liver tumors. material & methods: from january 2007 to december 2007, a total of 346 liver metastatic nodules in 87 patients (62 colon cancer, 13 breast cancer, 3 gastric cancer, 3 islet cell tumor, and 6 others) admitted to receive rfa were studied. the detection ability of liver metastasis was compared between ceus and conventional us using enhanced ct as reference standard. the mean numbers of treatment session of rfa were compared between patient treated with ceus assistance and historical controls matched for size and number of tumors. results: the detection rate was 78.6% with conventional us and 96.4% with ceus (p=0.0004). 83 nodules in 25 patients were not detected by conventional us and detected after injection of sonazoid. in addition, 12 nodules in 2 patients were detected not by ct but only by ceus. the mean number of session was 1.5±0.5 as compared to 2.0±1.0 in the historical controls (p<0.001). conclusions: ceus with sonazoid is useful for detection of liver metastasis. sonazoid is an excellent supportive agent in rfa of liver metastasis. background/aims: carcinogenesis of intrahepatic cholangiocarcinoma (icc)-associated liver fluke infection accumulated genetic and epigenetic alterations. cholangiocarcinoma cell line (kku-m213) is adenosquamous carcinoma which rare variants and not commonly found in icc. however, interactions of liver fluke-associated icc proceed to genetic alterations in adenosquamous carcinoma that have been not elucidated. objectives: to analyze the whole genome-wide genetic alterations in kku-m213 using microarray comparative genomic hybridization. methods: dna of kku-m213 and matched-sex reference were differentially labeled with fluorescence dries (cy3 and cy5) and mixed together with cot-1 dna. the mixture was hybridized on array with spotting 2,464 human bacterial artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13.1, 4q21.1, 4q21.2, 5p tel, and 5p15.3, whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3. conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13. 1, 4q21.1, 4q21.2, 5p tel, and 5p15.3 , whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3 . conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. acknowledgements: this work was supported by faculty of medicine, kku, thailand (grant no. i51117 background/aims: we studied the clinical efficacy of arterial chemoinfusion therapy through an implanted port system for patients with intrahepatic cholangiocarcinoma (icc). thirty patients with unresectable icc or intrahepatic recurrence of icc after surgery were studied. comparison was made between patients who received arterial chemoinfusion therapy through an implanted port system with adriacin and lecithin-added lipiodol emulsion in 5 patients and 5-fluorouracil (5-fu) in 7 patients. eighteen patients were treated without port system. results: disease was stable in 5 patients with adriacin and lecithin-added lipiodol emulsion and in 3 patients with 5-fu. disease was progressed in 4 patients with 5-fu. the mean survival period was 20.8 months in patients with adriacin and lecithin-added lipiodol emulsion, 9.3 months in patients with 5-fu, and 10.5 months in patients without port system (p=0.02, p=0.04). conclusion: arterial chemoinfusion therapy through an implanted port system is useful for patients with intrahepatic recurrence of icc after surgery. pe038 s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh. india background: a number of dietary factors have been involved in the pathogenesis of cholelithiasis. cholesterol overfeeding is the primary means of inducing supersaturated bile and cholesterol gallstones in animal models.aim of the study was to investigate the rate of epithelial cell death and proliferation in gallbladder during gallstones formation. methods: balb/c mice was divided into two groups control in this group animals were fed normal chow diet, high fat diet group in this group (2% cholesterol,0.5% sodium cholate, 5% butter fat and 15% coconut oil) mixed with chow diet was fed to the mice for 4 weeks. cell apoptosis and proliferation was assayed in gallbladder epithelial cells. histological analysis of gallbladder sections were done with hematoxylin and eosin staning. results: mice fed high fat diet had apoptotic as well as necrotic epithelial cells. rate of proliferation was enhanced after 24 and 48 hrs in mice fed high fat diet group as compared to the control group. the histopathological section of control gallbladder has normal morphology whereas gallbladder wall thickness was markedly increased; epithelial cells appeared more elongated in mice fed high fat diet. conclusion: results obtain show that high fat diet markedly induced biliary epithelial cell proliferation and biliary epithelial cell apoptosis. it has been determined that when there is an injurious stimulus that leads to apoptosis, it is later followed by reparative proliferation and when there is no injurious stimulus, apoptosis occurs late in the course as part of remodeling. background: obstructive jaundice can be caused by malignancy. the treatment can be drainage by biliary stenting. in advanced malignant jaundice, the stent placement is often difficult. objective: to evaluate the success rate of malignant obstructive jaundice evaluation of ercp and success rate of stent placement. methods: retrospective study based on data of ercp from october 2004 until july 2008. results: we evaluated 139 patients who has done ercp examination, 131 (94,2 %) patients have clinical diagnosis of obstructive jaundice. there were 73 (55,7%) male and 58 (44,3%) female, age range 20 -84 (median age was 51). there were no malignancy in 66 (50,4 %) patients; malignancy in 48 (36,6%) patients and 17 (13%) patients need further evaluation.. from 114 patients, 57 (50 %) patients attempted to have stent placement, 50 (43,9 %) patients do not and 7 (6,1 %) patients have no data. we done descriptive study on 57 patients attempted to have stent placement, 32 (56,1 %) patients succeed in stent placement whereas 25 (43,9 %) failed. malignancy was showed to be a factor of stent failure (malignancy: 23 fail and 10 success (30,3 %) vs non malignancy: 2 fail and 22 success (91,7%)). conclusion: ercp can identify the cause of obstructive jaundice in 87 % patients. the success rate of stent placement was 56,1 %. the success rate of biliary stenting in malignant obstructive jaundice was 30,3 % whereas in non-malignant cases was 91,7 %. papillary carcinoma was the most frequent cause of malignant obstructive jaundice. background: in hydatid disease of the liver cystobiliary fisula (cbf) constitutes an entity characterized by the occurrence of a life-threatening cholangitis with increased morbidity. aim: to study the different diagnostic and therapeutic aspects of cystobiliary fistula in hydatid disease of the liver. patients and methods: fourteen patients with complicated cysts were divided into 2 groups; group a: nine patients presented with cholangitis, and group b: five patients had history of jaundice. in all patients, the diagnosis of cbf was confirmed by erc (endoscopic retrograde cholangiography). preoperative endoscopic sphincterotomy (es) was done in group a with retrieval of hydatid daughter cysts. seven patients (subgroup a1) were subsequently submitted to surgery entailing endocystectomy in 5 and hepatic resection in two. the remaining 2 patients in group a (subgroup a2), were managed by endoscopic therapy only. patients of group b (n=5), were not submitted to preoperative es and were subsequently managed by hepatic resection in one patient and endocystectomy in four. results: there was no mortality in the studied group. postoperative bile leak occurred in four cases in group b. in contrast, none of the patients who were submitted to preoperative es (subgroup a1) had bile leak. all patients received albendazole treatment. conclusion: erc is important in confirming the diagnosis of cbf. also, therapeutic erc has a place in the treatment algorithm of cbf as it was found to be a safe and a reliable therapeutic alternative especially in high risk patients for surgery. v. singh 1 , g. singh 1 , g.r. verma 1 , v. gupta 1 , s. ghosh 1 , r. gupta 1 , r. kapoor 1 , n. sharma 1 , a. bhalla 1 , s.k. mahi 1 1 background: endoscopic palliation in malignant hilar biliary obstruction requires ercp. however, contrast injection leads to cholangitis. recently, contrast-free metal stenting with or without mrcp has shown encouraging results. however, mrcp and metal stents are costly. there have been no reports on the use of air cholangiography in these patients. methods: we prospectively studied the role of air cholangiogaphy assisted unilateral plastic stenting in these patients. results: ten patients with unresectable malignant hilar biliary obstruction were studied. air cholangiography detected type ii obstruction in 8 and type i in 2 patients which is similar to mrcp. all patients underwent unilateral plastic stenting. a successful endoscopic drainange was achieved in 100% patients. cholanngitis occurred in none and there was no 30-day mortality. no major complications were observed. conclusion: air cohlangiography assisted plastic stenting in these patients is a safe and effective method of palliation. however, it requires a larger study. introduction: a description of igg4-related sclerosing cholangitis (igg4-sc) without pancreatic lesion has recently been reported. in addition to imaging, diagnosis relies on findings of elevated serum igg4 and immunodetection of invading igg4-positive cells. here we report a case of igg4-sc with only slight common bile duct abnormalities and normal pancreatic findings. case study: a 65-year-old man suffering from cephalalgia, general malaise and muscle ache was admitted to our hospital. his blood examinations on admission revealed eosinophilia, mild anemia, liver dysfunction and an igg level of 2820 mg/dl (igg4 374 mg/dl). although ercp did not reveal typical stenosis or irregularities of the bile duct wall, visualization of peripheral bile ducts was slightly impaired. echography revealed thickening of the intrahepatic bile duct and gallbladder walls as well as adenopathy. due to a gradual increase in pleural effusion and a progression of anemia, oxygenation was begun on the seventh day of illness. based on the combination of eosinophilia, elevated serum igg4 levels, image findings and a negative result for helminth, igg4-sc was suspected. liver biopsy was performed on the ninth day of illness and steroid therapy was initiated, after which symptoms and laboratory findings improved. the igg4-positive plasmocytic infiltrate present around the portal region at the time of biopsy disappeared within eight months of treatment. summary: this case displayed two unusual features that are not generally observed with igg4-sc: complications due to hemolytic anemia, and destruction of the peripheral bile duct with little damage to the common bile duct. introduction: various systemic diseases have been reported to be associated with igg4. although steroids are effective in the treatment of igg4-related diseases, there are some reports on relapses with their treatment, and cases are often difficult to differentiate from malignant diseases. we encountered a case of autoimmune pancreatitis with sclerosing cholangitis (aip-sc), in whom ca19-9 was elevated with episodes of exacerbation and an elevated serum igg4 concentration. igg4 staining was also useful for the diagnosis. case study: an 81-year-old woman noticed tumors beneath the bilateral jaw and was found to have an elevated level of ca19-9 (304) seven years previously. her left submandibular gland was removed and diagnosed as sclerosing sialadenitis. four years previously, she was diagnosed as having diabetes mellitus complicated by a recurrence of ca19-9 (419) elevation and liver dysfunction. cholangiocarcinoma was suspected based on ercp, but was not confirmed by histologic findings of bile duct biopsy. elevated igg4 and other test results established the diagnosis of aip-sc, so steroid therapy was initiated, after which symptoms and laboratory findings improved. this recurrence of ca19-9 elevation (634) was diagnosed as a relapse of aip-sc based on an increased igg4 level and histologic findings. summary: some papers have reported that igg4-positive cells are found in liver tissue in this disease, but such cells were not detected in the liver specimens in our case. this might be because intra-liver sites may have differed in the degree of morbidity, and long-term steroid therapy might have suppressed inflammation in the liver tissue. s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh, india background: cholelithiasis, a gallstone disease is major cause of morbidity affecting millions of people throughout the world. aim of the present study was to investigate the predisposing factors that lead to the formation of gallstones. methods: the study was carried out on gallstones, bile and serum of patients. gallstones and bile were divided into three groups' cholesterol, pigmented and mixed gallstones. blood of the patients was divided into two groups with gallstones and without gallstones patients. trace elements and various biochemical estimations were carried out. clinical history of the gallstones patients was recorded from the hospital records. results: trace elements analysis in bile and gallstones showed that calcium is the main element in all the three types of stones. iron was the main element in mixed gallstones. in pigmented gallstones magnesium and zinc were the major trace elements. liver function tests and lipid peroxidation levels in sera were significantly increase whereas, antioxidant enzymes concentrations in sera were significantly decreased in patients with gallstones. clinical history of the gallstones revealed the cases had jaundice, diabetes mellitus and estrogen replacement therapy respectively. conclusion: results suggest that trace elements in gallstones and bile as well as clinical history of patients with chronic cholelithiasis could be the underlying factor in the pathogenesis of gallstones. the concentration of products derived from the free radicals reactions increases with degree of inflammation. such a condition increases risk of bile saturation which would further contribute to the progress of gallstones formation. background and aims: diseases of the biliary tree and gallbladder are being described with increasing frequency among patients with the acquired immunodeficiency syndrome (aids).therefore there is a need to do a research about the risk factors of gallbladder diseases in hiv/aids patients. so it can be useful to clinicians to predict the possibility of a patient having gallbladder disease and consider the options of further plans. the aim of this study was to find the prevalence and varieties of gallbladder diseases in hiv/aids patients. methods: a cross sectional study was performed in patients with hiv/aids who visited ciptomangunkusumo hospital, jakarta. the risk factors (route of transmision,cd4,arv,hepatitis) and clinical presentations were studied.ultrasonography examinations were performed to detect gallbladder annormalities. results: 68 patients with hiv/aids match the study criteria. there were gallbladder abnormalities in 22 (32.4%) subjects, which 19 (27.9%) had acalculous cholecystitis and 3 (4.4%) had cholecystitis with cholelithiasis. on bivariate analysis, there was a significant association between abdominal pain, jaundice and the use of arv to gallbladder abnormalities (p = 0.000; 0.000; 0.004; 0.012). however, there was no association between age, sex, transmision route of hiv, hepatitis and cd4 to gallbladder abnormalities. conclusion: hiv/aids patients are susceptible to opportunistic gallbladder infection. acalculous cholecystitis is the most frequently encountered gallbladder abnormalities of hiv/aids patients in this study. poster exhibition -hbv poster session, hall 5b long-term stopping therapy t.b. trung 1 , p.h. phiet 1 1 university medical center, hochiminh city, vietnam background: among the approved nucleos(t)ide analogues therapies for chronic hepatitis b, lamivudine was used widely, sometime inappropriate in practice due to high safe and low price but lamivudine is associated with the highest rate of drug resistance. objectives: the aim of the study was to determine the ymdd variants after long-term stopping treatment in lamivudine-resistant patients using more sensitive technique. methods: 16 blood samples from lamivudine resistant patients were collected after long-term stopping therapy. the ymdd variants are detected using technique pcr restriction fragment length polymorphism (pcr-rflp) at hcmc university medical center results: after stopping lamivudine treatment 25 months (6-72 months) ymdd mutants were detected in 14 (87,5%) of 16 patients. among them 13 (92.9%) had the most important m204v/i mutant, 1(7 1%) had accompanying l180m mutant. it means that once drug resistant mutants have been selected, they are archived for the long time even if treatment is stopped. many of patients have the features characterized for the patients in immune tolerance phase (young age, hbeag positive, normal alt). the treatment of this group is not strongly recommended due to low efficacy and high risk of drug resistance. conclusion: the most important m204v/i mutant was still detected with significant portion of the virus population after long-term stopping therapy in lamivudine resistant patients. the options of retreatment for this patients when necessary are limited due to cross-resistance. the management of chronic hepatitis b should be followed strickly the recommendations of specialized association to avoid this problem. background/aim: whether liver stiffness measurement (lsm) using transient elastography is reliable to assess liver fibrosis in the settings of severe acute exacerbation of chronic hepatitis b (chb) is uncertain. methods: we prospectively recruited consecutive patients with severe acute exacerbation of chb (alanine aminotransferase or alt >10x upper limit of normal). the relationship of alt levels and lsm were serially assessed and liver biopsy was performed after alt normalization. results: eleven patients (10 male, median age 43 years) were followed up for 25 weeks; 9 patients received anti-viral therapy. overall, lsm was positively correlated with alt levels (r=0.67, p<0.001). at initial presentation, the median serum alt and lsm was 1136 (581-2210) iu/l and 26.3 (11. 1-33. 3) kpa. a progressive reduction in lsm was observed during subsequent visits in parallel with the reduction of alt levels. even after the normalization of alt at week 12, lsm of 9 patients continued to drop at week 25. at the last visit, the median alt was 27 (11-52) iu/l and lsm was 7.7 (4.7-10.8) kpa. among the 5 patients who had liver biopsy performed at week 25, 4 patients had f2 fibrosis (lsm 5.7-8.1 kpa) and 1 patient had f3 fibrosis (lsm 8.6 kpa). conclusions: lsm using transient elastography may misdiagnose liver cirrhosis in patients suffering from severe acute exacerbation of chronic hepatitis b. lsm should be assessed after normalization of alt levels in order to accurately assess the degree of fibrosis. h.c. lai 1 , s.w. lai 1 , k.f. liao 1 , c.s. liu 1 , t. lin 1 , c.c. lin 1 1 china medical university hospital, taichung, taiwan background: in 2007, chronic liver disease was the seventh leading cause of death in taiwan. hepatitis b and hepatitis c are two major causes of chronic liver disease in taiwan. the purpose was to investigate the seroepidemiology of hepatitis b surface antigen (hbsag) and hepatitis c virus (hcv) antibody in taiwan. method: this was a hospital-based cross-sectional study. we analyzed viral hepatitis data from 2695 subjects who received health checkups at one medical center in taichung from 2003 to 2004. all subjects were divided into three age groups, including 20-39, 40-64 and 65. this study emphasized the prevalence of hbsag and hcv antibody by gender and age. the statistical analysis was performed by t test and 2 . result: there were 1526 men (56.6%) and 1169 women (43.4%). the mean age was 49.2 (standard deviation 12.2, range 20-84). the overall prevalence of hbsag was 14.7%, with statistically significant difference(ssd) between gender (17.4% for men vs 11.2% for women, p <0.001). the prevalence of hbsag was decreased with age in men, with ssd (p <0.001), and also decreased in women, without ssd (p =0.08). the overall prevalence of hcv antibody was 5.2%, without ssd between gender (4.8% for men vs 5.7% for women, p =0.272). the prevalence of hcv antibody was increased with age both in men and in women, with ssd (p <0.001). conclusion: we hope this study can provide the epidemiological data for further studies of hepatitis b and hepatitis c virus infection in taiwan. s.m. wu 1 , x. zhou 2 1 wuhan medical treatment center, 2 center for gene diagnosis, zhongnan hospital, wuhan university, china e-selectin is revealed to facilitate leukocyte adhension to the endothelium and migration into inflamed tissue in inflammatory diseases. chronic hepatitis b virus infection is regarded as a chronic inflammatory process. to examine the possible involvement of e-selectin in the etiology of chronic hbv infection, we analyzed two polymorphisms of e-selectin and determined the plasma souble e-selectin levels in patients with chronic hbv infection and controls. the frequency of c allele of the a561c polymorphism was significantly increased in patients with lc campared with controls. no significant positive association was observed between the g98t polymorphism and chronic hbv infection. but in patients with lc, divided according to the child-pugh classification, the frequency of t allele was of significant difference between child'class a and class b plus c. plasma levels of soluble e-selectin were significantly increased in patients with chronic hepatitis and liver cirrhosiscompared with controls. in the liver cirrhosis group, levels of se-selectin were significantly decreased from child' class a to class c. in each group, patients with c allele of the a561c polymorphism showed higher soluble e-selectin levels than those with a allele. this is the first report describing the association between e-selectin polymorphisms and hbv-related hepatic fibrosis. our data showed the a561c polymorphism of e-selectin gene is associated with disease progression in patients with hbv infection and controls the expression of plasma soluble levels, the g98t polymorphism may be related to fibrotic severity in patients with liver cirhosis. background: chronic hepatitis b (chb) patients with high serum hbv-dna and normal serum alanine aminotransferase (alt) levels might be considered for treatment if histopathological findings show fibrosis stage 2 or more. however, to our knowledge there is no recommendation with regard to the therapeutic agents for this group of patients. objective: this study was aimed to evaluate the efficacy of nucleoside analogues (entecavir or telbivudine) in treating chronic hepatitis b patients with high serum hbv-dna and normal serum alt levels. patients and method: this was an open-label study in chb patients with high level serum hbv-dna levels between january 2007 and october 2008. patients were included if they showed normal serum alanine aminotransferase (alt) level at two measurements within a 3-month interval and had fibrosis stage > 2 on liver biopsy specimens. patients were treated with entecavir 0.5 mg/day or telbivudine 600 mg/day. the primary endpoint was the reduction or undetectable of serum hbv-dna at 24 week and 48 week of treatment, while the secondary endpoint was hepatitis b e antigen (hbeag) seroconversion. results: during a 2-year period, 37 chb patients with high level serum hbv-dna with normal alt two times with 3 months interval underwent a liver biopsy. twenty-eight (75.7%) of 37 pts showed fibrosis stage 2 on histological findings (metavir score). twelve of these 28 patients received nucleoside analogues, 7 (58.3%) of them were men. patients' median age was 42 (range: 24-52) years. there were 5 patients with stage-2, 6 patients with stage-3 and 1 patient with stage-4 fibrosis. eleven (91.7%) patients had genotype b virus. at baseline, the mean serum alt level was 32 + 11.8 u/l and mean hbv-dna level was 2.48 x 10 6 iu/ml, ranging from 1.23 x 10 3 to 2.4 x 10 7 iu/ml. six patients received entecavir and the other six received telbivudine therapy. undetectable hbv-dna was achieved by 9 (75.0%) patients at week-24 and 2 (16.7%) patients at week-48 of treatment. one patient who had the highest hbv-dna level had viral load reduction to 1.6 x 10 4 iu/ml at week-48 of treatment. two out of 5 patients with positive hbeag achieved hbeag seroconversion at week-48 of treatment. conclusion: this preliminary study has shown that nucleoside analogues might be considered in the treatment for chronic hepatitis b patients with high serum hbv-dna and normal serum aminotransferases levels. j. chen 1 , x.j.. wu 1 , y. wang 1 , g.q. wang 1 1 department of infectious diseases, peking university first hospital, beijing, china background: the dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are new members of cd28/b7 family, as co-stimulatory molecules expressing on t cells and antigen present cells (apcs). their engaging can downregulate the t cells function, including proliferation, cytokines secretion and cytotoxicity. in periphery blood, pd-1 was upreguated on virus specific-t cells, leading to the impairment of t cells. blocking the pd-1/pd-l can improve the function of t cells. methods and patients: 21 patients with chronic hepatitis b (chb) were treated by pegylated ifn -2b (pegintron from schering-plough, once a week, 0.5 or 1 g/kg/weight). the periphery blood were taken at 0 weeks, 4 weeks, 8 weeks, and 12 weeks. periphery blood mononuclear cells (pbmc) were isolated from fresh heparinized blood by ficoll-hypaque (density: 1.077g/l) density gradient centrifugation. then the cells were incubated with apc-conjugated anti-pd-1 antibodies. the pd-1 expression on lymphocytes was detected by flow cytometry (fcm). results: the pd-1 expression on lymphocytes at 0 weeks was 14.47±5.8%, at 4 weeks was 9.68±3.75%, at 8 weeks was 6.95±2.39%, at 12 weeks was 6.08±1.31% (p<0.05). conclusion: treatment with ifn -2b can downregulate the pd-1 expression on lymphocytes and may partially restore the function of t cells. to investigate the effects of nucleoside analogs therapy in hepatitis b related acute-on-chronic liver failure, we treated 55 hbv related acute-on-chronic liver failure patients with entecavir. as control, the remaining 74 were not treated with nucleoside analogues. results show the survival rate of entecavir therapy group has no significantly difference with none-treated group (p>0.05). although entecavir greatly reduced hbv replication during different therapy times (p<0.001), the meld score and liver function (alt, albumin, bilirubin, prothrombin time) had no significant changes (p>0.05). further more, we analyzed the meld score and liver function in different hbv-dna level patients .no significantly difference was observed (p>0.05). there is no significant correlation between hbv-dna level and meld score in different therapy times (p>0.05).the hbv-dna level between patients with over 3 months and less than 3 months survival patients showed no significant difference either (p>0.05). however, meld score and some parameters of liver function (albumin, bilirubin, prothrombin time) showed significant difference (p<0.05). these results suggest hbv-dna loading may not be a direct factor to increased liver injury and suppression of hbv replication may not reduce the severity of liver failure in hbv related acute-on-chronic hepatitis. s. firdoos 1 , u. adeeb 1 , a. mehmood 1 , m. gill 1 1 islamabad specialists clinic, islamabd, pakistan background: before the availability of etv, it was common to use adv for treatment of chronic hepatitis b patients. primary nonresponse and suboptimal response is a common problem with adv treatment. methods: we wanted to study the outcomes of entacavir therapy in this subset of patients. study was conducted between april 2007 to april 2008. we enrolled 30 chb patients who had non response to 12-24 weeks of 10 mg adv therapy. non response and suboptimal response was defined as non dimunition of at least one log of hbvdna from baseline after 12 weeks of therapy and persistence of 3 log10 after 24 weeks of therapy respectively.they were switched to 1mg entacavir before breakfast daily for at least 12 months.they had serial alt cbc and hbvdna measured every 12 weeks. results: out of 30 patients 20 male and 10 were female. only 8 patients were hbeag(+).mean hbvdna level prior to adv exposure was 6.5 log copies/ml.mean duration of exposure to adv was 26 weeks.5 patients lost to f/u.we did intention to treat analysis. 15 out 30 (50%) patient has, undetectable level of hbvdna after 12 weeks of therapy labelled as group 1.6 out of 30 (20%) had hbvdna level reduced by a mean of 3 log 10 copies/ml labelled as group 2.on week 24 treatment analysis all 15 patients from group 1 was hbvdna undetectable, 2 additional patients from group 2 had undetectable hbvdna. conclusion: entacavir therapy results in rapid suppression of hbvdna levels in majority of patients with primary nonresponse or partial non response to adv therapy. background: except for serum alt level, baseline factors predictive of therapeutic response to lamivudine in patients with hbeag-positive chronic hepatitis b remain largely unknown. we thus studied the influence of pre-therapy viral factors on end-of-treatment responses to lamivudine therapy. methods: a total of 116 treatment-naïve hbeag carriers who had pre-therapy serum alt level> 5xuln and received lamivudine for 18 months reimbursed by the national health insurance were prospectively enrolled. hbeag seroclearance and combined hbeag seroclearance, alt normalization as well as undetectable hbv dna at the end of therapy were defined as primary and secondary endpoint, respectively. the pre-therapy viral factors including viral load, genotype, precore stop codon (pc)/ basal core promoter (bcp) status, and pre-s deletion were determined to correlate with therapeutic endpoints. results: the frequency of patients with detectable pc mutation (g1896a), bcp mutation (a1762t/g1764a), and pre-s deletion at baseline was 22.4%, 21.6%, and 12.1%, respectively. after completing 18-month lamivudine therapy, overall hbeag seroclearance rate was 56.0%. patients with hbeag seroclearance had a higher prevalence of baseline pc mutation than those without (30.8% vs, 11.8%, p= .015). by multivariate analysis, the odds ratio of patients with pc mutation to develop hbeag seroclearance was 3.33 (p= .024). in addition, the presence of pc mutation also correlated with the combined response. conclusions: for hbeag-positive chronic hepatitis b patients with serum alt> 5xuln, pc mutation could predict a higher hbeag seroclearance rate at the end of 18-month lamivudine therapy. the efficacy of adefovir dipivoxil against all patterns of lamivudine resistant hepatitis b d.j. kim 1 , y.d. park 1 , y.g. kwon 2 , h.g seo 1 1 daegu fatima hospital, 2 kunngpook national university hospital, daegu, korea background: our aim was to evaluate the efficacy of adefovir dipivoxil (adv) and determine patient-dependent or laboratoroy variables that are predictive of hbeag loss and ivr for hepatitis b patients resistant to lamiduvine. also we evaluated the activity of adv against all patterns of lamivudine-resistant hbv. method: 179 hbv-infected patients with lamivudine resitance received adv for 6 months. quantitative hbv dna, hbeag/anti hbeag, alt was checked every 3-6months. the hbv polymerase of 161 patients were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. result: there is no significant difference in all patterens of hbv mutation about hbv dna reduction at 24w, 48w, 72w. there is no significant difference in all patterens of hbv mutation about alt normalization at 24w, 48w, 72w. conclusion: adefovir dipivoxil demonstrated similar potent anti-hbv efficacy regardless of the different patterns of lamivudine-resistant hbv mutations. g. novelli 1 , m. rossi 1 , v. morabito 1 , f. pugliese 1 , p. berloco 1 1 la sapienza university, rome, italy background: hepatitis b (hbv)-related end-stage liver disease is one of the most common indication for liver transplantation (lt). a number of patients dying while on the waiting list or removed because of being too ill is progressively increasing. we valued the possibility to improve the model end-stage liver disease (meld) of patients awaiting liver transplantation using a albumin dialysis: molecular adsorbent recirculating system (mars). methods: we treated 34 patients (19 male and 15 female) with a mean age 49.5. inclusion criteria: serum bilirubine > 15mg/dl, meld 25, inr > 2.1, encephalopathy grade ii. all patients were treated with mars mean 9±2.5 hr cycles and mean 9 treatments (range 3-15). all patients received standard medical treatment in addition to mars dialysis. the patient survival was valued at six months. results: we obtained a significant change of cytokines levels as interlukine 6 (p< 0.02) and tumor necrosis factor alfa (p<0.01) in association with an improvement of kidney, hepatic and hemodynamic parameters. at the end of mars treatments we observed a significant reduction of meld score (p<0.003). the results of meld show a rebound effect between the end of treatment and the follow up at six months without returning at starting values (p<0.005). twenty patients lived and 14 dead for clinical complications. conclusion: the improved meld score with mars gave patients on lt waiting list more time of survival, thus allowing them more opportunity for liver transplantation. entecavir for treatment of lamivudine-refractory patients chronic hepatitis b h.t. dat 1 , p.t.t. thuy 1 1 medic medical centre, ho chi minh, vietnam lamivudine treatment is associated with frequent development of resistant hepatitis b virus. this incidence especially is higher in longer time of treatment and loss of treatment benefit. entercavir is a new antiviral agent shown its high efficacy even in cases of mutations with lamivudine resistance. in this study, we evaluate the efficacy, the safety of entercavir in treatment of lamivudine-refractory patients chronic hepatitis b. sixty chronic hepatitis b patients with evidence of lamivudine resistance were randomly divided into two groups in proportion of 3:1. group i (n=45) used entecavir 1mg/day, group ii (n=15) used lamivudine 100mg/day. treatment time was 48 weeks. histology, alt, hbvdna were evaluated in the end of the treatment. age, sex, alt, hbvdna, genotype, hbeag were analyzed to evaluate their influences to the treatment. the results have showed hbvdna<2000 copies/ml in entecavir group 37.78% vs. 0% lamivudine group (p<0.01). hbvdna negative in entecavir group was 17.77% and incidence of seroconversion of hbeag was 8.82%. alt was normal in entecavir group 77.77% vs. 26.66% in lamivudine group (p<0.001).histologic improvement in entecavir group was 37.77% vs.6.66% in lamivudine group (p<0.05). patients with hbeag negative, genotype b, low viral load were shown better results. entecavir was shown to be efficacious in treatment for chronic hepatitis b patients experienced with lamivudine resistance. entercavir is safe, with almost no side effects. factors such as hbeag negative, genotype b, low viral load seems to be better in response to treatment. recurrence or mutation of entecavir resistance should be studied further in future. j.m. kim 1 , s.k. hwang 1 , b.h. choe 1 1 department of pediatrics, kyungpook national university hospital, daegu, korea backgrounds: by analyzing the characteristics of children with chronic hepatitis b who have lost hbsag by long-term lamivudine treatment, the selection of target patients could be relevantly predictable in the treatment of chronic hepatitis b in children. methods: a total of 75 hbeag positive children (< 18 y-o) were recruited who have visited kyungpook national university hospital from mar. 30, 1999 to may 8, 2008 . they were treated with lamivudine for at least 6 months. hbeag seroconversion occurred during lamivudine treatment in 49 out of 75 children. they were divided into hbsag clearance and non-clearance group. parameters influencing treatment results were analyzed according to hbsag loss. result: thirteen out of the 49 (26.5%) patients with hbeag seroconversion were classified as hbsag clearance group, while 36 (73.5%) as non-clearance group after lamivudine treatment. twenty five of 49 patients with hbeag seroconversion were under 6 years old, in 10 (10/25, 40%) of whom hbsag loss occurred as well. twenty four of 49 patients were over 6 years old, in 3 (3/24, 12.5%) hbsag loss occurred, that showed significantly difference (p-value= 0.029, or: 4.667, ci: 1.094-19.902) compared to younger group. age was significantly lower in hbsag clearance group (5.1±4.3 years) than non-clearance group (8.2±5.0 years) (p=0.043), but no difference was observed in other parameters. anti-hbs appeared in 12 patients. conclusion: in the treatment of hbeag positive chronic hepatitis b with lamivudine, age was significantly lower in hbsag clearance group than non-clearance group. background: dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are members of cd28/b7 family, was reported to transfer inhibitory signal, leading to the dysfunction of t cell. background: hepatitis b viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. mutations of hbsag allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus in reduced to undetectable levels. methods: immunohistochemical analysis of tissue samples from 56 patients with chronic hepatitis b (chb), 12 acute hepatitis b (ahb) patients and 10 health controls was performed. results: pd-1 was positively expressed on lymphocytes infiltrating the portal area.pd-l1 expression was the same as pd-1,also expressed in interlobular.pd-l2 expressed on kupffer cells and dendritic cells.pd-1-,pd-l1-,and pd-l2-positive cells express index of chb patients were much more than that of health controls and ahb patients(p 0.05).between groups in chb,the expression rate increase with the disease progression (p 0.05). methods: 58 chronic hepatitis b patients with both positive for hbsag and hbsab were studied.serological markers of hbv were detected by elisa and microparticle enzyme immunoassay. hbv dna levels were determined by fluorescent quantitative pcr, s gene fragments were directly sequenced, liver function was analyzed by automatic biochemistry analyzer au400. correlation test was conducted to evaluate their dependablity. conclusion: overexpression of pd-1 and pd-l within liver might be involved in inhibiting the immune response and be a mechanism of chronicity in hbv infection. results: the level of hbsag and hbsab was 254.4±68.3 s/n and 39.4±38.1 miu, respectively. hbv dna was detectable in 46 patients. fifty-one mutations of s gene were detected in 38 patients, and the relating amino acid substitution was at the sites of 36, 39, 47, 63, 77, 89, 90, 115, 126, 129, 139 and 154. eight (15.7%) out of 51 mutations were located at the "a" determinant region in 14 patients, while no mutation was found at the sites of 124, 137 and 147. however, the mutation did not affect hbv replication. hbv dna was positive correlated with hbeag. conclusions: change in hbsag antigenicity due to s gene resulted in concurrent hbsag and hbsab. the existence of hbsab did not affect hbv replication. the damage of liver failure in those patients was slight. background: hbv infection is common in bangladesh. we often encounter young patients incidentally detected with hbeag negative chronic hepatitis b (chb) in our clinical practice. however the characteristics of these patients is yet to be studied in this country. the aim of this study was to study the characteristics of young bangladeshis incidentally detected with hbeag negative chb. methods: we did percutaneous liver biopsies of 36 chb patients aged between 8 to 20 years. they were all hbeag negative with persistently normal or raised serum alt values. we did pre-core mutation (pcm) study in 4 patients who were randomly selected. results: 56% patients had significant necro-inflammation (hai-ni >3), while significant fibrosis (hai-f >2) was seen in 17.6%. serum alt (cut off 42 u/l) was raised in 38.2%, while high hbv dna load (>10 5 copies/ml) was observed only in 26.5%. pcm was negative in all 4. conclusion: although chb patients between 10-20 years of age are supposed to be in immune clearance phase, which is characterized by low hbv dna and hbeag positivity, the study shows that hbeag negative chb is an entity that can also be seen in this age group and a significant percentage of such patients may have considerable hepatic involvement. this challenges our current concept about immune clearance state of hbv infection, although much larger study is needed to draw any specific conclusion. background: hbv infection is common in bangladesh, but characteristics of young patients incidentally detected with chronic hepatitis b is yet to be studied in this country. methods: we did percutaneous liver biopsies of 88 chb patients aged between 8 to 20 years. results: significant necro-inflammation (hai-ni >3) was seen in 79.6% patients with hbeag positive and 56% patients with hbeag negative chb, while significant fibrosis (hai-f >2) was seen in 20.3% and 17.6% patients in these two groups respectively. serum alt (cut off 42 u/l) was raised in 37% hbeag positive and 38.2% hbeag negative patients, while in these two groups 87% and 26.5% patients respectively had high hbv dna load (>10 5 copies/ml). conclusion: hbeag negative chb is an entity that can also be seen in young population. a significant percentage of both hbeag positive and negative patients may have considerable hepatic involvement. profile of hbeag +ve chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: inactive hbv carriers constitute the major reservoir of hbv. present management guidelines provide inadequate treatment modalities. they are recommended for regular check-up; treatment is only recommended when patients exhibit evidence of liver damage. this is due to lack of information about their extent of liver damage. aim of this study was to assess extent of liver damage in hbeag +ve patients, unaware of their infection. methods: in this retrospective study, records of 206 hbeag +ve chb patients from our pool of 561 chb patients were reviewed. they were tested for hbsag, hbeag, hbv dna, anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 78.2% (161/206) patients were males and 21.8% (45/206) females. they were between 8-45 years of age. alt was raised >2times unl in 17% (35/206). 92. 2% (190/206) patients had high hbv dna (>10 5 copies/ml), while low hbv dna (<10 5 copies/ml) was seen in 7. 8% (16/206) . in high hbv dna group, significant necro-inflemmation (hai-ni >7) was seen in 48. 9% (93/190) and significant fibrosis (hai-ni >3) in 24. 7% (47/190) . figures were 37.5% (6/16) and 31.3% (5/16) respectively in low viral load group. none tested positive for hcv infection. conclusion: study indicates that machinery should be developed to characterize undetected hbv carriers in developing countries by conducting multi-center clinical studies. we have shown that considerable number of patients, unaware of their hbv infection, suffer from progressive liver damage. the overall strategy of management of chronic hbv infection should also be revisited. high viral load does not necessarily represent significant liver damage in patients with chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: in general, it is assumed that patients with chronic hepatitis b virus (hbv) infection with high viral load exhibit increased liver damages. treatment guidelines also emphasize on reducing viral load. these observations were mainly accumulated from developed countries. >80% chronic hbv carriers live in the developing nations, but little is known about relationship between hbv viral load and extent of liver damage in these countries. in this study, we addressed this issue. methods: in this retrospective study we reviewed records of 306 chb patients from our pool of 561 patients. all had high hbv dna (>10 5 copies/ml). 62. 1% (190/306) were hbeag +ve and 37.9% (116/306) hbeag -ve. they were alsotested for anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 51.1% (97/190 ) hbeag +ve patients with high hbv dna had non-significant hepatic necro-inflammation (hai-ni <7); this figure was 53.4% (62/116) in hbeag -ve patients. non-significant hepatic fibrosis (hai-f <3) was observed in 75. 2% (143/190) and 69.8% (81/116) in hbeag +ve and -ve patients respectively. none tested positive for hcv. conclusion: correlation doew not exist between viral load and liver damage in chb in bangladesh. many with both hbeag +ve and -ve chb with high hbv dna do not have significant hepatic necro-inflammation and fibrosis. further study may be needed to find out influence of other factors on liver damages in chb in bangladesh. most of these patients have not been characterized and treatment modalities have not been defined for them. background/aims: expression of intrahepatic hepatitis b core antigen (hbcag) is related to the immunopathogenesis of hepatitis b virus (hbv) infection. the role of hbv genotype and basal core promoter (bcp) mutation in expression of hbcag was investigated. methods: seventy hbeag-positive chronic hepatitis patients (genotype b in 52 and c in 18; bcp t1762/a1764 mutation in 16) were enrolled. clinical, virologic and histologic features were compared with regard to localization and expression of intrahepatic hbcag. the effects of hbv genotype and bcp t1762/a1764 mutation on the expression of hbcag were further evaluated by in vitro assays. results: cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic hbcag were found in 38 (56.7%), 25 (37.3%) and 4 (6.0%), respectively. fifty-eight (80.6%) of these patients expressed a high level of hbcag. in multivariate analysis, cytoplasmic localization of hbcag correlated only with low serum viral load (p=0.045) and bcp mutation (p=0.04). high expression level of hbcag also correlated with high serum viral load (p=0.015) and bcp wild-type sequence (p=0.037). in vitro assays supported that hbv bcp mutant had lower subcellular expression of hbcag compared with bcp wild-type strain. conclusions: hbv bcp mutation and viral load but not genotype contributes to the expression of intrahepatic hbcag. hepatitis b virus (hbv) genotypes show distinct geographical distributions and virological and clinical differences. in some of genotypes, specific substitutions and mutations have been described in association with hepatitis b e (hbe) protein expression and viral replication. in this study, genetic characteristics of hbv genotype e (hbv/e) were investigated using clinical samples obtained from 12 hepatitis b e antigen (hbeag)-positive, and 11 anti-hbe-positive asymptomatic carriers (ascs) in west-africa. full-genome analysis of isolated hbv strains revealed strong association between precore (pc) mutation and hbeag to anti-hbe seroconversion. furthermore, using 53 partial genome sequences, correlation among hbeag/anti-hbe status, viral load and key mutations were analyzed. the data showed that pc mutation is associated with hbeag seroconversion and enhanced viral replication efficiency. comparison between hbv/e and hbv/d strains reveals these two genotypes to have an identical sequence in their core-promoter-upstream and basic core promoter (curs/bcp) regions. it has been known from the previous phylogenetic studies, that hbv/d and hbv/e cluster together in trees reconstructed on x and precore/core orfs. in addition, this study, demonstrates that in spite of the high sequence similarity of curs/bcp region, the seroconversion-related mutation patterns are different between hbv/e and hbv/d in asc. further studies are needed to clarify the clinical significance of the regulatory sequence similarity between hbv/e and hbv/d. necro-inflammation and fibrosis p. siddappa 1 , p. kar 1 , b. das 2 , r. gondal 1 , m. asim 1 1 maulana azad medical college, 2 icpo, new delhi, india background: chronic hepatitis b(chb) is an important cause of morbidity and mortality. methods: pilot study involving 30 patients of chb, were equally randomized to receive either adefovir or lamivudine for 6 months. quantification of serum and hepatic hbv dna levels by real time pcr and liver biopsy done at start and end of 6 months. results: after 6 months there was significant and comparable reduction in serum and hepatic hbv dna viral load and liver biopsy showed significant reductions in hai scores in both the groups. serum alt which was elevated to 2 or more times normalized in both the groups. in the adefovir group 2 patients became hbeag negative and 2 patients who were hbeag negative at the start of therapy remained so. in the lamivudine group one patient became hbeac negative and 2 patients who were negative at the start of therapy remained so. in the adefovir group 4 patients became hbv dna (qualitative test) and in the lamivudine group 2 patients became hbv dna negative. there was strong correlation between serum and hepatic hbv dna levels both before and after the completion of therapy. conclusion: both the drugs bring about biochemical, histological and serological improvement with significant reduction in viral load in serum liver after 6 months without complete clearance of virus. there was not enough evidence to show therapeutic advantage of one drug over the other. the serum and hepatic hbv dna levels correlate well with eachother before and after treatment. aim: assessing efficacy and safety of treatment of chronic hepatitis b in children with pegylated ifn. materials and methods: 13 children (9 boys and 4 girls) aged 11-17 years with chb treated with peg-ifn alfa-2a, 100 g/m 2 /week during 48 weeks, 5 hbeag-positive and 8 hbeag-negative children, 4 previously treated with recombinant interferon. no child had liver disease greater than grade 2, stage 2. serum hbv dna was quantified at baseline, tw 4, ("rvr") tw 24, tw 48 (etr) and w 72 (svr) with rt pcr method (roche taqman). alt activity, haematology and adverse events were monitored. results: after 4 weeks treatment median hbv dna level decreased from 7.8x10 3 iu/ml at baseline to 1.43x10 2 iu/ml (p<0.01). "rvr" -undetectable hbv dna at tw4 was observed in 6/13 children and associated with lower pretreatment alt levels <25 iu and pretreatment viral load <750 iu/ml. all children with "rvr" were hbeag-negative pretreatment. at tw 24 and tw 48 seven children including all with "rvr" had undetectable hbv dna. 5 children achieved svr (undetectable serum hbv dna in w 72), among them 3 with "rvr". in 2/6 children with "rvr" hbsag disappearance was observed since tw 48. leukopenia was reported in 7 children, thrombocytopenia in 3. no adverse events were observed following dose modifications. conclusions: 1. peg-ifnalfa-2a is a good therapeutic option for children with chb, in particular with hbeag-negative chb 2. low pretreatment viral load and "rvr" seem to be predictive factors of efficient therapy. control by investigating the sanitizing modes among appliances used in the public service places (psp) and hbsag among appliances and practitioners worked in those places. methods: 63 beauty parlors, barber shops and bathing centers selected by stratified randomization sampling, 682 workers were investigated in questionnaire. the hbsag in appliances of psp and employee was detected by ria. results: the rate of hbsag among appliances of psp was 2.13%. the rate of hbsag in large-, medium-and small-sized appliances was 0.63%, 2.67% and 3.70%. the rate of hbsag has different( 2=6.68 p 0.05). the rate of hbsag among appliances of beauty parlors, barbering shops and footbath inns was 2.97%, 0.61% and 3.42%. different appliances had different rate of hbsag, such as the rate of acne needle and the forceps was 5.13% and 4.17%. the positive of hbsag amongworkers in psp was 7.13%. the rate of hbsag among workers in large-, medium-and small-sized psp was 7.34%, 8.33% and 2.94%. the rate of hbsag among workers in beauty parlors, barbering shops, footbath inns and bathing centers was 9.01%, 6.37%, 4.35% and 7.29%. the hbsag rate among workers was different in different works, the rate was higher in tattoo workers (13.33%), pedicures workers (12.68%), massagists (8.03%). conclusions: it is important to enhance the sanitizing management in psp and improve workers kap) of hepb. and we should promote health education to enhance the knowledge of hepatitis b control and build up supervision consciousness. background: integration of hepatitis b virus (hbv) dna into host chromosomes is often found in chronic liver disease and hepatocellular carcinoma, which is likely an early event of hbv-related carcinogenesis. however, the molecular mechanism of integration remains unclear. here we describe a potential mechanism of hbv integration and identify that ku70 and ku80, the gatekeepers of non-homologous end-joining (nhej) repair pathway, can serve as targets for anti-hepatitis virus integration. methods: using i-sce endonuclease-based system, we induced a dna double-strand break (dsb) in human hepatoma cell line huh-7. the cells were then incubated with serum from patients with chronic hbv infection. pcr amplification and direct sequencing were used to detect the inserted sequence in the site of dsb. finally, we employed taqman-based real-time pcr assay to quantify the integrated hbv dna and evaluate the effects of shrna on hbv integration. result: when huh-7 were exposed to viral serum and incubated for several days, hbv dna was detected in integrated form at the exact site of dna damage. furthermore, small interference rna (sirna) targeted against gatekeeper genes for nhej can down-regulate nhej repair and even the frequency of hbv integration. conclusion: thus, this project provided us with the first direct evidence that dna double-strand breaks are potential targets for hbv integration. the study has also shown that shrnas targeted against gatekeeper genes for nhej can regulate the frequency of hbv integration. objective: to screen proteins of human pancreas cdna library interacting with hbsag protein. methods: the library was amplifed, purified and evaluated, and then the puried library plasmids were transformed into yeast strain y187. the reconstructed plasmid pgbkt7-hbsag was transformed into yeast strain ah109 and screened on the nutrient deficiency medium sd/-trp. the transformed ah109 mated with y187 containing the library plasmid. the diploid yeast cells were plated on nutrient deficiency medium sd/-trp/-leu/-his/-ade and sd/-trp/-leu/-his/-ade containing x--gal for selecting. the plasmids in diploid yeast cells were extracted and electrotransformed into e.coli dh5 . the plasmids in dh5 were extracted, sequenced and analyzed by bioinformatic methods. results: sixteen proteins interacting with hbsag were founded. conclusions: these results show that hbsag protein may be related with metabolism of glucose and lipid. comparison of the sensitivity and specificity of the elecsys ® hbsag ii assay with other available assays in china for detection of hbsag j.d. jia 1 , l. wei 2 , x.x. zhang 3 , y.l. mao 4 , l.l. wang 5 , z.l. gao 6 , j.l. hou 7 , j. zhang 8 , w. melchior 9 , w. van der helm 9, 10 1 beijing friendship hospital, beijing, china, 2 beijing people hospital, beijing, china, 3 ruijin hospital, shanghai, china, 4 beijing 302 hospital, beijing, china, 5 west china hospital, chengdu, china, guangzhou, china, 7 guangzhou nanfang hospital, guangzhou, china, 8 shanghai public health clinical centre, china, 9 roche diagnostics ltd, rotkreuz, switzerland, 10 conclusions: in this patient population the prevalence of hbsag positive and anti-hcv were much higher than reported in community studies. genotypes 1 and 6 accounted for most of hcv. these very high rates of viral hepatitis in a hospital setting challenge to healthcare providers in terms of patient management as well as caregiver's prevention. hepatitis b is one of the major diseases of mankind that kills about one million persons each year in the world. accoring to primary study about 3% of iranian population is chronic hbv carriers. among iranian cirrhotics, 70-84% has evidence of exposure to hbv and 51-56% is carriers. because increase demand of blood transfusion, high blood dependent patients and long term window period of hbv infection, any controlling hbv infection program in blood donors can enhance the blood safety and public health. pe078 in this descriptive study included all the blood donors that referred to dezful blood transfusion center during 2005-2008. all the blood donors screened for hbs ag by using enzyme immuno assay and repeatedly reactive (r.r) samples confirmed by hbc-ab or confirmatory (neutralization) tests. the data analyzed by using spss 11.5. we found that in the first year 1.052 % were repeatedly reactive and 1.045 % confirmed. the results for other years as the followed: 0.782 %(r.r) and 0.770 % confirmed and in the last 0.725 % (r.r) and 0.700 % confirmed. the repeated blood donors increase in this period (42.11 %, 50.15 % and 51.68 % respectively). aim: we aimed to evaluate the cost-effectiveness of telbivudine versus entecavir with reference to lamivudine by roadmap model. methods: decision analysis model was used to study the incremental cost-effectiveness ratios (icer), i.e. the additional cost (in usd) required to achieve undetectable hbv dna or hbeag seroconversion for a patient at 2 years in america and hong kong. entecavir was used as a continuous monotherapy. lamivudine and telbivudine would be shifted to entecavir if hbv dna was detectable at month 6 and continued otherwise with drug resistance treated by add-on adefovir. weighted event rates based on previous reports were estimated for analysis. according our study, although the prevalence was higher than other region in our province, the hbv prevalence showed good decrease after stablishment strategies such as of repeated blood donor recruitment , improvement the donor selection and other educational programs . good following up those strategies to enhance the blood safety recommended. results: telbivudine was generally cheaper than entecavir to achieve an incremental case of undetectable hbv dna from lamivudine at 2 years. entecavir was least effective and most costly for hbeag seroconversion. conclusions: telbivudine is a cost-effective alternative to entecavir particularly when its cost is low in hong kong. h. tang 1 , g.l. zhang 1 , y.x. li 1 , r.q. tian 1 , m. liu 1 , x. li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china micrornas (mirnas) are single-stranded noncoding rnas of 18 to 25 nucleotides that play critical roles in a wide spectrum of biological processes. we investigated whether the mirnas-silencing machinery influences hbv replication or antigen expression. on the basis of elisa and mtt, the effect of 328 mirnas on the hbsag expression and cell proliferation was examined. three micrornas efficiently inhibited hbsag expression without significant effect on the proliferation of hepg2 2.2.15 cells compared to lacz control. subsequently, bioinformatics analysis were used to predict targets for the three mirnas, and the prediction results were conformed by cdna microarray analysis. the target region in hbv genome and the 3'utr region of one cellular gene were identified by fluorescent reporter assay, semi-quantitative rt-pcr and western blot. the results demontrated that mirna may play an important role in replication and gene expression of hbv. hepatitis b virus (hbv) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. although considerable progress has been made, the pathogenesis of hbv infection is still elusive. there's an urgent need to elucidate the mechanisms of hbv-host interactions, to discover novel biomarkers for diagnosis and prognosis and to develop therapeutic targets for anti-hbv treatment. herein, we applied a two-dimensional gel electrophoresis and maldi-tof/ms based comparative proteomics approach to globally analyze the host response to hbv by using an inducible hbv-producing cell line hepad38. of the 23 differentially expressed proteins identified, glucose regulated protein 78 (grp78) was one of the most striking proteins elevated by hbv replication, which was confirmed by real-time pcr and western blotting. knockdown of grp78 expression by rna interference resulted in a significant increase of both intracellular and extracellular hbv virions in hbv-transfected hepg2 cells. reversely, grp78 overexpression led to hbv suppression. the expression levels of hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) were determined by enzyme linked immunosorbent assay (elisa). immunofluoresce further revealed a positive correlation between the expression levels of grp78 and hbsag in both hbv-transfected hepg2 cells and hbv-infected human liver tissues. altogether, these data demonstrate for the first time that grp78 is an endogenous anti-viral factor in hbv-transfected hepg2 cells and may serve as a potential prognostic indicator of viral status in anti-hbv therapies. background/aims: to evaluate the predictors of response to long-term treatment of adefovir dipivoxil (adv) in patients with emerging lamivudine (lam)-resistant hepatitis b e antigen (hbeag)-positive chronic hepatitis b (chb) patients. methods: one-hundred-thirty-four lam-resistant hbeag-positive chb patients were treated with adv for a median of 28.0 months (range, 18 -54 months), following lam therapy for a median of 25.5 months (range, 5 -66 months). 65 patients (48.5%) were switched from lam to adv monotherapy, 43 (32.1%) were switched to adv with 1 month of lam overlap therapy, and 26 (19.4%) were switched to adv with 3 months of lam overlap therapy. the influence of baseline parameters on treatment response to adv in patients with lam-resistant hbeag positive chb was analyzed. result: during the follow-up period, 28 (20.9%) of 134 patients achieved complete response, defined as normalization of alt level, negative hbv dna by a digene hybrid capture assay and achievement of hbeag loss. sixteen (11.9%) patients achieved hbeag seroconversion. twenty-eight (20.9%) patients developed adv-related mutations during adv treatment. in multivariate analysis, virological response at 3 months (or=9.70, 95% ci: 32.63-35.78, p=0.001), defined as serum hbv dna levels less than 5 log10 copies/ml or a reduction in serum hbv dna levels greater than 2 log10 copies/ml after 3 months of adv therapy, independently predicted complete response. conclusions: virological response at 3 months was the strongest predictor of adv response in lam-resistant hbeag-positive chb patients. background/aims: to explore the effects of hbv dna level hbv genotype/subgenotype on the pathogenesis of severe liver diseases in chongqing. methods hbv dna level was analyzed in patients with severe liver diseases in retrospect,and hbv genotype/subgenotype hbv dna level and hbeag were determined in patients with hepatocellular carcinoma (hcc,n=78 ), liver cirrhosis(lc, n=58),chronic hepatitis b(chb, n=20) and acute on chronic liver failure(aclf, n=42). results hbv level from high to low with chb were lc, aclf and hcc in turn(p 0.05). hbv genotype was mainly genotype b.the rate of genotype b and c were 51. 3% and 47.4 respectively in hcc patients, 60.3% and 39.7 in lc patients, 55% and 40 in chb patients, 55% and 40 in aclf patients. the percentage of genotype b/c in aclf patients was higher in compared with other groups. but the distribution of hbv genotype among groups was not statistically different(p 0.05).subgenotypes of genotype b were almost ba but one. subgenotypes of genotype c were mainly ce in chongqing area, and there was no statistical difference among the 4 groups (p 0.05). conclusion: hbv dna level seems not to be a determining factor at end point of severe liver disease. both genotype b and c of hbv can lead to severe liver diseases, and there are more mixed infections by different genotypes in aclf. the efficacy of switching to entecavir (etv) monotherapy in japanese lamivudine (lvd)-experienced patients. background: this study aims to determine the efficacy of switching to 0.5mg etv daily in chronic hepatitis b (chb) patients previously treated with lvd. method: retrospective analysis of chb patients (n=134) previously on 100mg lvd daily and switched to 0.5mg etv daily. results: lvd-experienced patients were divided into three groups based on hbv viral load at time of switching to etv (<2.6 log10 copies/ml; 2.6-5.0 log10 copies/ml and >5.0 log10 copies/ml). detection of lvd-resistant virus at the time of switching was higher in the group with hbv dna 2.6 log10 copies/ml (76% in both 2.6-5.0 and >5.0 log10 copies/ml groups versus 23% in <2.6log10 copies/ml group) and was higher in patients treated with lvd for 3 years (52% versus 24% for patients on <1 year of lvd). a year after switching to 0.5mg etv daily, hbv dna undetectable rates were 100% (42/42), 94% (17/18) and 43% (6/14) for <2.6, 2.6-5.0 and >5.0 log10 copies/ml groups, respectively. alt normalization occurred in more than 90% patients at the end of the first year of switching to etv for all three patient groups. only one patient in the 2.6-5.0 log10 copies/ml group, who had lvd-resistant mutants at the time of switching, developed etv resistance during follow-up. conclusion: switching from lvd to etv maintains or improves viral suppression and alt normalization, especially in patients with viral load <5.0 log10 copies/ml. background/aims: we investigated the association between on-treatment hbsag decline and sustained response in patients treated with pegasys±lamivudine. methods: hbsag levels were measured retrospectively pre-treatment and at weeks 12, 24, 48 and 72 using the abbott architect hbsag assay in sera from 542 patients (83% asian) treated with pegasys alone (180 g qw; n=271) or combined with lamivudine (100mg qd; n=271) alone for 48 weeks as part of a large multinational trial. response was measured 6 months post treatment. results: more patients treated with combination therapy had >1 log decline in hbsag from baseline to week 48 ( figure) . hbsag level <1500 iu/ml at week 12 was associated with higher rates of response to pegasys±lamivudine 6 months post treatment ( figure) . data comparing hbsag and hbv dna as on-treatment predictors of response will be presented. conclusion: on-treatment hbsag monitoring may be useful for predicting response in patients treated with pegasys. y. wakui 1 , j. inoue 1 , y. ueno 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university graduate school of medicine, sendai, japan background/aim: chronic hepatitis b patients are clinically treated with nucleot(s)ide analogues and ifn-. nucleot(s)ide analogues have problems including drug resistance in continuous treatment, and ifn-has disadvantages of limited effectiveness and side effects. therefore, novel antiviral drugs are still needed. in this study, the suppressive effect to the replication of hbv was examined in vitro by using bezafibrate and rosiglitazone, which are ligands of peroxisome proliferator activated receptor (ppar) and , respectively. methods: the cytotoxicity of bezafibrate and rosiglitazone to hepg2 cells was examined with mts assay, and the concentration of 50% cytotoxicity (cc50) was calculated. hepg2 cells were transiently transfected with the plasmid containing 1.3-fold hbv genome of a genotype b strain. after 24 hours of transfection, rosiglitazone and bezafibrate was added to the cells. using the medium at day 3 after the addition of drugs, hbv dna was quantified with real-time pcr. results: the cc50 of bezafibrate and rosiglitazone in hepg2 cells were 250 m and 150 m, respectively. the amount of hbv-dna in the medium was decreased when the density of bezafibrate was over 200 m, but the density demonstrated considerable cytotoxicity. in contrast, rosiglitazone of 5 m, which showed no cytotoxicit, decreased the amount of hbv dna. the 50% effective concentration (ec50) was calculated to be 9.8 m. conclusions: in this study, it was suggested that the replication of hbv was inhibited by rosiglitazone of the density without cytotoxicity. the mechanism is uncertain and being investigated now. q. zheng 1 1 center for liver diseases, the first affiliated hospital, fujian medical university, fuzhou, p. r. china background: the objective of this study was to evaluate the early virologic response for prediction of achievement of hbeag seroconversion and hepatitis b virus (hbv) dna negativity after two years of lamivudine treatment in chronic hepatitis b (chb) patients. methods: in this retrospective study, adult patients with chronic hepatitis b (255 hbeag-positive and 122 hbeag-negative) were treated with lamivudine (100 mg/day), and followed-up up to 72 months. response and resistance to the treatment were assessed during the treatments with lamivudine. results: it was found that gender, age, baseline levels of alt and hbv dna, serum hbv dna at week 24 (p =0.019 , or = 0.442) were closely related to the achievement of hbeag seroconversion, undetectable hbv dna level and emergence of drug resistance after 2 years of lamivudine treatment. hbeag positive patients with baseline serum hbv dna in 10 6 -10 8 copies/ml and serum hbv dna 10 3 copies/ml at week 24 showed high response rate of alt normalization rate (91.7%), undetectable hbv dna rate (84.5%), hbeag seroconversion rate (55.2%), as well as low drug resistance rate (25.4%) after 2 years of treatment. similarly, hbeag negative patients with serum hbv dna 10 3 copies/ml at week 24 could achieve high 2-year response rate of alt normalization rate (72.41%), undetectable hbv dna rate (82.3%), and low drug resistance rate (15.5%). conclusion: serum hbv dna 10 3 copies/ml at 24-week provide the best prediction of 2-year lamivudine treatment response. background/aims: unlike oral antivirals, a finite course of (peg)interferon can induce sustained post-treatment response in patients with chronic hepatitis b (chb), with increasing rates of hbsag clearance observed in patients who respond during post-treatment follow-up. hbsag clearance is considered to be the closest outcome to a cure, being associated with improved histological outcome, reduced incidence of hcc and increased survival. methods: in a randomised multinational study, patients (hbeag-negative) received 180µg pegasys+placebo (n=177); 180µg pegasys+100mg lamivudine (n=179); or 100mg lamivudine (n=181) for 48 weeks, and were assessed 6 months post-treatment. from this initial study, 230 of those who had received pegasys±lamivudine and 85 patients who had received lamivudine monotherapy participated in a long-term observational study to investigate post-treatment response. hbsag clearance at yearly post-treatment follow-up visits up to 5 years post-treatment was analysed. results: hbsag clearance in patients treated with pegasys±lamivudine increased post-treatment (3% at 1 year to 6%, 8%, 11% and 12.2% at years 2, 3, 4 and 5). at year 5, 28 pegasys-treated patients (12.2%) had cleared hbsag compared with 3 (3.5%) of lamivudine-treated patients (p=0.022). 16/28 pegasys-treated patients had anti-hbsag (hbsag seroconversion). detailed analysis of the 5-year follow-up data will be presented. conclusion: the ability of a finite course of pegasys to induce sustained response with increasing hbsag clearance rates in responders during post-treatment follow-up supports its use as first-line therapy in hbeag-negative patients with chb. background/aims: recent studies suggest that quantification of hbsag levels early during treatment can be used to predict post-treatment response to pegasys. elecsys ® hbsag ii (roche) is a sensitive assay for the detection of hbsag. this assay can be used for the quantification of hbsag levels using a simple dilution algorithm. we compared results obtained using the elecsys ® hbsag ii method with those of a commonly used quantification assay. methods: hbsag levels obtained using the elecsys ® hbsag ii assay were compared with those obtained using the abbott architect ® hbsag assay for a total of 40 samples from patients infected with hbv genotypes a (n=8), c (n=1), d (n=29) and f (n=2). samples were diluted 1:100 in diluent provided by the manufacturer. samples with hbsag levels >250 iu/ml were retested at a final dilution of 1:1000. samples with hbsag levels <0.05 iu/ml were retested undiluted. results: overall, hbsag levels measured with the two assays correlated well (r 2 =0.9718) over a wide range (3-3x10 5 iu/ml). discrepancies in hbsag levels >±20% were reported for a minority of the samples (n=15), mainly distributed evenly above and below the ideal line (n=11). in the four low titre (range 3-9x10 3 iu/ml) samples with greatest discrepancy elecsys ® underestimated values (in two cases by >50%). conclusion: the elecsys ® hbsag ii assay provides a simple and reliable means for determining hbsag levels. this simple assay format could be used to provide useful information during on-treatment monitoring of hbsag levels in patients with chronic hepatitis b undergoing therapy. conclusions: hbv/a has been increasing in chb patients in japan as the consequence of ahb, spreading in the younger generation through promiscuous sexual contacts, thrust by an inclination of hbv/a to induce chronic hepatitis. the spread of hbv/a infection in japan should be prevented by universal vaccination programs. introduction: chronic viral hepatitis is common in end-stage renal disease (esrd), from endemic hepatitis b (chb) and nosocomial hepatitis c (chc). reduced outcomes post-renal transplant were reported thus chb and chc cirrhosis became contraindications to listing. however, these predated effective anti-viral therapies. we reviewed outcomes of patients with chronic viral hepatitis following assessment for renal transplantation. methods: prospective database of esrd patients with viral hepatitis referred for renal transplantation was reviewed. results: 110 patients were assessed. 23 patients underwent kidney transplantation. two were cirrhotic and had liver/kidney transplantation; both died within 6 months. 21 were non-cirrhotics, of whom 20 are alive. 14/20 have functioning allografts; predictors were normal alt and low viral load. of the 87 non-transplanted, 31 had cirrhosis; 17/31 received anti-virals. mortality was 35% -7 liver-related (3 hepatoma,1 bacterial peritonitis,3 sepsis -5 inactive cirrhosis); 4 non-liver related (2 cerebral,1 haemorrhage,1 renal -3 inactive cirrhosis). 12/20 surviving cirrhotics received anti-virals. in non-transplanted non-cirrhotics, mortality was 18%; 80% of survivors had inactive disease. 23 chb patients received lamivudine; 11 adefovir (lamivudine resistance). 11 chc patients received ifn-based therapy. conclusion: excellent outcomes are achieved in esrd patients with chb/chc post-renal transplant, in absence of cirrhosis. normal alt/non-detectable viral load can predict graft function. however, cirrhosis is associated with high mortality on dialysis whereas non-cirrhotics with inactive disease do well. the role of kidney transplantation in cirrhotics with suppressed viral replication needs to be reassessed. the truncated hbc149 interferes with replication of hepatitis b virus j.c. han 1,2 , x.b. pan 1,2 , l. wei 1,2 , k. deng 1,2 1 institute of hepatology, 2 peking university people's hospital, china hepatitis b virus (hbv) capsids play an important role in production of progeny virus and other elements of the virus life cycle. misdirection of capsid assembly and formation of aberrant particles may be an effective approach to interfere with virus replication. hbv capsids can be assembled in vivo and vitro from the dimeric hbv core protein (hbcag). the interaction of single and dimeric hbcag with some truncated hbcag is verified in vitro. the truncated hbcag consists of the first 149 amino acids and lacks the c-terminal, 34-residue rna-binding domain. method: we transiently transfected hepg2.2.15 with pcdna3.1hbc149 by fugene6.after 48 and 96h, hbvdna, hbeag and hbsag in culture supernatant were detected and cell subjected to southern blot and immunofluorescence analysis. result: the level of hbsag and hbeag had gentle change, we found that hbvdna decreased at 96h after transfection( 10 3 10 4 copies/ml p<0.01) ,but replication intermediates obviously decreased from 48h. some positive signal of hbcag located around the nuclear and conglomerated in cytoplasm compared to the control. conclusion: the truncated hbc149 can inhibit replication of hepatitis b virus. misdirection of capsid assembly and formation of aberrant particles could be an important cause. y.p. li 1 , r.c. li 1 1 objective: to assess the long-term efficacy of recombinant yeast derived hepatitis b vaccine in infant s born to hbsag and hbeag carrier mother. methods: a total of 273 neonates born to hbsag, hbeag both positive mothers were vaccinated with 5 ,5 ,5 g doses of recombinant yeast derived hepatitis b vaccine by 0 ,1 , and 6 months schedule. they were all followed for 129 years after the primary vaccination. results: twelve infant s (6.63 %) become hbsag positively conversed in 9 year after primary vaccination ,and the positive rate of hbsag in 1-9 year was 0.72 %-6.8 % ,17.18 % of child in no/ lowly respond become hbsag positively. at the ninth year, the positive rates of anti-hbs were 60 % above. anti-hbs positive rates and immunity level were higher at 3-5 year old by repetition immunity than others. conclusion: the recombinant yeast derived hepatitis b vaccine have good immunogenecity and long-term protective efficacy to hbv interruption of perinatal transmission , a booster dose seems necessary in aged 3-4 years to the mother with hbsag and hbeag.it is high risk tobecome hbsag positively in the baby of norespones to hepatitis b vaccine. chb patient group-initiated programme to improve awareness, adherence and treatment outcomes in asia pacific n. leung 1 1 founding chairperson of asiahep background: worldwide, over 400 million people live with chronic hepatitis b (chb); 300 million in asia pacific. regional survey data from 1,500 patients in 10 countries showed a lack of knowledge and understanding of chb, its severity and impact on quality of life. this initiative aims to coordinate patient groups in the region and devise programmes to improve knowledge and healthcare outcomes. methods: the patient groups met in hong kong in may 2008 and identified common needs to: (1) improve educational resources; (2) raise awareness; (3) increase diagnostic yield; and (4) enhance treatment compliance through education about the need for sustained viral suppression to reduce long-term complications. results: a patient engagement programme was developed for people with newly diagnosed or known chb. the programme comprises: -detailed information about chb -a health-tracking tool for self-monitoring of blood tests and treatment progress -detailed information for carers/family -a patient-physician communications video (including role-play) -mobile phone text messages providing advice and compliance/appointment reminders conclusion: this programme was developed to address the needs of patients and clinicians. improved knowledge and long-term support, particularly for patients on antiviral medication, is expected to improve quality of life. the programme encourages clinicians and patients to develop enduring therapeutic partnerships to promote optimal outcomes. acknowledgement: the chb patient group meetings and the patient engagement programme are supported by an unrestricted educational grant from glaxosmithkline. serum hbv rna level reflects the potency of nucleos(t)ide analogue y.w. huang 1,2 , k. chayama 3,4 , m. tsuge 3,4 , s. takahashi 3,4 , t. hatakeyama 3,4 , m.y. lai 2,5 , h.l. you 1 , j.t. hu 1 , c.j. liu 2,5 , p.j. chen 2,5 , d.s. chen 2,5 , s.s. yang 1 , j.h. kao 5, 6 1 liver unit, cathay general hospital medical center, 2 background and aims: serum hbv rna is detectable in patients treated with lamivudine (lmv) or entecavir (etv) (hatakeyama, 2007 and huang, 2008) . the aim of this study was to determine the clinical significance of serum hbv rna levels in patients treated with nucleos(t)ide analogues of different potency. methods: serum hbv rna was serially determined in 20 patients treated with nucleos(t)ide analogues for 48 to 52 weeks (9 with adefovir (adv), 5 with lmv, and 6 with etv). serum hbv rna was quantified by reverse transcription of hbv nucleic acid extract with subsequent real-time pcr. results: hbv rna was detectable in 11 patients as follows: 2 of 9 in adv (22%), 3 of 5 in lmv (60%), and 6 of 6 in etv (100%) (p = 0.002). mean log serum hbvdna levels at baseline were 10.1 ± 0.6 for adv, 6.6 ± 2.0 for lmv, and 9.5 ± 0.9 for etv, which were comparable between less potent adv and most potent etv (p = 0.14). during antiviral therapy, peak log hbv rna level of patients with etv was significantly higher than that of those with adv or lmv (3.2 ± 0.9 vs. background: in the phase iii clinical trials, clevudine 30mg for 6 months showed potent antiviral activity along with a marked post-treatment antiviral effect. the objective of this study is to compare the anti-hbv activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis b (chb) patients in a randomised way. methods: the patients are received clevudine for 24 weeks and then combination of clevudine and vaccine for another 24 weeks or clevudine alone for 48 weeks. eligible patients were treatment-naïve hbeag(+) chb patients with hbv dna levels 500,000 copies/ml. the primary endpoint is the proportion of patients with hbeag loss. preliminary results are presented here. results: thirty-one patients have completed week 24 visits and from them, 15 patients (11 in clevudine alone and 4 in combination group) have completed week 36 visits. at week 24, 26% of patients had hbeag loss. at week 36, 46% in clevudine alone and 25% in combination group (3 months on combination after clevudine monotherapy) had hbeag loss. at week 24, 63% of patients had negative hbv dna by amplicor pcr (<300 copies/ml). at week 36, all of patients in both groups had negative hbv dna by pcr and 73% in clevudine alone and 80% in combination group had normal alt. conclusion: clevudine demonstrated good serologic response as well as significant viral suppression and alt normalization. with this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. background/aims: to determine the reasonable number of clones for hbv quasispecies analysis. methods: 16 chronic hepatitis b patients were enrolled with hbvdna levels range from 4~10 log copies/ml. hbvdna was extracted. hbv reverse transcriptase (rt) gene encompassing the overlapping surface s gene was amplified by polymerase chain reaction, then cloned and sequenced. ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. the characteristics of hbv quasispecies including shannon entropy and genetic distance were calculated. results: the shannon entropy and genetic distance of 10 clones group was higher than those of 20 and 30 clones group, either in rt gene or in s gene (p<0.01). while the shannon entropy and genetic distance of 20 clones group showed on difference with those of 30 clones group, neither in rt gene nor in s gene (p>0.05). the number of different quasispecies detected in 30 clones group was higher than that of 20 and 10 clones group (p<0.01). the shannon entropy and genetic distance in three different clones group had no correlation with hbv dna levels (p>0.05). conclusion: although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than 20. the information contained in 20 clones per sample could well represent the quasispecies characteristics. the clone number was not necessary modulated according to different hbv dna levels. background: recent studies reported that basal core promoter mutation (a1762t and g1764a) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and hcc. but the effect of the double mutations on the activity of enhancer ii/basal core promoter is still uncertain. objectives: to evaluate the influence of nt1762 a/t and nt1764 g/a mutations on hbv enhancer ii/basal core promoter activity. methods: the pcr fragments of hbv enhancer ii/basal core promoter (nt1601 to nt1815) from the serum-derived genotype b hbv dnas of one hbv carrier aged 66 and one hbv related hepatocellular carcinoma patient aged 26 were introduced into the pgl3-basic-vector from promega via restriction sites of xho i and hind iii. the nt1762 a to t and t to a, the nt1764 g to a and a to g mutations were carried out by genetailor site-directed mutagenesis system from invitrogen. the promoter activity was evaluated by comparing firefly luciferase measurement with renilla luciferase as the internal control using the dual-luciferase reporter assay system from promega. results: the luciferase reporter assay results indicated that the 1762 t to a combined with 1764 a to g mutations increase (p<0.001) while the 1762 a to t combined with 1764 g to a mutations decrease (p<0.05) the hbv enhancer ii/basal core promoter activity significantly. conclusions: associated with increased risk of hepatocellular carcinoma, a1762t and g1764a double mutations of hepatitis b virus reduce the enhancer ii/basal core promoter activity. background/aims: a substantial proportion of chronic hepatitis b (chb) patients with mildly elevated alanine aminotransferase (alt) have significant fibrosis. we evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. methods: one hundred five chb patients with alt less than two times the upper limit of normal underwent liver biopsy. multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. results: there were 27 patients in the low normal alt group, 21 in the high normal alt group, 37 in the low elevated alt group, and 20 in the high elevated alt group. fifty eight patients (55.2%) had significant fibrosis ( stage 3) and 35 (21.0%) had significant inflammation ( grade 3). the age, platelet count and grade of inflammation were factors associated with significant fibrosis. progressive liver disease was observed in 23 (27.4%) of the 84 followed-up patients. the stage of fibrosis, alt group and antiviral therapy were significant predictive factors for progressive liver disease. conclusion: liver biopsies should be recommended in patients over 35 years with mildly elevated alt levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. background: four nucleos(t)ide analogues (nas) are currently approved for the treatment of hbv infection in china. however, long-term benefits are limited by the emergence of drug-resistant viruses. methods: patients accepted the examination based on physician's instruction. hbv reverse transcriptase gene was amplified from serum via nested pcr and sequenced directly. results: well-recognized drug-resistant mutations were detected in 567 of 2,000 patients. in patients receiving na monotherapy, corresponding drug-resistant mutations were detected in 214/381 for lamivudine (lam), 35/333 receiving adefovir (adv), 0/57 for entecavir (etv), and 9/17 for telbivudine (l-dt). the mutations were detected in 272/615 patients receiving 31 kinds of sequential/combined usages of the 4 nas. m204i (32%), m204v+l180m v173l (32%), and m204i+l180m (21%) were identified as major mutant patterns of lam monotherapy. n236t a181 substitution was the dominant adv-resistant mutation. t184 substitution was the dominant etv-resistant mutation always accompanied with lam-resistant mutation. l-dt-resistant mutation was m204i l180m exclusively. adv-resistant mutation was frequently seen in lam-resistant patients receiving adv sequential therapy rather than those receiving adv add-on therapy. controversial lam/adv-resistant mutations including a181t, v214a, q215s and i233v were detected in some patients singly or with the well-recognized drug-resistant mutations. interestingly, the drug-resistant mutations were also observed in a few of patients naïve to nas. conclusions: the exploration of hbv drug-resistant mutation profile in large clinical samples furthers our understanding of hbv drug-resistant status in china with implications for administrating anti-hbv therapy more reasonably. toll-like receptor (tlr) 2, tlr4 and cd4+cd25+cd127low/-regulatory t cells correlate with hepatitis b virus infection y. zhang 1 , j.q. lian 1 , c.x. huang 1 , x. wei 1 , j.p. wang 1 , p.z. wang 1 , x.f. bai 1 1 center of infectious diseases, tangdu hospital, the 4th military medical university, xi'an, china background: tlrs play a crucial role in sensing and initiating innate antiviral response and tregs actively suppress immune response, contributing to viral persistence and chronic tissue damage. in this study, we determined tlr2 and 4 expression and treg frequency, as well as their function in the effect of hbv infection. methods: tlr2 and tlr4 expression on monocytes and circulating cd4 + cd25 + cd127 low/-tregs were determined by flow cytometry in 16 ahb, 42 chb, 22 asc and 20 nc. spearman correlation was performed to investigate associated variables on treg or tlrs. pbmcs were stimulated with hbeag or hbcag and the tlrs profile was examined. result: tlr2 expressions were up-regulated in chb and asc, while tlr4 were increasingly expressed in ahb and asc. treg frequency in chb was significantly higher than that in nc. in chb, the increased tlr2 negatively correlated with hbv dna loads and treg frequency negatively correlated with tlr4 expressions. tlr2 was up-regulated after hbeag stimulation in both nc and chb. conclusion: increased tregs may be associated with chb and there might be possible interactions between hbeag, tlr signaling and the innate immune response, which may partially explain the mechanism of hbv infection induced immuno-tolerance. (6.7±1.35) . hbv-dna was quantitatively determined by polymerase chain reaction (pcr) technique, and hbv genotype was determined by pcr microwave gene chip technique. antiviral efficacy was assessed using measuring the following scales: the alt normalization rate, hbv-dna negative conversion rate and the hbeag/anti-hbe seroconversion rate. results: among 55 serum specimen, hbv genotype distribution was 38 genotype c, 14 genotype b, and 3 genotype non-b or c respectively. in genotype b, alt normalization rate was 78.57%(11 cases), hbv-dna negative conversion rate was 35.71%(5 cases) and the hbeag/anti-hbe seroconversion rate was 14.28%(2 cases). in genotype c, alt normalization rate was 76.31% (29 cases), hbv-dna negative conversion rate was 47.37%(18 cases) and the hbeag/anti-hbe seroconversion rate was 18.42%(7 cases). the efficacy of adefovir dipiroxil showed no significant differences between genotype b and c in the treatment of chronic hepatitis b p>0.05 . conclusion: adefovir dipiroxil is an effective antiviral drug. hbv genotype is irrelevant to the antiviral efficacy of adefovir dipiroxil in treatment of patients with chronic hepatitis b. the effect of anti-hbv drugs on albumin and bilirubin levels, and platelet count h. yoshida 1 , h. taniguchi 1 , r. nagano 1 , k. sakitani 1 , e. seki 1 , t. serizawa 1 , y. ito 1 , h. mizuno 1 , y. mitsuno 1 , r. nakata 1 , m. omata 2 1 japanese red cross medical center, 2 university of tokyo, japan background/aim: we assessed the efficacy of anti-hbv drugs on the liver function. methods: patients with hbv-related disease followed at our center between 2005 and 2007 were enrolled. lamivudine (100mg), lamivudine (100mg) +adefovir (10mg), or entecavir (0.5mg) was administered to the patients with detectable hbv dna and elevated alt. liver function (alt, alb, and t.bil) and platelet count were observed. alt, alb, t.bil, and platelet count of treated group at pretreatment, year 1, and year 2 were compared with untreated group. results: eighty six patients with positive hbsag were enrolled between jan 2005 and dec 2007. seven patients (2 acute infection, 1 overlap infection with hcv, 7 lost of follow up) were excluded. in total 76 patients were followed up for a median follow up of 26 (range 2-39) months. of 76 patients, 32 received anti-viral treatment. twenty one patients were treated with lamivudine, 4 with lamivudine+adefovir, and 7 with entecavir. the mean of levels of pre-treatment-year1-year2 were alt:129-37-43 (u/l), alb:4.0-4.4-4.4 (g/dl), t.bil:1.0-0.8-0.7 (mg/dl), and plt:14.3-15.4-16.0 (x10 4 mcl) respectively. markers of untreated group (n=44) (at baseline-year1-year2) were alt:33-40-35 (u/l), , t. bil:0.8-0.8-0.9 (mg/dl), and plt:18.7-18.0-18.5 (x10 4 mcl) respectively. although all of four markers in treated group were significantly worse than untreated group at baseline, all of four markers did not showed significant difference from untreated group at year2. conclusion: treatment with anti-hbv drugs showed the efficacy not only transaminase levels, but also on albumin, bilirubin, and platelet count improvement-improvement of "hepatic reserve" which is valuable for prevention of cirrhosis. background: currently, hbeag-negative chronic hepatitis b(chb) is increasing. but there are still controversial on the treatment of hbeag-negative chb with alt 2×uln. we have investigated the clinical efficacy of nucleotide analogues(nas) in the treatment of hbeag-negative chb with alt 2×uln. methods: the data of patients who were treated by nas for more than 2 years and with alt 1 2×uln (n=87) , alt 1×uln(n=43) and alt 2×uln(n=88) were collected, and 12w and 24w virologic response, 48w and 96w complete response, virologic breakthrough and clinical resistance were analyzed. results: compared with the base line, hbv dna level in all three groups were significantly decreased (p 0.01), and there was no significant difference between alt 1 2×uln group and alt 2×uln group. the viral load was significant decreased in alt 1×uln group at 12w, 24w and 36w (p<0.05). virologic response at 12w and 24w complete response at 48w and 96w was 71.3%, 82.8% , 82.8% and 86.2% respectively in alt 1 2×uln group and was 51.2%, 60.5%, 65.1% and 74.4% respectively in alt 1×uln group. there was no significant difference between alt 1 2×uln group and alt 2×uln group. virologic response at 12w and 24w and complete response at 48w were significant decreased (p<0.05) in alt 1×uln group. there was no significant difference among the three groups in virologic breakthrough and clinical resistance. conclusion: hbv replication can be satisfactory inhibited by nas in hbeag-negative chb patients with alt 2×uln, which suggests that in these patients the indication of alt is different from hbeag-positive patients. quantitative hbeag assay as a predictive factor of hbeag seroconversion induced by peg-ifn -2a therapy to hbeag-positive chronic hepatitis b y.y. zhu 1 , j. dong 1 , y.t. chen 1 , j. chen 1 , j.j. jiang 1 1 liver diseases research center, the first affiliated hospital of fujian medical university, fuzhou, fujian, china rp, 350005 background: to find predictive factor for hbeag seroconversion in the treatment of hbeag-positive chronic hepatitis b (chb) by peg-ifn -2a. methods: hbeag-positive chb patients were given peg-ifn -2a treatment for 48 weeks. clinical data were collected every 3 months. receiver operator characteristic (roc) curve was employed to calculate positive predictive value (ppv), negative predictive value (npv), sensitivity and specificity. results: sixty-five patients completed peg-ifn -2a therapy. among them, 17 (26.15%) were found hbeag seroconversion and 19 (29.23%) were found hbeag loss at cessation of therapy. none of age, gender, alt level and hbv dna load at baseline had relationship with hbeag seroconversion. hbeag level of baseline was correlated to hbeag seroconversion, with p value as 0.003 (table 1) . according to roc curve, supposed auc as 0.705 and p value as 0.042, the ppv, npv, sensitivity and specificity of hbeag level as 61 at 12 week were 0.361, 0.862, 0.765 and 0.521, respectively. supposed auc as 0.828 and p value as 0.001, the ppv, npv, sensitivity and specificity of hbeag level as 15 at 24 week were 0.452, 0.912, 0.824 and 0.646, respectively. the hbeag level (s/co) and decreased degree (percentage) at 12 week and 24 week were significant related to hbeag seroconversion (table 2) . conclusion hbeag level at baseline and at 12th and 24th week and its decreased degree (percentage) during the treatment course could be used as predictive factor for hbeag seroconversion. background: it is well documented that perinatal transmission is the major cause of chronic hbv infection in china. the aim of this study was to evaluate the efficacy of interruption of hbv intrauterine infection with hepatitis b immunoglobulin (hbig) in pregnant women with hbeag positive. methods:: a prospective randomized controlled trial was adopted. each subject in the trial group (28 cases) was given 200 iu hbig intramuscularly every 4 weeks from 28-week of gestation, while each subject in the control group (24 cases) received placebo in the same way. the cord blood of newborns were collected for detecting hbsag, hbeag and hbv-dna. results: for newborns, hbeag positive rate in trial group was 21.4%(6/28).hbeag positive rate in control group was 79.2%(19/24). there was significant difference in hbeag positive rate of newborns between the two groups( p < 0.01, rr = 0.27). hbv-dna positive rate in trial group was 25%(7/28). hbv-dna positive rate in control group was 83.3%(20/24). there was significant difference in hbv-dna positive rate of newborns between the two groups( p < 0.01, rr = 0.30). hbv-dna load of 7 cases of newborns in trial group was lower than that of their mothers(t = 28,p = 0.02). there was no significant difference in hbv-dna load between women and their newborns after delivery in control group (t = 81.5,p > 0.1). conclusion: it is effective and safe to prevent hbv intrauterine infection with hbig from the 28(th) wk in pregnant women with hbeag positive. ), especially, the cirrhosis and hcc cases obviously more in both hbeag and anti-hbe patients are negative than hbeag-negative but anti-hbe positive patients (.36.7% vs 26.7%; 7.9%vs5.5%, p ) .the prevale nce of pre-core g1896a mutate have no significant difference regardless of hbv serum marker status or the state of illness. conclusion: recent 5 years the hbeag-nagative chronic hepatitis b patients are gradually increasing in yunnan province. while the hbeag disappear but no anti-hbe serum transfer, and the virus still active replication -it may be a crucial phase determined the diseases outcome, which should be pay more attention by physicians. the clinical significant of pre-core g1896a mutate remain unknow. efficacy of interferon for chronic hepatitis b patients with normal or paranormal alt z. liu 1 , j.z. guo 1 , y.j. lin 1 , y.j. zhang 1 , z.w. lang 1 1 beijing ditan hospital, beijing, china background: we reported interferon treatment for 4 cases with normal or paranormal alt but in which liver histologic exam showed g2-4 and/or s3-4. methods: 4 patients were male with an average age of 28 years.mean alt was 46.7 iu/l and hbv dna level was 3~5 log10copies/ml. two patients were hbeag positive; one patient was both negative for hbeag and anti-hbe and one patient was anti-hbe positive. liver biopsy showed g2~g4 and s3~s4 respectively. 3 patients were treated with ifn-alpha, liver biopsy was repeated after 1 year.only one patient had received combination therapy with ifn and adefovir after 10 months treated ifn monotherapy and liver biopsy was taken after 1.5 years. results: all patients got normal alt after 1 year treatment. hbv dna was undetectable in 3 patients. 2 patients with initial positive hbeag cleared. but 3 patients still were anti-hbeag negative.liver biopsy showed change fromg4 s3-4 to g2 s2-3 in 1 patient; fromg2 s3 to g3 s4 in 1 patient and no change in the other 2 patients. conclusions: though alt and hbv dna improved after 1 year treatment, histological improvement is not satisfying. 1 patient's improvement in liver histology may be due to seroconversion before treatment and adding adefovir after 10 months of interferon therapy. after 8 months of combination therapy we did liver biopsy again. the other 3 patients were hbeag negative, but hbeab were also negative, liver biopsy was taken 1 year later without combination of nucleoside analogs. evaluation of long term efficacy of hepatitis b vaccination r.c. li 1 , j. gong 1 , j.y. yang 1 1 objective: to evaluate the long term effectiveness of preventive hbv infection and to monitor the incidence of hepatitis b in children to see possible impact on the program of long an that was launched in 1986. methods: (1) set up a surveillance systemof hepatitis ,to evaluate the possible impact on incidence of hepatitis b. (2) to serologically evaluate the effect of the program, a stratified random sampling of 1000 subjects in 1987 birth cohorts was recruited for long term follow up at the age 1-20 years. (3) cross-sectional seroepidemiolgical survey was carried out in the county in 1985 before the program and 20 years later. hbsag , anti-hbs and anti-hbc were tested by ria. results: the average coverage of hepatitis b vaccine was 89. 1 %. at 20 years after vaccination, the seropositivity for hbsag in population of 1-20 years has decreased from 16. 0 % to 2. 3 %, the annual effectiveness was 89. 7 %. hbv accumulated infection rate was 3. 5 %, protective rate was 96. 2 %. the incidence of acute hepatitis b was 1. 60 per 100,000 in population aged 1 -20 years , it decreased by 91.3 % as compared with the incidence of 18.38 per 100,000 in same age group in 1985 -1987. conclusion: mass hepatitis b vaccination program in long an county has proved to be effective in control of hbv chronic infection and incidence of acute hepatitis b. background and aims: although the evolution of viral quasispecies may be related to the pathological condition of disease, little is known about this in hepatitis b virus (hbv), especially during hbeag seroconversion. methods: nucleotide sequences of hbv precore/core genes from 5 time points were analyzed in four cohorts of chronic hepatitis b, interferon-induced seroconverters (is, n=9), interferon non-responders (in, n=9), spontaneous seroconverters (ss, n=9) and non-seroconverters (sn, n=9), followed during 60 months on average. only patients with genotype c were used. viral diversity was then estimated after nucleotide genetic distance was assessed and phylogenetic trees were constructed. results: analysis of 1800 nucleotide sequences showed that the nucleotide genetic distance of seroconverters (is and ss; 9x10 -3 substitutions/site and 8.5x10 -3 subsititutions/site, respectively) was similar to that of non-seroconverters (in and sn; both 7x10 -3 substitutions/site) before seroconversion. compared to that of nonseroconverters (in and sn; substitutions/site and 7.5x10 -3 substitutions/site, respectively) the viral diversity of seroconverters (is and ss; 13x10 -3 substitutions/site and 12x10 -3 substitutions/site, respectively) was significantly higher after seroconversion (p<0.05) and it was higher after seroconversion in seroconverters compared with that berore seroconversion (p<0.05) while it almost didn't change in non-seroconverters irrespective seroconversion. phylogenetic trees also showed that complex trees appeared in secoconverters and relatively simple in nonseroconverters. conclusions: the distinctly higher viral diversity after seroconversion in hbeag seroconverters could be related to increased hbv-specific t-cell responses and escape mutant which arise from stronger selective pressure caused by host immune activity. adefovir dipivoxil 10mg (adv) resistance at 5 yrs in chinese hbeag+ve chronic hepatitis b (chb) j.l. hou 1 , y.z. wang 2 , x.q. zhou 3 , j.q. niu 4 , y.m. wang 5 , h. wang 6 , y.m. mao 7 , k.f. barker 8 1 nanfang hospital, guangzhou, prc, 2 jinan infectious disease hospital, jinan, prc, 3 ruijin hospital, shanghai, prc, 4 1st hospital of jilin university, changchun, prc, 5 xinan hospital, chongqing, prc, 6 people's hospital, beijing, prc, 7 renji hospital, shanghai, prc, 8 glaxosmithkine r&d, london, uk background: long term adv provides clinical and histological improvement in chb, but may lead to emergence of treatment associated resistant mutations. we report on adv resistance data from chinese hbeag positive subjects treated for 5 years. methods: 480 hbeag positive chb subjects were randomized in an initial 52 weeks controlled adv study (with a 12 weeks placebo period in half of patients) and then offered open label adv treatment for a further 208 weeks. a total of 474, 456, 443, 421 and 390 subjects completed the 1 st , 2 nd , 3 rd ,4 th and 5 th yr, respectively. at the end of each year samples were analysed from those subjects with protocol-defined hbv dna breakthrough for the rtn236t or rta181v adv mutations associated with resistance. sera from subjects with breakthrough were analysed at all subsequent yearly timepoints whenever possible. results: at the end of the 1 st yr, none of the 45 subjects with hbv dna breakthrough had either mutation. sera were available for analysis from 137, 199, 228 and 187 subjects with viral breakthrough at the end of the 2 nd , 3 rd , 4 th and 5 th yr, respectively, with new mutations identified in 6, 20, 24 and 20 subjects at the same timepoints. of the cumulative 70 subjects at the 5 th yr analysis 31 had rtn236t, 29 had rta181v, and 10 had both mutations. conclusion: treatment with adv in chinese hbeag positive chb subjects for up to 5yrs resulted in a cumulative rate of 14.6% (70/480) adv resistance-associated mutations with hbv dna breakthrough. background and aims: to evaluate the predictive significance of rapid virologic response (rvr) for achieving an end-of-treatment virologic response (er) or hbeag seroconvertion and the predicting indicator of nonresponse (nr). methods: 205 patients with chronic hepatitis b were treated with adv and prospectively observed to 48 weeks. we assessed the values of virus load reduction at weeks 4, 8, and 12 weeks to predict the er and hbeag seroconversion. the association between less reduction of viral load at 12 and 24 weeks and nonresponse was also analyzed. results: of 146 etv-treated patients enrolled in etv-901, 98 met criteria for inclusion into 5 year etv treatment analyses. the proportion of patients achieving efficacy endpoints through 5 years of etv therapy is presented the table. results: after 48 weeks of therapy, serum hbv dna levels decreased with a median 4.07±2.47 log10 copies/ml. twenty-three(11.2%) of patients had er. twenty-six (12.7%) patients achieved hbeag seroconversion. hbv dna <4 log 10 copies/ml at week 4 predict both er and hbeag seroconversion. hbv dna> 4log10 copies/ml at 4 weeks but decline to <4log10 copies/ml at 12 weeks or 24 weeks both can predict er and hbeag seroconversion. less than 2 log 10 hbv dna reductions at 24 weeks might predict nr. conclusions: the majority of patients experienced durable serum hbv dna suppression (94%) and alt normalization (80%) after 5 years etv therapy. conclusions: the virologic response within 4 weeks could be useful for prediction of er and hbeag seroconversion of adefovir therapy. failing to evr might not predict nr. objectives: to determine the accuracy of hbcigm in diagnosing ahb and the correlation between hbcigm and liver inflammation (alt), bilirubin & biosynthetic functions (albumin,pt). result: a total of 233 patients were included: in 40 patients, adv was added on lam (add-on therapy), and in 193 patients, lam was switched to adv (switch therapy). during 16.5 months of follow-up, 25 patients developed adv resistance (rta181v and/or rtn236t) and all had undergone switch therapy. the cumulative probability of adv resistance at the 24th month was 17.5%. although add-on therapy induced no adv resistance, it failed to show significant superiority over switch therapy (p=0.220). in multivariable analysis, female (odds ratio [or], 2.75; 95% confidence interval [ci], 1.22-6.17; p=0.014), liver cirrhosis (or, 2.39; 95% ci, 1.07-5.33; p=0.033), and age >45 yr (or, 3.31; 95% ci, 1.14-9.66; p=0.028) were independent risk factors of adv resistance. methods: a retrospective cross-sectional study involving patients with hbcigm positivity between june 2006-december 2007, satisfying the definition for ahb and chbf,and fulfilling the exclusion criteria was performed. hbcigm test were done by using microparticle enzyme immunoassay (meia) and results were expressed as an index value.hbcigm positivity was defined as index value of >1.00 results: 74 patients were positive for hbcigm and 60 fulfilled the criteria(33 ahb, 27 chbf).hbcigm was significantly higher in ahb compared with chbf(median 3.46 vs 1.70;p <000.5).the hbcigm arbitary index value of 2.76 was highly sensitive(97%) and specific(85%) in diagnosing ahb with high accuracy(auroc 0.964;95% ci:0.925-1.003;p<0.0005).among patients in both groups, there was a weak, but significant negative correlation between hbcigm and pt above control(r = -0.309,p =0.016).however, among patients with chbf,the negative correlation between hbcigm and pt above control was moderately strong(r = -0.557,p =0.003).there was also a weak, but significant positive correlation between hbcigm and albumin in with chbf(r = +0.434,p =0.024). conclusion: adv add-on therapy developed no adv resistance during the observation period. therefore, add-on therapy is recommended to lam-resistant chb patients with genotype c who have any risk factors for development of adv resistance: female, liver cirrhosis, and age >45 yr. hbcigm-hepatitis b core igm antibody ahb-acute hepatitis b,chbf-chronic hepatitis b flare alt-alanine transaminase,pt-prothrombin time pe117 detection of emerging drug resistance mutations associated with major approved hbv antivirals using a novel line probe assay (lipa). j. doutreloigne 1 , f. shapiro 1 , r. maertens 1 , e. van assche 1 , e. sablon 1 1 hepatitis diagnostics unit, innogenetics nv, belgium background: in study etv-022, etv demonstrated superior virologic, histologic and biochemical benefit compared to lamivudine (lvd). this study (etv-022/-901) presents efficacy and safety results for patients who received 5 years continuous etv treatment. background/aims: an increasing number of antiviral drugs are being used to treat chronic hepatitis b virus (hbv)-infected patients. however, induced viral escape mutants -some potentially cross-resistant -lead to viral non-responsiveness and treatment failure. effective treatment strategies must therefore take possible drug resistance (dr) into account with respect to monitoring and selection of alternative drugs. we evaluated the use of an updated inno-lipa hbv dr v2+v3 reverse hybridization assay versus sequence analysis to detect resistance mutations. methods: the study evaluates etv-treated nucleoside-naïve hbeag (+) patients who completed etv-022 and enrolled into etv-901 with a treatment gap 35 days. the proportion of patients with hbv dna <300copies/ml, alt normalization, hbeag loss or hbeag seroconversion was evaluated at week 240. background: etv resulted in improved liver histology compared to lvd at 1 year. histologic data for patients on etv for a median of 6 years is evaluated. methods: 273 clinical samples (from untreated hbv patients or treated with different antivirals; hbv genotypes a-h) were tested for mutations with the lipa assay and sequencing. for lipa, samples were extracted with the qiaamp® dna blood mini kit (qiagen), and then tested on the lipa strips. sequencing-derived reference data were subjected to phylogenetic analysis (kodon version 3.10 applied maths, neighbour joining, with kimura-2 parameter). sequential samples from 9 patients were evaluated as well. methods: etv-treated patients completing etv-022 or etv-027 received etv (1.0mg daily) in etv-901. primary endpoints included 2-point decrease in knodell necroinflammatory score, no worsening of knodell fibrosis score and improvement in ishak fibrosis score (ifs) ( 1-point decrease) vs. baseline. secondary endpoints included proportions with hbv dna<300 copies/ml, alt normalization, and ifs normalization in patients with advanced fibrosis/cirrhosis. results: quasi-perfect concordance (> 99.6%) was obtained between the two assays for the samples tested. no indeterminate results were observed. for one sample, lipa provided additional information (wild-type/mutant mix), whereas sequencing showed only wild type. for sequential samples, lipa was clearly able to detect emerging treatment-resistance mutations associated with viral breakthrough. results: etv treatment led to significant histological improvements and improved ifs in 96% (55/57) and 88% (50/57) of patients respectively. of 10 patients with baseline fibrosis/cirrhosis (ifs 4), all demonstrated 1-point improvement in ifs (median change of -3). conclusions: lipa accurately detects the complex quasispecies nature of hbv and can help unravel the dynamics of emerging hbv resistance during treatment with different antiviral drugs. like its predecessor, it is useful for the monitoring and early detection of drug resistance. conclusions: long-term etv therapy in nucleoside-naïve chb patients results in durable virologic suppression, continued histologic improvement and regression of fibrosis/cirrhosis. precore (pc, g1896a) and basal core promoter (bcp, a1762t and g1764a) mutations of hbv are important for predicting the risk of hepatocellular carcinoma (hcc). we developed a new mass spectrometry-based assay using restriction fragment mass polymorphism (rfmp) to detect a1896 and t1762/a1764 mutations, and applied it to analyze their clinical significance in type b liver diseases (n=336), including 35 hccs, 118 liver cirrhosis (lc), 157 chronic hepatitis b (chb), and 25 hbsag-positive with low level viremia (inactive hbsag carrer, ihc). we devided patients into 4 major groups according to the presence of wild (w) or mutant (m) genes in bcp/pc regions; w/w, w/m, m/w and m/m gene types. each proportion was 9.5%, 3.6%, 41.4% and 23.5%, respectively. mixed infection (x) was also found as minority; 4 w/x, 28 m/x, 15 x/w, 4 x/m and 13 x/x. disease distributions (hcc, lc, chb and ihc) in each group were as follows; [w/w (n=32)] 3.1%-6.3%-90.6%-0; [w/m (n=12)] 0-16.7%-58.3%-25%; [m/w (n=139)] 9.4%-42.5%-46%-2.2%; [m/m (n=79)] 11.4%-45.5%-20.1%-13.9%. these results suggest that, in korea where only genotype c has been identified, bcp dual mutation is predominant (>73.2%), while bcp wild alone is only 13.1%. especially, a1896 mutation alone without bcp mutation (w/m type) is uncommon, while bcp mutation alone without a1896 mutation (m/w type) is most common. it might be suggested that prognosis of wild type in bcp and pc region (w/w type) is much better than that of m/w or m/m types. background/aims: entecavir is a potent inhibitor of hbv dna polymerase, which has been shown to be safe and effective for the treatment of chronic hepatitis b (chb) patients. the aim of this study was to evaluate the virologic, biochemical, and serologic responses of entecavir through 1 year in chb patients. methods: from 2007 may to 2008 october, we reviewed 82 patients (mean age 46±12 years, male:female=57:25) who were diagnosed as chb patients (hbeag (+) 64). forty-seven patients (57.3%) had been treated with 0.5 mg of entecavir and 35 (42.7%) with 1 mg of entecavir, respectively. mean follow-up period was 25±17 weeks. hbv dna was quantified by bdna assay with a lower limit of detection of 141,500 copies/ml. results: median hbv dna levels before therapy was 7.64 log10 copies/ml and the median decreases from baseline in hbv dna were -4.19, -4.31, -5.09, -5.31, and -5.59 log10 copies/ml at 4 (n=39, p<0.001), 12 (n=62, p<0.001), 24 (n=42, p<0.001), 36 (n=22, p<0.001), and 52 (n=15, p =0.053) weeks of follow-up, respectively. at baseline, overall median alt was 106 iu/l and the proportions of patients with normal alt were 13%, 48%, 56%, 71%, 67%, and 69% at baseline (n=82), 4 (n=42), 12 (n=63), 24 (n=44), 36 (n=24), and 52 weeks (n=16) after entecavir therapy, respectively. thirteen cases (15.9%) of hbeag seroconversion were noted. background: hepatitis b virus (hbv) infection is a major risk factor for the progression of liver diseases. because its clinical course varies, it is difficult to detect the predictive factor for the prognosis of patients with hbv infection. the aim of the present study was to determine the risk factors for the occurrence of hcc. methods: a total of 620 patients who tested positive for hepatitis b surface antigen and were referred to chiba university hospital between february 1985 and march 2008 were included in the study, and their following characteristics were analyzed: age, gender, the status of hbeag, alt, hbv-dna level, and plt. result: hcc was detected in 30 cases during the follow-up period (5.4 ± 5.1 years). multivariate analysis revealed that age [compared with young patients: odds ratio (or) = 1.07, 95% confidence interval (ci) = 1.03-1.11] and plt level (compared with patients with low plt level: or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. in patients with age more than 35 years, the hbv-dna level (compared with <5.0 log copies/ml: or = 3.29, 95% ci = 1.40-11.5) and plt level (or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. conclusion: advanced age and low plt level were the risk factors for hcc occurrence in patients with hbv infection irrespective of the plt level at baseline. in patients with age more than 35 years, viral load was also a risk factor for hcc. results: before treated by lps, the total mapk p38 level of pbmcs have no significant difference among the healthy control group, different stage groups with hbv infection, however, after treated with lps, the phosphorylated mapk p38 (ptpy180/182) in healthy control group are significant elevate than hbv infected groups(120.0±52 vs 80.2±84.8, p<0.05). in the two groups which hbsag, hbv dna are positive, alanine aminotransferase elevate than normal and hbsag positive, but hbv dna lower under the detect limited level, after treated by lps the ptpy180/182 although lower than healthy control yet, but significant elevated than themselves before treated by lps(86.6±38.6 vs117.8±21.3, 63.3±24.7 vs 93.1±21.8; p<0.05).otherwise, in the group of both hbsag and hbv dna are positive, but alt is normal, before and after treated by lps, the level of ptpy180/182 have no significant difference. conclusion: mapk p38 is a important signal transduction pathway which involving in inflammation and immune response, especially, mapk p38 activated up-regulate the ifn-gamma mrna. according to the result shown, we propose a hypothesis, hbv infection and virus active replication inhibit the mapk p38 activated, consequent on host immunotolerance and hbv persistence, thus, mapk p38 may be as a potential therapeutic target to break immnotolerance and establish host anti-viral states. van der helm 10 1 siriraj hospital, bangkok, thailand, 2 ramathibodi hospital, bangkok, thailand, 3 phyathai hospital, bangkok, thailand, 4 singapore general hospital, singapore, 5 national university hospital, singapore, 6 changi general hospital, singapore, 7 cheil general hospital, korea, 8 hwasun jeonnam university hospital, korea, 9 st mary hospital, korea, 10 roche diagnostics ltd, rotkreuz, switzerland background/aims: hepatitis b virus (hbv) surface antigen (hbsag) is one of the most important markers for diagnosis of acute and hbv infection. high sensitivity of hbsag assays can reduce the diagnostic window during course of disease. in addition, the presence of hbv mutants may be affected by the performance of the hbsag kit. therefore, the technical performance of the elecsys ® hbsag ii assay was explored, using samples (including recombinant mutants), at multiple sites in three countries. methods: nine hbsag screening centers in thailand, korea and singapore compared the sensitivity of elecsys ® hbsag ii assay with that of their routine testing procedure -abbott architect ® (7 centers), abbott axsym ® (1 center) and bayer advia ® centaur hbsag assays (1 center) using preselected seroconversion panels (n=5), recombinant hbv mutant panels (n=13) and routine clinical practice samples (n=1,863). results: the sensitivity of elecsys ® in seroconversion samples was equivalent to the architect ® assay, but more sensitive than the axsym ® and advia ® centaur assays (26 vs 23 and 24 vs 18 positive bleeds, respectively). there was concordance between the elecsys ® and architect assay results with respect to potentially cross-reactive samples (99.74%). the elecsys ® and architect ® assays detected all recombinant mutant samples, whilst axsym ® and advia ® centaur failed to detect three and nine samples, respectively. conclusion: elecsys ® hbsag ii assay was not only highly sensitive and specific when compared with established hbsag screening assays, but also reliably detected hbsag mutants. therefore, this attractive assay is suitable for hbv diagnosis and assessing safety of blood products. background: recent studies have shown a higher rate of adefovir-resistant mutation in lamivudine-resistant chronic hepatitis b (chb) patients treated with switch-to therapy than those treated with add-on therapy. we compared the clinical efficacy of adefovir monothrapy and lamivudine-adefovir combination therapy in lamivudine-resistant chb. methods: a prospective cohort study was performed in 49 patients with lamivudine-adefovir combination therapy and 77 patients with adefovir monotherapy for lamivudine-resistant chb over 18 months. result: biochemical response was achieved in 41 patients (83.7%) treated with combination therapy and in 77 patients (92.8%) treated with monotherapy (p=0.101). virologic response was observed in 19 patients (38.8%) in combination therapy and in 26 patients (31.3%) in monotherapy (p=0.383) and treatment periods for virologic response was significantly shorter in patients with combination therapy than in monotherapy (8.9±4.8 months vs. 13.9±5.0 month, p=0.001). cumulative rate of virologic response was significant higher in patients with combination therapy than monotherapy (p=0.033). hbeag loss was found in 6 patients (16.2%) in combination therapy and 17 patients (20.5%) in monotherapy (p=0.485). biochemical breakthrough was found in 17 patients (20.5%) with monotherapy significantly more frequent than 3 patients (6.1%) with combination therapy (p=0.026). genotypic resistance to adefovir was developed in 1 patient (2.0%) in combination therapy and 9 patients (10.8%) in monotherapy conclusion: to achieve a complete virological response and reduce the risk of adefovir-resistant mutants in lamivudine-resistant chb patients, adefovir in combination with lamivudine is preferable. background/aims: adefovir dipivoxil (adv) effectively inhibits both wild-type and lamivudine (lam)-resistat chonic hepatitis b virus (chb) replication. the aims of this study were to determine the factorts associated with antiviral effect of adv in lam-resistant chb. methods: one hundred-eighteen lam-resistant chb patient (74.6% hbeag-positive) were treated with adv plus lam (n=96) or adv monotherapy (n=22) for a mean of 33.0 months. restriction-fragment mass polymorphism analysis was used for detection ymdd and adv mutants. results: fifty-eight patients (49.2%) achieved complete response(cr) defined as hbv-dna levels <2000 copies/ml and alt normalization. twenty-eight patients (23.7%) achieved initial vilologic response(ivr) defined as hbv-dna levels <10 4 copies/ml within the first 6 month of treatment. 47 (53.4%) of 88 hbeag-positive patients exhibited hbeag loss and 31% seroconverted to anti-hbe ab. five (4.23%) patients developed adv-related mutations. factors associated with ivr were pretreatment level of alt (p=0.00), ast (p=0.00), pretreatment hbv dna level (p=0.03), hbeag negativity (p=0.01) and hbeab positivity (p=0.00). factors associated with cr were ivr (p=0.00), hbeab positivity (p=0.01), pretreatment level of alt (p=0.01), ast (p=0.02) and y-glutamyl transferase (p=0.04). age, sex, presence of liver cirrhosis, pretreatment hbv dna level and the type of ymdd mutants were not related to an cr during adv treatment. conclusions: adv therapy achieved cr in more than 49% of lam-resistant chb. factors associated with cr were ivr, hbeab positive status, high base line alt, ast, ggt levels. q.j. sheng 1 , y. ding 1 , x.g. dou 1 1 department of infectious disease, shengjing hospital affiliated to china medical university, shenyang, 110004, china objectives: to study a kinetics of hepatitis b virus during 12-week and 24-week of treatment with enticavir (ent); to compare the detecting results of hbvdna levels from different detection reagents. methods: thirty-seven cases of chronic hbv infections were selected randomly, treated with daily dose of ent 0.5-1.0mg (0.5mg for nucleoside-naïve patients, 1.0mg for lamivuding-refractory patients). evaluation indexes: serum hbvdna, hbv serological markers, and liver function tests. hbvdna levels were measured by pcr assay, using both domestic reagents and roche cobas amplicor,. the lower limits of measure level of hbvdna were 1000 copies/ml and 300 copies/ml, respectively. results: mean baseline of hbvdna was 5.24log10copies/ml for detection using domestic reagents and 5.70log10copies/ml for that using roche cobas amplicor, (p>0.05). the ratios of cases with undetectable (<1000 copies/ml) hbvdna at week-12 and week-24 were 46.2% and 72.9%, respectively. the ratios of cases with undetectable (<300 copies/ml) hbvdna at week-24 were 54.5%. among the cases whose hbvdna were lower than 1000 copies/ml(using domestic reagents), the ratio of hbvdna lower than 300 copies/ml(using roche cobas amplicor) was 94.7%. conclusions: ent can suppress hbv dna rapidly no matter the patients with alt elevation or not. there is a concordance on hbvdna levels detection between domestic reagents and roche cobas amplicor. background the study on the effect of nucleoside analogue therapy on the quantity of hepatocellular cccdna and tdna and sera hbv dna, hbsag to probe reliable marks for evaluation of therapy endpoint. methods the quantity of hepatocellular cccdna and tdna and sera hbvdna were assayed by fq-pcr, and sera hbsag by elisa in 4 chb patients over 2 years nucleoside analogue therapy satisfied the china criteria of therapy endpoint (therapy group)and 12 chb patients without antiviral therapy and sera hbvdna<10 3 copies/ml(control group). results: the quantity of hepatocellular cccdna, tdna and sera hbvdna, hbsag in therapy group were lower than that of control group,but low level hepatocellular cccdna in therapy group could be detected. conclusion: long term nucleoside analogue therapy may consume hepatocellular cccdna with decreasing of hepatocellular tdna and sera hbvdna, hbsag; although the patients have satisfied the china criteria of therapy endpoint, low level of hepatocellular hbvcccdna were detected, cessation of therapy may cause relapse. peptides that lead nuclear entry of nucleocapsid of hepatitis b virus in hepg2.2.15 cells x.b. pan 1,2 , l. wei 1,2 , j.c. han 1,2 , k. deng 1,2 1 peking university hepatology institute, 2 peking university people's hospital background: the nuclear entry of nucleocapsid is a key step for the hbv life cycle and the formation of covalently closed circled dna (cccdna). it has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (nls). whereas hbcag was primarily distributed in cytoplasm and no marked cccdna was detected in hepg2.2.15 cells. methods: we designed peptides containing a cell-penetrating sequence (rrrrrrr) and a nucleocapsid binding sequence (gsllgrmkga) with/without a classic nuclear localization sequence (pkkkrkv) and these sequences were linked by a soft linker acp. hepg2.2.15 cells were treated with the peptides at levels of 0 m, 25 m and 50 m for 4 days. results: compared with that of control cells, the results showed hbv dna levels in culture medium decreased at least 2 log10 both in 50 m of peptide rrrrrrracpgsllgrmkga treatment group and rrrrrrracpgsllgrmkgaacppkkkrkv treatment group; whereas hbsag and hbeag increased at 1.34+0.21 folds and 2.15+0.37 folds respectively. the signal strength of cytoplasmic hbcag increased at about 2.5-fold in both groups. in rrrrrrracpgsllgrmkgaacppkkkrkv treatment group, nuclear hbcag increased about 3.2-fold and obvious cccdna signal was detected by southern blot. conclusion: our results implied that the nls of core protein likely does not expose to surface of nucleocapsid in hepg2.2.15 cells, the artificial peptide containning nls binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccdna. our study presents a tool for study on cccdna formation and nuclear entry of nucleocapsid. b. tang 1 , j. xia 1 , y.m. wang 2 , h.f. wang 1 1 liver failure treatment and research center, 302rd military hospital, 2 the dept. aims: to establish a reliable real-time fluorescence quantitative (rfq) pcr method to quantify hbv cccdna, basing on lightcycler system and taqman probe. methods: hbv genotypes a-g were aligned to obtain a conserved sequence, crossing rcdna gap, which was used to design cccdna primers and taqman-mgb probe. also another pair of primers for quantify hbv total dna (tdna) was designed, utilizing the same probe. to increasing specificity, we added plasmid-safe atp-dependent dnase (psad) digestion step just before cccdna pcr amplification. a standard curve from 9 standard plasmid samples, from 2.0×10 9 to 2.0×10 1 copies, was created to examine our system. 50 hbv cccdna samples with known-amount were quantified by creating standard curve from 5 standard samples. results: the standard curve had clear log-phase and excellent parallelism, which means nice and equal amplification efficiency in all reaction capillarys. the slope (regression coefficient) of standard curve was -3.141, mean square error was 0.0990 and regression coefficient was -1.0. all of these key indexes measured up. in 50 tests, we got right results if the starting templates of cccdna copies were between 10 2~1 0 9 copies. the range was superior to commercial hbv kits. by quantifying 16 samples containing different amounts of cccdna and rcdna, digested or undigested, psad digestion would eliminate 10 7 rcdna molecules, leaving 10 2 cccdna molecules untouched. the test specificity was maintained up to 1:100,000 ratio of cccdna:rcdna. conclusions: the rfq-pcr based on lightcycler system for hbv cccdna quantification is reliable, sensitive, with high specificity and low cost. background: to study the changes of toll-like receptor (tlr)3 on dendritic cells derived from peripheral blood mononuclear cells(modc) and its role in the pathogenesis of chronic hepatitis b(chb) and chronic severe hepatitis b(cshb). methods: the expressions of tlr3 on 10000 modc were stimulated by poly i:c, and then were determined by flow cytometry in 20 healthy controls, 28 patients with chb and 30 patients with cshb.the level of interferon ifn-was determined by elisa. the differences of expression of tlr3 on modc and serum ifn-among the three groups of study subjects were determined by student-t test.the correlation between tlr3 and ifn-were determined by linear correalation test. results: the values of mean fluorescence intensity(mfi) of tlr3 on modc of the healthy controls, patients with chb and cshb were 1593.00±349.65,1369.56±287.08,and 1203.96±192.40.the serum ifn-(pg/l) of respective groups was 172.66±37.96,107.98±31.15 and 72.06±29.58. there was a gradual decrease of these values from the group of healthy controls to the group of patients with chb and cshb .significant positive correlations between tlr3 and serum ifn-were found. conclusion: tlr3 may have a role in the pathogenesis of chb and cshb. background/aim: to evaluate the efficacy and safety of entecavir treatment in patients with hbeag-positive chronic hepatitis b who had not previously received a nucleoside analogue. methods: fifty-five patients received 48-week entecavir 0.5mg/d therapy. serum hbv dna load was measured with quantitative real-time-pcr. alanine aminotransferase (alt) activity, hbeag, anti-hbe-antibodies, hbv dna level in serum were evaluated at baseline, week 2, 12, 24 and 48 during therapy. evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. results: hbv dna levels declined sharply by around 3 log10 copies/ml during the first two weeks, with a highly significant reduction (p<0.0001) at week 2 and thereafter, as compared to those at baseline; 31%, 51% and 78% of the patients had undetectable serum hbv dna levels at week 12, 24 and 48 respectively. highly significantly decreasing serum alt (p<0.0001) occurred during the first 2 weeks of the study. at week 48, alt levels were normalized in 84% of the patients. hbeag seroconversion (hbeag negative, hbeab positive) was achieved in 7.3% and 14.5% of patients by 24 and 48week. at the end of 24 th and 48 th weeks, complete response (alt normalization and hbv dna and hbeag loss) was observed in 11% and 15%, respectively. there was no evidence of drug resistance or adverse effect in chb patients treated for up to 48 weeks. conclusion: entecavir treatment through 48 weeks was well tolerated and resulted in continued benefit for patients with hbeag-positive chronic hepatitis b. aim: to assess the associations of single nucleotide polymorphisms of the mxa gene promoter and sustained treatment response of chronic hepatitis b or c patients with interferon treatment by meta-analysis of individual dataset from all studies published till date. methods: to clarify the impact of mxa gene promoter polymorphisms on sustained treatment response of chronic hepatitis b or c patients with interferon treatment, we performed a meta-analysis of the published data from eight studies comparing the frequencies of mxa gene promoter polymorphisms at nt -88 g/g, -88 g/t, -88 t/t and nt -123 c/c, -123 c/a , -123 a/a alleles in individuals with interferon treatment. as we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. results: the sustained treatment response rate was higher in patients with the nt -88 g/t and nt -123 c/aalleles in the mxa promoter snp . the meta-analyses yielded summary estimatesodds ratio (or) were 2.07 [95%ci (1.58, 2.7), p <0.00001] and 1.9 [95%ci (1.32, 2.73), p =0.0006] of the nt -88 g/t and nt -123 c/a alleles, respectively. conclusion: mxa gene promoter polymorphisms at nt -88 g/t and nt -123 c/a may be useful as a marker to predict the sustained treatment response of chronic hepatitis b or c patients with interferon treatment, and further investigation regarding their real significance is warranted in a large series of patients. background: to determine whether hbv with the same characteristics causes dissimilar mutations in different hosts. methods: full-length hbv genome was amplified and linked with pmd t18 vector. positive clones were selected by double-restriction endonuclease digestion (ecori and hindiii) and pcr. twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (s)]. bioeditor, clustal x and mega software were used to perform phylogenetic and mutational analysis. potential immune epitopes were determined by the stabilized matrix method (smm), smm-align method and emini surface accessibility prediction. result: all of the 27 sequences were genotype c, the inner-divergence for the mother and son was 0%-0.8%.13 specific nucleotides differed from the other pubished genotype c isolates were co-exist in the mother and her son. aa 1-11 deletion in pres1 was the dominant mutation in the mother (14/18). the 1762t/1764a double mutation existed in all clones of the mother, 3 of them were also coupled with g1896a mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential hla class i epitopes and one b cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the p gene. conclusion: the son was infected hbv from his mother, and discrepant mutation occurred in the mother and her son during infection. background/aims: nucleos(t)ide analogues have been recognized as an effective treatment for chronic hepatitis b. this randomized, double-blind trial compared the efficacy and safety of telbivudine and lamivudine after 104-week therapy in patients with compensated chronic hepatitis b in taiwan. methods: we analyzed 114 taiwanese patients from globe trial receiving telbivudine 600mg (n=58) or lamivudine 100mg (n=56) once daily for 104 weeks. the primary efficacy endpoint was therapeutic response with serum hbv dna <5 log10 copies/ml and either hepatitis b e antigen (hbeag) loss or alanine aminotransferase (alt) normalization. results: the therapeutic response at week 104 was 74.8% in telbivudine group versus 50% in lamivudine group (p=0.005). more patients with telbivudine achieved nondetectable serum hbv dna (<300 copies/ml) (p=0.024) and alt normalization (p=0.021) at the end of treatment. the cumulative resistant rate was significantly lower in those with telbivudine treatment (p=0.0032). the rate of hbeag seroconversion was comparable in both groups (p=0.407). although a lower percentage of patients in lamivudine group (83.9%) reported adverse events than those in telbivudine group (89.7%), the difference was not significant. conclusions: telbivudine demonstrates a significantly greater efficacy and a lower resistant rate than lamivudine in treatment of chronic hepatitis b in taiwan. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. aim: to define the potential role of pd-1/pd-lpathway in different hbv infection status; we examined the expression of pd-1on cd8+ t cells in pbmc of patients with chb and aehb infection. methods: the pd-1 level on cd8+ t lymphocytes and the number of hbv specific cd8+ t lymphocytes in patients and healthy controls were analyzed by flow cytometry. pcr was used to measure the serum hbvdna levels. results: the level of pd-1 expression on cd8+ t cells in chb patients was higher than that in aehb patients and healthy individuals. compared to aehb patients, lower frequency of hbv-specific cd8+ t cells was detected in chb patients. there was an inverse correlation between the strength of hbv-specific cd8+ t-cell response and the level of pd-1 expression. besides, there was a significant positive correlation between hbv viral load and the percentage of pd-1 expression on cd8+ t cells in chb and aehb subjects. however, pd-1 expression was not associated with alt levels. conclusion: our results confirm previous reports that hbv specific cd8+ t-cell response in the peripheral blood is more intense in patients with aehb than in chb with persistent viral infection. moreover, there is a negative correlation between the level of pd-1 and the intensity of virus specific cd8+ t cell response. observed in 10.9% of patients with a mean age of 51.8±12.9. the ethnic composition was 53.1% chinese; 27.3% malay; 14% indigenous sabahans; 2.9% indigenous sarawakians; 1.8% indians and 0.8% others. chinese patients were on average, older (mean 45.6±14.5 years), indians patients had higher mean alanine transaminase and indigenous sarawakian patients had the highest rate of cirrhosis (p<0.0001). during the study period, 20.7% of patients were on treatment and they were significantly older than those who were not on treatment (mean age 44.6 ± 14.5 vs 40.7 ± 14.3). lamivudine was the first agent used in 86.9% of cases. conclusions: in malaysia, chb remains a public health issue and significantly afflicts males in the productive age groups and of chinese ethnicity. the observed differences among ethnic groups could point to different disease severity which needs to be addressed in the local treatment guideline and policy. background/aims: liver stiffness measurement (lsm) has been validated for predicting fibrosis stage in patients with chronic hepatitis c. however, studies on lsm for chronic hepatitis b (chb) are few, and the relationship between histologic findings and liver stiffness needs to be further elucidated. this study was conducted to assess the association of histologic activity on liver stiffness in addition to fibrosis in patients with chb methods: thirty three patients who had taken liver biopsy and lsm at korea university ansan hospital between march 2008 and october 2008 were enrolled. necroinflammatory activity and fibrosis stage were assessed by metavir system. activity, fibrosis, and the sum of both score were included for the correlation analysis with lsm results: among 33 patients, 29 (87.9%) were male, and median values were as follows: age, 42 (20~54); ast, 49 iu/l (19~627); alt, 62 iu/l (8~778); total bilirubin, 1.03 mg/dl (0.4~2.39); lsm, 10.1 kpa (4.2~43.5). fibrosis stages were f1 in 4 (12.1%), f2 in 9 (27.3%), f3 in 12 (36.4%), and f4 in 8 (24.2%) patients. spearman correlation coefficient with lsm were 0.457 (p=0.008) for activity, 0.694 (p<0.001) for fibrosis stage, and 0.724 (p<0.001) for the sum of activity and fibrosis. in linear regression analysis, only the sum of activity and fibrosis remained to be significant. conclusions: not only fibrosis but also activity was an important factor for determining lsm for chb. it would be more appropriate to consider both activity and fibrosis for interpretation of lsm in patients with chb background: hbeag seroconversion is a key goal of chb therapy. hbeag kinetics may predict hbeag seroconversion during treatment. we aim to develop a robust hbeag quantitative method as the value of hbeag quantitation is undefined and data is limited. methods: we evaluated two commercially available qualitative hbeag assays (abbott architect, siemens centaur) for their linear range and validated them against paul-ehrlich institute (pei) standards. hbeag levels were determined from samples of untreated and telbivudine-treated chb patients. results: as a pre-requisite for quantitative use, the linear range for the architect (0.5-43 peiu/ml) and centaur (0.05>5 peiu/ml) assays were defined. architect was selected for further investigation. hbeag levels of 44 untreated patients (mean hbv-dna 9.8 log10 copies/ml, mean alt 166.7 iu/ml) varied from 0.4 to 1073.5 peiu/ml (median 161.7 peiu/ml). in 23 patients (mean hbv-dna 9.9 log10 copies/ml, alt 166 iu/ml) treated with telbivudine for 12 weeks, baseline hbeag levels varied from 1.6 to 664 peiu/ml (median 150.7 peiu/ml). after 12 weeks of telbivudine treatment, median hbeag level was 4.4 peiu/ml, with 76% decline from baseline (median decline 95.1 peiu/ml, range 1.4-657.6 peiu/ml). individual hbeag decline from baseline varied but occurred in all patients and was not correlated to baseline or decline from baseline hbvdna. conclusion: hbeag quantitation is feasible and robust with architect hbeag assay. hbeag decline occurred in all telbivudine-treated patients, and was not correlated to hbvdna. whether the magnitude of hbeag decline is predictive of future hbeag seroconversion merits further investigation. experience from the combined globe (nv-02b-007/cldt600a2302) and 015 (nv-02b-015) study clinical safety database. c. avila 1 , r. laeufle 2 , w.b. bao 3 1 novartis pharma ag, fabrikstrasse 6, basel, switzerland, 2 novartis pharma ag, basel, switzerland, 3 novartis pharma, east hanover, us background: creatine phosphokinase (ck) is a commonly used marker of muscle damage and is elevated by many factors (e.g. exercise, injury, drugs). normal ck levels are affected by muscle mass and elevated levels are described during the natural course of chb. 22% of patients in the globe study had pretreatment grade 1-4 ck elevations. methods: we reviewed data from this combined study clinical safety database, and describe the experience of ck elevation and its relationship to adverse event reports of muscle related symptoms. results: the frequency of new onset of grade 3-4 ck elevations in telbivudine-treated patients (combined database itt population) was 1.8% (15/847), 6.3% (53/838), 3.2% (27/826) and 5.1% (40/786) from weeks 0-24, 24-52, 52-76 and 76-104 respectively. the frequency of grade 3-4 ck elevations for all patients from week 0-104 was 12.6% (107/847). the majority of grade 3-4 ck elevations were asymptomatic, rarely resulted in discontinuation or interruption, spontaneously declined within 1 or 2 visits and were not associated with more frequent muscle-related adverse events. cumulative data from this combined database showed no relationship of the degree of increased ck to acute or persistent muscle disease. conclusion: ck elevations are associated with hbv disease and were also common during the globe and 015 trials and were not predictive of the development of muscle related symptoms. onset of muscle-related symptoms should prompt clinical and treatment review, including concomitant medications. backgrounds: leptin plays a crucial role in the regulation of energy balance and body weight control by activating the long form of the leptin receptor (ob-rl). epidemiologic studies showed that obesity is one of the factors associated with hbv related hepatocellular carcinoma. methods: huh7 cells were transiently transfected with 1.3 copies of hbv-replicon plasmid. after 48h, cells were harvested and total rna of the cells were extracted and reverse-transcribed into cdna. long form and short form leptin receptor (ob-rl, ob-rs) mrna transcription levels were assayed by real-time pcr respectively. and mrna transcription levels and protein expression of ob-rl and ob-rs in hepg2.2.15 cells were also detected. results: after transfected by 1.3 copies hbv-replicon plasmid, the mrna transcription level of ob-rl was inhibited significantly (**p<0.01), but the mrna transcription level of ob-rs did not change, and the ob-rl protein expression was reduced. in hepg2.2.15 cells, the mrna transcription level of ob-rl was also significantly lower than the mrna transcription level of ob-rl in hepg2 cells, while the mrna transcription of ob-rs in hepg2.2.15 and hepg2 cells didn't show significant difference. besides, the protein expression level of ob-rl in hepg2.2.15 was also lower than it in hepg2 cells. conclusion: hbv replication down-regulated the expression of long form leptin receptor in cell cultures, which could in part explain the clinical observation of obesity in association with development of serious sequelae in hbv infections. the results of entevavir treatment in patients with chronic hepatitis b s. kose 1 , g. akkoclu 1 , m. turkeri 1 , a. gozaydin 1 1 the ministery of health tepecik training and research hospital, izmir purpose: we evaluated the short and long term effectiveness of entecavir. patients and methods: those patients had received diagnosis of chronic hepatitis b. their pretreatment transaminases, hbsag, anti-hbs, hbeag, anti-hbe, hbv-dna were checked and a liver biopsy and a resistance test for lamivudine (lam) and adefovir (adv) were performed. a total of 44 patients who were taking entecavir for at least 24 weeks were included in the study. findings: the biochemical and virologic response were observed in 88.8 % at 3 and 6 months and in 75 % at 12 months. in 9 hbeag positive patients who had received therapy previously, the biochemical response was observed in 88.8 % at 3 and 6 months and in 100 % at 12 months. the virologic response in 77.7 % at 3, in 88.8 % at 6, and 100 % at 12 months. posttreatment hbeag seroconversion did not develop. in 9 hbeag negative patients the biochemical and the virologic responses were observed in 88.8 % at 3 months and 100 % 6 and 12 months, respectively. in 17 hbeag negative patients had received therapy previously, the biochemical response was observed in 76.4 % at 3, in 100 % at 6 and 12 months. the virologic response in 94.1 % at 3, in 100 % at 6 and at 12 months. conclusion: in our study, a higher therapy-response rate was achieved, especially in hbeag negative patients. in hbeag positive patients biochemical and virologic response rates were high. background/summary: patients with liver disease are known to have a higher prevalence of glucose intolerance. preliminary studies suggest that viruses can be an additional risk factor for the development of diabetes mellitus. individuals with type ii diabetes have an increased prevalence of cirrhosis, and a proportion of patients with acute and chronic liver disease develop diabetes mellitus. there is now emerging epidemiological data to suggest that hepatitis c virus (hcv) infection may also contribute to the development of diabetes reported to be higher than expected compared with the general population. while these investigations suggest an epidemiological association between hcv infection and diabetes, large controlled studies are required to observe association between hbv infection and diabetes. the present study was designed to study the relative proportion of diabetes mellitus in patients suffering from hepatitis b virus (hbv) infection. background: in easl 2008, we reported significant liver disease among hbeag negative patients with serum hbv-dna level <4log10copies/ml (i.e. 2.0x3log10iu/ml) and alanine aminotransferase (alt) <55iu/l. this reflects fluctuating nature of these levels. the aim of this retrospective study is to demonstrate the frequency of fluctuation among this group of chb patients. methods: clinical records of hbeag negative treatment naïve chb patients with at least one serum hbv-dna <4log10copies/ml were reviewed. results: there were 194 (62.5% male, median age 48.0 years) chb patients with negative hbeag and hbv-dna <4log10 copies/ml (roche cobas amplicor pcr assay, lod<300copies/ml). 32 had serial hbv-dna measurements within 2 years; 7 of them (21.9%) had increase serum hbv-dna level by >1log10 copies/ml; 2 patients had associated serum alt elevation from normal (normal range <55 iu/l), 4 had persistent normal alt, and one had persistently raised alt. 5 (15.6%) had serum hbv-dna level decrease by >1log10 copies/ml. another 20 patients had hbv-dna levels fluctuating within 1log10 copies/ml. 159 hbeag negative patients with single hbv-dna measurement showing <4log10 copies/ml had serial serum alt measurements within 3 years. 35 (22.0%) patients had intermittent / persistently raised alt; while 124 (78.0%) patients had persistently normal alt. conclusions: hbeag negative chb is common among chinese. serial serum hbv-dna and alt measurements are necessary to detect fluctuating levels and progressive liver disease that may require antiviral therapy. background: to determine the best vaccination strategy, a model that reflects the country-specific infection profile is needed. methods: a model was built in order to obtain the age-specific infection frequency q(t) for neonates(n), infants(i), children(c), and adults(a). the infected group can either become hbsag(+) or anti-hbs(+)* based on f(t). q(t) can be found from p(t) = [q(t) x (1 -f(t)) x crs + q(t) x f(t) x (1 -cras)], where p(t) represents the proportion of the late anti-hbs(+)** group and crs/cras denote natural conversion of hbsag/anti-hbs. to test the model, cross-sectional serologic marker data in korea were used. because f(t), crs, and cras were known(f(n)=0.1, f(i)=0.5, f(c)=0.8, f(a)=0.95, crs=0.015, cras=0.02), in order to determine q(t), only p(t) values were needed, which were evaluated from logistic modeling using the glm() function of s-plus. results: the infection frequencies during neonate, infant, children, and adult periods in non-vaccinees were 15.4%, 33.3%, 16.6%, and 34.7%, respectively. each group's likelihood of infection compared to adults was then: neonates 170.2 times more likely, infants 33.5 times, and children 0.9 times, making a strong case for neonatal and infantile vaccination for the studied region. conclusions: the hbv infection model can be used for determining the most cost-effective strategy for hb vaccination in nations where longitudinal data are not available. and where longitudinal data are available, it can be used to determine the appropriate time of transition of vaccination strategy to maintain cost-effectiveness. the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b x.c. pan 1 , f. yang 1 , m. chen 1 1 objective: to investigate the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b. methods: 36 patients with hbeag positive chronic hepatitis b were recruited and receiving telbivudine treatment for 9 months. before and during 3 6 9 months of treatment , flow cytometry was used to detect the proportion of peripheral blood tregs; real-time pcr was used to detect the levels of hbv dna in surum, markers of hepatitis b virus infection were detected by elisa assay and levels of alanine aminotransferase in serum were measured. results: the proportion of peripheral blood tregs in patients with chb was significantly higher than that in healthy controls and decreased over 6 or 9 months of treatment to a level comparable to that of healthy controls. after 3 months of treatment, the rate of alt normalization in patients which the proportion of peripheral blood tregs was unreduced was significantly lower than that in patients which the proportion of peripheral blood tregs was reduced (p<0.01). 3, 6 or 9 months of telbivudine treatment resulted in negative hbeag in 4(11%) patients, 7(19%) patients or 9(25%) patients respectively. within 9 months of treatment, 7 (19%) patients seroconverted from hbeag to anti-hbe , in which the proportion of peripheral blood tregs had decreased to a level comparable to that of healthy controls over 3 or 6 months of treatment. conclusion: during antiviral treatment with subsequent reduction of the viral load or alt levels, the proportion of tregs decreased to a level similar to that of normal healthy controls. in addition, seroconversion from hbeag to anti-hbe was prone to be established in patients which the proportion of tregs decreased quickly at the early phase of antiviral treatment with telbivudine. background: in china a part of patients with alt <1.5×uln and hbv dna >10 5 copies/ml will advance into hepatic cirrhosis even hepatoma. so these patients should not only be monitored but also be treated. this study was made to determine the safety and efficacy of combining therapy of pegylated interferon alpha 2a (peg-ifn -2a) and entecavir in treating naive patients with alt <1.5×uln and hbv dna>10 5 copies/ml. methods: nine patients with hbsag positive over 6 months and alt<1.5×uln hbv dna >10 5 copies/ml were taken as research subjects. before treatment,liver biopsy was used to assess histological damage. patients were treated with peg-ifn -2a 180 g /week for 48 weeks, and in the first 12 weeks entecavir 0.5 mg/day was applied, then it was stopped. results: 1 liver biopsy showed that 7 patients had mild inflammation. 2 after 12 weeks' treatment , hbv dna level in all patients decreased to less than 10 4 copies/ml, and after 24 weeks' treatment(12 weeks after entecavir was stopped)hbv dna in all patients was less than 10 3 copies/ml. 3 normal alt was seen in all patients after 12 weeks' treatment and 24 weeks' treatment. 4 none of the patients had peripheral neuropathy with combining treatment. conclusions: 1. bulk of patients with alt <1.5×uln and hbv dna>10 5 copies/ml had mild inflammation and need treatment. 2 combing treatment of peg-ifn and entecavir was safe and effective to this group. 3 it proved that it was safe for patients to stop treatment with entecavir after short time use. background & aims: quantification of serum hbv dna levels is important to monitor viral replication in chronic hepatitis b (chb) patients. both abbott realtime hbv and roche cobas amplicor hbv monitor are updated fully automatic commercial assays for hbv dna quantification. the aim of this study is to compare the performance of these two assays on the hbv dna quantification in chb patients. methods: serial serum samples from 30 chb patients were collected at the baseline and at days 4, 7, 10 and 14 and weeks 3, 4, 8 and 12 after the commencement of therapy. genotype was determined by sequence alignment. abbott and roche assays were employed for hbv dna extraction and quantification according to the instructions of manufactories. results: hbv dna quantification results of abbott assay was significantly correlated with those of roche assay (r=0. 972, p<0.0001). for genotype c, the difference in hbv dna levels [median (range): 1.22 (-0.16-2.36) log units] measured by these two assays was significantly higher than that for genotype b [0.66 (-0.52-1.63) log units, p<0.0001]. moreover, the difference in serum hbv dna levels after 12 weeks antiviral treatment [1.18 (-0.21-1.76) log units] measured by these two assays was significantly higher than that in baseline serum hbv dna levels [0.68 (-0.12-1.24) log units, p<0.0001]. conclusion: the quantification results of abbott realtime hbv showed a good correlation with those of roche cobas amplicor hbv. but the performances of these two assays have significant difference in the quantifications of serum hbv dna levels in genotype c patients and in patients after 12 weeks antiviral therapy. background: guidelines suggest hepatitis b virus (hbv) vaccination to all hepatitis c virus (hcv) infected patients and healthcare workers. we attempted to find out hbv vaccination status in our hcv infected population, and healthcare workers. methods: prospective survey of 100 consecutive hcv infected patients and also doctors and paramedical staff in our hospital. results: major sources of viral infection in study patients (58 males; average age 40 years -range 18 to 65 yrs) were reused syringes (38 pts). twenty had a household member infected with hcv. twenty were co-infected with hbv. eighty five of hcv infected patients were not vaccinated.against hbv. twenty five of them (29%) had financial reasons and 45 patietns (52%) had lack of awareness. out of 30 doctors, 12 and 15 did not know about their hbv and hcv status respectively, but none was known to have either of these infections. four (13%) were not vaccinated against hbv. out of 29 paramedical staff, 1 was hcv positive, 11 each were unaware of their hbv and hcv status, and remaining were negative for these markers. thirteen of them (44%) were not vaccinated against hbv. conclusion: thirty eight percent hcv infected patients were infected by reuse of syringes. eighty five percent were not vaccinated against hbv, out of which 52% had no awareness about it, whereas 29% could not financially afford it. a significant number of paramedical staff and some doctors were also not vaccinated background: early prediction of efficacy could decrease unnecessary interferon exposure of patients with chronic hepatitis b. methods: a multi-center clinical study. patients were injected interferon alpha 2b 5 million iu subcutaneously every other day for 24 weeks and 24-week follow-up was followed. results: 53 patients (44 male) were enrolled, 27.5 ±9.1 years old. 24 hours after administration, hepatitis b virus (hbv) load decreased significantly (6.96±1.03log copies/ml, p<0.05) from baseline (8.00±0.93 log copies/ml). hbv load was 4.94±1.47 and 5.25±1.58 log copies/ml at week 24 and 48, respectively. at the two points upwards, complete response rate was 4.2%(2/48) and 7.9%(3/38), partial response rate was 35.4%(17/48) and 34.2%(13/38), respectively. at week 4 and 12, hbv dna levels of complete responder and partial responder were lower than those of non-responder (p<0.05) at week 24. at baseline, on hour 12, day 2, 3, week 2, 4 and 12, hbv dna levels of complete responder were lower than those of non-responder at week 48 (p<0.05). multiple linear regression showed that baseline hbv dna was the independent variables to predict the response at week 24 and 48. conclusions: interferon alpha 2b was effective in treating patients with hbeag positive chronic hepatitis b. it could decrease the hbv dna level rapidly. early hbv dna levels were predictive to response at the end of treatment and follow-up. baseline hbv dna level was the independent predictor of the response at the end of treatment and follow-up. aim: to investigate features of pd-1 expression on peripheral tcells and pd-l1 expression in liver in chronic hepatitis b (chb) patients in immune clearance phase. methods: pd-1 expression on total peripheral t cells were evaluated by using flow cytometry. immunostaining was performed according to the envision chemmate methods. the degree of pd-l1 expression was scored and assessed according to the percentage and staining intensity of positive cells. results: compared to health control, the percentage of total peripheral t cells expressed pd-1 was elevated in chb with repeatedly increasing alt level. no specific association between the percentage of pd-1 positive and the mean fluorescence intensity mfi of pd-1 expression on total t cells with serum viral load were found. but alt level was correlated with the mfi of pd-1 expression on total cd8+t cells significantly. pd-l1 is up-regulated on hepatocytes by viral infection, and high expressed in fibrosis section. conclusion: the mfi of pd-1 on cd8+t cells plays important role in regulating the immune-host interaction in chb in immune clearance phase. and pd-1 expression on t cells is correlated with high immune inflammatory refection. aim: to study the quantity, characteristic of hbv-specific t-cell and the extent of liver damage in chronic hepatitis b (chb) patients with different hbeag status. methods: 103 chb patients were enrolled and divided into two groups according to the hbeag status, and the liver damage index were analyzed. the frequency and foxp3 expression of cd4 + cd25 + regulatory t cells (treg) were measured, as well as the frequency and phenotypic molecules expression of hbv-pentamer+ t-cell. hbv specific t-cell responses including cellular proliferation and ifn-production, with or without anti-pd-l1 and/or anti-ctla-4 blocking, were also observed. results: the demographic characters, serum alt, ast levels, the frequency and foxp3 expression of cd4 + cd25 + treg were similar, while the serum hbv dna levels were higher in hbeag+ patients (p <0.05). the liver necroinflammation was comparatively more severe in hbeag-patients (p =0.056), but the median percentage of liver cirrhosis was much higher in hbeag+ patients (p <0.05). the difference of hbv-specific t-cell frequency was not significant between two groups, while the expression levels of pd-1 and ctla-4 on hbv-specific cd8 t cells were significantly higher in hbeag+ patients (p both <0.05). combined using of anti-pd-l1 and anti-ctla-4 mab significantly increased the cellular proliferation in either hbeag+ or hbeag-patients, but only markedly enhanced the ifnproduction in hbeag+ patients. conclusion: hbeag persistency could probably induce higher expression of pd-1 and ctla-4 on the hbv-specific t cells and result in t-cell impairment, high hbv dna load and high percentage of liver cirrhosis in hbeag+ chb patients. hepatocyte apoptosis in patients with chronic hepatitis b y. liu 1 , k. wang 1 1 background: to investigate the relationship between hepatocyte apoptosis and the level of inducible nitric oxide synthase (inos) in hepatic tissue in the patients with chronic hepatitis b chb . methods: we observed 37 cases with chb and 10 normal controls. transferase-mediated-utp-biotin nick-end labling ( tunel) technique was used to detect apoptosis cells and immunohistochemical staining were also performed to investigate the expression of inducible nitric oxide synthase (inos) in biopsy samples .the serum level of alt hbv-dna grading of necroinflammatory activity and staging of fibrosis were also assessed. results: hepatocytes in all chb liver tissues were positively stained by tunel in various degree. in contrast, control tissues did not show dna fragmentation. a significant correlation was seen between apoptosis index (ai) and necroinflammatory grading ((r=0.404, p=0.015) and serum inos level r=0.465, p=0.004 . it did not correlate with fibrosis stage and serum alanine aminotransferase level. conclusion: the oxidative stress.in patients with chb may reflected the apoptosis of hepatocyte. apoptosis involves in liver injury of chb,but with no significant correlation to serum level of alt. objectives: to investigate the genotype-dependent development of lamivudine resistance in hepatitis b virus (hbv). methods: 215 patients with chronic hepatitis b who had been treated with lamviudine for more than 1year, and become lamviudine resistance were analysed for the hbv genotypes and cumulative rate of rt region mutant with standard dna sequencing technology. results: among the 215 patients, 60 patients were infected with hbv genotype b (hbv/b)(27.9%), and 155 with genotype c (hbv/c)(72.1%). in the hbv/b patients, 16/60(26.7%) were of subtype ba, and 44/60(73.3%) were of of subtype bj. the cumulative type and ymdd mutation rates in patients with genotype c were showed as l180m+m204v (67/155,43.2%) > l180m+m204i (52/155,33.5%) > m204i (36/155, 23.3%), while in patients with genotype b as l180m+m204v 36/60(60.0%) > m204i(24/60, 40.0%), none of l180m+m204i. conclusions: our results indicated that in patients with lamivudine resistance, hbv genotype c (hbv/c) were higher than genotype b (hbv/b). in both genotypes the combined mutations (180+204 sites) were found more than the single 204 site, showed some significance for monitoring lamivudine resistance. background & aims: il-35, a novel identified inhibitory cytokine specifically produced by regulatory t cells (tregs), is an ebi3-il-12 heterodimer encoded by epstein-barr-virus-induced gene 3 (ebi3) and interleukin-12 alpha (il12 ). the aim of the study is to determine the expression levels of il-35 in peripheral blood mononuclear cells (pbmcs) of chronic hepatitis b (chb) patients in different phases. methods: a total of 36 treatment naïve chb patients, including 17 in immune-tolerant phase [group 1, alt: 21 (12-51) u/l, serum hbv dna: 2.48 x 10 9 (6.30 x 10 6 -1.20 x 10 10 ) copies/ml] and 19 in immune-clearance phase [group 2, alt: 221 (71-1530) u/l, serum hbv dna: 5.10 x 10 8 (1.62 x 10 6 -1.77 x 10 10 ) copies/ml] were enrolled in the study. the relative mrna expression levels of ebi3, il12 and foxp3 were determined by semi-quantitative pcr. results: the significant correlations were observed between the expression of ebi3 and il12 (r=0.661, p<0.001), ebi3 and foxp3 (r=0.388, p<0.05), il12 and foxp3 (r=0.431, p<0.01). the relative expression levels of ebi3 and il12 in pbmcs were significantly higher in group 2 when compared with those in group 1 (1.46 ± 0.23 vs 0.80 ± 0.10 and 1.26 ± 0.19 vs 0.65 ± 0.09, p<0.05, respectively). furthermore, the relative expression levels of ebi3 and il12 in group 2 were significantly correlated with alt levels (r=0.473, r=0.474, p< 0.05, respectively), but not with serum hbv dna levels. conclusions: the expression levels of il-35 in pbmcs were significantly higher in chb patients in immune-clearance phase than that in immune-tolerant phase. increased il-35 expression levels were associated with liver injury. background: there are a number of oral antivirals approved for chronic hepatitis b. lamivudine, the first oral nucleoside analog, is associated with increased rates of drug resistance with prolonged use--from 20% at one year to 50% at three years. therefore, an alternative or add-on treatment is necessary. adefovir, an oral nucleotide analog, is used either in combination with lamuvudine or as monotherapy in lamivudine-resistant chronic hepatitis b. we did a meta-analysis to compare the efficacy of adefovir in combination with lamivudine versus adefovir alone in the treatment of lamivudine-resistant chronic hepatitis b infection. methods: a comprehensive literature search was performed using the following databases: medline, cochrane, and embase. a total of 3 randomized controlled trials were retrieved and analyzed. outcomes measured were virologic response, biochemical response and resistance rates. results: meta-analysis on virologic response showed that combination treatment with adefovir and lamivudine is as effective as adefovir monotherapy (or 1.04, 95% ci 0.50-2.15, p=0.92). likewise, in terms of biochemical response, both regimens were equally effective (or 1.06, 95% ci 0.47-2.40, p=0.90). one study showed statistically significant increase in adefovir resistance rate in the monotherapy arm compared to combination arm (p= 0.0182) after the first year of therapy. conclusion: in patients with lamivudine-resistant chronic hepatitis b infection and compensated liver disease, adding adefovir to lamivudine is as effective as switching to adefovir alone in terms of virologic and biochemical response. r. safadi 1 , q. xie 2 , y.g. chen 3 , y.k. yin 4 , l. wei 5 , s.g. hwang 6 south korea, 7 bnai zion medical center, haifa, israel, 8 beijing friendship hospital, beijing, china, 9 novartis pharma ag, basel , switzerland background: ldt produces greater viral suppression than lam. we investigated whether patients receiving lam can benefit from switching to ldt. methods: hbeag positive and negative persistently viraemic patients (median hbv dna 5.0 (ldt), 5.3 (lam) log 10 copies/ml) and lam treated for 3-12 months, were randomized to either switch to ldt or continue lam. we report the benefit of ldt switch assessed by primary treatment failure (tf, <1 log hbv dna decline) and viral breakthrough (vb, >1 log above nadir). results: 17% (21/122) of the ldt switch and 15% (18/124) continuing lam patients had pre-existing m204 mutations at screening. tf was 5% (ldt) versus 21% (lam, p<0.05). in patients with >24 weeks prior lam treatment, tf was 10% (ldt) versus 41% (lam). 83% ldt tf (5/6) was associated with resistance at screening versus 56% lam tf. in ldt switch with <24 weeks prior lam, no ldt tf occurred versus 12% lam. in hbeag positive, tf occurred in 6% (ldt) versus 29% (lam). among hbeag positive with >24 weeks prior lam treatemtn, vb was 19% (ldt) versus 44% (lam, p<0.05). differences were not significant for hbeag positive with >24 weeks lam or for hbeag negative regardless of duration of prior lam treatment. conclusions: early switch to ldt is associated with better virological outcomes in these patients. persistent viraemia for >6 months on lam treatment is associated with a high risk of tf and vb. for these patients, genotypic analysis is recommended prior to screening. objective: the aim of this study is to evaluate the proper endpoint in the treatment of chronic hepatitis b with antivirals by investigating the viral rebound ratio after one year's nucleosides or (three months) sustained treatment with lamivudine, adefovir, entecavir, or interferon when viral response and seroconversion response have been finished . methods: eag positive chronic hepatitis b naïve patients with alanine aminotransferase (alt) more than 2 uln were assigned to receive 100 mg of lamivudine, 10mg of adeforvir, or 0.5mg of entecavir once daily, respectively. patients in the interferon group were administrated with 5,000,000 iu of 2a interferon on every other day, and the therapeutic duration lasted for another three months after eag-ab seroconversion appeared. hbv dna and eag-ab in the serum were tested during the off-treatment period of 12 months. results: thirty four patients in lamivudine group of 148 cases got eag-ab seroconversion after treatment with 20±5 months of average duration, and the viral rebound ratios in the off -treatment 6 an 12 months follow up period were 20.6 7/34 and 40.1 15/34 , respectively. in adeforvir group were 19 4/21 and 33. 7/21 . in enticavir group were 20 3/15 and 46.7 7/15 . in interferon group was 16.2 6/37 in the off-treatment 6 months follow up period. conclusions: we conclude that eag-ab seroconversion in the treatment of eag positive chronic hepatitis b patients is the goal but not an endpoint of therapy physicians should aim at. to gain everlasting effect, longer duration of treatment may be needed. background: universal hepatitis b(hb) vaccination of hbsag negative people (especially infants) is widely recommended and practised. objective: to assess whether there is robust evidence of protective efficacy to back such practice. methods: this cochrane review included randomised trials identified from six databases through detailed electronic searches. trials comparing hb vaccine versus placebo/another vaccine, in hbsag negative persons were included without any restrictions. the primary outcome was hb infection (developing hbsag or anti-hbc). robustness of evidence was assessed through comparison of available-case analysis versus intention-to-treat(itt) analysis using four different models: (i)assuming unfavourable event for all missing data, (ii)assuming favourable outcome for all missing data, (iii)best-case-scenario and (iv)worst-case-scenario results: twelve trials were eligible among 2964 citations; all were methodologically poor (high risk of bias). data from four trials could be included in meta-analysis. efficacy of vaccination varied with the type of data analysis. available-case analysis suggested efficacy in reducing risk of developing hbsag (rr=0.17;95%ci=0.09-0.31;n=1341) and anti-hbc (rr=0.42;95%ci=0.31-0.57;n=1235). itt analysis results varied depending on the model chosen (table) , but liberal approaches suggested high efficacy, whereas conservative approaches did not. the available evidence on efficacy of hb vaccination in hbsag negative people is not robust; there are serious limitations in quality and quantity. background: open-label rollover study (etv-060) assessed histologic improvement in chb patients on at least 3 years etv therapy. methods: 100% nucleoside-naïve patients and 98% lamivudine (lvd)-refractory patients from etv-053 and etv-052 studies, respectively, entered etv-060 study and received etv at 0.5/1mg for greater than 96 weeks. improvement in knodell necroinflammatory (ni) score and knodell fibrosis score at weeks 48 and 148 were studied. results: at week 148, 95% of nucleoside-na ve patients and 56% of lvd-refractory patients achieved hbv dna <400copies/ml. furthermore, 95% of nucleoside-na ve patients and 93% of lvd-refractory patients had normalized alt levels. mean platelet counts in both naïve and lvd-refractory patients were improved at weeks 48 and 148 compared with baseline. conclusions: naïve and lvd-refractory chb patients showed significant improvement in liver histology after 3 year etv therapy, and improved dna and serum alt levels. results: 84.7 percent of patients were younger than 30 years old, 15.3 percent were older than 30 in this study. 48.0% patients' mothers were hbsag positive. high levels of serum hbv dna were founded in all patients, >10 7 copies/ml were 78.6%. only 5 cases (5.1%) whose liver inflammation grade were g0, the rest patients were mild inflammation, in which g1 were 64 cases (65.3%), g2 were 29 (29.6%); there were 56 patients (57.1%) had no signifecant liver fibrosis, the rest 42 cases (42.9%) had different fibrosis, among those s1 were 23 cases (23.5% , s2 were 14 14.3% , s3 were 5 5.1% , none of patients had cirrhosis. the fibrosis stages of higher alt level were markedly severer than lower alt in patients with normal alt p < 0.01 . conclusions: most of patients with chronic hepatitis b virus in immune tolerant phase present mild inflammation in liver, part of them have already appeared fibrosis, so some patients determinated by clinics are actually not in immune tolerant phase. although alt testing are in the normal range, but the possibility of liver fibrosis is increased in patients with relative higher alt level, so liver pathology should be recommended to judge illness correctly. background/aims: hepatitis b virus infection (hbv) is a global health problem. in bangladesh, 5-7% of people are hbsag positive. this study was carried out to evaluate the efficacy and safety of peginterferon alfa-2a in chronic hepatitis b patients. methods: a total of 60 patients with chronic hepatitis b, 32 (53.3%) were hbeag positive (group a) while 28(46.7%) were hbeag negative (group b) were included in this study after meeting the following criteria: age 18 to 60 years, hbsag positive for more than 6 months, serum hbv-dna was >5 log(10) copies/ml and alt more than two times the upper normal limit. they were given peginterferon alfa-2a (180 microgram once weekly) for 24 weeks and followed for an additional 24 weeks. results: after 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hbvdna levels below 20,000 copies per milliliter was significantly higher in hbeag positive patients (59 percent and 43 percent, respectively) than among hbeag negative patients (45 percent and 33 percent). loss of hepatitis b surface antigen occurred in 3 patients in group a, as compared with 1 patients in the group b (p<0.01). adverse events including pyrexia, fatigue, myalgia, headache and haematologic abnormalities were similar in both groups. conclusions: patients with hbeag positive chronic hepatitis b had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a. background: the effect of hepatitis b vaccination on individuals with isolated anti-hbc in endemic areas is not clear. we investigated the prevalence of individuals positive for anti-hbc only and their antibody response after hepatitis b vaccination in a single healthcare center. methods: the study included 1,812 healthcare workers. after screening for hbsag and anti-hbs, the individuals negative for both hbsag and anti-hbs were examined for anti-hbc and were vaccinated with a recombinant hepatitis b vaccine at 0, 1, and 6 months. the serum anti-hbs level was measured after the vaccination. results: of the subjects, 334 (220 females) were negative for both hbsag and anti-hbs. forty (2.2%) subjects had isolated anti-hbc, including more males (60.0% vs. 30.6%) and older people (45.7±8.2 vs. 37.3±8.5 years), compared with individuals negative for all of the viral markers. the anti-hbs seroconversion rate and anamnestic response in the individuals with isolated anti-hbc after the first vaccine injection were 60% and 27.5%, respectively. in the 294 persons who were negative for all hepatitis b viral markers, the seroconversion rate after the first vaccination was 52.6%. the anti-hbs seroconversion rate did not differ between the isolated anti-hbc positive individuals and those negative for all hepatitis b markers (89.5% vs. 96.6%) after the full course of vaccination. conclusions: serum hbsag and anti-hbs tests are sufficient for screening before hepatitis b vaccination, especially in healthcare workers. objective: to understand the quantity and distribution of cd83 + mature dendritic cells in patients with hepatitis b virus in immune tolerant phase. methods: there were 30 immune tolerant phase patients with hepatitis b virus infection (fibrosis stages were s0), 10 immune clerance phase patients, 10 non-active status patients and 5 healthy controls involved in our research. the quantity and distribution of cd83 + mature dendritic cells in liver were determined by immunohistochemical staining. result: the liver inflammation grades were between g1-g2 in patients who in inmmune tolerant phase and non-active status, moreover, patients in immune clerance phase were between g2-g4. there were a small amount of cd83 + dendritic cells in healthy liver tissue, scattered in portal areas and hepatic lobules. the quantity and distribution of cd83 + dendritic cells in patients who in inmmune tolerant phase and non-active status were similar to the healthy, and the quantity were no difference among them p 0.05 .the number of cd83 + cells in patients of immune clerance phase was significant increased compared with other groups, there were differences among them p 0.05 , the cd83 + cells mainly distributed in portal areas infiltrated with inflammatory cells and hepatic lobules with inflammatory necrosis. conclusion: cd83 + mature dendritic cells are involved in liver immune response in patients of inmmune clerance phase, is likely to related to hepatitis b virus clearance. lack sufficient mature dendritic cells may be one of the mechanisms of immune tolerance. background: local hospitals provide obstetric services including antenatal care to women normally living in the mainland china, whose prevalence of hepatitis b carrier is unknown. objectives: compare prevalence of hbv carrier of pregnant women from the mainland china with local counterparts and discuss the implications of results. materials and methods: antenatal serological results were retrieved from corporate laboratory information system databases. pregnant women from the mainland china were identified by a specific set of temporary-allocated identity number during january 2007 -october 2008. results: 7491 pregnant local residents and 1397 pregnant women from the mainland china underwent antenatal serological tests for hepatitis b surface antigen. positive hepatitis b surface antigen results were more frequent in pregnant women from the mainland china (12.7%) than in local pregnant women (8.17%) (p<0.001). discussion: because infected pregnant women can transmit the hepatitis b virus to the infant at delivery, specific management could entail maternal medication, injection of hepatitis b immune globulin to the infant at birth and immunization later on. however, early repatriation to the mainland china, which is common, will make completion of immunization program difficult. these babies will be at a higher risk to be infected by hbv, particularly when breast-fed by hbv carriers. their return to hong kong later will dilute the effects of local immunization program. the volume of work derived from the provision of obstetric services to women from the mainland chinese is larger with regard to medication, counseling and immunization for babies born to hbv carriers. immunosuppressive or anticancer therapy k. hirano 1 , t. kodani 1 , s. sato 1 , y. narita 1 , t. kikuchi 1 , t. genda 1 , k. iijima 1 , k. ogawa 1 , t. ichida 1 1 background/aim: we compared the prevention of hbv reactivation in (hbsag)-positive patients with hbsag-negative patients who were positive for antibody to (anti-hbc) and/or (anti-hbs) undergoing immunosuppressive, anticancer or molecular target therapy. methods: from sep 2004 to nov 2008 hbsag-positive patients and 22 anti-hbc and/or anti-hbs-positive patients were enrolled in this study. we compared with 2 groups about background disease, age, blood examination, and nucleoside analogues. results: in hbsag-positive patients mean age were 56.6±10.2 years old, median ast levels were 30 (18-706) iu/l, and median alt levels were 36 (13-544) iu/l for 8 (47%) haematological disease and 9 (53%) collagenosis disease. in anti-hbc and/or anti-hbs-positive patients mean age were 71.3±9.6 years old, median ast levels were 20 (9-106) iu/l, median alt levels were 16.5 (5-247) iu/l for 17 (77%) haematological disease and 5 (23%) collagenosis disease. serum hbv-dna levels >5.0 log copies/ml were 8 (47%), 2.6~5.0 were 7 (41%), <2.6 were 2 (12%) in hbsag-positive patients, and serum hbv-dna levels <2.6 were all cases in anti-hbc and/or anti-hbs-positive patients. 14 (82%) of hbsag-positive patints received nucleoside analogues (7 lam and 7 etv), and 12 (55%) of anti-hbc and/or anti-hbs-positive patients received nucleoside analogues (8 lam and 4 etv). mean duration of treatment for 14.2 months in hbsag-positive patients, and for 4.1 months in anti-hbc and/or anti-hbs-positive patients, the resistance virus occurred to 3 (75%) of 4 hbsag-positive patients treated with lam for collagenosis disease more than two years. conclusion: when hb carriers of collagenaous disease undergoing immunosuppressive therapy required the nucleoside analogues more than two years, we recommended treatment to prevent hbv reactivation with etv. background: adefovir dipivoxil is used for the initial treatment of chronic hepatitis b or rescue treatment of lamivudine-resistant chronic hepatitis b, and exhibits excellent antiviral activity. however, the presence of resistance to adefovir dipivoxil was more frequently in lamivudine-resistant chronic hepatitis b patients than in lamivudine-naïve patients during adefovir dipivoxil monotherapy. but the rate of adefovir resistance related mutations is little known in lamivudine-resistant patients before adefovir dipivoxil treatment. the aim of this study was to investigate the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus in lamivudine-resistant patients not treated with adefovir dipivoxil. methods: the existence of adefovir resistance-related mutations was examined in 240 lamivudine-resistant chronic hepatitis b patients with breakthrough hepatitis and 100 antiviral-naïve chronic hepatitis b patients. both polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) and directly sequencing of pcr product were used to detect resistant viruses. results: rta181t mutants were detected in only two sera of lamivudine-resistant patients, while none in the antiviral-naïve chronic hepatitis b patients. there was no rtn236t detected in the two groups. conclusion: our results suggest that the rta181 mutant virus were present in a few lamivudine-resistant chronic hepatitis b patients before they have been treated with adefovir dipivoxil, but the rtn236t mutant was not detected in any of the two groups. the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus was low in such lamivudine-resistant patients before adefovir dipivoxil treatment. objective: in this study, we tried to detect and identify the special protein of hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. to find new opinion on the developing of chronic liver disease. methods: the sera of health adult, hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were respectively detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms). the arrays of every group were analysised by clustering analysis and to establish disease predictive model. then the sample was eluted with different ph tris, trypsinization on-chip, mass determination and peptide database comparison. results: according cm10 chip we find 18 protein with obviously deviation (p<0.05) among hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. clustering analysis for the data from seldi-tof-ms confirmed 42 differentially expressed proteins. then we developed disease predictive mathematic models ( decision tree model, dt model ) with average validity up to 73.9 . the 5805 da protein peak was identified to be chondroitin sulfate synthase 2 (chss2), which is a potential molecule involved in the pathologic process and a potential serum marker for the hbv related hepatic diseases as well. conclusions: our results suggest that seldi-tof-ms is a usefull technique for differential expressed proteins screening and analysis in hbv related chronic liver disease. chss2 may be useful during the developing of hbv related chronic liver disease. backgound: clevudine is a new nucleoside analogue with potent antiviral activity in chronic hepatitis b patients. however, the efficacy and safety of clevudine in cirrhotic patients are not well recognized. this study was conducted to evaluate the early virologic and biochemical response rate as well as safety of clevudine in cirrhotic patients with chronic hbv infection. methods: 46 patients with chronic hbv infection who visited korea university ansan hospital and guro hospital between may 2007 and may 2008 were included. 24 patients had chronic hepatitis b (group a) and 22 had liver cirrhosis (group b). early virologic response was defined as hbv dna less than 200 iu/ml at week 12. early biochemical response was defined to be normalization of alt (<45 iu/l) at week 12. result: pretreatment hbv dna levels were higher in group a compared with group b (8.06 log iu/ml vs 7.09 log iu/ml, p=0.06). pretreatment alt levels were not significantly different between the two groups (166 iu/l vs 139 iu/l, p=0.725). the rate of early virologic response was significantly higher in group b compared with groups a (72.7% vs 33%, p=0.01). the rate of early biochemical response were not significantly different in both groups (75% vs 72.7%, p=0.725). conclusion: clevudine is considered to be safe and effective in cirrhotic patients with chronic hbv infection as well as chronic hepatitis b patients. long term safety and efficacy need to be evaluated in the future. objective: the aim of this study was to evaluate the role of nucleos(t)ide analogues against hbv reactivation in immunosuppression. methods: non-active hbsag carriers suffering from cancer, autoimmune diseases and needing the treatment of immunosuppressants or cytotoxic chemotherapy were enrolled in the study. the outpatients or in-patients from april 2007 to july 2008 were enrolled. the nucleos(t)ide analogues were used in cancer patients 1-2 weeks before chemotherapy, and the duration lasted 6-12 months according to patients' compliance after completion of chemotherapy. patients with other diseases used nucleos(t)ide analogues in 1-3 months before using glucocorticoids or other immunosuppressive agents, and continued to use for 6-12 months after accomplishing the course of immunosuppressant treatment. the characheristics and clinical manifestations about hbv reactivation were investigated. results: of the thirty two patients in prospective group, twenty two patients suffered from cancer, eight patients suffered from idiopathic thrombocytopenic purpura, two patients suffered from chronic nephritis. the amount of hbv dna was detected in the first, third, sixth and 12th month after the use of nucleos(t)ide analogues. after chemotherapy or immunosuppressant treatment, only 9.4% (3 / 32) of them suffered from hbv reactivation, which presented with hbv dna positive and abnormal liver function. conclusion: non-active hbsag carriers would appear potential incidence of hbv reactivation during use of chemotherapy or immunosuppressant. nucleos(t)ide analogues could be used in early phase as prophylaxis for reactivation of hepatitis b in immunosuppression and to improve clinical prognosis. background: hbv therapies are evolving toward combination antivirals. this study evaluated the combination of clevudine (clv), a potent nucleoside analog, with tenofovir dipivoxil (tdf). methods: a phase i, single-arm, multi-dose study in 18 healthy adult volunteers to evaluate pharmacokinetic and safety interactions between clv and tdf. subjects received 21 days of clv 30mg followed by 7 days of clv 30mg +tdf 300mg. pk profiles were obtained on days 1, 21 and 28. clv auc and cmax were compared on days 21 and 28. day 28 tenofovir pk was compared to historical data. safety assessments were conducted throughout. results: 18 subjects were enrolled (13m/5f);17 completed the study. the mean (range) age was 37y (22-49) and body mass index (kg/m2) was 27.6 (22.9-31.9). 22 aes were reported by 6 subjects, with 10 aes reported during clv-only dosing and 12 aes reported during clv+tdf dosing. aes included nausea (2) and pharyngolaryngeal pain (2). the majority of the aes were mild. there were no clinically significant changes in ecgs or laboratory parameters. comparisons of clv auc and cmax on day 21 and 28 revealed no significant impact of tdf upon the plasma clv exposure (d28/d21 auc ratio=0.98, d28/d21cmax ratio=0.89). there is no significant effect of clv on tenofovir when comparing auc and cmax of tdf to historical values. conclusion: safety and pharmacokinetic results demonstrate that clv and tdf may be safely co-administered, supporting the further study of this drug combination for the treatment of chronic hbv infection. s. kuznecovs 1,2 , i. kuznecovs 1 , k. jegina 2 , g. kuznecova 1 1 background: dolichyl (dol), the main lipid intermediator of dolichyl phosphate cycle (dpc) has been reported to be elevated in urine of patients with multidrug resistance in cancer. drug resistance poses a major threat to nucleoside analogue-based therapies for chronic hbv infection. methods: with focus on a risk predictor for susceptibility to the development of hbv drug resistance the present study was carried out to estimate urinary levels of dol in chronic hbv infection. the samples obtained every week before and during the course of treatment from 42 patients with hbv. the occurrence of exacerbations of chronic hbv were registered for 2 years. dol in urine was assayed by hplc method. results: the normal amounts of dol in healthy persons urine (n=1500) are 6,0 + 10,0 mkg/mmol creatine. during the period of observation 36 (86%) of patients treated with nucleoside analogue-based therapies were diagnosed with exacerbations due to resistance of hepatitis b virus to antiviral drugs. from this group of hbv patients 35 (98%) have had elevated urinal dol excreation (45,8±5,2 g/ml vs . 8,2±1,9 g/ml, p<0.0001) in more than 3 months of observation. conclusion: there is a reason to suggest that elevated urinal dol detected in patients with exacerbations during hbv treatment may evidence of possible defect of host mechanism of drug resistance development to nucleoside analogue-based therapies. the interest drawn to the employment of dol as a predictor for exacerbation of chronic hbv is explained by the role of dpc in p-glycoprotein regulation in human hepatocytes. background: a significant proportion chb patients treated with adv have a suboptimal response, increasing the risk of disease progression and development of resistance. we report clinical results from patients who either failed or relapsed following adv therapy and were subsequently switched to etv. methods: study etv-079 was a randomized, open-label study comparing antiviral efficacy of etv (0.5mg/day) vs adv (10mg/day) in nucleoside-naïve hbeag-positive patients. after up to 96 weeks of treatment in etv-079, 24 patients treated with adv (13 suboptimal responders) rolled over into study etv-901 (1.0mg/day). hbv dna viral suppression and safety was evaluated during 48 weeks of etv treatment. results: at entry to etv-901, the median hbv dna was 5.72log10 copies/ml. median exposure to etv (1.0mg) in etv-901 was 46 weeks and 18 patients currently remain on study therapy. at week 24, the mean reduction in hbv dna was 4.5log10 copies/ml and 8/16 (50%) reached hbv dna levels <300 copies/ml. nine patients have achieved week 48 and all have achieved hbv dna <10 4 copies/ml and 8/9(89%) had hbv dna levels <300 copies/ml. no patients experienced virologic breakthrough on etv. the safety profile of etv in adv-treated patients remained consistent with the previously reported experience. conclusions: the majority of patients who were suboptimal responders or virologic rebounders following adv treatment in study etv-079, experienced rapid reductions in hbv dna levels when switched to etv. hbv dna levels continued to decline to undetectable levels with 48 weeks of etv treatment. y. li 1,2 , t. han 1 1 tianjin third central hospital, 2 aim:to quantify hepatitis b virus (hbv) total dna and covalently closed circular dna (cccdna) in liver biopsies and sera which from chronic hepatitis b(chb) liver cirrhosis of hepatitis b(lc) and hepatitis b relevance hepatocellular carcinoma(hcc) patients, and analysis hbv replication under the circumstances of different diseases. methods:total hbv dna and cccdna in serum and liver biopsy samples were measured in 21 chb 23 lc and 25hcc patients by the real-time pcr assay. results: the levels of total hbv dna in serum,intrahepatic total hbv dna, intrahepatic cccdna, as well as the proportion of intrahepatic cccdna in total hbv dna decreased progressively in chb,lc and hcc ,moreover chb had significantly higher levels of total hbv dna in serum and liver biopsy samples than lc (log [total serum hbv dna] p = 0.024;log [total intrahepatic hbv dna] p = 0.034); chb and lc had significantly higher levels of intrahepatic cccdna and the proportion of intrahepatic cccdna in total hbv dna than hcc(p <0.01); cccdna couldn't be detect in all patients'serum. in chb ,the levels of serum's total hbv dna,intrahepatic total hbv dna and cccdna in hbeag-positive group had significantly higher than the hbeag-negative group(p <0.01) ,but in lc only intrahepatic total hbv dna had statistical difference between hbeag-positive and negative group (p=0.026) , no statistical difference between hbeag-positive and negative group in hcc. conclusions: the replication activity of hepatitis b virus in chb,lc were higher than hcc, hbv reproduction reduced significantly in hcc. duplication of hbv in lc was lower than chb but had no statistical difference. the levels of hbv reproduce in hbeag-positive group was higher than hbeag-negative group of all three desease. background: clevudine is a pyrimidine analogue with potent and sustained antiviral activity against hbv in the 24 week therapy. the present study assessed the efficacy and viral resistance of 48 week clevudine therapy in patients with chronic hepatitis b. method: a total of 42 patients (26 hbeag positive and 16 hbeag negative) who were received clevudine 30 mg once daily for 48 weeks were included in this analysis. serum hbv dna was quantified by real time pcr assay. result: at week 48, median reductions of serum hbv dna from baseline were 4.98 log10 iu/ml (5.00 log10 iu/ml for hbeag positive and 4.95 log10 iu/ml for hbeag negative) and 76.2% of patients showed undetectable serum hbv dna (<60 iu/ml) (65.4% for hbeag positive and 93.8% for hbeag negative). the normalization of alt levels (<35 iu/l) was achieved in 81.0% (88.5% for hbeag positive and 68.8% for hbeag negative). 15.4% of hbeag positive patients showed hbeag loss or seroconversion. hbv dna negativity at week 48 was associated with hbeag negativity (p =0.037), hbv dna <2,000 iu/ml at weeks 4 and 24 (p =0.019 and 0.001, respectively). two hbeag positive patients showed viral breakthrough with m204i mutation during 48 week. conclusion: clevudine therapy in patients with chronic hepatitis b showed potent virologic responses at week 48, especially in those with hbeag negativity and complete early virologic response (hbv dna <2,000 iu/ml at weeks 4 and 24). but clevudine resistance can occur in hbeag positive patients. background: serum alanine aminotransferase (alt) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. current standards for "normal" alt level were defined by using populations that included persons with subclinical liver disease. there is no study regarding normal level of alt and its modulating factors in healthy thai people. objective: to definitions of normal ranges for serum alt level in thai people. design: prospective observational study setting: phramongkutklao hospital and army institute of pathology pramongkutklao medical center(a.i.p.), bangkok, thailand participants: 200 persons who were first-time blood donors from august through december 2007 were negative for anti-hepatitis c virus(hcv), negative hbsag(hbv), and had no contraindications to donation. measurements: univariate and multivariate analyses examined associations between clinical and laboratory factors and alt levels. normal ranges for alt were computed from the population at lowest risk for liver disease. results: serum alt activity was independently related to body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. normal ranges for serum alt level in thai people upper limits for men 26 u/l and for women 21.67 u/l. conclusion: in men serum alt is strongly associated with body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. the normal range of alt should be defined for male and female separately. background: the open-label rollover study etv-060 was conducted after etv phase ii clinical study etv-047 for nucleoside-na ve adult chb patients in japan. in this analysis, we report etv long-term efficacy and safety in patients who were switched from 24-week lvd treatment to etv therapy. methods: ninety-seven percent (33/34) of lvd-treated patients from etv-047 were rolled over into etv-060 treated with 0.5mg of etv. thirty patients completed 96 weeks of etv therapy and were evaluated for hbv dna level, alt normalization, hbeag seroconversion, resistance and safety. results: comparing to baseline before switching to etv, after 96 week of etv treatment, the proportion of patients achieving undetectable hbv dna (<400 copies/ml) increased from 21% to 90%. increases were also observed for alt normalization (81% to 90%) and hbeag seroconversion (10% to 19%). three patients had detectable hbv dna at week 96 after etv treatment and samples from two were tested for resistance. neither demonstrated substitutions associated with etv or lvd resistance. five patients had grade 3-4 laboratory abnormalities, including increased ast/alt and increased lipase levels. conclusions: switching patients from lvd therapy to etv resulted in increased proportions of patients achieving hbv dna suppression, alt normalization and hbeag seroconversion, with no evidence of etv resistance. etv was well tolerated during treatment. backgrounds/aims: hepatitis b virus (hbv) reactivation in patients undergoing chemotherapy hampers an adequate administration of cytotoxic agents and even causes an treatment failure. prophylaxis failure occasionally results from viral breakthrough or withdrawal flare. the aims of this study were to identify predictors of anti-viral prophylaxis failure and to determine the optimal strategy for anti-viral prophylaxis. methods: cancer patients with positive hbsag who underwent cytotoxic chemotherapy in a tertiary medical center from january 2005 to june 2008 were included. prophylactic lamivudine was started with initiation of chemotherapy, continued during the chemotherapy, and discontinued within 6 months after the completion of chemotherapy. all patients were followed up even after withdrawal of lamivudine. results: 115 patients were enrolled. twenty-nine patients (23.7%) had hematologic malignancies and eighty-six (76.3%) had solid tumors. median follow-up duration was 15.9 months and twenty-six patients (22.6%) experienced the prophylaxis failure: viral breakthrough (11 patients, 9.6%), withdrawal flare (15 patients, 13.0%). ymdd mutation developed in four patients. withdrawal flare occurred at a median 2.5 months after discontinuation of lamivudine. using log-rank test and cox multi-variate analysis, our results showed that the type of underlying malignancies (hr 2.46, 95% ci, 1.11-5.43; p=0.026) and baseline hbv dna titer (hr 3.91, 95% ci, 1.63-9.39; p=0.002) were significant independent risk factors for antiviral prophylaxis failure. conclusion: cancer patients with high viral load of hbv and hematologic malignancies may need more prolonged and potent anti-viral prophylaxis to avoid interruption or delay of chemotherapy. back ground: the usefulness of hepatitis b virus (hbv) dna and hbv core-related antigen (hbcrag) was evaluated for timing hepatitis flare after viral breakthrough or withdrawal of antiviral treatment in chronic hepatitis b. method: a total of 32 events of hbv reactivation due to withdrawal of lamivudine (lam) or emergence of mutants resistant to lam or adefovir dipivoxil (adv) virus were analyzed in 25 patients with chronic hepatitis b (20 men, median age 56 years [range: 30-66]). they were followed monthly for serum alt, hbv dna and hbcrag before, during and after the treatment. result: high alt flare (alt > 100 iu/ml) after viral breakthrough or withdrawal was related with baseline hbeag positivity (p=0.016), hbcrag level at hbv dna elevation (p=0.038) and duration from hbcrag elevation to salvage therapy (p=0.044). in multivariate analysis, hbcrag > 4.0 log u/ml (or 22.9, 95%c.i. 1.7-304.1, p=0.018) and salvage therapy after 8 weeks from hbcrag elevation (or 10.6, 95%c.i. 1.6-71.4, p=0 .015) were selected as related factor with high alt flare. after appearance of resistant-virus or withdrawal of lam or adv therapy, hbv dna re-elevated without increase of hbcrag, then hbcrag elevated with hbv dna. re-elevations of alt occurred in 27 of the 32 (84%) events. in 23 of the 27 (85%) events, alt re-elevated within 8 weeks from the start of hbcrag increase. conclusion: hbcrag was useful for timing the re-elevation of alt after hbv dna re-elevation induced by drug-resistant virus or withdrawal of lam or adv therapy. background and objectives: the aim of the study was to observe the efficacy of a patient's therapy for switching lamivudine + placebo to adefovir dipivoxil (adv), and modeling the viral dynamics. methods: the studied object was a chinese chb patient with lamivudine mutation. used the lvd + placebo for 12 weeks' therapy. then switched adv for another 95 weeks. after that stopping the treatment and following up for 24 weeks. based on our modified basic virus infection model, we introduce a personalized model consisting of four variables: x, y, v, e , representing uninfected cells, infected cells, free virus, and ctl cells, respectively. results: selected the model parameters, the simulation data of hbv dnas of our model are good in agreement with the clinical ones. observe that after 24 weeks' treatment cessation, the benefit (hbv dna < 250 copies/ml) for suppressing hbv replication can still be kept. numerical simulation show that if the patient's immune functions can be kept after therapy stops, it needs 9 years to replace all infected cells by normal ones. conclusion: for lvd mutation patients, lvd+ placebo to avd therapy scheme may help patients to suppressing hbv replication. further researches are promising. acknowledgments: this work is jointly supported by the nnsf of china background: g-a-1896 pre-core mutants (p-c-mt) cause hbeag-negative chronic hepatitis (chb) in genotype d infected mediterranean adults. we studied their emergence during chronic hbv infection in children. methods: eighty consecutive hbsag carriers (50/30 males/females, age 11y, range 0.2-17y) with vertical (66%) or intra-familial (16%) transmissions were followed-up for 12.5y (range 1-25 y). hbv genotype and hbeag status were determined at the admission, hbeag/anti-hbe every 2 years thereafter. during the follow-up, hbv-dna was measured in 185 sera (1-7 sera/patient) (cobas-amplicor, roche); p-c populations were characterized by direct sequencing (ds), by oligo-hybridization (oha) and allele-specific-pcr (as-pcr) with 30%, 10% and 0.1% sensitivities, respectively. results: seven children were genotype a and 73 d; 70 (87.5%) were hbeag-positive. fifty-five (78.6%) underwent hbeag/anti-hbe seroconversion (median age 13y, range 1.3-27y). baseline hbv-dna (cp/ml) was lower in seroconverters (7.9+1.9 vs 9,4+0,6, anova p=0.012). ds/oha p-c-mt were 4.2% at the admission and 45.8% after follow-up; as-pcr p-c-mt 33.3% and 50% respectively. after seroconversion 47 (85.5%) became inactive carriers, 6 (14.5%) lost hbsag (5 genotype d/p-c-wt); 8 p-c-mt had chb. hbv-dna (cp/ml) was lower in 31 p-c-wt than in 14 p-c-mt inactive carriers (3.42+1.05 vs 4.58+0.91; anova p=0.007). conclusions: in genotype-d infected children p-c-mt is selected progressively after hbeag/anti-hbe seroconversion to become predominant in hbeag-negative chb. early and efficacious immune control of hbv replication avoids p-c-mt selection and leads to hbsag loss. , a, , d, k, u, m, n , k1 ,k2 and k3. here k1, k2, and k3 are the rate of ctl production and dead, killing virus, respectively. results: the patients with hbv dna levels less than 1000 copies/ml were reported in 17.5% (7/40). a patient whose hbv dna levels were higher than 40000 copies/ml can keep treatment benefits even stopping the therapy for over ten weeks. the simulation data of our model are in agreement with the patient's hbv dna data. our simulation also shows that it needs to spend about 18 years for clearing all infected cells. conclusion: the simulation result implies that some chinese patients may need long term's therapy to clear all infected cells. patients' ctls assays are needed to confirm the effectiveness of the personalized modeling, and help doctors to decide whether stop the drug treatment even patients' hbv dnas are higher than undetectable levels. background/aim: recent reports have shown that programmed death 1(pd-1) expression is associated with t cell exhaustion and persistent viral infection. we studied longitudinally 28 chronic hepatitis b(chb) patients undergoing treatment with nucleos(t)ide analogues or pegylated interferon-(peg-ifn-) in 12-16 weeks to determine the relationship between pd-1 expression levels on t cells and early reduction of viral load induced by treatment. methods: our investigations were focused on three points: baseline (time point 1, t1), treatment weeks 4-6(time point 2, t2) and treatment weeks 12-16(time point 3, t3). pd-1 expression on total cd4 and cd8 t cells in chb patients during antiviral therapy was detected by flow cytometry. serum hepatitis b virus (hbv)-dna load was measured by real time polymerase chain reaction. results: between t1 and t2, pd-1 expression on total cd8 (p<0.01) and cd4 t cells (p<0.01) dropped concurrently with treatment-induced hbv-dna decline(p<0.01). between t2 and t3, however, only the hbv-dna levels reduced significantly (p<0.01). conclusion: early suppression of hbv replication induced by antiviral treatment results in a significant decrease in pd-1 expression on total cd8 and cd4 t cells in chb patients. c. zhao 1,2 , w. zhang 2,3 , x.c. tian 1 , c.y. fang 2,3 , h.j. lu 2,3 , p.y. yang 2,3 , y.m. wen 1,2 background/aims: hepatitis b virus (hbv) is still regarded as one of the major causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. the interactions between hepatitis b surface antigen (hbsag) and host cells still remain largely unknown and need to be explored in detail. methods: differential protein expression profiles of hepg2-s-g2( stably expressing hbsag cell line) and hepg2-neo-f4 ( control cell line) were compared using two dimensional gel based differential proteomic approach. cell proliferation assay and survival assay were used for further studies on the candidate protein. results: compared with the control down regulation of 44 proteins and up regulation of 38 proteins were found in hepg2-s-g2 cell. all these regulated proteins were identified by ms/ms and could be fell into several categories including metabolism-associated, immune-response-related, protein modification, signal transduction and others. among them, a group of proteins in putative pathways associated with apoptosis were found out and discussed, including glucose-regulated protein 78kd (grp78/bip), heterogeneous nuclear ribonucleoprotein (hnrnp), far upstream element-binding protein (fusebp), rho gdp dissociation inhibitor (gdi), cystatin b and some scaffold proteins. grp78, an important chaperone protein involved in multiple functions in host cells, was consistently decreased in hepg2-s-g2 and in huh7 cell transiently transfected with hbsag expression plasmid. decreased crp78 inducing by hbsag or blockage of rnai consistently led to the less resistance to staurosporine-induced cell death. conclusions: these results revealed a possible pathogenesis induced by hbsag via grp78. background/aim: to evaluate the efficacy of adefovir dipivoxil alone and in combination with lamivudine in treating patients with lamivudine-refractory hbeag-positive chronic hepatitis b. methods: eighty-five hbeag-positive patients who had received lamivudine treatment for various periods and had a lamivudine-resistant liver function abnormality, documented ymdd mutations and persistent viremia were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. the primary efficacy measure was virological response. the secondary efficacy measure was serological response (hbeag loss rate and hbeag seroconversion rate) and alt normalization rate. results: after 24 weeks of therapy, mean reduction of hbv-dna level, the percentage of patients with hbv-dna lower than 5 log10 copies/ml and the percentage of patients with hbv-dna level decrease of more than 2 log10 copies/ml in patients of adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups were significantly higher than those in patients of lamivudine group (2.58, 2.21 log copies/ml vs. 1.02 log copies/ml, 92.3%, 88.5% vs. 33.6%, 76.9%, 75.8% vs. 28.8%; p < 0.001, respectively). at the end of 52 weeks, mean reduction from baseline in serum hbv-dna level at was 0.08, 4.25, and 4.12 log10 copies/ml in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. alt normalization rates were significant hihger in adefovir dipivoxil/lamivudine and adefovir dipivoxil recipients than those in lamivudine recipients (62%, 55% vs. 8%, p < 0.001, respectively). a similar pattern was observed in hbeag loss among three groups. conclusions: adefovir dipivoxil is an effective treatment option for patients with lamivudine-refractory hbeag-positive chronic hepatitis b. aim: to find the prevalence of hbv virologic flare as defined by hbv dna viral load of > 2,000 iu/ml in inactive chronic hepatitis b (hbv dna less than 2,000 iu/ml), hbeag-negative patients who have not received any treatment and to identify if there are any predictors that can predict virologic flare. methods: we retrospectively analyzed medical records of the patients who have attended hepatitis clinic, siriraj hospital from january 1, 2002 to february 28, 2008 . the patients were eligible if they were naïve to any treatment and hbv dna less than 2000 iu/ml at entry. co-infection with hiv and/or hepatitis c virus were excluded. hbv dna measurement determine by roche amplicor ® (detection limit of 60 iu/ml). hbv virologic flare was defined as hbv dna more than 2000 iu/ml during follow up period. result: there were 84 patients with mean follow up time was 598 days with annual prevalence of hbv virologic flare of 12.8, 4.8, and 4.2% for the first, second and third year of follow up, respectively. initial hbv dna level was the only predictor that can predict reactivation. no patients with hbv dna at entry below detection limit developed flare and the patients with hbv dna above 740 iu/ml had 22 times higher chance to develop flare during follow up. conclusion: hbv dna flare is not uncommon in inactive chronic hepatitis b patients. most of the virologic flares occur in the first year. the most important predictor or virologic flare is higher hbv dna at beginning. background: multi-drug resistant hbv developed with multiple antiviral agents. there existed difficulty in dealing with multi-drug resistant hbv. methods: retrospective analysis of 23 consecutive patients who exhibited chronic hepatitis b associated with multiple drug-resistant mutations to lamivudine and adefovir during antiviral treatment. multiple drug-resistant mutations were detected in those patients by dna direct sequencing. result: before multiple drug-resistant hbv emerged, 20 patients accept sequential antiviral therapy, 3 patients accept na monotherapies. there were 16 cases of rta181t/v rtm204v/i mutation, 2 cases of m204v/i +n236t mutation, 4 cases of a181t/v+m204v/i +n236t mutation, 1 case of l180m+a181t/v mutation. 14 cases received rescue therapy of interfronand hbv dna level of 8 cases decreased; other 9 cases received combination treatment and hbv dna level of 4 cases decreased. conclusion: the main reason of multiple drug-resistant mutations was sequential antiviral therapy. another reason may be pre-exist drug-resistant mutation before nucleoside or nucleotide analogue treatment. de novo combination of antiviral agents should be recommended. combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant hbv. interfron may be one choice for hbv of multiple drug-resistant mutations. background: to furnish basis for an accurate evaluation of hbeag negative chronic hepatitis b (e chb), the present study studies the clinical features and hepatic pathology, and analyzes the relation between the data and the grade and stage of hepatic pathology in e chb. methods: a study is performed in 120 chinese e chb patients (106 men and 14 women; mean age sd, 34.3 9.4 years). the relationship between the clinical features and the grade and stage of hepatic pathology was analyzed by spearman's rank correlation test or kruskal-wallis test by applying stata 7.0 software. result: negative correlation is shown between the grade and leucocyte count methods: 80 patients with hbeag-positive compensated chb with hbv dna >6 log10 copies/ml, serum alt 2 x uln were divided two groups:one treated with telbivudine and the other treated with entecavir. results: baseline characteristics were well balanced between treatment groups. at 12wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 50 52.5 (p 0.05), the rates of hbeag negative were 10 0 respectively, the rates of hbeag seroconversion were 20 5 respectively; at 24wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 80 70 (p 0.05), the total rates of hbeag negative were 20 15 respectively, the total rates of hbeag seroconversion were 27.5 17.5 respectively(p 0.05).no adverse reactions were found in both groups conclusion: there was no significant difference in hbv dna undetectable rates between two nucleotide analogs in short-term (24 weeks).the telbivudine group has better effect in hbeag seroconversion rate than the entecavir group in early stage,but no statistical significance. j.w. song 1,2 , z. xin 1 , j.x. tang 1 , l. yao 1 , b. wu 1 1 sun yat-sen university, 2 zhuhai sinochips biosci. co.,ltd., china background: to develop a equipment free, and can be widely used in clinical practice biosensor-based microarray for hepatitis b virus pre-c/bcp mutation assay. methods: a thin film optical biosensor were applied for amplification the microarray signal in situ. and hbv sites 1762, 1764,1768,1770 and 1896 were selected as the targets and the microarray were be fabricated. the 5 mutated plasmids contained 1762, 1764,1768,1770 and 1896 sites and 30 hbv sera were be tested in our study and all the plasmids and sera pcr products were be assayed by really time pcr and sequencing. results: the biosensor based microarray signal can be easily record by digital camera or even by the naked eyes and the detection signal for positive discriminated from negative were sharply contrasted as whole yes or no and it looks be significantly superior to classical microarray technique; 2. the sensitivity of the detection limitation of sera hbv load is 2x10e3 copies/ml with 95% reproducibility. the concordance index of 50 times negative and mutated plasmids were 98%(kappa=0.932). 3. 30 sera samples of hbv>10e4 load and 20 sera of hbv negative tested by pcr fragment sequencing were showing very good agreement between sequencing with our biosensor based microarray and the concordance index kappa was 0.7619. conclusion: our biosensor-based microarray for pre-c/bcp mutation assay were a both sensitive and accurate method. and its advantages of equipment free, sharply contracted signal of positive vs negative and easily be perform in testing were make it be a promised assay for clinical application. objective: to investigate the frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b, we assayed the differences among hbsag-positive and healthy subjects by flow cytometry. the results might offer some experimental evidence to explain the high rates of hbv persistent infection in vertical transmission of hbv from hbv-infected mothers. methods: 12 newborns born from hbsag positive mothers were recruited , 10 healthy subjects being used as a control group. the cord blood and peripheral blood of mothers were collected respectively .frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b were analyzed by flow cytometric analysis. result: the number of cd4 + cd25 + regulatory t cells/pbmcs in the cord blood of newborns born from hbsag positive mothers 4.49 ±1.18 significantly exceeded that in normal controls 2.26 ±0.97 ,p 0.001 ;and newborns born from hbsag positive mothers presented a much higher fraction of cord blood cd4 + cd25 + /cd4 + 9.62 ±2.34 than those in normal controls 7.93 ±1.43 ,p p=0.0025 p 0.05 . conclusions: the results indicate that the proportion of cd4 + cd25 + regulatory t cells in hbsag positive mother cord blood was higher than those of healthy cord blood. objective: to study the clinical features of chronic severe hepatitis b with negative hepatitis b e-antigen (hbeag) and positive hepatitis b e-antigen (hbeag) methods: a total of 353 in-patients with chronic severe hepatitis b were recruited into the study and divided into two groups according to the hbeag status. the serological chemistry data, hepatitis b virus (hbv) dna quantification data were detected, and morbility of cirrhosis, its complications and prognosis were also studied. results: of the 353 in-patients, 236(66.8%) patients were hbeag-negative. 117(33.2%) patients were hbeag-positive. the ratio of hbeag-positive patients was significantly higher than that of hbeag-positive patients (p<0.05).the average age of hbeag-negative patients was older than that of hbeag-positive patients (p=0.048). the serum hbv dna level of hbeag-negative patients was significantly lower than that of hbeag-positive patients (5.49±2.02) vs(6.64±1.41) log copies/ml (p<0.01).the ratio of patients who had a serum hbv dna level less than 5log copies/ml in hbeag-negative patients was significantly higher than that in hbeag-positive patients (41.8% vs 11.9% ,p=0.000). there was no significant difference in serological chemistry data, morbility of cirrhosis and its complications on infections, ascites, hepatoencephalopathy, gastrointestinal hemorrhage, as well as prognosis of the patients between those two groups. conclusions: the study suggested that serological chemistry data, morbility of complications and prognosis of the disease of hbeag-negative patients mimics that of hbeag-positive patients. the hbeag-negative patients had a higher level of age, while a lower level of serum hbv dna. to reduce the incidence of liver failure, more frequent monitoring and earlier antiviral therapy prone to be reasonable for chronic hepatitis b patients with negative hepatitis b e-antigen. background: the emergence of lam-resistant virus greatly limits the efficacy of therapy and induces the liver injury. the aims of this study were to assess the related factors of lam-resistant mutation in hbeag positive chb patients. methods: thirty-five patients carrying lam-resistant with hbeag positive were enrolled in this study. all of them underwent percutaneous liver biopsy, histological findings and had detectable viral load. age, viral load, levels of alt, types of mutation and hbv genotype was monitored. result: the median year of mutation found was 23months. 85.71% were genotype c and 14.29% were genotype b. the mutation of l80i, l80v, g173l, l180m, m204v and m204i were detected. the emergence rates were 34.3%(12/35), 25.7%(9/35), 17.1%(6/35), 60%(21/35), 57.1%(20/35), 54.3%(19/35) respectively. the rate of patients with two or three mutation were much more than one or four mutation. 62.9% patients were found to have significant histological findings, even 5 had established cirrhosis. two had no histological finding. one had rtl80i and rtm204i. the other had rtl80v, rtl180m and rtm204v. the number of resistant mutation has no significant finding with histological finding, basic alt level and basic viral load. conclusions: the emergence rate of l180m, m204v and m204i were higher than that of l80i, l80v, g173l in hbeag positive chb patients with lam-resistance. most of them have two or three lam-resistant mutation regardless of histological finding severity, level of basic alt and viral load. we must select the efficacious method to treat the patients with lam-resistant. objective: to investigate the therapeutic efficacy of foscarnet sodium in the treatment of patients with severe chronic hepatitis b. methods: forty four patients were randomly divided into foscarnet sodium treatment and placebo groups.each group consisted of 22 patients, 22 patients in foscarnet sodium group were treated with foscarnet sodium twice daily 3.0g given by intravenous infusions ,in addition to general therapy for 28 days.the other 22 cases were treated without any form of antiviral therapy as control.all patients were followed up for 6 months.the hbv markers, quantification of hbv-dna, serological chemistry data were measured at baseline , during therapy period and the end of follow-up period . results: clinical symptoms were improved in two groups patients, meanwhile alanine aminotransferase (alt) and total serum bilirubin (tbil) decreased. compare alt and tbil at the end of trentment, there were no significant differences between the two groups (p>0.05). in foscarnet sodium treatment group, the level of serum hbv-dna descreased from (6.993±0.898) log copies/ml to( 4.033±1.286) log copies/ml (p<0.05), the rate of hbv-dna descrease of more than two log was 81.1% 18/22 . in the control group, the level of serum hbv-dna descreased from( 7.068±0.938) log copies/ml to (5.188±1.926 )log copies/ml, the rate of hbv-dna descrease of more than two log was 45.4% 10/22 .a comparison of serum hbv-dna showed significant differences between the two groups(p<0.05) conclusion: foscarnet sodium administered can inhibit hbv replication in treating severe chronic hepatitis b.it can rapid lower the level of serum hbv-dna obviously.but the relapse rate was 47.0% in foscarnet sodium treated at the end of follow up period objective: evaluation of efficacy and safety of five years trail of entecavir for chronic hepatitis bpatients failed with lamivudine therapy in the chongqing area. methods: thirty-two eligible patients were enrolled who had documented lvd failure.in the double-blind phase,patients were randomized(4:1)to etv1.0mg/d (n=28)and placebo (n=4) for 12 weeks.in the open-lable phase ,patients received etv 1.0mg/d for 240 weeks.hbv-dna level,liver function tests,hbv serology and safety assessments were conducted. results: the mean reduction in hbv dna levels at week 12 was 4 05 logl0 copies ml in etv group compared to 0.08logl0 copies ml in placebo group(p< 0 05). the mean of hbv dna levels after 240 weeks of etv treatment decreased to 2.58logl0 copies m1 the proportion of hbv dna<3log10copy/ml raised from 0 at baseline to 6.25% at week 8,to 15.6% at week 24,to 50% at week 96,and raised to 57.14% at week 240.there were two patients with hbsag seroconversion and four patients with hbeag seroconversion at the end of study. the mean of alt became normal at week 12 and remained normal throughout week 240.there was one patient who had a severe adverse event during the trail. conclusion: the findings from this study demonstrated the antiviral activity and safety of etv in adults with chb who have failed lvd pe198 showing delayed response on t cells as increased on day 7.the mrna expression of il-5 and il-2 showed no response to hbv vaccine but highly regulated in tt after day 7(p=0.00,0.001).myd88andtraf 6(p=0.042)upregulated in hbv vaccine group followed by of ifn-(0.012)no change of ifn found in tt conclusions: i) hbv vaccine stimulates innate response by day 3 which potentiates further cascade,peripheral dendritic cells plays significant role in generating immune flare follows myd88 pathway and releases ifn-.ii) whereas t cells marjory involved in tt showed delayed immune response.iii) identification of key factors at different time points may prove to be a novel model to study the initial events after vaccination. objective: to compare th1/th2 cytokines' dynamic change and its clinical significance in hepatitis b e antigen-positive patients treated with telbivudine. methods: twelve hepatitis b e antigen-positive patients treated with telbivudine.the blood sample was collected at baseline, week 4, week 8, week 12, week 24 and week 48 and stored at -70c; serum il-2, il-4, il-6, il-10, tnf-and ifn-were tested at each time point by cytometric bead array (cba), compare th1/th2 cytokines' dynamic change at different time point in each group and compare th1/th2 cytokines' dynamic change cross four different groups: complete response, partial response, non-response and break through . results:the level of th1 type cytokines in complete response group are obviously higher than the group of partial response non-response and breakthrough,but the level of th2 type cytokines are lower than the group of partial response, non-response and breakthrough. conclusions: th1/th2 cytokines is essential for the regulation of the immune function of the body. after treated with telbivudine, the level of th1-type cytokines in the complete response group increased significantly, while the level of th2 cytokines declined trend. a. soamni 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background: chronic infection with hepatitis b virus causes spectrum of manifestations ranging from asymptomatic carrier state (often inactive with low replication) to the development of cirrhosis-related complications.the characterization of asymptomatic state has not been done in this part of the country, which forms important objective of present study. methods: 61 incidentally detected asymptomatic hepatitis b surface antigen positive (idahs) subjects having hbsag positivity for >6 month presenting to our liver clinic were enrolled after appropriate consent. detailed clinical, laboratory and sonographic evaluation was done. they were divided into two groups according to presence or absence of e antigen. group a -hbeag + (n=48) group b -hbeag -(n=13) results: most of our patients (49%) were young adults (21-30 years) with male to female ratio of 3.6:1. approximately half of our patients were detected during routine medical checkup, followed by family screening of contacts. most of our patients were asymptomatic, and fatigue was most common symptom found in 16%. all demographic and biochemical parameters other than ast & alt were comparable in both groups. among hbeag negative 48 (79%) subjects, hbv dna level >10 5 copies/ml was found in 31%. subjects with positive hbeag as compared to non-replicative infection (antihbe positive and hbv dna negative) had more frequent elevation of transaminase levels (62% versus 31%, p<0.05). antihbe antibody was positive in all hbeag negative subjects. mean age of seroconverted (antihbe positive) individuals was a decade older than hbeag positive. conclusion: from our study we can suggest that ongoing liver disease is present in approximately one-thirds of incidentally detected asymptomatic hepatitis surface antigen positive subjects previously referred to as carrier state. hbsag testing should be mandatory in all routine medical checkup and family and sexual contacts of index case should be screened. background and objectives: this research was carried out to determine the prevalence of hbcab among the hbsag negative first-time blood donors who had referred to khorramabad and borujerd centers for blood donation. materials and methods: this study was established on a descriptive cross-sectional basis in which hbsag test (elisa) was primarily performed on all of the donors having referred to khorramabad and borujerd blood centers; then, out of all those referred 1000 subjects, who were first-time and hbsag negative, were selected for furthur investigation. the information concerning age, gender, job, blood transfusion, and hbv vaccine injection was included in the questionnaire of the study. hbcab (total & igm) and hbsab tests were performed on the selected donors. data were collected and finally the prevalence rate of hbcab was determined. results: the results of the study showed that out of 1000 hbsag-negative first-time blood donors, only 47 were hbcab+, from which 27 were hbcab (total)+, and 3 were hbcab (igm)+. 18 were both hbsab+ and hbcab+, and 53 were seropositive only for hbsab. conclusions: it was demonstrated that the first-time blood donors who are seronegative for hbsag marker will be easily identified through hbcab test if they are in the so-called core window period of the virus. meanwhile, this group of donors have been implicated as high-risk for transfusiontransmitted hbv infection. so, detecting this marker will remarkably reduce the chance of latent cases of hbv infection and help promote blood safety. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. method: 20 hbv transgenic mice were randomly divided into physiologic saline group and matrine injection group. another10 normal mice at the same species and age with hbv transgenic mice were regarded as the normal group. the mice in matrine injection group were administrated at dosage of 82.2 mgkg -1 d -1 by intraperitoneal for 30 days. the mice in physiologic saline control group and normal group were administrated normal saline with the same volume at same time. the contents of hbv dna in serum and liver were quantitated by pcr. and the spleens were separated for cultivating dendritic cells. the surface molecules of dendritic cells were tested by flow cytometry. ifn-mrna and tnf-mrna in liver were tested by rt-pcr. result: there was no significant difference of the serum hbv-dna level between physiologic saline and matrine injection groups. the content of serum hbv-dna after treatment showed a significant decrease in two groups. the content of serum hbv-dna in matrine injection dropped significantly as compared with that in the physiologic saline group. but there was no significant difference in the content of hbv-dna in liver between physiologic saline and matrine injection groups. the expression level of mhc-ii on dendritic and hepatic ifn-r mrna and tnf-a mrna showed a significant decrease in hbv transgenic mice than normal mice. in comparison with physiologic saline group the expression level of them in matrine injection group showed a significant increase. conclusion: matrine injection was effective on depressing hbv-dna in hbv transgenic mice. its antiviral action may be achieved through regulating mhc-ii on dcs surface and promoting the production of antiviral factor such as ifn-and tnf-. purpose: to stimulate non-specific immune response capacity as the main content of the study to explore the hbv-dna and non-specific immune responses in the relationship between the low response capability, methods: 190 cases of asymptomatic carriers, double-blind, randomized into mycobacterium fu 36, lamivudine and traditional chinese medicine for the treatment group, mycobacterium fu36 with traditional chinese and lamivudine with traditional chinese medicine were in the control group, a total of 12 weeks of treatment, follow-up six months after the termination of treatment. results: different treatment of hbv -dna effect of the existence of significant differences; p> 0.01, the performance of different types of asymptomatic carriers negative rate of hbv-dna there is a significant difference; p> 0.01, as well as the performance of the different types of asymptomatic carriers continued application a treatment plan presented hbv-dna rebound rate there is a significant difference; p> 0.01, conclusion: hbv-dna and non-specific immune responses in response to the lower capacity, anti-hbv therapy is not associated with non-specific immune response capacity or improve is the anti-hbv drugs alone can not solve the asymptomatic carriers in anti-hbv therapy where the cause of the problem, solve the asymptomatic carriers in the anti-hbv treatment although the need for anti-hbv drugs with non-specific immune activation synchronous drugs on the basis of the joint application , but the simultaneous combination of two drugs rather than as a result of hbeag and hbv-dna can hbeag-positive asymptomatic carriers receive hbv-dna negative effect of the results. background: adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine (lam)-resistant hepatitis b virus (hbv) replication and resistance to this drug is infrequent compared with lam. in this study, we tried to identify factors affecting the emergence of resistant mutants after adefovir monotherapy in lam-resistant chronic hepatitis b (chb) patients. methods: the subjects were 87 chb patients with lam-resistance who had received adefovir for more than 12 months (range 12-39 months). the initial viral response (ivr) was defined as hbv dna <4.0 log copies/ml. the adefovir resistant mutant was assayed at baseline and every 6 months during adefovir administration. results: ivr was observed in 38% of patients. the cumulative emergence rates of adefovir resistance were 2.6% at 6 months, 10.4% at 1 year, 14.6% at 2 years and 21% at 3 years. in univariate analysis, factors contributing to the emergence of adefovir resistant virus were baseline hbv dna > 6 log copies/ml (p=0.003) and ivr (p<0.0001). the presence of precore mutation and type of ymdd mutants were not related. in multivariate analysis, only ivr was an independent factors affecting the emergence of adefovir resistant virus (p<0.0001). conclusion: ivr is a useful predictor for emergence of adefovir resistant mutants after adefovir monotherapy in lam-resistant chb patients. for ivr-negative patients, the change of therapeutic options such as add-on lam or switch to other drugs should be considered because of the high incidence of the emergence of adefovir resistant mutants. background: elevated hbv dna is strongly associated with the risk of disease progression. this study investigated the early viral suppression effects of etv and lvd in nucleoside-naïve chinese patients with active hbeag (+) chb. methods: this open-label study was conducted in 5 major hospitals in china. at study entry all patients had hbv dna levels 10 7 copies/ml, elevated alt (1.3-10xuln) and compensated liver function. patients received either 0.5mg etv or 100mg lvd daily. hbv dna measurements were taken at baseline and at weeks 2, 4, 12 and 24 during treatment, using roche cobas amplicor assay (llod 300 copies/ml). results: a total of 97 patients were enrolled; 42/50 etv patients and 40/47 lvd patients completed 24 weeks of treatment. at baseline, mean hbv dna levels were 8.45 0.81 in etv group and 7.67 1.76 log 10 copies/ml in lvd group (p<0.05). the mean change in hbv dna from baseline (log10 copies/ml) was -2. 96±0 ngo's/funding-agencies representative at apasl2009-conference need to address-this-issue. we ngo-representatives from developing-nations need exposure to research treatments used by european/american experts. do we all failed in addressing socio-economic issues of cancer-sufferers? we need to address these socio-economic issues of affected population in resource-poor-nations. background: a garlic derivative s-allylcysteine (sac) has anti-cancer effect in human prostate and colon cancers. we aimed to investigate the effect of sac and combination of chemo-drug on tumorigenesis and metastasis of liver cancer. methods: the orthotopic liver tumor model using a metastatic liver cancer cell line mhcc97l labeled with luciferase gene was applied. sac was given at day 7 after tumor implantation at 1mg/g/day, or 1mg/g/day combined with low dose cisplatin for 5 weeks. tumor growth and metastasis were monitored by xenogen in vivo imaging system. hepatic stellate cell (hsc) activation and tumor-associated macrophage (tam) in the tumor tissue were detected by -sma and ed1/ed2 staining. tumor micro-vessel density (mvd) and apoptosis were also analyzed. in vitro functional tests including proliferation assay, cell cycle analysis and apoptosis analysis were performed. results: tumor growth was inhibited by sac combined with cisplatin treatment at different time points accompanied by lower incidence of lung metastasis compared with other groups. the observation of xenogen ivis was confirmed by histopathological examination. the hsc activation by -sma staining in the liver tumors was suppressed by sac and cisplatin treatment accompanied with less tam infiltration. consistent with in vivo study, in vitro functional study also demonstrated that sac not only induced cell cycle arrest, apoptosis, and inhibited tumor cell proliferation, but also sensitized the anti-cancer effect of cisplatin. conclusion: sac treatment inhibited liver tumor growth and metastasis by inhibiting tumor cell proliferation, inducing apoptosis and sensitization of chemotherapy. background: anti-angiogenic therapy would be a promising approach against hepatocellular carcinoma (hcc). although a sorafenib has survival benefits in patients at advances stages of hcc, there seem to be several serious concerns to employ this agent for chemoprevention against hcc. branched-chain amino acid (bcaa) reportedly inhibits the incidence of hcc in patients with insulin resistance (ir). however, the possible mechanism is still obscure. the aim of the current study was to examine the effect of bcaa on hepatocarcinogenesis under the condition of ir, especially in conjunction with angiogenesis. methods: the effect of bcaa on the development of liver enzyme-altered pre-neoplastic lesions and angiogenesis in the obese diabetic otsuka long-evans tokushima fatty rats was examined. we also performed an in-vitro study to elucidate the possible mechanisms involved. result: treatment with bcaa markedly inhibited the glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions along with suppression of neovascularization in the liver. the hepatic expression of the vascular endothelial growth factor (vegf), a potent angiogenic factor, was also attenuated. bcaa treatment significantly suppressed the glucose-and insulin-induced in-vitro angiogenesis in the presence of vegf. these results indicate that bcaa exerted a chemopreventive effect under the condition of ir via suppression of vegf-mediated angiogenesis. conclusion: since bcaa is widely used in the clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against ir-based hcc in the future. background: krüppel-like factor 8 (klf8) is a member of transcription factors. whether and how klf8 signaling pathways contribute to hepatocellular carcinoma (hcc) development and progression is unknown. this study investigated role of klf8 in hepatocellular carcinoma cell line hcclm3 proliferation, invasiveness and epithelial to mesenchymal transition (emt). methods: the expression of klf8 in different liver cell lines was detected by quantitative real-time pcr and immunocytochemistry. we used small interfering rna (sirna) to down-regulate klf8 expression in hcclm3. the change of proliferation and invasive ability of klf8 down-regulated hcclm3 was investigated by mtt reduction assay and trans-well invasive assay respectively. the change of proliferation, invasiveness and emt related gene in klf8 down-regulated hcclm3 was evaluated by quantitative real-time pcr. result: klf8 protein expressed predominantly in the nuclei of cancer cells and its expression is positively correlated with metastatic potential of these cell lines. hcclm3 has the highest klf8 level. decreased klf8 expression can notably inhibit the proliferation (p<0.001, n=6), mobility and invasiveness of hcclm3 (p<0.001, n=8). we found that the mrna level of n-cadherin, fibronectin and vimentin is much higher than that of e-cadherin in hcclm3. the expression of cyclin d1, focal adhesion kinase (fak) and fibroblast markers including n-cadherin and fibronectin was obviously suppressed in klf8 down-regulated hcclm3. conclusion: klf8 plays an important role in the process of hcclm3 proliferation, invasiveness and emt. background: insulin resistance (ir) has shown to play an important role in the progression of chronic liver diseases, including liver fibrosis development and hepatocellular carcinoma. the aim of this study was to elucidate the possible mechanisms of ir on the liver fibrosis development and hepatocarcinogenesis using obese diabetic otsuka long-evans tokushima fatty (oletf) rats. methods: to induce liver fibrosis, 1.0ml/kg of pig serum was injected twice a week for 4 weeks in the oletf and leto rats. in the hepatocarcinogenesis model, glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions were induced by a single injection of 200mg/kg of diethyl nitrosamine (den). we also performed in-vitro studies to examine the mechanistic insights. results: the liver fibrosis development and gst-p-positive pre-neoplastic lesions were both markedly accelerated in oletf. in the fibrosis experiment, -smooth muscle actin-positive activated hepatic stellate cells (hsc) also increased in oletf along with augmentation of the hepatic collagen content and transforming growth factor-b1. in the den model, the neovascularization was up-regulated in oletf almost in parallel with the pre-neoplastic lesions development and a potent angiogenic factor, the vascular endothelial growth factor. our in-vitro study showed that both glucose and insulin stimulated the proliferation of the activated hsc and augmented the neovascularization. conclusion: these results indicated that the ir status directly accelerated the liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated hsc proliferation and hepatic neovascularization, respectively, in the rat. n. wakui 1 , t. ikehara 1 , r. takayama 1 , m. takahashi 1 , k. shiozawa 1 , h. nagai 1 , m. watanabe 1 , k. ishii 1 , k. iida 1 , y. igarashi 1 , y. sumino 1 1 case: a 68 years old man diagnosed with chronic hepatitis c regularly visited our hospital. in april of 2005, ultrasonography revealed a tumor 20mm in diameter in s2 of the liver and another tumor 15mm in diameter in s6/7 of the liver. the patient was hospitalized for further examination. computer tomography (ct) revealed that the tumor localized in s6/7 presented a pattern of hypervascular hepatocellular carcinoma (hcc). for the tumor localized in s2, the following were revealed. 1) contrast-enhanced ultrasound findings: a tumor vessel passed from outside the tumor to the center of the tumor in the early vascular phase, then radiated in a wheel-like shape at the center of the tumor; parenchymal phase perfused imaging in the area produced a similar imaging obtained from the area surrounding the liver. 2) ct: no tumor was detected. 3) spio-mri (t2 weighted imaging): iso-low intensity images were obtained. although these imaging findings indicated fnh, the patient was hcv positive. in order to disprove the possibility of hcc, a biopsy was performed on the tumor at s2 in the liver. the resulting diagnosis was well-differentiated hcc. discussion: until now, a characteristic finding of fnh has been spoke-like vasculariation, which is considered diagnostically quite important. however, some recent cases of hcc have been reported to present fnh-like vascularization. from now on, when evaluating a tumor that presents spoke-like vascularization underlining chronic hepatitis, the possibility of hcc should be considered and a close examination may be needed. chronic infection with hcv is problem .clinical management of chronic hcv depend on extent liver fibrosis .liver biopsy gold stander an invasive procedure responsible for severe complications and sample variability interpretation. serum biomarkers for inflammation/fibrosis investigated to wave liver biopsy. diagnostic accuracy panel of non-invasive serum biomarkers for hepatic fibrosis (fibrosure , apri score, forn's score) versus liver biopsy. 20 hcv patients subjected for: apri, forn's , fibrosure scores pcr quantitative hcv-rnaliver functions .lipid profile cbc . ultrasound guided liver biopsy. forns score; auroc (0.917) with 95% ci(0.791-1.042) for(fof1) vs. (f2f3f4) while(0.688)with 95% ci(0.464-0.91)for(fof1f2) vs.(f3f4). cutoff(>6.9)sensitivity for significant fibrosis(f2f3f4)and extensive fibrosis (f 3f4)were (100%) and with low specificity ,with accuracy(40%) and (20%)respectively.-apri score; auroc(0.792)with 95% ci( 0.568 -1.015)comparing(f0f1) vs.(f2f3f4)while was(0.875)with 95% ci(0.703 -1.047)for( f0f1f2)vs.(f3f4).cutoff(<0.5) had low sensitivity and specificity(100%)with accuracy(60%)for significant fibrosis and(80%)for extensive fibrosis.-fibrosure(fibro-acti test); showed best auroc(1.00)in different fibrotic stages with 95 % ci (1.00-1.00).cutoff(>0.59) sensitivity(50%)for significant fibrosis and(100%)for extensive fibrosis while specificity(100%)in all fibrotic stages. the ppv (100%)for significant and extensive fibrosis .npv and accuracy(75%, 80%)respectively for significant fibroses,while it was (100%) for extensive fibrosis respectively.significant correlation between liver biopsy and fibro-test(p0.002)and acti-test(p0.000).significant correlation between liver biopsy hepatitis activity score and apri (p 0.047)and forns score (p0.000). conclusion: forns score wasn't considered since does not discriminate between significant and extensive fibrosis. low sensitivity of apri prohibtes detection of minmal fibrosis and allow undetermined results. fibrosure classified all cases of chronic hcv sufficient to wave liver biopsy pe215 introduction: hcc is the 6 th common cancer. global increase of hepatitis b and c infection, the incidence of hcc steadily increasing. egypt seroprevalence of hcv in nile delta 20 -35%. afp had limited sensitivity 60% and specificity 90% for small hcc. gpc-3 oncofetal protein over expressed in hcc. evaluating validity of glypican-3 as early detector of hcc.:10 healthy controls and 40 hcv positive patients:10 patients chronic hepatitis c virus infection.10 patients compensated cirrhosis [child-pugh class a and b].10 patients decompensated cirrhosis [child-pugh class c]. 10 patients hcc. liver functions: alt, ast, bilirubin(t), albumin, gt.tumor markers: afp and gpc-3.viral markers: hcv antibodies, hbs ag and hbc ab. the median value of gpc-3 in hcc, dc, cc significantly higher than chronic hepatitis and control groups. no significant correlation between afp and gpc-3. auroc of afp 0.85 & auroc of gpc-3 0.84. the diagnostic sensitivity of afp (20 ng/ml) 70% with ppv 53.8%. the specificity85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) 100% with ppv 27%. the specificity 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved specificity to 87.5%. conclusion:gpc-3 although a serological test for early detection of hcc, showed limited specificity, where detected in different stages of chronic liver disease,it is oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 improve prediction accuracy of hcc patients in those showing seronegativity to afp. results: patients with hcv infection (n=388) were significantly older (mean age, 69 years) than patients with dual virus (n=75, 65 years) and hbv infection (n=752; 60 years) (p< 0.0001). the male-to-female ratio for hbv, dual virus and hcv group was 5.2, 3.4 and 1.3, respectively (p< 0.0001). patients in the hbv group more often had higher total tumor volume (mean, 409 cm 3 ) than the dual virus group (244 cm 3 ) and hcv (168 cm 3 ) group (p< 0.0001). no significant differences of the severity of liver cirrhosis, performance status, cancer staging and tumor cell differentiation were noted among the three groups. patients in the hcv group had a significantly poor survival in comparison to the hbv group only in the subset of patients with small tumor volume (< 50 cm 3 ) in the cox proportional hazards model (relative risk: 1.44, p=0.041). conclusions: dual hbv and hcv virus infection does not accelerate the speed of hcc formation in patients with chronic hepatitis b, and appears to have a modified course of carcinogenesis pathway diverted away from the biological behavior of hbv and hcv infection. background: patients presenting with hcc is not infrequent in our clinical practice. the aetiology vary ranging from hbv, hcv, nash and alcohol. the aim of this study was to see the aetiology of hcc in bangladeshi patients. methods: in this retrospective study, records of 976 patients who attended our opd between july 2004 to august 2008 were reviewed. patients having hepatic sol and/or heterogeneous echotexture of liver on usg and/or ct scan were included. diagnosis of hcc was confirmed at usg guided fine needle aspiration cytology with or without elevated serum afp (>500 ng/ml). results: of the 976 patients, 75% (732/976) had hbv infection. hcv infection was diagnosed in 17% (165/976). nash was responsible for 5% (49/976) cases, alcohol in 1% (10/976), while in the rest 2% (20/976) cases no specific aetiology could be established. conclusion: the study shows that hbv is the commonest cause for hcc in bangladesh followed by hcv. background: the aim of this study was to determine whether the hepatitis b virus (hbv) dna viral load and antiviral therapy is associated with hepatocellular carcinoma (hcc) recurrence. methods: this retrospective study involved 93 patients who underwent hepatic resection or radiofrequency ablation for initial hcc curative treatment. the patients were divided into four groups. fifteen patients with low serum hbv dna levels ( 4 log10 copies/ml) at the time of initial hcc treatment received antiviral therapy (lamivudine, adefovir, dipivoxil, entecavir) before hcc appeared (pre antiviral therapy group; pre-tg). thirty-four had low serum hbv dna levels without antiviral therapy (low virus group; lvg). fourteen had high serum hbv dna levels and received antiviral therapy after hcc appeared (post antiviral therapy group; post-tg). thirty patients had high serum hbv dna levels without antiviral therapy (high virus group; hvg). results: the cumulative hcc recurrence rates at 3 years in the hvg, lvg, pre-tg, and post-tg groups were 68.9%, 42.2%, 44.3%, and 57.1%, respectively. there were significant differences in the hcc recurrence rates between the hvg and lvg groups (p = 0.016), and between the hvg and pre-tg groups (p = 0.013). the recurrence rate was lower, though not significantly, in the post-tg group than in the hvg group (p = 0.18). conclusions: not only hbv dna viral load but also antiviral therapy is associated with hcc recurrence. antiviral therapy before hcc appears is important for patients with high serum hbv dna levels to prevent hcc recurrence. background/aims: few reports have described methods for predicting prognosis in unresectable hepatocellular carcinoma (hcc) patients, especially those treated by repeated transcatheter arterial chemoembolization (tae). to determine risk factors for death and determine prognosis in patients treated with repeated-tae, we evaluated clinical data. methodology: we retrospectively analyzed clinical parameters of 224 unresectable hcc patients treated with repeated-tae from january 1997 to december 2007. tae was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. factors affecting survival were evaluated using multivariate analysis after univariate analysis. next, we combined the score for each significant factor into a single prognostic score, after which the results were compared with jis and clip score methods. results: multivariate analysis revealed that bilobular hcc, alpha-fetoprotein ( 400 ng/ml), tumor invasion of the portal vein, tumor size ( 10 cm), and albumin (<2.8 g/dl) were related to poor prognosis, using those 5 factors, we developed a new prognostic scoring system. the 50% survival period was 29.2 months for all subjects, while it was 54.6, 29.2, 15.0, 5.6, and 1.0 months for those with scores of 0, 1, 2, 3, and 4 or over, respectively (p<0.0001), using our new system. clip score was not useful to predict prognosis, while jis score was better. however, subjects with jis scores of 1 and 2 were difficult to differentiate. conclusion: our scoring system was easy to perform and the results showed that repeated-tae was effective for unresectable hcc with a score of 2 or less. local ablative therapies and intrahepatic pressure c. kawamoto 1 , a. yamauchi 1 , k. kaneko 1 , n. miyagi 1 , k. kani 1 , t. aoyama 1 , k. yakabi 1 1 saitama medical center, saitama medical university, japan background: some of the unexpected recurrence observed after radiofrequency ablation (rfa) might be caused by increased intratumoral pressure. the present study examined the relationship between local ablative therapies and intrahepatic pressure. methods: a. basic study: under general anesthesia, laparotomy was performed on 19 pigs. a leveen needle and a percutaneous ethanol injection (pei) needle were inserted into the liver and intrahepatic pressure was monitored using an invasive blood pressure monitor. ablation was performed as follows: 1. rfa. 1) single-step method: after fully deploying the electrode, the power was initially applied at 30 w, then increased in increments of 10 w/min until power roll-off. 2) multi-step method: the array was deployed in 8 steps. at each step, the power was fixed at 30 w until power roll-off. 2. pei. injection of ethanol (2 ml). b. clinical study: we examined the multi-step rfa and pei for hcc. under local anesthesia, intratumoral pressure was monitored. 1. rfa. 39 patients with a mean tumor size of 15.3±4.9 mm were studied. 2. pei. in 10 patients with a mean tumor size of 21.4±8.8 mm, 5 to 10 ml of ethanol was injected per session. results : a. basic study: the intrahepatic pressures were: single-step method, 154.5±30.9 mmhg; multi-step method, 24.1±18.2 mmhg; and pei, 12.0±8.5 mmhg. b. clinical study: intratumoral pressure was 39.5±27.9 mmhg for rfa and 12.2±9.0 mmhg for pei. conclusion: these results suggest that consideration of intrahepatic pressure is crucial in local ablative therapies. background: a late evening snack (les) is recommended for liver cirrhosis. however, no clinical study has evaluated the nutrition status and the effect of les in cirrhotic patients with hepatocellular carcinoma (hcc). we investigated the effect of les undergoing hepatic arterial infusion chemotherapy (haic) in patients with hcc. method: nineteen patients with hcc were enrolled. ten patients were les group, and nine were control group. in the les group, the patients received les supplementation with a branched-chain amino acid (bcaa)-enriched nutrient mixture. in the control group, the patients received ordinary food. there were no significant differences in relation to age, gender, etiology, child-pugh scores, tumor stage, clinical responses to haic between two groups. blood biochemical data, nutrition status using an indirect calorimeter were evaluated at before and at the end of chemotherapy. results: the non-protein respiratory quotient (nprq) and molar ratio of branched-chain amino acid to tyrosine (btr) were significantly improved in the les group but not in the control group. there were no significant differences in the area under the concentration curve for glucose between before and the end of chemotherapy in two groups. background & aims: hepatocellular carcinomas (hccs) often show hypoor mixed vascularity, and the prognosis of these relatively hypovascular hccs is not fully elucidated. cytokeratin (ck) expression profiles may also be useful prognostic indicators, and specifically ck19 may reflect metastastic potency in hccs. this study was to assess the prognostic implication of tumor vascularity and its relation to ck19 expression in hcc patients. methods: a total of 150 patients who underwent surgical resection for hcc were enrolled. tumor vascularity was evaluated according to arterial enhancement pattern on ct scans and ck19 expression was evaluated using tissue microarray methods. clinicopathologic data were analyzed using kaplan-meier and cox proportional hazard model. results: during follow-up period, 91 (60.7%) patients experienced tumor recurrence. forty-five patients (30%) had hypovascluar tumor at the time of diagnosis, and they showed significantly higher positivity for ck19 expression (p=0.001) and shorter disease-free survival (p=0.023) than patients with hypervascular hccs. in addition, recurred tumors in these patients showed more frequently hypovascular pattern than in patients with hypervascular hccs (p=0.001). hypovascularity at initial diagnosis and microvascualr invasion were independent poor prognostic factors predicting survival. following treatment of recurred hccs, hypovascular tumors showed poor response to transarterial chemoembolization (tace), which resulted in shorter overall survival than hypervascular tumors (p=0.057). conclusions: these results demonstrate that tumor hypovascularity in hccs is associated with positive ck19 expression, early tumor recurrence, poor tace response and poor survival. therefore, tumor vascularity may also be a prognostic indicator in hcc patients. background: hepatic stellate cells (hscs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma(hcc). in this study, we determine gene expression changes in two different models of hscs activation and investigate whether induction-activated hscs(ihscs) gene expression changes are different from culture-activated hscs(ahscs). methods: hscs were isolated by density centrifugation and exposed to conditioned medium from rat hcc cell lines c5f. twenty-seven thousands and one hundred gene expression between quiescent hscs(qhscs), ahscs and ihscs was analyzed by microarray and confirmed by real-time rt-pcr and western blot. results: sixteen hundreds and seventy-one probe sets were differentially expressed in ahscs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors. seven hundreds and eleven probe sets were differentially expressed in ihscs. induction-activated hscs showed specific gene expression patterns including raf1, rac2, adam17, wnt6, mmp-9 and tnf, suggesting that hcc cells can specifically induce hscs activation. induction-activated hscs might play a important role in invasion and metastasis of hcc. conclusions: induction-activated hscs gene expression patterns are different from ahscs. culture-activated hscs does not properly regulate gene expression in hscs, suggesting that ihscs may be considered the model for the study of hscs biology in hcc. background: hepatocellular carcinoma (hcc) is a hypervascular tumor, and angiogenesis is important for tumor growth. ephrin receptors are related with vascular system development and the polymorphism of ephb1 in the carcinogenesis of digestive tract has been reported. our aim was to examine the polymorphsims of ephb 1 with the occurrence of hepatocelluar carcinoma in korean population. methods: genomic dna was extracted from 182 patients with hepatocellular carcinoma (hcc), 266 healthy subjects. ephb 1 polymorphism was determined by polymerase-chain reaction-based assays, and the association with hcc was investigated. results: with regard to ephb 1 polymorphism, a/a genotype at rs11926992, t/t genotype at rs7644369, a/a genotype at rs6776570, t/t genotype at rs3821502 and g/g genotype at rs6766459 were significantly associated with hcc but these were not associated with clinical characteristics of hcc. conclusions: five out of seven polymorphisms on ephb1 gene were statistically associated with hcc, in the korean population. therefore, more studies of ephb1 gene polymorphisms including various risk factors should be performed to use as genetic markers of hcc occurrence. background: we aimed to compare the results of hepatectomy for hcc in patients older than 70 years old with those for younger patients. methods: clinicopathological data and outcomes for 155 elderly patients and 333 younger patients with hcc who underwent hepatectomy between 1992 and 2007 were retrospectively compared. results: although postoperative delirium was more common in the elderly group, there were no significant differences between the 2 groups with regard to operative morbidity, hospital death, disease-free survival, and overall survival. the overall recurrence rate was significantly higher in the elderly patients with alcohol abuse than in younger patients with alcohol abuse. multivariate analysis revealed that preoperative alcohol abuse was a prognostic factor for elderly patients. conclusions: elderly patients with preoperative alcohol abuse should be followed closely, even after r0 surgery, because alcohol abuse is strongly correlated with postoperative recurrence and worse survival. background: little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. methods: among 410 patients who underwent curative resection between 1992 and 2005, 180 patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group a), while 46 patients were only transfused with packed red cells (group b), 43 patients were only transfused with fresh frozen plasma (group c), and 141 patients had no transfusion (group d). results: group c had significantly fewer postoperative complications and a shorter hospital stay than group a. preoperative coagulation was significantly worse in group c. survival was significantly better in groups c and d than in group a. conclusions: perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma. background: this study investigated risk factors for postoperative liver failure after resection of hepatocellular carcinoma to detect markers that could identify candidates for hepatectomy. methods: perioperative risk factors for liver failure after hepatectomy were analyzed in 191 patients with hepatocellular carcinoma. results: liver failure occurred postoperatively in 16 patients, 3 of whom died. the hyaluronate/gsa-rmax ratio was a risk factor for postoperative liver failure by univariate analysis and was the only risk factor according to multivariate analysis. all 3 patients who died had a hyaluronic acid/gsa-rmax ratio 500 mg min/dl. conclusions: to reduce postoperative liver failure, preoperative planning should employ various measures of the hepatic functional reserve, including tests of both parenchymal and nonparenchymal liver function. the hyaluronate/gsa-rmax ratio can predict liver failure after hepatectomy, and a ratio greater than 500 mg min/dl is a relative contraindication to liver resection. the patient was a 73-year old japanese man with chronic hepatitis c(ch-c) who achieved a sustained virological response(svr) to interferon(ifn) therapy. as a result the liver functions were normalized and the histological findings of the liver also improved. however, 13 years after svr, mild liver dysfunction was noticed along with a marked increase of tumor markers. several modalities revealed huge liver tumors about 13 cm in greatest diameter in the left lobe invading the bile ducts and another tumor about 3 cm diameter in segment v. we performed liver biopsy and confirmed that this tumor was well-differentiated hepatocellular carcinoma (hcc). only mild fibrosis development could be observed in the adjacent non-cancerous lesions. we successfully treated these tumors with transcatheter arterial chemoembolization and stereotactic radiosurgery. recent studies revealed that the risk of developing hcc still exists even after svr. since most of hcc that develop in patients with svr are usually detected within 5 years, several investigators speculate that hcc is already present but too small to be detected at the time of completion of ifn therapy. this speculation is not the case in our patient, since svr was achieved 13 years ago and no hcv-rna could be detected when hcc appeared. therefore, another possible mechanism should be considered. an annual follow-up with strict surveillance program for hcc should be performed for more than 10 years after the completion of ifn therapy. background/aims: in order to investigate the role and importance of oxidative stress as to carcinogenecity of hepatocellular carcinoma (hcc) we analyze the expression of 8-hydroxydeoxyguanosine (8-ohdg) in the liver tissue of the hcc patients with and without hepatitis viral marker. methods: patients undergoing hepatic resection for the first hcc from 1995 to 2004 were enrolled into the study. only the cases that took no alcohol or small amount of alcohol were enrolled. 24 cases were negative for hepatitis b surface antigen (hbsag) and antibody to hepatitis c virus (hcvab) (nbnc group). 24 were positive for hbsag and negative for hcvab (b group). 21 were positive for hcvab and negative for hbsag and antibody to hepatitis b core antigen (c group). staining with hematoxylin and eosin (h&e) and berlin-blue, and immunohistochemical staining for 8-ohdg were performed using the non cancerous liver regions. the degree of 8-ohdg immunostaining was expressed as the labeling index, which means the percentage of positive hepatocytes per 1000 hepatocytes. results: the labeling index of 8-ohdg for nbnc group is 19.3(±55.3), significantly lower (p=0.014) than that for b group 49.7(±89.5), and also lower (p=0.016) than that for viral group (b group and c group)(35.4±71.8). the labeling index of 8-ohdg had no correlation with grading, staging, fatty and iron deposit among all cases. conclusions: there is possibility that oxidative stress might not associate with the carcinogenesis of hcc in some cases without hepatitis viral infection. background: no effective chemopreventive agent has been approved against hepatocellular carcinoma (hcc) yet. since neovascularization plays a pivotal role in hcc, an angiostatic agent is considered as one of the promising approaches. recently, it has reported that vitamin k2 (vk) and angiotensin-converting enzyme inhibitor (ace-i) exert anti-angiogenic activity. the aim of the current study was to elucidate the combination effect of the clinically used vk and ace-i on cumulative recurrence after curative treatment, especially in consideration of neovascularization. methods: vk (menatetrenone; 45 mg/day) and/or ace-i (perindopril; 4 mg/day) were administered for 36 to 48 months after the curative therapy for hcc. the cumulative recurrence and several indices were analyzed. results: a 48-month follow-up revealed that the combination treatment with vk and ace-i markedly inhibited the cumulative recurrence of hcc in association with suppression of the serum level of vascular endothelial growth factor (vegf); a central angiogenic factor. the serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with vegf. these beneficial effects were not observed with single treatment of vk or ace-i for 36 months. conclusions: the combination treatment of vk and ace-i may suppress the cumulative recurrence of hcc after the curative therapy, at least partly through suppression of the vegf-mediated neovascularization. aim: the aim of this study was to clarify the cilnicopathologic features and management of hepatocellular carcinoma (hcc) patients surviving more than 5 years after hepatectomy. materials & methods: retrospective study was carried out on 823 hcc patients who underwent curative hepatectomy between 1973 and 2002. clinicopathologic factors in 5-year survivors and patients who died within 5 years were compared. the prognostic factors affecting survival were examined among the 5-year survivors. results: there were 290 patients who survived for more than 5 years after initial hepatectomy, and 83 of those patients survived for more than 5 years after hcc recurrence. the overall 3-, 5-, 10-and 20-year survival rates were 57.4%, 40.9%, 17.2%, and 8.5% respectively. in multivariate analysis, absence of underlying cirrhosis, solitary tumor, alfa-fetoprotein less than 1000 ng/ml, and absence of microscopic vascular invasion were favorable independent factors associated with 5-year survival. negative hepatitis c virus antibody status was favorable independent factor associated with longer disease-free interval and survival after tumor recurrence. multimodal treatments such as repeat hepatectomy or percutaneous ablation led to improved survival after recurrence, compared with the survival after transarterial chemoembolization (p<.05). conclusions: the results suggest that patients without underlying cirrhosis who have a solitary hcc that does not demonstrate vascular invasion or high afp levels might survive for longer than 5 years after the initial hepatectomy. close follow-up and multimodal treatment could contribute to prolongation of survival in such patients, even if cancer recurrence occurs. the history of the use of carbon ion radiotherapy (cirt) for treating hepatocellular carcinoma (hcc) goes back to 1995, when clinical trials were initiated at the national institute of radiological sciences. we have already reported that cirt used for the treatment of hcc is safe and effective, and that it causes only minor liver damage. in a phase ii clinical trial, the local control and cumulative overall survival rates were 94 % and 35 % at 5 years, respectively. however, the patients with tumor adjacent to the gastrointestinal tract are thought to be ineligible for cirt because of the high risk of radiation injury of the digestive organs. in order to extend the indication of cirt, we have challenged the cirt for such patients under the use of spacers. a case was a 67-year-old female with 8cm tumor in segment 4. in radiological findings, the tumor revealed typical enhancement pattern for hcc, and was near the ec junction. she had been judged ineligible for hepatectomy because of the high retention rate of indocyanine green. she could undergo the 42.8 gye/2-fraction cirt after the placement of gore-tex soft tissue patch under the laparoscopic procedure. up to the present date, no adverse effect due to the spacer has been occurred, and an apparent anti-tumor effect has been observed. this method seems to have a promising efficacy for extension of the indication of cirt to the patients with tumors adjacent to the gastrointestinal tract. background: previously we reported that high ubiquitination was marker of human hepatocellular carcinoma. on the basis of these finding, we firstly analyzed the effect of bortezomib(proteasome inhibitor) on human hcc cell line. we also reported that hhm/dip1/gcip1 was early marker for human hepatocarcinogenesis. hhm was suggested to be a new tumor suppression gene, but the mechanism was not well confirmed. we analyzed change of hhm signal by bortemib. method and result: we used hcc cell line (huh7, hlf, hepg2) . the inhibitory effect of bortezomib was evaluated using mtt assay. 20nm bortezomib significantly inhibited proliferation of hcc cell line. the inhibitory effect by 20nm bortezomib was similar with 10 m cisplatin. on the other hand, bortezomib has no inhibit effect in isolated hepatocyte from rat. in this condition, we analyzed the expression of cyclin d1, phospho-rb and hhm in hcc cell line by western blot analysis. expression of cyclin d1, phospho-rb decreased, but hhm was increased with time. next we analyzed cell cycle by facs. bortezomib induced hcc cell line into cell cycle arrest in g2/m. the transcriptional activity of hhm was also activated by bortezomib administration using ptimer-promoter-hhm plasmid. conclusion: bortezomib has specific anti-proliferative effect on hepatocellular carcinoma. the induction of hhm by bortezomib might be related with cell cycle arrest. bortezomib will be a useful drug for hcc. neovascularization is required for carcinogenesis of non-alcoholic steatohepatitis: experimental and clinical study m. kitade 1 , h. yoshiji 1 , r. noguchi 1 , k. kaji 1 , t. namisaki 1 , y. aihara 1 , h. background/aim: non-alcoholic steatohepatitis (nash) may progress to liver cirrhosis, and finally hepatocellular carcinoma. recent study suggested that development of hepatic angiogenesis correlates the risk for hepatocarcinogenesis in liver cirrhosis patient. we therefore examined the role of angiogenesis in the hepatocarcinogenesis of nash in both experimental and human study. methods: as an experimental nash model, zucker (z) rats, which naturally develop leptin receptor mutations, and their lean littermate (l) rats were fed a choline-deficient, amino acid-defined (cdaa) diet. in human study, 11 patients with nash-related cirrhosis or pre-cirrhosis, regarded as high risk group of hepatocarcinogenesis, and 13 with simple fatty liver (fl) were enrolled and underwent clinico-pathological examinations. immunohistochemical analysis of 4-hydroxy-2-noneal (4-hne) and cd34 were employed for detection of reacrive oxidative stress (ros) and angiogenesis in the liver tissues, respectively. results: in experimental nash model, both groups showed marked steatohepatitis by feeding cdaa diet. in sharp contrast, the development of glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions and hcc could be observed only in the l-rats. the hepatic neovascularization was also significantly increased only in the l-rats. in human study, both nash and fl exerted a marked elevation of ros. in sharp contrast, significant development of hepatic neovascularization was observed only in nash, whereas almost no neovascularization could be observed in fl. conclusion: in conclusion, these results suggested that neovascularization might play a important role in hepatocarcinogenesis in nash. background: paternally expressed gene 10 (peg10), which was an imprinted gene with an active paternal allele but silent maternal allele, was highly expressed in a great majority of hepatocellular carcinoma(hcc). the aim of this study was to generate transgene mice expressing peg10 in the liver under the control of mouse albumin (alb) promoter and study the integration, transcription, expression of peg10 gene in the transgenic mice methods: the linearized 1716bp transgene fragments, which contained alb promoter and structural gene of peg10, were microinjected into fertilized eggs of mice. then manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. all the newborn mice were screened and identified by pcr detecting genomic dna in tail tissue. as the transgene was driven by the alb promoter, we examined its expression in the liver of transgenic mice by rt-pcr and western blotting. results: the transgene fragment was microinjected into the male pronucleus of 3741 fertilized oocytes. the injected eggs were implanted into oviducts of 94 pseudo-pregnant foster mothers, of which 22 mice became pregnant and give birth to 108 offspring. 65 of them died from unknown reason. among the 43 offspring, 3 were identified to carry peg10 cdna as demonstrated by pcr, and peg10 transgene could be expressed successfully in the liver of the established transgenic mice. the ratio of transgene integration were 6.97% (3/43) by pcr. conclusions: the peg10 transgenic mouse model should be valuable for studying the in viro function of this imprinted gene in hcc. background/aims: brivanib alaninate is the l-alanine ester prodrug of bms-540215, an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. it is being developed in treating hepatocellular carcinoma (hcc), a disease highly prevalent in asia-pacific region. this analysis investigated whether bms-540215 exposure was different between asian and non-asian subjects. methods: a population pharmacokinetic (ppk) model was developed with data collected in 125 subjects (78 non-asian, 47 asian) with advanced and metastatic solid tumors (including hcc) from 2 clinical studies. potential effects of the following covariates on model parameters were examined: age, gender, race, and baseline body weight. model-based simulation was performed to examine bms-540125 exposure in asian and non-asian patients following brivanib doses of 800 mg qd (phase iii dose). results: the ppk of bms-540215 was characterized by a 2-compartment model with first-order absorption and elimination. clearance was found to slightly increase with body weight (p<0.01). however, effects of age, gender and race on clearance were not statistically significant. the median of apparent clearance in asian was 9.5% lower than that of non-asians, which was adequately explained by 16% lower body weight in asians. there was substantial overlap in steady-state bms-540215 auc of asian and non-asian patients, simulated based on their observed body weight distributions in these patient groups. conclusions: bms-540215 pk can be adequately described by a linear 2-compartment model; exposures in asian and non-asian subjects are similar following brivanib doses of 800 mg qd. background/aims: hepatic resection is the standard treatment for hepatocellular carcinoma. in some patients with multiple hcc, one-block resection can not be feasible due to either the tumor location or the reserved liver function. in this study, we attempted to analyze the outcome of multiple-site resection or combined resection and rfa in patients with multiple hcc. the prognostic factors for postoperative survival were also investigated. methods: among 507 patients who received resection from january 1996 to august 2006, 58 patients had a radiologically detected multiple hcc. patients with multiple hcc were divided into: group a, patients treated with one-block resection (n=40) and group b, patients with multiple-site resection or combined resection and rfa (n=18). results: in group b, 6 received multiple-site resection and 12 underwent combined resection and rfa. the clinicopathological variables and postoperative complication rate were not significantly different between the two groups. the 5-year disease-free survival rates for group a and b were 24.1% and 18.3%, respectively (p=0.386). the overall survival rates were also not significantly different (36.9% vs. 39.4%, p=0.528). the multivariate analysis revealed that radiological tumor number 3, edmondsons-steiner grade (iii-iv) and indocyanine green retention rate at 15 minutes> 10% were adverse prognostic factors for overall survival. conclusions: active treatments including multiple-site resection and combined resection and rfa showed similar treatment outcomes compared with one-block resection in patients with multiple hcc. the prognosis after treatment was associated with tumor number, tumor grade and icg r15. background: nasopharyngeal carcinoma (npc) is endemic to southern china. mortalities are mostly associated with secondary metastases. novel treatments for npc metastases are thus urgently needed. we aim to test the efficacy of a physiologically stable gold compound, gold (iii) meso-tetraarylporphyrin 1a (gold-1a), in treating intrahepatic npc metastasis in athymic mice. methods: twenty million of c666-1 human npc cells were injected into the livers of athymic mice to induce primary tumors. gold-1a was administrated by intraperitoneal injection. survival times, tumor volumes and degrees of metastasis of the animals were evaluated. intratumoral microvessel density was determined by immunohistochemical staining for cd34. tube formation by ms1 mouse endothelial cells were conducted with an in vitro angiogenesis assay kit. gene expression level was determined by semi-quantitative reverse transcription-polymerase chain reaction. cell proliferation was performed by methylthiazolyldiphenyl-tetrazolium bromide assay. result: gold-1a prolonged the survival and inhibited intrahepatic and lung metastasis of the tumor-bearing animals. the compound induced tumor tissue necrosis and reduced tumor microvessel formation. in in vitro studies, gold-1a inhibited tube formation and proliferation of ms1 cells, and downregulated the expression of stanniocalcin 1 (stc1), which plays roles in angiogenesis. furthermore, our preliminary data showed that overexpression of stc1 in ms1 cells rescued cells from gold-1a-induced death. conclusion: gold-1a is a novel anticancer agent that prolongs survival of the npc metastases-bearing mice. it inhibits intrahepatic and lung metastasis in vivo and inhibits angiogenesis in vitro, in part via downregulation of stc1. tbx3 is a transcriptional repressor that is important for embryonic development. overexpression of tbx3 was found in a large variety of cancers, including breast cancer, ovary cancer, cervical cancer, lung cancer, bladder cancer and liver cancer. tbx3 promote carcinogenesis by bypass cellular senescence via suppression of p14 arf . our resent studies revealed that two key motifs composed of 7 + 3 residues are essential for its transcriptional repression. based on this finding, we designed a set of peptides to block its transcriptional repression activity and tested their antiviral effects. we found that tat-tagged peptides (taps) effectively transduced hepatoma hepg2 and bel7404 cells at almost 100% efficiency and inhibited cell growth in a dose dependent manner. further studies revealed that the tap treated cells underwent up-regulate apoptosis via suppression of p14 arf both at mrna and protein levels, demonstrating the potential of novel taps for anti-hcc treatment in the future. safety and long-term outcomes of radiofrequency ablation therapy in elderly and cirrhotic patients with hepatocellular carcinoma k. kakisaka 1 , h. kuroda 1 , k. kasai 1 , y. takikawa 1 , k. suzuki 1 1 iwate medical university background and purpose: a tendency of the aging in patients with hepatocellular carcinoma (hcc) is predominantly seen in japan. in fact, the mean age of patients with hcc in our institute in 1990 was 60.1 years old, while that in 1999 was 67.8 years old. it is not still remained whether the percutaneous radiofrequency ablation (rfa) therapy in elder patients with hcc is safety and equal in therapeutic usefulness compared to the non-elder patients with hcc. subjects and methods: two hundred six cirrhotic patients with hcc (376 tumor nodules) received rfa therapy curative intent since august, 2006 were enrolled. we divided all patients into two groups: over 65 years (elder group: n=135) and under 65 years (non-elder group: n=71), and compared the patient's characteristics, tumor factors and survival rate and causes of death in two groups. results: the characteristics of patients, tumor factors, cumulative survival rate and recurrence rate were not revealed in two groups. although in elder group two patients complicated aspiration pneumonia and respiratory depression due to sedation under rfa respectively, total occurrence rate of complications did not differ between two groups. conclusion: rfa therapy is safety and effective even in elder patients with hcc, although their care is necessary to prevent any complications which are often occurred during the rfa therapy. background and purpose: the aim of this study is to evaluate whether administration of the branched-chain amino acid (bcaa) enriched nutrient (namely, aminoleban en, ostuka pharmaceutical company, japan) might improve protein-energy malnutrition (pem) status and quality of life (qol) in cirrhotic patients with hcc receiving rfa therapy. subjects and methods: thirty-five cirrhotic patients with hcc who had received rfa therapy from october 2005 to october 2006 in our institute were randomized into two groups: diet with supplementation of aminoleban en (en group: 20 patients, 420 kcal/day) and diet only (control group; 15 patients). the total intakes of calories (30-35 kcal/kg) and protein (1.0-1.5 g/kg) were equal between tow groups. the primary end point was event-free survival rate (development of liver cancer, rupture of esophageal varices, or progression of hepatic failure) and second end points were serum albumin levels and the health-related qol by shortform-8 questionnaire (sf-8). results: total intakes of calories and protein were similar during the one year after rfa. no significant differences in event-free survival rate were seen between two groups. however, decreased serum albumin levels and one (general health perception) of domains in sf-8 were significantly improved in en group compared to the control group. conclusion: supplementation of bcaa-enriched nutrient may improve the impaired liver function and qol after rfa therapy. large scale prospective study should conduct to confirm these results near the future. backgrounds and aims to investigate the effects of selective cox-1 and cox-2 inhibitor on proliferation and apoptosis of hcc cell. methods hep3b and snu 387 cells were treated with ns-398 and sc-560. mtt assay, caspase 3/7 activity assay and tunel assay were performed. cox protein and mrna expression were measured by western blot and real time rt-pcr. results in hep3b cell line, cox-1, cox-2 (50, 100, 200 um) and combination (25+25, 50+50, 100+100 um) treatment after 24hr showed a significant dose dependent inhibitory effect on cell growth (p<0.05). cox-1, cox-2 (100 um) and combination (50+50, 100+100 um) treatment after 24hr significantly increased caspase 3/7 activity (p<0.05) and induced apoptosis (p <0.05). however, the combination treatment could not showed a additive effect to cox-1 or cox-2 inhibitor (p>0.05). in snu 387 cell line, cox-1 inhibitor and combination treatment showed a inhibitory effect on cell growth (p <0.05) similar to hep3b cell line but any of treatment could not induce apoptosis significantly (p >0.05). in cox protein and mrna expression, snu 387 cell line showed significant cox-1 predominency (p=0.037) but hep3b cell line showed cox-2 predominency (p=0.032). conclusions in hcc cells, no additive effect of the combination treatment of cox-1 and cox-2 inhibitors could be anticipated. the apoptosis inducing effect of cox inhibitor could be different between hcc cell lines. more studies for the mechanism of different response to cox inhibitor between cell lines is needed. background: the aim of this study was to determine the maximum tolerated dose and recommended dose of combination chemotherapy with mitoxantrone and uracil/tegafur (uft) (phase i part), and to clarify its efficacy (tumor response, overall survival, and progression free survival) and safety in patients with advanced hepatocellular carcinoma (hcc) at the recommended dose (phase ii part). methods: patients eligible for study had histologically confirmed, chemo-naïve advanced hcc, who were unsuitable for resection, local ablation therapy or transcatheter arterial chemoembolization. the therapy consisted of mitoxantrone dosages (6, 8 and 10 mg/m 2 /day) intravenously on day1 and oral administration of uft 300 mg/m 2 on day 1 through day 21. the treatment was repeated every four weeks if there was no evidence of tumor progression or unacceptable toxicity. results: a total of 25 patients were entered into the study. all had a good ecog performance status score of 0-1. in phase i part, dose limiting toxicities occurred in all three patients (two patients: grade 4 neutropenia, one patient: grade 3 creatinine elevation) given mitoxantrone at dosage of 10 mg/m 2 /day, and the recommended mitoxantrone dosage was 8 mg/m 2 /day. among 19 patients administered at the recommended dosage, one patient (5.3%) achieved a partial response, 8 patients (42.1%) had stable disease and 10 patients (52.6%) had progressive disease. one-year survival proportion, median survival and median progression free survival were 26.3%, 8.4 months and 2.5 months, respectively. the most common toxicities were grade 3-4 leucopenia (15.4%) and neutropenia(10.3%). conclusion: mitoxantrone 8 mg/m 2 with uft 300 mg/m 2 /day is recommended dose. this regimen is generally well tolerated, but appears to have little activity for advanced hcc. these findings do not support its use in practice, and further trials with this regimen in patients with advanced hcc are not recommended. the study assessed the benefits of 3-d reconstruction of spiral ct scans for the diagnosis of and surgical guidance to large liver tumors or tumors at the hepatic hilum. we retrospectively analyzed 33 cases of children with such tumors treated in past 5 years.the patients were examined by 3-d reconstruction using 64 slice spiral ct. in 28 cases, the volume of tissue removed exceeded 1/3 the entire volume of the liver. in 5 cases, the excised tissue represented less than 1/3 of the total liver volume, but the location of the tumor was adjacent to major hepatic vessels. pathological diagnoses included hepatoblastoma (n = 20), hepatocellular carcinoma (n = 4), mesenchymal hamartoma (n = 4), teratoma (n = 1) and adenoma (n = 4). all children had curative resections with tumor-free microscopic margins. 3-d ct imaging can provide high quality images and accurate location of the tumors. it could help the surgeon identify the tumor borders accurately and devise a safe surgical strategy. with its help the surgeon could identify vital hepatic blood vessels before operation, and can avoid massive hemorrhaging during operation. background: to investigate the association between c-509t polymorphism of transforming growth factor (tgf)-1 gene and hbv-related hepatocellular carcinoma (hcc). methods: patients with hbv infection (196 cases were hbv carriers, 379 cases were hcc) and 299 healthy volunteers were enrolled. the polymorphism of tgf-1 gene c-509t was identified by polymerase chain reaction-restriction fragment length polymorphism method. the concentrations of plasma tgf-1 were measured by enzyme linked immunosorbent assay (elisa). tgf-1 mrna expression was quantified by real-time pcr. a recombinant construct containing -509c>t variant as promoter and cat as reported gene was transfected into hepg2 cells. the reporter gene cat was detected with elisa. results: the ct genotype at position -509 of tgf-1 gene prevailed in all three groups, the frequency of genotype cc and allele c at -509 in hcc were significantly higher than those of the hbv carriers and controls. the plasma tgf-1 concentration among the three genotypes did not show any significant difference in three groups. however, both the tgf-1concentration and liver mrna levels were statistically higher in patients with cc genotype than in those with tt genotype in the hcc group. reporter gene cat was elevated when hepg2 were transfected with -509c-cat recombinant construct compared to that with -509t-cat one (p<0.05) conclusion: the presence of c allele at position -509 may play an important role in the development of hbv-related hcc through influencing tgf-1 expression both at mrna level and protein level. background: to assess diagnostic value of n-glycan markers in identifying hepatocelluar carcinoma (hcc) from liver fibrosis after hbv infection. methods: a total of 273 cases of hbv related liver fibrosis (n=128) and hcc (n=145) patients as well as matched healthy controls (n=120) were recruited. routine liver function and tumor markers were detected by automatic biochemistry or immunological analyzer. n-glycome of serum protein was profiled by dna sequencer-assisted fluorophore-assisted carbohydrate electrophoresis with a capillary electrophoresis-based abi3130 sequencer. results: the abuncance of a single agalacto biantennary glycan (ng1a2f, peak 4) was increased in liver fibrosis and decreased in hcc, while that of a branching triantennary glycan (na3fb, peak 9) was decreased in fibrosis and increased in hcc. the efficacy of the log ratio of above two n-glycan abundance [log (p9/4)] was similar to afp in differentiation hcc from fibrotic patients. with logistic regression analysis, the accuracy and sensitivity of the diagnostic model combining afp with n-glycan analysis(cscore b) were increased 7-10% compared to afp. log(p9/4) was even more powerful in monitoring the progresison of hcc with the specificity improved 16% and accuracy improved 8% compared to that of afp. besides, log(peak 9/4) was correlated well with other tumor markers and tnm stages. conclusions: the log ration of the abundance of a branching triantennary glycan (na3fb, peak 9) to a single agalacto biantennary glycan (ng1a2f, peak 4) and the model combining afp with n-glycome markers are promising in hcc diagnosis and progression monitoring. the low incidence of tumor seeding and post-procedure bleeding after radiofrequency ablation (rfa) of hepatic tumors has been attributed to the use of thermocoagulation of the tract, which results in necrosis, upon electrode withdrawal. however, different investigators use different techniques with no experimental evidence of the effectiveness of a particular technique. objective: we aimed to compare the necrotic zone produced using different electrode withdrawal techniques. methods: eighteen tract ablation zones were created in ex vivo porcine livers by withdrawing an internally-cooled rfa electrode (cool-tip radiofrequency system, valleylab) 1-2 mm/second using energy-dependent (20 vs.40 vs.60 vs.80 watts) and temperature-dependent (80 vs. 90 0 c) techniques. horizontal mathematical modeling suggests an impractical number of radiofrequency ablation (rfa) zones needed in order to ablate a medium-large hepatic tumor. however, overlapping rfa zones may increase the necrotic diameter disproportionately to that deduced from single ablation alone. objectives: to compare the necrotic diameter in single (group1), dual overlapping (group2) and dual non-overlapping (group3) ablation. methods: single (n=5) and dual (overlapping n=18; non-overlapping n=6) ablation zones were created in ex vivo porcine livers using cool-tip rfa electrodes. necrotic diameter was measured at the midpoint (maxd) of the single and the two distinct rfa zones of the dual ablation groups and compared with the necrotic diameter at the tip of the second ablation (maxd-o), corresponding to the point of overlap in group2. the rfa electrode was withdrawn 2.5 and 4.1 cm before re-ablating for group2 and group3, respectively. results: despite no difference in end-rfa temperature between 3 groups (group1=75.6+7.4cvs.group2=73.9+7.3cvs.group3=74.8+4.8c; p=0.873), maxd was significantly greater (p=0.011) in group2 (4.1+0.7cm) as compared to group1 (3.3+0.6cm) and group3 (3.3+0.4cm), with no difference between group1 and group3(p=1.00). further proof of synergism between two overlapping ablations is that the maxd-o in group2(4.3+0.6cm) was larger than maxd of group1 (p=0.042) and group3 (p=0.028), and was similar to maxd of group2 (p=1.00). conclusions: overlapping two rfa zones results in incremental increase in necrotic diameter compared to single and dual non-overlapping ablation. this may explain the discrepancy in the number of ablation zones needed between clinical and mathematical modeling studies. background: hepatocellular carcinoma (hcc) is the fourth most common cancer worldwide, main etiological factors being chronic infections with hepatitis b and c viruses. the present study was undertaken to evaluate the association of glutathione-s-transferase (gst) t1 and m1 null genotypes and microsomal epoxide hydrolas e(mephx) polymorphisms with hepatitis virus related hcc risk in indian population. subjects and methods: three groups of subjects were considered viz. control (n=169), chronic viral hepatitis (n=174) and hcc (n=63). pcr-rflp was used for this polymorphic study. genotype distributions between categories were compared using the 2 test; odds ratios (ors) and 95% ci were calculated to express the relative risk. results: presence of gstm1 null genotype significantly (p<0.05) decreased the risk for hcc development among chronic viral hepatitis subjects. however, gstt1 null genotype was associated with an increased risk for hcc by 2.23 and 1.42 times among control and hepatitis subjects respectively. in case of mephx, tyr113his and his113his genotypes significantly (p< 0.05) reduced the risk of hcc development in both viral hepatitis and control subjects. in case of mephx exon 4 genotypes, arg139arg imposed an approximate 2 fold risk for hcc development in the two groups. combination of heterozygous mutant genotypes at mephx exons 3 & 4 also imposed around 2 fold risk (non-significant) for hcc. conclusions: polymorphic forms of gst and mephx share an association with viral related hcc risk in indian population and should be further evaluated as the candidate genes to determine individual susceptibility for viral related hcc. background : the association between type 2 diabetes mellitus (dm2) and hepatocarcinoma (hcc) has been identified in the last ten years. methods: to clarify the temporal relationship between dm2 and hcc and the possible effects of antidiabetic therapy on hcc risk, we recruited 465 patients with hcc compared with 490 control subjects without liver diseases and 618 cirrhotic patients. results: prevalence of dm2 was 31.2% in hcc, 23.3% in cirrhotic and 12.7% in control group. in univariate and multivariate analysis, the odds ratio (or) for hcc in diabetic patients were respectively 3.12 (ci 2.2-4.4; p <0.001) and 2.2 (ci 1.2-4.4; p= 0.01). or in univariate analysis were higher in male than in female patients. in 84.9% of the patients dm2 pre-exists the diagnosis of hcc from a mean time of 141.5 months. moreover, the insulin treatment was more frequent in diabetic hcc patients than controls and we report an or for hcc of 2.99 (ci 1.34-6.65; p= 0.007) in patients treated with insulin or sulfonylureas, and an or of 0.33 (ci 0.1-0.7; p= 0.006) in patients treated with metformin. conclusion: our study confirms that male patients with type 2 diabetes mellitus have a significantly increased risk of hcc independently of other cofactor such as hbv, hcv and alcoholic abuse. dm2 is a pre-existing disease in most hcc patients and suggests that insulin and sulphonylurea treatments in dm2 are associated with an increased risk of hcc development, while metformin may have a protective effect. background & aims: over the last few years, techniques that allow systematic analysis of chromosome aberrations at a genome-wide level were applied to hcc. the purpose of this study is to apply gene loss expression profiling in the attempt to discover new related genomic regions not revealed by loh or cgh, and search the new tumor suppression genes for hcc. methods: primary hcc and corresponding non-tumor liver tissues were obtained from surgery. serologically, 13 cases were with hepatitis b virus infection and 12 cases were with hepatitis c virus infection. four non-viral infected tissues from four patients receiving surgical resection for hepatic adenoma or focal nodular hyperplasia.affymetrix genechip, u133a, was used to compare the loss and gained gene expression in liver needle biopsy samples (n=54). results: after adjusting by chromosome arm length, 16p, 17p, 19p, 19q and 22q showed higher gene loss-expression ratio (>= 10 loss / cm) in the comparison between normal samples and tumor samples; 1q, 2p and 4q showed higher gene loss-expression ratio in the comparison between tumor and non-tumor tissues. more than 50 genes showed different loss expression level in this study. for example, cd10 was loss expression in all non-tumor samples comparing to four normal samples. ficolin 3 and ficolin 2 were loss expression in hcc samples with hbv infection and with hcv infection, respectively. conclusion: our results revealed the potential tumor suppression genes and the genomic region they harbored. further study is needed to validate the observation. background/aims: hepatocellular carcinoma is common malignancy in human, accounting for 1 million deaths in the world annually. caspase 8, as an initiator caspase, is involved in the induction of apoptosis. survivin, a novel inhibitor of apoptosis is related to the ability to inhibit caspases and involved in critical steps of onset and progression of hcc with unfavorable prognosis. methods: to explore the possibility that the epigenetic alteration of caspase 8 and survivin genes is implicated in the development and progression of hcc, promoter methylation of two genes was analyzed in 73 cases of primary hcc by methylation specific pcr. the relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathologic parameters was also investigated. results: the methylation of caspase 8 (34.2%, 25/73) and survivin (32.9%, 24/73) demonstrated a negative correlation with immunohistochemical expression of capsase 8 (47.9%, 35/73) and survivin (43.8%, 32/73) (p=0.042 and p=0.001 respectively). methylation of caspase 8 and immunohistochemical expression of its gene product was significantly correlated with apoptosis (p=0.026 and p=0.032). survivin nuclear immunoreativity revealed significantly correlated with proliferative activity of tumor cells (p=0.001). by survivial analysis, the negative caspase 8 expression and positive survivin expression showed worse prognosis in hcc, that was statistically insignificant (p>0.05). conclusion: in conclusion, caspase 8 and survivin may contribute an important regulatory mechanism for tumor cell proliferation and apoptosis, and may be prognostic predictors in hcc. injection was recently reported to be effective against hcc with pvi, though the therapy is not always applicable for the patients with arterial abnormality. therefore we tried combination therapy of transcatheter arterial cisplatin embolization and radiation, and will report the effectiveness and toxicity of the therapy. methods: the combined therapy was conducted in 7 hcc patients with pvi. transcatheter arterial embolization with 1mg/kg cisplatin powder (ia call) was performed against intralobar lesions, followed by external radiation targeted for pvi (60gy in 2 gy fractions). the following variables were evaluated with the survival rate: gender, age, viral etiology, child's class, performance status, and location of pvi. results: one (16%) patient showed complete response and another two (33%) partial response. two (33%) showed no change, and one (16%) showed progress of disease. the survival rates at six months among overall patients were 85.7%. adverse events were limited to nausea and appetite loss. one of the patients with partial response underwent curative resection, and is still alive without any recurrence for 530 days. conclusions: the combination therapy of cisplatin embolization and radiation is safe, effective and also feasible to the patients with arterial abnormality. this therapy is suggested to be a useful alternative therapy for the patients with extensive pvi. recently, the injection port has been used for hepatic arterial infusion chemotherapy (hai) in japan. hai is usually used for the treatment of multifocal bilobar tumors of the liver or hccs combined with portal vein tumor thrombosis (pvtt), not amenable to tace. this study examined the efficacy and toxicity of repeated hepatic hai using lipiodol suspension mixed with cisplatin powder. methods: from april 2005 to september 2008, 20 patients with inoperable advanced hcc were enrolled in this study. all received cisplatin powder (50mg) and lipiodol (2ml) suspension, with an intervening 4 weeks interval. the drugs were delivered from an injection port. 13 patients had hcc with pvtt, and 7 had hcc without pvtt. 11 patients with liver function of child grade a, 7 of grade b, and 2 of grade c were enrolled. result: the mean number of hai given during the follow-up period was 4.1 times. we found complete response in 1 case, partial responses in 6, no change in 6, and progressive disease in 7. the overall response rate was 35.0%. the 1-year survival rate was 36.7% and the 2-year survival rate was 12.1%. although 3 patients had cisplatin-induced anaphylaxis, no severe adverse events (hepatic failure and renal failure) were observed. conclusion: chemo-lipiodolization using cisplatin powder delivered via an injection port provides some clinical benefits without severe adverse events in patients with far advanced hcc. background: recently, the antitumor efficacy of angiogenesis inhibitors is expected in the treatment to hepatocellular carcinoma. the gene expression relevant to the vascularization, which is a target of these inhibitors, has a difference according to each case and it is thought that it influences the therapeutic effect of them. however, there are still few reports of mrna expression of vascular endothelial growth factor (vegf) receptors in hcc. methods: the relative mrna level of vegf and its receptors (kdr and flt-1) was analyzed using quantitative rt-pcr in 51 patients with hcc. matched samples of hcc (t) and non-tumor liver tissue (nt) were obtained by fine needle (21gauge) biopsy. results: gene expression level of vegf and flt-1 was significantly higher in hcc than nt (vegf; p<0.001, flt-1; p<0.001). according to the clinicopathological findings, gene expression level of vegf and kdr in hcc was significantly high in hypervascular hcc compared to hypovascular hcc (vegf; p=0.002, kdr; p=0.042). additionally, flt-1 tended to be expressed higher in hypervascular hcc than hypovascular hcc (p=0.09). moreover, gene expression level of vegf, kdr and flt-1 tended to be higher in advanced-stage hcc than early-stage hcc. conclusion: not only vegf but kdr and flt-1 were highly expressed in hypervascular and advanced hcc. aims: fibrinogen-like protein 2/fibroleukin (fgl2) has been reported to play a vital role in the pathogenesis in mhv-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo-and xeno-graft rejection by mediating "immune coagulation". fgl2 functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. therefore, this study was designed to examine the role of fgl2 in tumor development. methods: tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (hcc) model on nude mice (established from high metastasis hcc cell line mhcc97lm6) were obtained. results: hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human hcc nude mice. hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, nk cells, and cd8 + t lymphocytes and vascular endothelial cells. hfgl2 mrna was localized in cells that expressed hfgl2 protein. fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. in vitro, il-2 and ifn-increased hfgl2 mrna by 10-100 folds and protein expression in both thp-1 and huvec cell lines. one-stage clotting assays demonstrated thp-1 and huvec cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. conclusion: the hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction. . the therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least 2 cycles, when responses to treatment were evaluated by eastern cooperative oncology group criteria. results: of 146 patients treated (male, 79%; median age, 64 years), 64% had child-pugh a, and 31% had b. 90% had either metastasis or vascular invasion. 71% had metastasis and 49% had vascular invasion. on the basis of independent assessment, three (2.1%) patients achieved a complete response, thirteen (8.9%) had a partial response, and 42 (28.8%) had stable disease. there was no grade 3/4 drug related toxicities. median overall survival was 6.9 months. conclusion: combination therapy of ifn +5-fu has modest efficacy in hcc. background: amt is a mixture of approved pharmaceuticals in low therapeutic doses (human insulin and chlorpheniramine) and herbal components (aqueous camomile extract). preclinical and phase i data in healthy volunteers showed a favourable safety profile for amt. this pilot study should examine efficacy and safety of amt in the patients with advanced hepatocellular carcinoma (hcc). methods: thirteen patients with advanced hcc (tnm stage iii-iv), who did not respond to existing therapy, were treated with i.m. amt at 0.066 ml/kg up to a maximum volume of 5 ml twice daily for 1-10 months. primary study objectives: clinical benefit response (cbr). secondary objectives: safety of amt, tumor response according to who-recist criteria, quality of life (qol) and iimmunomodulatory effects. the effects were evaluated by cytokine production of pbmcs before and after the treatment. results: there were no significant safety issues. four and 3 patients showed positive and stable responses for cbr, respectively. tumor response was 1 pr, 6 sd and 6 pd. even in the patients with pd, 2 and 1 patients showed positive and stable responses for cbr. qol data showed clear improvement. immune monitoring demonstrated effects of amt on the functional immune parameters in about half of patients. in the patients with pr, histological examination showed tumor necrosis and many lymphocytes including plasmacytes infiltrating in the tumor. conclusion: these results suggest that a promising rate of patients with advanced hcc respond clinically to the amt treatment without significant safety issue and amt has some immuno modulatory capacities. background/aims: dysplastic nodules are important due to premalignant potential. the aim of this study was to evaluate the electron microscopic findings of liver dysplastic nodule in patients with liver cirrhosis. methods: a total of 5 patients (mean age: 64±9 years old, male 4) with dysplastic nodules which suspected as malignant nodule (mean size 1.38±0.22 cm) was enrolled from 48 cases of liver cirrhosis undergone ultrasonography-guided biopsy from december 2006 to january 2008. the etiologies of liver cirrhosis were as follows; alcohol (1 patient), hepatitis b virus (2), and hepatitis c virus (2). results: hepatocytes showed rosette formation of regenerative hepatocyte or degeneration. the nucleus was round or oval shaped and the nucleus membrane was irregular. the nucleolus was prominent and clear, the mitochondria were crowded to one side in the cytoplasm with megamitochondria. glycogen granules and lipofuscin pigments were abundant. sinusoid formation was poorly developed and collagen fiber bundles were increased. the hepatocytes of rosette formation and bile ductules cell made of canal of hering, which was dilated and microvilli was decreased. the number of canal of hering was 7, which was composed of 1.7±0.8 with hepatocyte and 2.1±0.7 with bile ductule cell, respectively. there was no oval cell in the canal of hering, which was relatively well developed. schwann cells were clustered together in nerve plexus. therefore, these electron microscopic findings showed that dysplastic nodule was similar to early hepatocellular carcinoma. conclusions: this study showed that dysplastic nodule in liver cirrhosis is nearly identified to early hepatocellular carcinoma. dcp is an important risk factor for recurrence after radiofrequency ablation of single hepatocellular carcinoma -< 3cm in diameter r. kuromatsu 1 , a. takata 1 , n. fukushima 1 , s. sumie 1 , m. nakano 1 1 division of gastroenterology, department of medicine, kurume university school of medicine background and aims: the aim is to analyze the risk factors for local recurrence + intrahepatic metastasis after radiofrequency ablation (rfa) and hepatic resection (hr) for single hepatocellular carcinoma (hcc) < 3cm in diameter. methods: between 1999 and 2007, 219 patients with single nodule < 3cm in diameter and child-pugh grade a were treated by hr and rfa, and recurrence rate and survival rate using kaplan-meier method, and important risk factors for recurrence using cox's proportional-hazards regression model were analyzed. factors used for multivariate analyses were age, gender, viral marker, tumor diameter, afp, afp-l3, dcp, and platelet count. results: mean age was 66 years old, m/f ratio was 136/83, hr/rfa was 59/160, and mean observation period was 1194 days. five-year survival rates, and 2-year local recurrence-free + intrahepatic metastasis-free rates were not significant between hr group and rfa group (82/72%, 92/89%). in rfa group, the only independent risk factor for local recurrence-free + intrahepatic metastasis-free survival was dcp (p=0.0034). tumor diameter was not significant for recurrence. in hr group, there was no risk factor for recurrence. in pathological analyses of hr group, dcp had a tendency to associate with microvascular invasion (p=0.065). conclusions: rfa was effective for hcc < 3cm in diameter and dcp < 40 mau/ml. hepatic resection should be selected for single hcc with dcp > 40 mau/ml even though hcc <3cm in diameter. background: radiofrequency ablation (rfa) is now a common treatment for small hepatocellular carcinoma (hcc). however, critical complications after rfa such as rapid intrahepatic dissemination have been reported. in this study, we investigated the method how to estimate the malignant potential of small hcc by dynamic ct before rfa. methods and results: firstly, 61 hccs less than 3cm in diameter were analyzed. those tissues were classified into 4 groups as followed, small nodular type with indistinct margin (type e), simple nodular type (type 1), simple nodular type with extranodular growth (type 2), confluent multinodular type (type 3). in the type 2 and 3 groups, portal invasion over vp1 were observed more frequently than those in the type 1 and e groups. at the next step, these 61 hccs were classified into above-mentioned 4 types by two radiologists according to the shape of early stain or defect of delay phase of dynamic ct before operation. the accorded rate was 87% between those classifications. next, 45 patients, which had solitary hcc less than 3cm in diameter and treated with rfa, were classified into those 4 types by dynamic ct before rfa. the recurrence rate and prognosis of those patients were examined. in the type 2 and 3 groups, the recurrence rate was higher and significant worse prognosis was showed than those in type 1 and e group . conclusion: it was suggested that hcc with type 2 and 3 might process higher malignant potential and rfa should be carefully performed on those types of hcc. background/aims: hypoxia-inducible factor-1 (hif-1 ) is the central transcriptional factor in the cellular response related to various aspects of cancer biology, including proliferation, survival, angiogenesis, and extracellular matrix metabolism to hypoxia. il-8 became known to replace hif-1 functions in the other cancer cell lines. the aim of this study was to evaluate whether il-8 may induce angiogenic factors without hif-1 by inflammation signal of hypoxic condition. methods: hif-1 knockdown cell lines of hcc (huh7 and hepg2) were constructed by rna interference tools, and cultured under normoxia (21%o2, 24 hours) and hypoxia (1%o2, 24 hours) conditions. following transfection, the amounts of hif-1 , il-8, angiogenic factors and matrix metalloproteinase (mmp) were examined using rt-pcr and western blotting, respectively. results: the expression of hif-1 , angiogenic factors, mmp, il-8 was markedly enhanced in wild types that were cultured under hypoxia, and the hypoxic induction of angiogenic factors and mmp was partially blocked in hif-1 knockdown hcc cell lines. nf-b inhibitor suppressed angiogenic effects by blocking il-8 activity. conclusion: these data suggest that il-8 induced tumor angiogenic factors in hif-1 knockdown hcc cell lines. background there were some reports that liver caner related to the levele of sera hbvdna our research focused on the relationship between the quantity of hepatocellular hbv cccdna , tdna and liver cancer. methods the samples included the liver tissue of 24 chb patients (chb group) and the para-liver cancer tissue of 26 primary liver cancer patients (phc group) the quantity of hepatocellular hbvcccdna, tdna were assayed by fq-pcr in both groups. result: the quantity of hepatocellular hbv cccdna in chb group was 11.56±16.14 copys/cell higher than phc group(3.96±9.27 copys/cell), p=0.011; the quantity of hepatocellular hbv tdna in chb group was 57.38±91.64 copys/cell higher than that of phc group(35.07±93.03 copys/cell),p=0.13.; conclusion: the quantity of hepatocellular hbvcccdna, tdna can not be used as predictors of liver cancer for hepatitis b patients. hepatic cancer predominantly occurs in males. this is almost a commonsense to most of us. but the detailed mechanisms underlying such phenomenon are still not well-known. the average age of liver cancer patients are about 30-40 years old. so most female patients have udergone pregnancy at least one time. pregnancy is a very important event before or during the development of liver cancer in females. in this special period, not only sex hormones secrete in a strange manner, but also immune system functions in a special module which is very different from normal. so it is urgent to investigate the impact of process of pregancy on the development of hepatic cancer. in this study, 100 female sd rats are randomly divided into two groups: pregance group and controll group. rats in both groups are injected iv diethylnitrosomine(a chemical carcinogen). in pregnance group, rats are raised together with male rats in 3:1 ratio(3 female, 1 male) to make every rats undergo pregnancy. while in controll group, rats are coupled with spermaduct-ligated male rats. the size and amount of hepatic cancers in pregancy group are smaller and less than those in controll group. the survial rate is also significantly higer than that in controll group. we conclude that the process of pregnancy exerts an inhibitory role in the development of chemical induced hepatic cancer in rats. acknowledgement: this project was sponsed by the national natural science foundation of china. the number of the grant is 30600727: the use of alpha-fetoprotein measurement in detection of recurrent hepatocellular carcinoma after living donor liver transplantation n. yamashiki 1,2 , y. sugawara 1,3 , s. tamura 3 , r. tateishi 2 , h. yoshida 2 , j. kaneko 3 , y. matsui 3 , n. kokudo 1,3 , m. omata 2 1 organ transplantation service, 2 department of gastroenterology, university of tokyo, 3 department of surgery, university of tokyo background: the recurrent hepatocellular carcinoma (rhcc) after liver transplantation (lt) can occur in 10 to 20 % of transplant recipients despite with a careful patient selection. for the surveillance of rhcc, frequent measurement of alpha-fetoprotein (afp) and annual ct scan is commonly used. however, the usefulness of afp is not clear. we report the update of our experience using our surveillance protocol. methods: between 1996 and march 2008, 330 adult living donor lt were performed at the university of tokyo. among them, 100 recipients with hcc in their explanted liver were subjected to analysis. we used monthly measurement of afp and des-gamma carboxy prothrombin (dcp) with annual dynamic ct scan. results: 83 met milan criteria pre-operatively and 12 did not. 5 were incidental hcc. rhcc was experienced in 9 patients at 10 (2-24) months after lt. recurrence sites were graft (1), lung (2), bone (3), and multiple organs (3). rhcc was first suspected from elevation of tumor markers in 8; afp in 4, dcp in 2, and both afp and dcp in 2. rhcc was confirmed with ct scan (7) or mri (2) in 4 (0-6) months after the first sign of rhcc. when the cutoff level of afp>20ng/ml was used, the sensitivity and specificity for rhcc were 66% and 100%. six cases were treated surgically of which two achieving prolonged survival. conclusions: although the confirmation of the rhcc sites required multiple imaging studies, afp measurement was useful as for the first sign of rhcc. purpose: to evaluate the therapeutic effect of heated (60 c) lipiodol via hepatic artery administration in vx2 rabbit liver cancer model. materials and methods: thirty male new zealand white rabbits were randomly divided into 3 groups with 10 rabbits for each group. vx2 carcinoma cells were surgically implanted into the left liver lobe. the tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of tumors detected by ultrasonograph reaching 2 to 3 cm. under the anaesthesia, transcatheter hepatic arterial embolization was performed and doxorubicin-lipiodol (37ºc) (1 ml), lipiodol (60ºc) (1 ml) and control (physiological saline (37ºc) (1 ml)) were injected into hepatic artery of the 3 different groups. one week later, the volume of tumor was measured by ultrasonograph again. the serum of all rabbits was collected before injection and at 4 and 7 days after injection and the level of aspartate aminotransferase (ast) was checked. the survival period of 3 groups of rabbits after treatment was also recorded. during the last course of their disease, the rabbits were given some analgetics to relieve suffering. results: the tumors' growth rate in lipiodol (60ºc) background/aims: hepatocellular carcinoma (hcc) is one of the male-dominant cancers, and hepatitis c virus (hcv) is one of the causes of hcc. it was reported that androgen receptor (ar) is expressed in hcc and its surrounding tissues. androgen signaling and ar may be involved in hepatocarcinogenesis. in this study, we investigated whether hcv interacts with androgen signaling in human hepatocytes. methods: hcv protein expression vectors were co-transfected with ar-expression vectors and ar-responsive element-driven reporter vector into immortalized human hepatocytes (ihhs) and human hepatoma cell lines. kinase inhibitors were used to examine the activation of the akt, mapk, and jak/stat3 pathways. real-time pcr and western blotting were performed. cell culture grown hcv (hcvcc) were also used, and angiogenesis was evaluated by tubule formation assays in human coronary microvascular endothelial cells in the presence of 5 -androgen-1 -ol-3-one. results: hcv enhances ar-responsive gene expression in the presence of androgen. hcv core protein has the strongest effects and induced ar activation associated with jak/stat signaling. hcvcc enhances vegf mrna expression and angiogenesis. conclusions: hcv core protein is an enhancer in androgen signaling and can be expected to play an important role in hcv-related hepatocarcinogenesis. background: to evaluate the therapeutic outcomes and the toxicity of the combination of arsenic trioxide and the chinese traditional jianpiliqi (jplq) formula in the treatment of advanced hepatocellular carcinoma (hcc). methods: patients with advanced hcc, not suitable for resection but with normal major organ functions, were enrolled to receive a therapeutic regimen consisting of intravenous arsenic trioxide (6 mg / m 2 ) administration from days 1-14, and an oral administration of jplq formula twice daily from days 1-28. each cycle was composed of 28 days and treatment could expand up to 4 cycles before evidences of intolerable toxicity or disease progression. result: one patient had partial response, one had minor response, 15 showed stable disease and 15 (46.9%) had disease progression. total disease control rate was 53.1%, median survival time was 5.8 months (2-23.9 ms), and time to progression was 4.2 months (1-16.9 months). the incidences of grade 1-3 abdominal distention and nausea/vomiting were 65.6% and 37.5%, respectively. increases in ggt occurred in 4 patients (2 grade 2, 1 grade 3, and 1 grade 4) and increases in serum creatinine in 2 patients (1 grade 3 and 1 grade 4), respectively. conclusion: compared with the single arsenic trioxide treatment reported in past literature, treatment by arsenic trioxide combined with jplq showed modestly higher anti-tumor activity and tolerable toxicity in patients with advanced hcc; its manageable toxicity and increased tumor response rate may offer a better treatment regimen, and deserve further investigation. aim: to investigate the effect of osteopontin (opn) expressions down-regulated by rna interference (rnai) on the invasion and metastasis of human hepatocelluar carcinoma (hcc). methods: hcc cell line (hcc-lm3) was transfected with the chemically synthesized small interfering rna (sirna) in study arm and with non-specific sirna in control arm. real-time pcr and western blotting were used to quantify the mrna and opn protein levels. the malignant phenotypes including cellular growth rates, colony formation and matrigel invasion activities of the hcc cell line were analyzed. results: in study arm opn mrna expressions decreased 75% and opn protein decreased 80% compared to those of blank arm. the number of formed colonies and migrating numbers of the cells in vitro decreased significantly (64.6% and 59.6% respectively) in study arm compared to these of blank controls (p<0.05). the parameters in the control arm did not differ from those of the blank arm (p>0.05). conclusion: the specific sirna was able to reduce opn expressions at both the mrna and protein levels and significantly diminished the invasiveness of hcc cells. methods: the expressions of mif and vegf in hcc and adjacent tissues were detected from 4 patients. specific sirna targeting mif gene was synthesized, and transfected into the hcc cell lines (plc and hepg2) in study group and non-specific sirna was used in controls. the mrna and protein expressions of mif and vegf were examined by pcr and western blot. results: mif and vegf mrnas were overexpressed in the hcc tissues compared with adjacent tissues (rq=8.91±1.85 and 3.00±0.86, p 0.01). the mrna and protein expressions of mif and vegf of hcc cell lines significantly decreased in study group compared with controls (p 0.01). vegf mrna levels decreased 75.6%±2.6%; 79.8%±1.2% in plc, and 73.6%±4.6%; 80.7%±2.2% in hepg2 cells when disposed with sirna 50nm and 100nm. vegf protein levels also significantly reduced in study group p 0.01 . conclusions mif and vegf mrnas were overexpressed in the hcc tissues in vivo, and mif sirna was able to knock down the expressions of mif and vegf in hcc cell lines in vitro. y.y. li, y.c. zhang, y.j. zhou, y.m. wei aim: to identify tumor-associated genes by constructing transcription profiles of pure hepatocellular carcinoma (hcc) tissues and normal liver tissues with the combination of laser capture microdissection and microarray. methods: hcc cells and normal liver cells from resection samples of 4 patients were laser capture microdissected. micro-rna was isolated from them for linear amplification then crna was tested with whole genome microarray. differentially expressed genes were screened. results: the quality control of this technique was satisfactory with rna integrity number>7, a260/a280 ratio for crna measurement=2.0~2.1 and good pictures for microarray. compared with normal liver tissues, hcc had 1361 differentially expressed genes, with 607 being up-regulated and 754 being down-regulated genes respectively. among the top ten ranked up-and down-regulated genes (total 20), 5 genes were known as hcc differentially expressed genes, 11, known as other tumors expressed genes previously. four unknown tumor related genes (depdc1b, aspm, fcn2 and bbox1) were detected in this study. conclusion: the combination with laser capture microdissection and microarray was effective in screening the differentially expressed genes of hcc. background/objective: young patients present with large hcc on initial presentation are not uncommon. our aim is to study the computed tomography(ct) imaging of hcc and the clinical features of this special group of patients. methods: 521 hcc patients had ct imaging of liver peformed in a three year period, 385 patients had ct imaging peformed at the time of initial hcc diagnosis in our centre and were selected. they were divided into three age groups: young patients with age </= 40 year-old(group 1), age 41 to 60(group 2), age >60(group 3) to study imaging and clinical factors. univariate and multivariate analysis by cox regression model done to look for prognostic predictive factors. results: infiltrative tumour in ct scans, symptomatic presentation, child's and tm staging are prognostic factors in hcc. conclusion: young hcc patients have larger infiltrative tumour in initial ct scans and more being symptomatic. age is not an independent prognostic factor. aim: to investigate the expression change of nk cells receptor nkg2d from human peripheral blood in patients with primary carcinoma of liver and study the relationship between nkg2d expression and cytotoxicity of nk cells. background/aims: lens culinaris agglutinin-reactive alpha-fetoprotein (afp-l3) is a specific protein produced by hepatocellular carcinoma(hcc), which is more valuable than afp in the diagnosis of hcc. aptamers are oligonucleotide ligands binding to target molecules sensitively and specifically, which are screened from a great capacity of synthetically oligonucleotide library by systematic evolution of ligands by exponential enrichment (selex). our aims were to select the aptamers against afp-l3 from a self-designed ssdna library for potential application in diagnosis of hcc. methods: a random ssdna library and its corresponding primers were designed and synthesized. aptamers against afp-l3 were selected by selex. individual aptamers were separated by polymerase chain reaction-single strand conformation polymorphism (pcr-sscp) analysis and characterized. results: a ssdna library of 78 nucleotides with 40 random nucleotides in middle were designed and used for the selection. the binding rate of library against afp-l3 was increased from 5.1% to 48.2% after 13 round selection. seven aptamers (s1 to s7) were isolated, and their sequences in random region and secondary structures were different from each other. all aptamers could bind afp-l3 in a different extent, and the dissociation constants of s4 and s7 are 650nmol/l and 132nmol/l. conclusions: aptamers for afp-l3 are successfully screened out and could bind afp-l3 specifically. methods: flow cytometry was used to determine the number of nk cells and the expression of nk cells receptor nkg2d from human peripheral blood in patients with 20 case primary carcinoma of liver 23 case hepatitis b cirrhosis 20 case hepatitis b and 20 healthy cases and enzyme mark instrument was used to detect cytotoxicity of nk cells in all cases. results: killing rate of nk cell for k562 cell,nkg2d expression level of nk cells, and the number of nk cells in the patients with primary carcinoma of liver decreased significantly p<0.05 compared with those in the healthy subjects and hepatitis b group ,and decreased a little compared with those in the hepatitis b cirrhosis (p>0.05).the activity of nk cells showed a obvious positive-correlation with the number of nk cell and expression level of nk cell receptor nkg2d. conclusion: the cytotoxicity of nk and the nkg2d expression of nk cells decreased significantly from human peripheral blood in patients with primary carcinoma of liver .the activity of nk cells is closely related to the nkg2d expression level of nk cells. enhancing the nkg2d expression level of nk cell may provide a new idea for adoptive immunotherapy of primary carcinoma of liver. and alpha-fetoprotein afp in serum and tissues for primary hepatic cancer(phc). methods: sixty-six phc and 32 cirrhotic patients were enrolled. in phc patients,male /female was 48:18, age was 55.4 ± 9.91.of them, 16 patients were defined as stage a-a. in cirrhotic patients, male /female was24:8, age was 53.3 ± 5.81. serumgpc3 was detected using elisa. serum afp was detected using electrochemiluminescence. the hepatic expressions of gpc3 and afp were measured using immunohistochemistry in 21 phc and 6 cirrhotic patients. results: the cutoff value of afp diagnosis for phc was 400 g / l or more, afp positive in phc patients was 28.8% (19/66); the cutoff value of gpc3 diagnosis for phc was 300ng / l or more, gpc3 positive was in 47.0 % (31/66), p = 0.031. in a-a stage phc patients,the positive of gpc3 and afp was 62.5% (10/16), 0(0/16), respectively,p = 0.000. in serum afp negative or positive patients, the positive of gpc3 was 46.7% (14/30) , 47.2% (17/36), respectively,p = 0.964. the relationship between gpc3 with age, sex, child-pugh grade, hbv infection, tumor size and metastasis were not observed.the positive expression of gpc3 and afp in hepatocellular carcinoma tissue was 85.7% (18/21), 4.8% (1 / 21), respectively, p = 0.000. neither gpc3 ,nor afp in the paracarcinomatous and cirrhotic tissue, was expressed. conclusions: diagnosis of glypican-3 protein for primary hepatic cancer is superior to afp.gpc3 can be regared as a early marker to diagnosis phc. objective: to investigate the effects and the possible mechanism of curcumin on the proliferation and the invasion of human hepatocellular carcinoma in vitro and in vivo. methods: hcclm3-rfp cell lines were maintained in dmem medium supplemented with 10% fetal bovine serum. the fluorescent areas of hcclm3-rfp were photographed daily and repeated in 4 consecutive days after curcumin treatment for obtaining cell growth curves. the cell morphologic changes were also observed. cell invasion experiment was performed with boyden chamber array. the rfp-expressing human hcc xenograft model in nude mice was established to study the anti-tumor effects of curcumin. the ctc was detected by facs. the expression of cyclind1 and mmp-2 was detected by sybr green real-time pcr. results: after incubation with 20 m, 10 m and 5 m curcumin respectively for 24, 48 and 72 hours, the growth of hcclm3-rfp was significantly inhibited and some morphologic changes were observed. the mean tumor size in nude mice treated with curcumin since day 21were significantly less than those of the control group(p 0.01). the mean metastasis area of lung and the number of ctc in curcumin group on day 35 were remarkably less than in the control group(p 0.01). the mrna levels of cyclin d1 p 0.01 and mmp-2 p 0.05 in curcumin group on day 35 were significantly lower than in the control group. conclusion: curcumin can inhibit the proliferation and invasion of hcclm3 cell line not only in vitro but also in vivo mainly by down-regulating the expression of cyclin d1 and mmp-2 in mrna levels. phosphorylated erk is a potential predictor of sensitivity to therapy with sorafenib in hepatocellular carcinoma -evidence from in vitro study z. zhang 1 , y.h. wang 1 1 background: sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (hcc) and thus sets the new standard for the first-line treatment of advanced hcc. however, it remains unresolved to predict the drug sensitivity in treating hcc with sorafenib. pretreatment perk level has been shown to be associated with favorable response to such therapy in a phase 2 preclinical study, indicating that perk may be a potential biomarker for treatment of hcc with sorafenib. methods: the effects of sorafenib and 5-fluorouracil on cell proliferation were evaluated by cell viability assay in four types of hcc cell line (smmc-7721, mhcc97-l, mhcc97-h and hcclm6), with different metastatic potential and basal perk expression. levels of perk expression were determined by immunocytochemical analysis and quantification, along with western blot analysis. correlation analysis was carried out between the ic50 values of drugs and mean optical density values of perk. results: the basal perk levels increased stepwise in cell lines in accordance with their metastatic potential. sorafenib inhibited erk phosphorylation at a concentration between 5 and 20 m dose-dependently, while no changes were observed after 5-fu treatment. correlation analysis between the ic50 values and mod values of perk revealed that the effects of sorafenib were significantly correlated with basal perk levels (spearman r=-0.8671, p=0.0003). on the other hand, the resistance to 5-fu were significantly associated with basal perk expression in these hcc cell lines (spearman r=0.7846, p=0.0009). conclusions: in this vitro study, perk was confirmed to be a useful biomarker predictive of sensitivity in treating hcc with sorafenib. the raf/mek/erk pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in hcc. background: to investigate the dynamic expression of igf-ii and igfbp-3 and its alteration of bcl-2 in hcc. methods: hcc models were induced with 2-faa on male sd rats. morphological changes of livers were observed and the dynamic changes of liver or serum igf-ii, igfbp-3, and bcl-2 were quantitatively analyzed by elisa. the expression and distribution of liver igf-ii were observed by immunohistochemistry. result: hepatocytes from granule-like degeneration to a typical hyperplasia to hcc and the progressing increasing of the levels of hepatic igf-ii after rats induced by 2-faa. the levels of igf-ii in hepatoma and sera were significantly higher than any of other groups. the positive relationship of igf-ii was found between liver and sera (p<0.01). the igfbp-3 levels in hepatoma were significantly lower than that in other groups (p<0.01) and the progressing increasing of the levels of hepatic bcl-2 expression during the course. the levels of bcl-2 in hepatoma tissues were significantly higher than those in normal and degeneration ones. the immunohistochemistry evidences indicated the positive expression and hepatocyte distribution of bcl-2 in rat hepatoma. conclusion: hepatic igf-ii, igfbp-3 and bcl-2 may participate in hepatocyte canceration and accelerate the occurrence and development of hcc. the expression of igf-ii and igfbp-3 could be useful molecular markers for early diagnosis and prognosis of hcc. background: this study was done to assess the etiological role of hepatitis b virus (hbv), hepatitis c virus (hcv) and aflatoxin b1(afb1) in development of hepatocellular carcinoma (hcc) in bangladesh. it was also investigated whether alpha-feto protein (afp) and protein induced by vitamin k absence or antagonist ii (pivka-ii) has any diagnostic advantage over each other methods: fifty five histologically proven hcc patients were tested for serological markers of hepatitis b and hepatitis c, and afb1-dna adduct. during the diagnosis, they were also investigated for liver function tests, afp pivka-ii. results: out of fifty five hcc patients, 41(74.5%) were found positive for serological markers of hbv, 21(38%) for hcv and 15(27%) for both. eight cases (14.5%) were negative for the markers of hbv and hcv. however, none had afb1-dna adduct above normal range. both pivka-ii and afp is strong marker for hcc with satisfactory level of sensitivity and specificity; but pivka-ii is more sensitive (92.7%) and afp is more specific (96%). conclusions: hbv and hcv is the major etiological agent responsible for the development of hcc in bangladesh. background: to investigate the influences on the malignant transformation of hepatocytes through the intervention of nf-b activation pathway. method: hcc models were induced with 2-faa on sd rats, thalidomide was administered intragastrically and rats were sacrificed fortnightly interval to the twelfth week. morphological changes were observed by he staining. nf-b expressions were detected by ihc. the relationship between nf-b expression and pathological characteristics in hcc and non-hcc were analyzed. results: rat hepatocytes showed vacuole-like denaturations at the early stages, then dysplastic nodules appeared at middle stage, and finally progressed to tubercles of cancerous nest, all of which were highly differentiated hcc. thalidomine can repress the morphologic change of liver cells. there were only punctiform denaturations at the early and middle stage; nodosity hyperplasy and minority atypical hyperplasia were found at the finally stage. the ihc results demonstrated that nf-b level was significantly higher than those in normal ones, and the nf-b level of livers in hcc was higher than those in thalidomide group. an increasing tendency of nf-b was found from normal to hcc. nf-b in hcc were significantly higher than those in nc. the nf-b levels with thalidomide intervence raised first and decreased later. nf-b expressions in hcc were higher than that in their non-cancerous tissues. no positive relationship presented between nf-b expression and histological differentiation grade or the number of tumor, and size of tumor. conclusion: decrease nf-b expression can inhibit hcc development and nf-b is expected to be a new molecular target of hcc therapy. method: the cellular distributions of vegf expression in hcc tissues were investigated by immunohistochemistry. the levels of total rna and vegf were quantitatively detected in hcc, their paracancerous, and distal cancerous tissues, respectively. simultaneitily, serum vegf were analyzed in patients with chronic liver diseases for clinical values. results: the positive expression showed palm-yellow or palm-brown granules and distributed in hepatocyte plasma of hccs. the incidence of vegf was 63.9% in hcc tissues, 78.3% in non-encapsulated hccs, and 90.9% in hccs with extrahepatic metastasis, respectively. no significant difference was found between hepatic vegf and hcc diameter or differentiation degree. the specific concentration (pg/mg liver) of vegf expression was significantly higher (p<0.01) in hcc than their paracancerous or distal cancerous tissues, respectively. the circulating vegf was abnormally elevated in hcc. if the cut off values was more than 280 pg/ml, the incidence of serum vegf was 88.4% in hcc, 14.3% in chronic hepatitis, and 10% in liver cirrhosis, respectively. the combined vegf and afp can increase positive rate up to 94.2% for hcc. conclusion: the vegf overexpression is a useful marker for vascular invasion and metastasis of liver tumors. background: hepatocellular carcinoma (hcc) represents a major health problem world wide. it accounts for 90% of all primary liver cancers and is the fifth most common malignancy (1). objectives: evaluation of radiofrequency thermal ablation versus transarterial hepatic chemoembolization with the effect of viscum (fraxini 2) on tumour recurrence. methods: 60 patients with hcc were enrolled in the study. group 1 include 30 patients and were treated with radiofrequency thermal ablation (15 patients of them received viscum 2 by subcutaneous route for 2 years). group 11 included 30 patients with hcc and were treated by tace (15 patients of them received viscum 2 subcutaneously for 2 years). results: group 1 patients showed total ablation in 70% with persistant inactivity during 2 years follow up. group 11 did not show significant difference from group 1 as regards relapse rate nor the performance status. complications as nausea, vomiting, fever, jaundice, and elevation of transaminases were significantly more encountered with tace. viscum 2 did not significantly arrest tumour recurrence. conclusion: non surgical patients with hcc can achieve curative treatment with radiofrequency with minmal side effects. tace is a palliative treatment option for large hcc. a new technique had been attempted to increase the field of radiofrequency ablation of expandable electrode needles in the treatment of hepatic neoplasms much larger than the routinely covered size of 5-7 cm according to the needle size overcoming the technical difficulties usually met with in the overlapping balls technique due to the hyperechoic focus that develops at the needle tip making reinsertion difficult and inaccurate. in this technique, two or three needles were inserted from the start into the mass with accurate estimation of the exact field of ablation of each needle trying to cover the whole extent of the mass before application of radiofrequency waves. 40 patients were included in the study, all presented with hepatic neoplastic mass lesion that range in size between 7 and 10 cm in its maximum diameter. all had a pretreatment helical (triphasic) ct study for accurate delineation of the whole extent and vascularity of the mass. two needles were sufficient to cover the whole extent of the mass in 23 patients (57%) while in the remaining 17 patients (43%) three needles were necessary. the procedure was done under general anathesia and ultra sound guidance, patients tolerated procedure well with smooth recovery. no major complications. follow up spiral (triphasic) ct was done 2 weeks after ablation revealed percentage of tumour necrosis of 90% or more in 30 patients (75%), 70-90% in 6 patients (15%) while in the remaining four patients (10%) the percentage was 50-70% necrosis. in conclusion this technique should be considered in the treatment of hepatic masses larger than the usual field of the needle. results: the median value of gpc-3 in hcc, dc, cc was significantly higher than chronic hepatitis and control groups. no significant correlation found between afp and gpc-3. auroc of afp was 0.85 & auroc of gpc-3 was 0.84. the diagnostic sensitivity of afp (20 ng/ml) was 70% with ppv 53.8%. the specificity was 85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) was 100% with ppv 27%. the specificity was 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved the specificity to 87.5%. conclusion:gpc-3 although it is a serological test for early detection of hcc, it showed limited specificity, where it is detected in different stages of chronic liver disease, as it is an oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 may improve the prediction accuracy of hcc patients in those showing seronegativity to afp. outcome of inoperable hepatocellular carcinoma patients receiving transarterial chemoembolization: retrospective analysis in an asian regional hospital w.m. yip 1 , k.f. li 1 , k.k. li 1 , m.l. szeto 1 1 background: hepatocellular carcinoma (hcc) is a common cancer worldwide causing substantial mortality. although surgical resection is a form of curative treatment in hcc, only a minority of patients is suitable for this treatment and the postoperative recurrence remains high. transarterial chemoembolization (tace) is a treatment option for inoperable hcc and it was proven by randomized control trials that tace can prolong survival in selected patients. the aim of this study is to evaluate the survival and the prognostic factors in patients with advanced hcc treated by tace. methods: seventy four patients with inoperable hcc diagnosed from january 1998 to december 2003 were analyzed retrospectively in this study. only patients with unresectable hcc or who refused operation were included. patients with advanced cirrhosis, extrahepatic metastasis or previously treated hcc were excluded. multiple host, tumor and treatment variables were analyzed in order to evaluate the predictive factors of favorable response to treatment and better survival. results: the median survival of the study patients was 213.5 days. the cumulative survival rates at 1 year, 2 year and 3 year were 28.4%, 12.2% and 6.8% respectively. by multivariate analysis, superselective cannulation performed in tace (hazard ratio: 0.47, 95% ci: 0.23-0.95, p=0.034), embolization with gelfoam (hazard ratio: 0.30, 95% ci: 0.11-0.80, p=0.017), treatment interval more than 45days (hazard ratio: 0.33, 95% ci: 0.15-0.72, p=0.006), child-pugh grade b (hazard ratio: 5.62, 95% ci: 2.11-14.97, p=0.001), and pre-treatment serum fp level (hazard ratio: 2.93, 95% ci: 1.50-5.73, p=0.002) were independent predictors of survival. conclusions: survival of patients with inoperable hcc is still grave despite treatment. this study provided information in predicting the survival of patients with inoperable hepatocellular carcinoma treated by transarterial chemoembolization. result: age <65, total bilirubin (tb) <2.0mg/dl, albumin (alb) 3.5g/dl, prothrombin time (pt) 70%, platelet counts (plt) 10.0 10 4 /mm 3 , single nodule, and type of treatment (surgery or local ablation therapy) were linked to increased survival at univariate analysis of clip 0-1 hcc patients. of clip 2-6 hcc patients, tb <2.0mg/dl, alb 3.5g/dl, des-gamma-carboxy prothrombin (dcp) <100mau/ml, absence of vascular invasion, and type of treatment were correlated with survival. the following factors were related to survival by multivariate analysis: clip 0-1 hcc patients; age, alb, single nodule, and absence of vascular invasion, clip 2-6 hcc patients; age, tb, alb, alpha-fetoprotein (afp) <100ng/ml, dcp, absence of vascular invasion, and type of treatment. conclusion: age, albumin, vascular invasion were significant predictors of survival both clip 0-1 and clip 2-6 hcc patients. clip 0-1 hcc patients: single nodule; clip 2-6 hcc patients: lower levels of tumor markers and patients receiving promising treatment had a better chance of prolonged survival. the role of gross classification as the predictor of microvascular invasion in hepatocellular carcinoma. s. sumie 1 , r. kuromatsu 1 , k. okuda 1 , e. ando 1 , a. takata 1 , n. fukushima 1 , m. sata 1 1 background; the presence of microvascular invasion (mvi) as the risk factor in hepatocellular carcinoma (hcc) is controversial. the aim of this study was to determine the outcomes and predictive factors after hepatic resection for hcc with mvi. methods; one hundred and ten patients who underwent curative resection for hcc were included in this retrospective study. the risk factors of these patients for recurrence-free and disease-specific survival were investigated, and the clinicopathological factors predicting the presence of mvi were also evaluated. result; multivariate analysis showed that cirrhosis and mvi were identified as independent risk factors for recurrence-free survival. the 2-year recurrence-free survival rates for patients with and without mvi were 44.6% and 76.7%, respectively. multivariate analysis showed that the number of tumors, presence of mvi, and im were identified as independent predictors of disease-specific survival. the 5-year disease-specific survival rates for patients with and without mvi were 59.3% and 92.0%, respectively. by univariate analysis, mvi was significantly associated with greater tumor size, gross classification, histological grade, and intrahepatic micrometastasis (im). gross classification proved to be the only independent predictive factor for mvi by multiple logistic regression analysis. the gross classification could be evaluated by preoperative imaging diagnosis. conclusion; mvi is strongly associated with recurrence and survival in hcc patients after curative resection. furthermore, gross classification of hcc can be helpful in predicting the presence of mvi. background: hcc is a common cause of cancer morbidity and mortality. pxd101 is a novel, low molecular weight, histone deacetylase inhibitor. this phase i study aims to determine dose limiting toxicity (dlt) and maximum tolerated dose (mtd). methods: patient eligibilities include unresectable disease, ecog 2, adequate organ functions. pxd101 was given intravenously on day 1-5 every 3 weeks; dose levels were: 600 (level 1), 900 (level 2), 1200 (level 3) and 1400 mg/m 2 /day (level 4). dlts are defined as grade 4 hematological toxicity or grade 3/4 non-haematological toxicity during cycle 1 (according to nci ctc v3), or treatment delay >2 weeks. the mtd is defined as the dose below which > 2 of 3 or > 2 of 6 patients experiencing dlt. results: 18 patients were entered; level 1 (3), level 2 (3), level 3 (6) and level 4 (6). grade 3/4/5 toxicities in cycle 1 included: raised alt 1/0/0, diarrhea 1/0/0, abdominal distension 1/0/0, anaemia 1/0/0. a total of 56 cycles were administered; overall grade 3/4/5 toxicities: raised alt 1/0/0, bilirubinaemia 1/0/0; cardiac ischaemia 1/0/0; diarrhoea 1/0/0, abdominal distension 2/0/0, anaemia 2/0/0; variceal haemorrhage 1/0/0; hypercalcaemia 1/0/0; hyperkalaemia 0/1/0; hyponatraemia 1/1/0; infection 2/0/0; liver dysfunction 0/2/0; muscle weakness 1/0/0; abdominal pain 1/0/0; prolonged qtc 1/0/0; syncope 1/0/0; seizure 1/0/0. there were 3 sd and 15 pd. conclusion: at the maximum dose of 1400 mg/m 2 /day, mtd has not been reached. pxd101 is very well tolerated. sponsor: the division of cancer treatment and diagnosis, national cancer institute, usa. tumor thrombus (pvtt) is prone to be produced in the portal vein near the main tumor nodule for hepatocellular carcinoma (hcc) patients and its molecular mechanism is still unclear. in this study, we first established a hcc cell line named csqt-01 from resected tumor thrombus in portal vein in a patient with histopathologically proved to be a moderately differentiated hepatocellular carcinoma . this cell line was composed of polygonal shaped cells and its peaks of the chromosome number was 69 and 77. study on stem cell biology in this cell line suggests that cd133 cells represent about one fourth of the tumor cell population and cd133(+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. with this cell line model and resected tumor thrombi specimen, we also studied the different expression of proteins in primary tumor and tumor thrombus and found 20 proteins expressed differentially between primary tumor and the pvtt. from these proteins, annexinv, prx , cycb were selected for further analysis to find potential biomarkers of pvtt in hepatocarcinogenesis. for clinical study, we recommended a new tumor thrombus type system ( type i iv) according to anatomic features of portal vein and tumor thrombus of hcc developing modes, then evaluate this type system to predict prognosis of hcc patients. the retrospective data of 406 hcc patients with pvtt underwent resection shows that the 1y, 2y, 3y overall survival rates were 44.7 , 26.3 and 19.7 for type i, 29.9 , 20.6 and 12.4 for type ii, 25.0 , 12.5 and 3.6 for type iii, 27.3 , 0 and 0 for type iv, respectively, suggests tumor thrombus type system may be helpful to determine treatments and prognosis of hcc patients with pvtt. polyprenol could decrease the risk of hepatocarcinogenesis in hbv g. kuznecova 1,2 , s. kuznecovs 1,2 , i. kuznecovs 1,2 background: over-expression of p-glycoprotein (pgp) is associated with liver cancer development from hbv . glycoprotein synthesis in malignant tissues is limited by dolichyl phosphate (dolp). the aim of the present study was to investigate the effect of polyprenol (pp) which provides a dolp substitute in regulation of n-glycosylation on pgp over-expression in the development of liver cancer in hbv infection. methods human hepatocytes, infected with hbv and human hepatocarcinoma hep 3b cell line were used. pgp was assessed by an immunohistochemical technique. dolp fractions were analysed by hplc methods. results it is confirmed that plasmatic membrans of hepatocytes cells contain 7,9 -9,4% of pgp (the total protein amount) as a resistance marker. hbv infected cells differ from normal hepatocytes in pgp content by 4-5 times and hep 3b cells differ by 10-12 times the study showed 5-fold dolp decrease in hbv infected cells and 10-fold dolp decrease in hep3b cells. the investigations demonstrate that the situation can be changed by treatment with dolp and pp. the dolp concentration in hbv infected hepatocytes was returned to the normal level. it is established that dolp in the concentration 10 -6 m aid 6-8-fold reducing pgp in membranes of hbv infected cells. background: metastasis is one of the most complicated and major pathological processes responsible for poor prognosis of hepatocellular carcinoma. snail was recently highlighted as a critical transcriptional factor for tumor metastasis. method & result: real time rt/pcr and western blot analysis demonstrated that snail mrna and protein, respectively, were induced by 12-otetradecanoylphorbol-13-acetate (tpa) in hepatoma cell hepg2. blockade of gene expression of snail by antisense oligodeoxynucleotide and/or sirna technique can prevent not only the tpa-triggered emt/cell migration and growth inhibition of hepg2 but also tpa-induced down-regulation of e-cadherin and up-regulation of p15ink4b. moreover, the tpa-triggered promoter activation of p15ink4b was also prevented. on the other hand, two of the hepg2 clone overexpressing snail, namely s7 and s15, had a scattered fibroblastic morphology and acquired higher motility than parental hepg2. also, the proportion of g0/g1 phase of s7 and s15 was higher than that of parental hepg2, consistent with the longer doubling time of both cells. semiquantitative rt/pcr analysis demonstrated a greatly elevated gene expression of snail accompanied with decreased e-cadherin and increased p15ink4b in both snail-overexpressing cells. on the transcriptional level, p15ink4b promoter activity was 2.6-fold higher in s7 as compared with parental hepg2. furthermore, electrophoretic mobility of dna fragments encompassing proximal p15ink4b promoter can be retarded by incubation of nuclear extract of s7. conclusion: our results demonstrated that snail play diverse trans-regulatory roles in hepg2. notably, we suggested that snail may upregulate p15ink4b gene expression by directly activating its promoter. a. schmitt-graeff 1 , r. fischer 1 , m. grosse-perdekamp 1 , o. skalli 2 1 universityhospital freiburg , 2 louisiana state university health sciences center, shreveport background/aims: synemin is an intermediate filaments (if) protein which affects the motility of several cell types by modulating the dynamic properties of alpha-actinin and f-actin. we have previously shown that synemin is expressed in resident hepatic stellate cells (hsc) and myofibroblasts (mf) in hepatic inflammation and fibrosis. in the present study we evaluated systematically the expression of synemin in a large cohort of western european hepatocellular carcinoma (hcc). methods: single and double immunolabelin for alpha-smooth muscle actin (sma), vimentin, cd31, cd34, cd68, cea, cd10, cellular retinol-binding protein1 (crbp-1) and synemin were performed on 75 paraffin-embedded hccs and 10 controls. results: synemin-positive hscs/ mfs were a hallmark of non-neoplastic fibrotic liver tissue at the border to the neoplastic lesion but were absent from normal controls. tumour cell plates of the trabecular and pseudoglandular types of hcc were covered by scattered synemin-positive cells outlining sinusoidal structures. a subpopulation of these cells showed features of pericytes while others resembled endothelium. this pattern correlated with the degree of differentiation and was not observed in poorly differentiated hccs which generally contained rare intratumoral mfs. conclusion: the presence of synemin-positive hscs/mfs in the vicinity of hccs suggests a possible contribution of mesenchymal cells to the promotion of liver carcinogenesis. since synemin expression is linked to motility, a migration of this cell type into the tumour and a differentiation in vascular mural cells may be implicated in sinusoidal remodeling and the expansion of the neoplastic population. a.s. butt 1 , a. ahmed 1 , s. hamid 1 , w. jafri 1 , h. ali shah 1 1 aim: to estimate the prevalence of viral marker negative hcc and to compare the clinical, biochemical, histological, radiological characteristics and initial treatment response among patients with viral marker negative and viral marker positive hcc. methods: medical records of patients diagnosed to have hcc visiting aga khan university hospital, karachi during january 1998 to december 2007 were reviewed. patients were divided in to nbnc-hcc(those who have negative hbsag and anti-hcv antibody)and viral hcc(those who have positive hbsag and anti-hcv antibody)group. results: out of 433 patients 68(15.7%) had nbnc-hcc. over all mean age was 57.48± 10.9 years and 69.5% were males. the proportion of hcc detected under surveillance was significantly smaller in nbnc-hcc group(p0.02). there was no difference in distribution of age, gender, bmi, child score, bilirubin, serum albumin, prothrombin time and alfa feto protein in both groups. however, patients with viral-hcc were found to be more thrombocytopenic(52.67±86.7vs.226.15±153.9,p<0.001) and had hepatopulmonary syndrome. on liver biopsy greater proportion of moderate to poorly differentiated hcc was observed in nbnc group(19.2%vs.7.9%,p<0.001). hcc measuring 5cm in diameter(60.3%vs.41.9%, p 0.01), non -solitary hcc(p0.038) and portal thromboses(p0.01) were strongly demonstrated in nbnc-hcc group. involvement of right hepatic lobe and extra hepatic tumor spread was greater in nbnc-hcc group but that difference was not statistically significant. out of 178 patients who underwent for liver transplantation(0.5%),tace(36.7%),resection(1%),ethanol ablation(2%) and chemotherapy(2%), poorer responses were observed in nbnc-hcc group (p 0.04). conclusion: hcc secondary to nbnc-cirrhosis is not uncommon. patients with nbnc-hcc tended not to be under surveillance that leads to diagnoses at more advanced stage and poor prognosis. background: hepatocellular carcinoma is a common malignancy in asia and is related to the high prevalence of chronic viral hepatitis. we examined the clinical features, treatments and survival rates in asian americans with hcc. methods: retrospective cohort study of 278 hcc patients who presented to the ucla liver cancer center in los angeles, california, usa from september 2000 to december 2007. results: two hundred and seventeen of 278 (78%) hcc patients were male, 58% and 30% had hbv and hcv infection respectively, and 73% had cirrhosis. hcc patients detected by surveillance had smaller tumor sizes, more within the milan and ucsf criteria, lower hcc tokyo system scores and had improved 1,3,5 year overall patient and disease free survival rates compared to hcc patients who presented with symptoms (p<0.01 to p<0.0001). by multivariate analysis, independent predictors of patient survival were tumor volumes greater than 5cm (hr1.48, p=0.04), afp per unit log10 increase (hr 1.12, p=0.02), hcc tokyo score per unit increase (hr 1.3, p<0.0001), liver transplantation (hr 0.14, p<0.0001), hepatic resection (hr 0.18, p<0.0001), rfa (hr 0.17, p<0.0001), tace (hr 0.44, p=0.0006), and hepatitis b infection (hr 0.71, p=0.03). factors associated with disease free survival were age per year increase (hr 1.01, p=0.03), meld per unit increase (hr 0.97, p=0.0049), liver transplantation (hr 0.23, p<0.0001), and hepatic resection (hr 0.39, p<0.0001). conclusion: hbv and hcv infection accounts for the majority of hcc in asian americans. hcc detected by surveillance resulted in treatments which improved overall patient and disease free survival. treatment of small ( 2cm) hcc tumours can be achieved by surgical resection and complete eradication always correlates with good patient's outcome, with low local recurrence and high survival rates. indeed, surveillance program for the early detection of small hcc tumour is imperative to facilitate curative treatment, and hence better survival. discovery of new blood-based biomarkers is obligatory and vimentin is a distinct novel small hcc tumour marker herein identified using proteomics. experimental design: a total of 76 liver tissues were evaluated by 2-de analysis. differentially expressed proteins were unequivocally identified by maldi-tof/tof and validation of the best candidate from protein to gene levels. indirect elisa assay was developed to detect soluble vimentin from 152 serum samples. results: vimentin was significantly over-expressed in small hcc tumours compared to non-malignant controls and maintained expression in >2cm tumours using 2-de analysis. blind verification displayed over-expression of vimentin in both transcripts and proteins levels. soluble vimentin was significantly detected at high level in small hcc as well as in overt hcc tumours. receiver operating characteristic analysis showed vimentin exhibited 40.91% sensitivity and 87.50% specificity in detecting small hcc at a cutoff of 245ng/ml. combined diagnostic performance of soluble vimentin and serum afp increases the detection sensitivity and specificity to 50.53% and 98.15%, respectively. conclusion: in this context, over-expression of vimentin is associated with the favourable 2cm sub-class of hcc thus may potentially be used as an effective serum-based diagnostic marker for cancer surveillance in high-risk cirrhotic patients. purpose: our recent comparative oncogenomic analysis in mouse model has identified yap (yes associated protein) as a novel oncogene in hcc. however, its clinical significance is unknown. in this study, we aimed to investigate the clinical values of yap as an independent prognostic marker in hcc. experimental design: a total of 177 hcc cases with retrospective clinicopathologic and follow-up data were recruited in this study. both tumor and adjacent non-tumor tissues were examined for immunoreactivity of yap expression by immunohistochemistry. clinicopathologic features and yap expression were investigated with pearson 2 test. hcc-specific disease free survival and overall survival with yap expression were analyzed by kaplan-meier curves and log-rank test. cox regression was used to test the independence and magnitude of the effects. results: yap was found over-expressed in hcc (62.1%) with nuclear expression pattern. positive yap immunoreactivity was significantly correlated with worse tumor differentiation grade (p=0.021) and high serum alpha-fetoprotein (afp) level >400 ng/ml (p<0.001). kaplan-meier plot and cox regression showed that yap was an independent predictor for hcc-specific disease free survival (hazard ratio, 1.653; 95% ci, 1.084-2.522; p=0.02) and overall survival (hazard ratio, 2.226; 95% ci, 1.312-3.778; p=0.003). conclusions: yap expression in hcc is correlated with tumor differentiation and serum afp level. it served as an independent prognostic marker for hcc. background: integrative analysis of global protein and mrna expression patterns could help researchers to understand cancer cell physiology without the need of any prior hypothesis. methods: we used a 2d-page approach to profile and compared the global protein expression profiles of hepatitis b virus-related hcc tissues, adjacent non-tumor liver tissues, normal liver tissues and hcc cell lines. subsequently, we established the bioinformatic tools for integrative analysis of gene expression and protein expression data. we compared the dysregulated protein list and the dysregulated gene lists obtained by meta-analysis of microarray gene expression data from 6 research centers in different countries. results: we identified 66 proteins dysregulated in hcc. hierarchical clustering analysis revealed that there was a progressive change of protein expression patterns from normal liver, adjacent non-tumor liver tissues, hcc tissue, then to hcc cell lines. according to the biological functions, the differential proteins could be classified into various groups, including heat shock protein, chaperone, kinase substrate, cell signaling, apoptosis regulation, transcription regulation, free-radical scavenger and metabolic enzyme. ontology analysis of the genes with consistent dysregulations at both mrna and protein levels identified specific pathways down-regulated during the progression of hcc. the inhibition of those pathways provides new insights in the hepatcarcinogensis and treatment strategies. results: in pre-s/surface regions, hcc patients had higher frequencies of pre-s deletions, amino acid substitutions at codon 4, 7, and 81 in pre-s1 genes, at the start codon in pre-s2 genes, and at codon 68 in surface genes. but they had a lower frequency of amino acid substitution at codon 2 in pre-s2 genes than those without hcc. in bcp/precore regions, hcc patients had higher frequencies of c or g1753, a1762/t1764, t1846, and a1899 than those without hcc. multivariate analysis showed that pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. the hbv with a complex mutation pattern (pre-s deletion, t1762/a1764, and a1899) rather than a single mutation was associated with hcc. patients with combined mutations of t1762/a1764 and pre-s deletion, t1762/a1764 and a1899, pre-s deletions and a1899, and t1762/a1764, pre-s deletions and a1899 had a 7.81, 7.7, 7.0, and 16.88 fold increased risk of hcc, respectively, compared to patients with wild-type at both or three genomic regions. conclusions: pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. combination of these viral mutations appeared increasing hcc risks. high peritumoral expression of placental growth factor in hepatocellular carcinoma is a poor factor for survival after curative resection h.x. xu 1 , x.d. zhu 1 , p.y. zhuang 1 , w.zhang 1 , h.chuan sun 1 1 background/aims: angiogenesis plays a significant role in the metastasis and recurrence of hepatocellular carcinoma (hcc). placental growth factor (plgf), which is one member of the vascular endothelial growth factor family, may have prognostic values in patients after curative resection of hcc. methods: expression of plgf was assessed by immunohistochemistry in tissue microarray containing paired peritumoral liver tissue and tumor from 105 patients underwent hepatectomy for histologically proved hcc. prognostic values of plgf and clinicopathological factors were evaluated. result: plgf staining was mainly on the cytoplasm of tumor cells or hepatocytes. the mean integrated optical densities of peritumoral and intratumoral density of plgf were 0.018±0.018 and 0.006±0.007 respectively. peritumoral plgf density was significantly higher than that in tumor (p<0.001), and this result was also validated in another cohort of 37 patients by quantitative real-time reverse transcription-pcr (p=0.007). intratumoral density of plgf was not correlated with common clinicopathological factors (eg, tnm stage, tumor size, microvascular invasion, intra-hepatic metastasis) or overall survival (os) (p=0.425) and time to recurrence (ttr) (p=0.419). however, peritumoral density of plgf, which was correlated with tumor size (p=0.003) and intrahepatic metastasis (p=0.004), was a prognostic factor for both os (p=0.015) and ttr (p=0.018). in multivariate analysis, peritumoral expression of plgf was also an independent prognostic factor for os (p=0.015, rr: 2.500 95% ci: 1.191-5.253 ) and ttr (p=0.045, rr: 2.013 95% ci: 1.014-3.996). conclusion: peritumoral expression of plgf in hcc patients is an independent risk factor for survival and recurrence, and may be a target of anti-angiogenic therapy in preventing post-operative recurrence. purpose: to further research rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection for treatment of advanced hepatocelluar carcinoma (hcc). methods: 25 cases were treated with transhepatic artery chemoembolization (tace) + radiofrequency ablation (rfa) + introportal vein chemotherapy (pvc) + percutaneous ethanol injection (pei) (four combined group) and this method was compared with 23 cases that were performed tace + pei (two combined group) . the serum level of afp was measured respectively after 2 and 6 months, ct scan and color doppler ultrasound were measured after treatment for six months. results: the serum level of afp declined in two groups after 2 months. for treatment after six months, afp in four combined groups was rose lower than two combined group (x 2 =5.06, p<0.05). ct and doppler ultrasound examination, four combined groups was superior than two combined groups to the control in tumor shrinkage (x 2 =8.29, p<0.01) and blood supply x 2 =6.81, p<0.01), relapse and mortality are also less. conclusions: rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection is a safe, effective combined method and has less complication in treatment of advanced hcc. poster exhibition -hcv poster session, hall 5b on the average, hepatitis c virus infects 1.2% of the population worldwide. in egypt, the prevalence rates reach 20% in some areas. ability of the virus to persist in about 75-85% of infected individuals is related to the virus higher mutation rate. six major hcv genotypes have been identified. genotype 4 seems to be confined to the middle east and central africa. extra hepatic syndromes have been reported in up to 1/3 of hcv patients. we aim in this study to determine the relationship between viral genotypes and specific extra hepatic haematological disease in patients with chronic hepatitis c. the study group included 60 selected patients with chronic hepatitis c having various haematological problems. we studied hepatitis c virus genotypes using rt-pcr. we found among 60 patients , 57 genotype 4 (95%) and 3 patients genotype 1a (5%).28 patients (46.66%) were diagnosed as chronic hepatitis c with associated thrombocytopenia, 16 patients (26.66%) were diagnosed mixed essential cryoglobulinemia(mec), 13 patients (21.66%) were diagnosed non-hodgkin's lymphoma, and 3 patients (5%) were aplastic anemia. positive serum cryoglobulins level was found in 20 patients (33.3%).no significant correlation was found between the level of viraemia and specific haematologic disease, biochemical liver markers or liver enzymes (p>0. 05). we did not find correlation between hcv genotype and specific extrahepatic haemological disorder in hcv infected patients. several environmental, genetic and immunological factors may contribute in disease progression. results: in the targeted area 111 shops of barbers were successfully interviewed and total 715 questionnaires were filled by both groups. the mean age were found in both groups of barbers (n=186) and clients (n=529), 28.47 years. the both groups showed that there are no any drugs which can protect us from diseases. both of the groups were not vaccinated for hepatitis b diseases. regarding the care providers the barbers replied that they prefer registered medical practitioners and the clients generally prefer the hakeems. those who knew hepatitis as liver disease, were 73 (39.2%), out of 186 barbers only 68 ( 36.6%) were knowing about hepatitis-b&c, when we enquired about routes of hbv& hcv transmission only (37%) replied correct routes of transmission in both groups. about hbv vaccination (49.7%) were aware, only (14.8%) were vaccinated against hbv. 60% barbers claimed for disinfection of instruments before shaving (88.9%) claimed for use of new blades. in the sero-surveillance the hbv found was very low and hcv became epidemic (5.7% -14.4 %) respectively. conclusion: the both groups need awareness for transmission. the use of new blade for the clients reduces the burden of hbv and hcv. the study highlights the roles of male sex, older age, and genotype 1b in the progression from chc infection to hcc. patients with higher hcv viral load potentially tend to develop hcc; however, hcc occurrence could be prevented using antiviral treatment. these two points need to be clarified further by a larger study population with longer follow-up period. an approach have recently been described that retroviral vectors encoding t cell receptor (tcr) genes are used to redirect the specificity of normal peripheral blood lymphocyte (pbl)-derived t cells to recognize the tumor antigens. the therapy in which t cells have been genetically modified with tcr genes to recognize hcv would represent a novel approach for the treatment of hcv infections and hcv-related malignancies. we have previously shown that hcv+ liver transplant patients that have received hla disparate liver allografts have hcv reactive t cells of host origin in their peripheral blood that are restricted by the donor hla molecules. initial studies indicate that the tcrs expressed by hcv reactive t cell clones from these patients have relatively high affinity for their ligands. we have cloned and expressed two tcrs which mediate recognition of the 1406-1415 and 1073-1081 epitopes from the hcv ns3 protein. the results indicate that these tcr transduced t cells can recognize the wild type epitopes, as well naturally occurring mutant variants of these epitopes. most importantly, the tcr transduced t cells could also recognize hcv+ hepatocellular carcinoma cells. these data suggest this high affinity hcv-specific tcr might have potential new immunotherapeutic implications. background: factors associated with svr in patients without an rvr remains unclear. methods: 200 hcv-1 (100 for 24 and 48 weeks, separately) and 150 hcv-2 (100 for 24 weeks, 50 for 16 weeks) patients were randomized to peginterferon-alpha-2a and ribavirin for analysis. results: multivariate analysis showed that treatment duration and a complete evr were the strongest independent factors associated with an svr. a higher svr rate and a lower relapse rate were observed in the standard regimen group than in the abbreviated group in patients who had a cevr (table 1). the best levels of viral loads in predicting cevr at week 4 were < 10 4 iu/ml (table 2) . conclusion: it was crucial to achieve a cevr with adequate treatment duration in patients who failed to achieve an rvr. our aim was to evaluate the impact of some biochemical, histological and viral factors on both evr and svr in patients with genotype 1 chronic hepatitis c (chc) treated with peginterferon plus ribavirin. patients and methods: we evaluated retrospectively 188 naïve patients with chc treated with peginterferon plus ribavirin at standard weight-based doses for 48 weeks. biopsies were assessed for inflammatory activity and fibrosis. steatosis was categorized by the proportion of hepatocytes per low-power field with fatty changes: >5%, >5-33%, 34-66%, >66%. biopsies were also assessed for stainable iron using the brissot scoring system. all patients were evaluated for metabolic syndrome (ms) using the ncep-atp iii criteria. results: evr was achieved in 115/188 pts (61.17%) while svr occurred in 82/115 (71.3%). after adjusting for sex and age, independent factors that negatively interfered with both evr and svr were: fibrosis score, steatosis, iron score, homa-ir index and viral load. after excluding the patients with ms criteria (n=32), evr was observed in 102/156 (65.4%) and svr in 82/102 (80.4%). factors that independently influenced both evr and svr were: fibrosis score, steatosis, iron score and viral load. conclusion: fibrosis, steatosis and iron scores, as well as viral load are independent parameters that can affect both evr and svr in genotype 1 chc patients, regardless the presence of ms. if ms is present, high homa-ir index can also additionally impair viral response. issue/argument: asia has rising cases of hepatitisb/c. alcohol/food-habits cause high prevalence in rural/tribal areas. lack of monitoring/follow-up complicates management. vaccines emerge as hope. clinical-trials of vaccines debated-issue. design of hepatitis-vaccine-trials in developing-countries complex ethical-issue. we focus on controversies identified in international/regional/local cme/pharma programs as vulnerability of volunteers to exploitation by foreign/local research-groups/funding-agencies. critical task is protect interests of vaccine-subjects in face of substantial-risks. determine if hepatitis-vaccine-volunteers will have access to treatment during trial. access to vaccine-trial-outcome. interaction with seniors 19 th apasl-congress from developed-countries will give voice to such burning-issues. methodology: researchers/pharmaceuticals/govt-policy planners need to develop forum to solve these problems. ngo's can play pivotal role. obligation on part of researchers to create mechanisms to offset anticipated risks of participation in controversial, risky vaccine-development. conclusion: counselling/right to withdraw from trial be made basic guideline. apart from monetary aspects unsuspected adverse reactions/deaths be properly evaluated/monitored. researchers need to evolve policy-guidelines to overcome barriers as variation in interpretation of essential ethical ideas, legal-system-differences, educational/economic-status. need to develop common consensus between research-community/pharma sector to reduce suffering of hepatitis-affected patients community. recommendations: researchers/ngos should come together at 19 th apasl-congress platform to form workgroup to settle these issues. we shall raise our this burning issue & present hepatitis-prevention-advocacy plan of our ngo graphically to apasl-2009 participants. results: 53% patients expressed that alternative-medicines-rx most important factor to cope with hepatitis. higher scores of qol (anova p < 0.001) correlated with alternative-medicines-rx. our ngo-initiative suggests that over 70% patients will need well trained specialist for home-based-care unit. conclusions: life-span/qol of hepatitis-sufferers depends on appropriate-palliative-care. ngo-personals should be trained in palliative-care-services. our data is being used for palliative care advocacy. field of spiritual/psycho-social/community support is fertile ground for further investigations. such use of complimentary indian medicinal plant extracts needs further evaluation in a large group in multicentre trial. treatment with adacolumn in patients with hepatitis c related who have undergone kidney transplantation: preliminary study g. novelli 1 1 la sapienza university introduction: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. background: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: the treatment was found to be safe without hemodynamic or infective complications. considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. m. sharaf-eldin 1 , h. el batae 1 , n. abd el-ghaffar 2 , w. rasheed 2 1 tanta faculty of medicine , egypt., 2 national research centre, cairo, egypt aim: we aimed to characterize serum cytokine levels of interleukin-1 beta (il-1 ) and interleukin -6 (il-6) in hcv infected patients & in patients with hepatocellular carcinoma (hcc) in comparison to control group and their possible use as markers of disease progression. patients and methods: sixty patients were divided into three groups: group i: twenty hcv infected patients without cirrhotic changes. group ii: twenty hcv infected patients with liver cirrhosis (lc). group iii: twenty hcv infected patients with hcc and 20 healthy subjects as control group. all patients and control group were subjected to biochemical and serological tests, anti hcv, hcv (rt-pcr) and cytokines measurements of serum il-1 & serum il-6 levels. results: showed a high statistically significant elevated serum il-6 and il-1 levels in patients with chronic hcv infection in comparison to control group. highly statistically elevated levels of il-6 and il-1 in liver cirrhosis and higher levels were found in hcc group in comparison to control group. the levels of il-6 and il-1 increased significantly in hcv infected patients as the disease progress. conclusion: serum il-1 , and il-6 levels are elevated in patients with hepatitis c-related liver diseases, especially in lc and hcc patients. their levels reflect hepatic dysfunction better than liver inflammation parameters; accordingly, we may use serum il-1 and il-6 as markers for liver disease progression in hcv-infected patients instead of invasive techniques. atsushi tanaka 1 , naoko hanawa 1 , mitsuhiko aiso 1 , yoriyuki takamori 1 , hajime takikawa 1 1 teikyo university school of medicine background and aim: pegylated interferon (peg-ifn) therapy is not indicated for many cases with hcv-related cirrhosis due to various adverse effects. however, patients with hcv cirrhosis are at high risk for development of hepatocellular carcinoma (hcc). thus we have introduced low-dose peg-ifn treatment for patients for compensated hcv cirrhosis. patients and methods: selection criteria for low dose peg-ifn is 1) compensated hcv-related cirrhosis, and 2) either the elderly (>65) or presence of thrombocytopenia (<8.0x10 6 / l). we have treated patients who met these criteria with low-dose peg-ifn, consisting of either peg-2a 90 g/1 -2w or peg-2b 0.5 g/kg/w+ribavirin 200mg/d. [results] twenty patients with compensated hcv cirrhosis (all patients genotype 1b) have been treated with low-dose peg-ifn (peg-2a:9, peg -2b+rib:11 . the age, platelet counts (x10 6 / l), and alt (iu/l) of 20 patients at baseline were 64.1±8.3, 7.8±4.6, and 90.4±68.6 respectively. all patients were well tolerated. low-dose peg-ifn has been continued 59.6±43.8 weeks on average. although viral response was not detected, biological response (br), defined as maintenance of alt within normal range, was obtained in 12 patients (12/20=60%). of note, neither development of hcc nor decompensated cirrhosis was observed in these 12 br cases. by contrast, hcc and decompensation developed in 5 and 1 patients respectively among 8 patients who failed to achieve br. conclusion: low-dose peg-ifn treatment was safe and well tolerable, and could potentially prevent hcc or decompensation in patients with liver cirrhosis when br was obtained. aims: to study the efficacy of peginterferon and ribavirin in treating chronic hepatitis c (chc) with genotype 6a in hong kong chinese. methods: to assess sustained virological response (svr) (serum hcvrna< 500iu/ml) at 6-months follow-up. results: nine patients with genotype 6a chc (included from jan2003 to dec2007) received peginterferon and ribavirin. mean age: 50 (range 28-64). mean alt before treatment: 96 iu/l (range 29-173 iu/l). seven patients had liver biopsy performed, only one showed stage 3-4 fibrosis and others showed active hepatitis without advanced fibrosis. mean serum hcv-rna: 9.17x10 5 iu/ml(range 9.0x10 3 -3.3x10 6 iu/ml). six patients had received peginterferon alfa-2b (1.5mcg/kg/week), other 3 received peginterferon alfa-2a (135mcg/week). ribavirin dosage ranged from 600mg-1000mg/day depending on body weight and baseline haemoglobin. treatment durations were 48-52 weeks in 7 patients, 24-26 weeks in 2 patients as one showed rapid virologic response at week 4 and the other was intolerant to side effect of peginterferon. eight patients had early virologic response at week 12 and one had >2log drop of hcvrna. eight patients had end-of-treatment response. eight patients (88.9%) achieved svr at end of follow-up. two patients who received only 24-26 weeks of combination therapy also achieved svr. the one who failed to achieve svr was at older age of 60 and had advanced fibrosis. conclusions: the efficacy of pegylated interferon and ribavirin in treating chinese patients with chronic hepatitis c genotype 6a can achieve high sustained virologic response rate of 88.9%. t. bharati 1,2 , p. kar 2 , a. mohammad 2 , k. mariappan 1 , j. annamalai 1 , r. introduction: hcv is a recognized cause of hcc. information on hcv genotypes in hcc are scanty in india. methods: a total of 154 hcc cases from delhi, 96 hcc cases from madurai and 246 cases of chronic hepatitis without hcc were controls in the study. rt-pcr for hcv rna and genotyping were carried out in all the cases results: in group-i, hcv rna was positive in 26.58% hcc cases in which genotype 3 was found in 57.1% genotype 1 was observed in 26.2% hcc cases. whereas 16.6% cases remained nontypable. in group-ii, hcv rna was positive in 23.95% hcc cases, with genotype 3 in 30.4% cases, genotype 1 in 52.2% cases and genotype 4 in 4.34% cases. however, 13.04% cases remained nontypable. out of the 246 control cases, 187 were ch and 59 were cirrhosis. in ch group, hcv rna was positive in 22.45% cases in which, genotype 3 was detected in 71.4% cases whereas genotype 1 was observed in 21.4% cases . however 7.1% cases remained nontypable. in cirrhosis group, hcv rna was positive in 37.2%cases. genotype 3 was found in 59.1% cases. while genotype 1 was present in 31.8% cases and 9.1% cases remained nontypable. conclusion: genotype 3 in delhi and genotype 1 in madurai were predominant. in hcc cases. our study demonstrates that no particular hcv genotypes were associated with hcc and genotype did not appear to influence the development of hcv-associated hcc. background/aims: the standard treatment for chronic hepatitis c infected with hcv genotype-1 is a combination of pegylated interferon alfa and ribavirin for a 48weeks. it is unclear if 24weeks treatment is possible for patients showing a rapid virologic response (rvr) without compromising the sustained virologic response (svr) in korea. method: between june 2005 and july 2008, among patients chronically infected with the hcv genotype-1 (hcv-1) who were treated with pegylated interferon alfa subcutaneously once weekly plus ribavirin (weight-based), 20 consecutive paients who had low pretreatment viral load ( 1.5x10 6 copies/ml) and rvr were treated for 24weeks and then followed up for 24weeks. the hcv rna was quantitatively assessed pretreatment, at 12weeks of treatment and was qualitatively assessed at 4weeks of treatment, the end of treatment (24weeks), 24weeks after end of treatment. rvr was defined as undetectable hcv rna at the 4weeks. results: baseline characteristics of patients was as followed; age (30-65years:mean 45years), bmi (21-27kg/m²:mean 23.5kg/m²), hcv rna titer (0.3-1.4×10 6 copies/ml:mean 0.5×10 6 copies/ml), alt (5-751iu/l:median 77iu/l). among the 20 patients, all patients (100%) had sustained virologic response (svr). conclusions: hcv-1 infected patients with a low baseline hcv rna concentration ( 1.5×10 6 copies/ml) who had hcv rna negative at week 4 of treatment may be treatment for 24weeks without compromising sustained virlolgic response. however, an additional trial will be needed to optimize the treatment duration. background/aims: acute hepatitis c (ahc) has a high chronicity rate of up to 50~84% if it is not treated. although the good treatment response to pegylated interferon (peg-interferon) therapy has reported, there is not definite guideline to treat of ahc in korea yet. the aim of our study was to investigate the clinical course and treatment outcome of ahc in single center of korea. methods: we performed a retrospective analysis of 35 patients who were diagnosed with ahc during the period from may 1996 to december 2007. the diagnosis of ahc was based on seroconversion to anti-hcv antibody or the clinical and biochemical diagnostic criteria satisfactory to ahc and on the presence of hcv rna in first serum sample. the spontaneous resolution was defined as loss of hcv rna in serum for 6-month in untreated group, and in treatment group, the sustained virological response (svr) was defined as a index of treatment success. results : thirteen of thirty-five patients were treated, six of thirty-five were untreated and observed clinical course, and sixteen patients were not followed up after diagnosis. in treatment group, nine of thirteen (69%) acquired svr, and two of six (33%) showed spontaneous resolution in untreated group. ten of thirteen treatment patients used conventional interferon, and another three patients used peg-interferon. conclusion : compared with untreated group, there was higher svr rate in treatment group (33% vs. 69%). so early interferon treatment in acute hepatitis c should be considered. background: this study was conducted to identify predictors of thyroid dysfunction and to determine whether virologic factors or treatment response affect thyroid dysfunction development during peginterferon (pegifn) therapy in chronic hepatitis c patients. methods: sixty chronic hepatitis c patients treated with pegifn -2a or -2b in combination with ribavirin from 1st july 2004 to 30th july 2008 were included in this study. treatment responses were evaluated and thyroid functions were assessed every 4 weeks. results: seventeen patients (28.3%) experienced thyroid dysfunction during treatment, and that occurred more frequently in women and in patients with a lower body mass index (bmi). the proportion of patients with a high viral load (a serum hcv rna titer >750,000 iu/ml) was significantly higher in the thyroid dysfunction group rather than in the euthyroid group(88.2% vs. 48.8%, p=0.005). among patients with hcv genotype 1, the rate of sustained virologic response was lower, and relapse occurred more frequently in the thyroid dysfunction group than in the euthyroid function group during pegifn-based therapy(svr, p=0.024; relapse, p=0.017). the female gender and the high viral load were independent predictors of thyroid dysfunction in multivariable analyses (female, or 9.44, p=0.009; high hcv rna titer, or 8.40, p=0.030) . conclusion: the risk of thyroid dysfunction during pegifn therapy for chronic hepatitis c was found to be higher for women and for those with a low bmi and a high viral load. background: in peginterferon alpha2b (peg-ifn 2b) and ribavirin (rbv) combination therapy for 48weeks for patients with chronic hepatitis c, it is still difficult to predict which patients will achieve sustained viral response (svr) at the completion of this therapy. aim: to predict svr and non-svr (relapse) at the end of this combination therapy by determining changes of serum hyaluronic acid (ha) levels. methods: eighteen patients were enrolled and their serum ha levels were measured before therapy, and after the 1st, 2nd, 3rd, and last trimesters during therapy. results: eleven patients achieved svr and 7 became relapsers. all patients showed higher ha levels in the 1st trimester than the pretreatment levels. in the svr group, 3 of 11 (27.2%) patients in the 2nd, 5 of 10 (50.0%) in the 3rd, and 9 of 11 (81.8%) in the last trimester showed lower ha levels than the pretreatment levels. by contrast, in the relapser group, none in the 2nd, 1 of 6 (16.7%) in the 3rd, and 1 of 7 (14.3%) (p<0.05) in the last trimester showed lower ha levels than the pretreatment levels. this study revealed that as the 48-week therapy went on, ha levels were more likely to fall below the pretreatment levels by the last trimester in patients achieving svr. however, ha levels of relapsers tended to continuously be above the pretreatment levels. conclusion: determination of changes of serum ha levels during peg-ifn 2b and rbv therapy predicts svr and non-svr at the completion of this therapy. results: the most frequent lymphomas were with high malignancy (40%), intermediate (32.5%) and low degree (27.5%). cryopathy was negative (87.5%). the presence of viral markers was performed soon as possible after the nhl diagnosis, at the same time (52.5%) or during the first year of evolution (32.5%). the prevalence of the hcv infection was 15%, comparable to the one in the control patients group (22.68%), admitted in a gastroenterological clinic. on the other hand, this prevalence is significantly increased compared to the one in the general romanian population (4.9%). the patients with nhl and hcv infection belonged especially to the low and intermediate malignancy degrees; the survival was influenced by the malignancy degree and not by the presence of hcv infection. the prevalence of hbv infection in the tested patients was 2.5%, being lower than that of hcv infection (2.5% vs. 15%, p = 0.113) but comparable to the one in the general population (2.5% vs. 6.3%, p = 0.525). conclusions: the prevalence of hcv infection in the patients having nhl was 15%, comparable to the one in the control group, but significantly increased compared to the one in the general population, leaving open the issue of a causal relationship between hcv infection and nhl. iron hepatic overload and hepatitis c d. damian 1 , m. grigorescu 1 , m.d. grigorescu 1 , t. zaharie 1 1 third medical clinic, cluj napoca, romania aim: evaluation of the prevalence and the degree of iron loading and the relationships with the clinical, biological and morphological changes. method: 212 patients with chronic hepatitis c were included, to whom we tested the blood iron level. in order to evaluate the hepatic iron accumulation we performed the perls staining, using a qualitative analysis and a semiquantitative scoring system (deugnier). results: from a total of 212 patients, 16.5% presented increased blood iron level (p = 0.000). the evaluation of the liver iron loading was performed in 54 patients, some having normal blood iron level (n = 34) and others (n= 20) increased (p = 0.007). the stainable iron was observed in 27 patients. the iron loading was usually low, the deposits were observed mostly at the sinusoid cells and the hepatocyte and less in the portal spaces, usually as a pale staining or of small, nonmerging granules. the total iron score deugnier was low. the increased blood iron correlated with the alt and ggt levels, the necroinflamatory activity and fibrosis. no correlationships between stainable iron and increased blood iron. the presence of liver iron accumulation only correlated with the fibrosis degree. conclusions: of the 212 patients to whom we tested the blood iron, 16.5% had increased levels. the perls staining was positive in 50% of the patients. the iron loading was mainly low, with a more frequent distribution in the sinusoid cells and in the hepatocytes and correlated only with the stage of fibrosis. response patients whose hcv rna became negative at 12 -16 weeks t. ide 1 , t. arinaga 1 , k. ogata 1 , i. miyajima 1 , k. kuhara 1 , r. kuwahara 1 , m. background/aim: chronic hepatitis patients whose hcv rna became negative at 4 weeks of peg-interferon/ribavirin treatment achieved excellent svr(sustained viral response) rate of almost 90%. however, in patients whose rna became negative after 20 weeks, the svr rate is very low. since many patients became rna negative at 12-16 weeks, it is important to clarify the characteristics of the patients. material and method: among 234 patients, 41 became rna negative at 12-16 weeks and the therapy completed (total 48-60 weeks). the characteristics were analyzed by using sex, age, weight, bmi, alt, gtp, hemoglobin, platelet counts, ccr, hyaluronic acid, the mutation of hcv core region (aa70, 91) and interferon sensitivity determining region, adiponectine, home-ir, rna dynamics, dose and the treatment period. result: the svr rate was 75.6%(31/41). because all 10 patients of non svr were female, we compared these 10 patients and 15 female svr. the platelets counts were low in non svr (non svr 15.0 ± 2.8 (x10 4 /mm 3 ) vs svr 20.2 ± 4.8 (p<0.05)). the mean dose of ribavirin was lower (447 ± 126 mg/day) in non svr (p<0.05) than in svr (625 ± 127). conclusion: as for the characteristics of the patients whose hcv rna became negative at 12-16 weeks but became non svr, female, low platelet count and low dose of ribavirin were important factors. in the patients who received reduced ribavirin doses, the idea to increase the ribavirin dose and to maintain it are necessary. (ex, use 400mg and 600mg alternately) pe329 background: chronic hepatitis c virus (hcv) infection poses a challenge for a growing number of infected patients who exhibit disease complications, including cirrhosis, hepatocellular carcinoma, and liver failure in china. the combination treatment of peginterferon alpha (peg-ifn alpha) plus ribavirin (rbv) is recommended as a standard care for hcv infections, which can improves hepatic markers and eradicates the virus in about 50% of patients. however, a significant number of patients do not respond to therapy or relapse following treatment discontinuation. several viral, hepatic, and patient-related factors influence response to therapy. methods: in our clinical practice, a total of 77 interferon-naïve patients (61% male; median age 47 years) with chronic hepatitis c include 11 cirrhotic patients (no genotyping) received peg-ifn alpha-2a 180 mcg/week plus rbv 900-1200mg/day for 48 weeks and follow up 24 weeks. results: show that the patients have more rvr and evr rate (54% and 90.9% respectively). while the svr (undetectable hcv-rna 24 weeks after treatment completion) rate is only 51.5% in conclusion: comparing with the data of clinical trail, the rvr, evr and eotr were higher, while svr was the same in chinese patient with chronic hepatitis c patients received the combination therapy of peg-ifn plus rbv. the reason of high relapse was still unknow. although optimal duration of retreatment and benefits and safety of maintenance therapy have not been determined, an extended duration is likely needed, even for the patients who achieved evr. s. nakamoto 1 , f. imazeki 1 , k. background/aims: recently amino acid (aa) substitutions in hepatitis c virus (hcv) core region (double wild (dw); arginine at aa 70, leucine at aa 91) were reported to be associated with sustained virological response (svr) in a combination therapy of peginterferon and ribavirin. we evaluated the viral factors influencing treatment response. methods: nucleotide sequences of core region were determined directly in 104 patients with genotype 1 and high viral load ( 100 kiu/ml) treated with peginterferon-alpha 2b and ribavirin for 48 weeks. rapid virologcal response (rvr) was defined as more than 2 log decrease of hcv-rna during the first four weeks of therapy and early virological response (evr) as that during the first 12 weeks. svr was defined as negative hcv-rna 6 months after the end of treatment and non-virological response (nvr) as less than 2 log decrease of hcv-rna during the treatment. results: dw at aa 70 and 91 was shown in 17/44 (35%) patients with rvr and in 5/44 (11%) with non-rvr (p=0.003), in 25/72 (35%) with evr and in 1/28 (3.6%) with non-evr (p=0.001), in 16/37 (43%) with svr and in 6/44 (14%) with non-svr (p=0.003), and in 1/24 (4%) with nvr and in 25/79 (32%) with non-nvr (p= 0.005). in multiple logistic regression analysis, dw was significantly associated with rvr, evr, svr and nvr. conclusions: dw at aa 70 and 91 in hcv core region was closely associated with virological response in a combination therapy of peginterferon and ribavirin. medicine and hepatology, henry dunant hospital, athens, greece, 3 biometrics, ist gmbh, mannheim, germany, 4 background: among patients with chronic hcv treated with pegylated interferon and ribavirin, the highest sustained virologic response (svr) rates are achieved in patients with a rapid virological response (rvr). here we investigate how the time taken to become hcv rna undetectable influences the probability of relapse during untreated follow-up. methods: data from 569 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180µg/week plus ribavirin 1000/1200mg/day were included in the intent-to-treat analysis. response was classified as rvr, complete early virological response (cevr) slow responder and non-evr. results: there was a correlation between the time required to become hcv rna undetectable and the relapse rate after stopping treatment. patients with an rvr had the lowest relapse rate (4%); this increased among patients with slower responses. conclusion: there was an inverse correlation between the time taken to achieve a virologic response and the probability of relapse. background: rapid virologic response (rvr; hcv rna <50iu/ml) at week 4 of treatment with pegylated interferon plus ribavirin can be used to predict the probability of achieving an svr. patients with detectable hcv rna at week 4 have a lower probability of achieving an svr than those with an rvr; further subdivision of these patients may be useful in predicting outcomes. methods: we conducted a retrospective analysis including 569 genotype 1 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180 g/week and ribavirin 1000/1200 mg/day. patients were categorized as rvr and non-rvr. those without an rvr were further subdivided into detectable but unquantifiable, 3, 2, 1 or <1 log10 drop in hcv rna. the proportion of patients with undetectable hcv-rna at week 12 and achieving an svr was calculated within each category. results: rvr and non-rvr patients had an 88% and 43% rate of svr respectively. among non-rvr patients, rates of svr depended on the categorical response at week 4: detectable but unquantifiable hcv rna, 77%; 3 log10 drop in hcv rna, 61%; 2 log10 drop, 43%; 1 log10 drop, 27%; and <1 log10drop, 13%. independent of week 4 response, undetectable hcv rna at week 12 was also highly predictive of svr. conclusions: patients achieving an rvr have high rates of svr. among patients who do not achieve an rvr a more precise prediction of svr can be achieved by considering the extent of viral load reduction at week 4 and week 12. retrospective japanese validation study of fibrotest and actitest in patients with chronic hepatitis c. n. nagata 1 , t. mine 1 1 background: fibrotest (ft) and actitest (at) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis c virus. the aim of this study was to perform a validation study the discordances between ft and at(ft/at) and liver biopsy in patients with chronic hepatitis c in japan. methods: 117 serum samples of chronic hepatitis c patients sended at -80°c at the biochemistry department of pitié salpetrière hospital were analysed between july and august 2007. ft/at components were assessed on thawed sera for 110 patients. 96 from 110 patients had liver biopsy at the moment of serum analysis. liver biopsy fibrosis and activity scores were assessed by a pathologist in japan according to metavir scoring system. for each individual test-ft/at the following statistical analysis were performed result: ft observed auroc for the diagnosis of advanced fibrosis was 0.81 and after adjustment according to the prevalence of different stages of fibrosis the auroc was 0.89. this difference could be explained by the non-homogenous distribution of different stages of fibrosis (low prevalence of extremes stages of fibrosis -f0 and f4-and high prevalence of adjacent intermediate stages -f1 and f2). the observed auroc of ft for the diagnosis of precirrhosis and cirrhosis was 0.86 and the observed auroc of at for the diagnosis of moderate to severe activity was 0.74 . conclusion: these results are similar to those observed in all independent validations worldwide. background: accurate monitoring of hcv-rna level throughout anti-hcv therapy is key factor for predicting sustained virological response (svr). real-time detection polymerase chain reaction (rtd-pcr) based methods are sensitive, have wide dynamic range of quantification and carryover contamination caused by classical pcr. aim: to compare rtd-pcr based assays; cobas ampliprep/cobas taqman (cap/ctm) and recently developed abbott realtime hcv for hcv rna quantification and measurements differences by 2 assays in different genotypes. methods: in total, 253 serum samples were used including, 135, 39, 24, 15, and 40 with genotypes 1b, 2a, 2b, 3a and 4 respectively were tested quantatively for hcv-rna by cap/ctm and abott realtime. results: good correlation between two assays as overall (r=0.96) with correlation coefficient (r) in genotypes 1b, 2a, 2b, 3a ranged between 0.99 to 0.98 and least in genotype 4 (r=0.78). mean differences between cap/ctm and abott realtime was significnat in genotypes 1b and 4. significantly hcv-rna genotype 4 underestimation by cap/ctm (4.3+0.9 log iu/ml) than abbott realtime (4.8+0.9 log iu/ml; p=0.01). in genotype 1b, significantly higher hcv-rna measurement by cap/ctm (5.7+1.7log iu/ml) than abbott realtime (5.0+1.4log iu/ml, p=0.001). two hcv genotype 4 samples showed measurement differences (cap/ctm minus abbott realtime) of -3.75 and -1.68 log iu/ml. studying genotype 4 sequences within 5 utr , target for cap/ctm rt-pcr amplification, revealed nucleotide polymorphisms at positions a107, a165, t203, a205, and a243. conclusion: different measurement efficiency by commonly used cap/ctm in different genotypes compared to abbott realtime. 6 new york, new york, usa, 7 vertex pharmaceuticals, 8 cambridge, ma, usa, 9 duke clinical research institute , 10 duke university, durham, nc, usa background: prove1 is a placebo-controlled study of 250 subjects with genotype 1 chronic hepatitis c randomized to 48 weeks of peginterferon-alfa-2a 180 ug/week (p) plus ribavirin 1000-1200 mg/d (r) (pr48, n=75), or 3 regimens of 750 mg q8h telaprevir (tvr) with pr: tvr/pr for 12wks followed by pr for 0wks (t12/pr12, n=17), 12wks (t12/pr24, n=79) or 36wks (t12/pr48, n=79). the impact of african american race (aa) and bridging fibrosis on sustained virologic response (svr) was examined. methods: subjects with cirrhosis were excluded from study. fibrosis was categorized as mild/minimal, portal, or bridging from biopsy within 2 years. itt analysis was performed. results: overall, svr was achieved by 41% of subjects in the pr48 group, 35% in t12/pr12 group, 61% in t12/pr24 group, and 68% in t12/pr48 group. subgroup analyses indicated svr was improved with tvr/pr (tvr/pr arms pooled) vs pr48 alone in aa subjects (44% (8/18) vs 11% (1/9)), and in subjects with bridging fibrosis (69% (22/32) vs. 26% (5/19)). adverse events leading to discontinuations were more frequent in the tvr/pr groups (21% vs. 10%). rashes, gastrointestinal events and anemia were more common in the t/pr arms, and rashes were more frequently severe (7% vs 1%). conclusions: tvr-based treatment for 24 or 48 weeks was associated with an increase in svr rates compared to pr48. subgroups with impaired response to standard peg-ifn/rbv therapy appeared to benefit from the addition of telaprevir. adverse events leading to discontinuation were more frequent in tvr-based regimens. background and aims: induction of type i ifns is a core issue in antiviral responses and must be tightly controlled. the protein kinase tbk1 is critically involved in virus-triggered type i ifn signaling. in previous studies, an alternatively spliced isoform of tbk1, termed tbk1s, was identified to be induced in both human and mouse cells. bound to rig-1, it is able to disrupt the interaction between rig-i and visa. this study was designed to observe the expression of tbk1s in hcv-infected patients. methods: total rna was extracted from samples of peripheral blood mononuclear cells obtained from 11 hcv patients, 9 hcv patients treated with ifn-/ribavirin and 9 healthy controls, and subjected to real -time pcr using the primer-probe sets for human tbk1s, tbk1 and ifn-genes. results: the tbk1s expression was significantly elevated in hcv-infected patients, while treatment of hcv-infected patients with ifn-/ribavirin resulted in down-regulation of tbk1s to the normal level. conclusions: the study strongly supports the idea that expression of tbk1s is correlated with hcv infection, and indicates that tbk1s may play an important role in the regulation of hcv infection.this work was supported by nsfc(30571643, 30672380, 30700702 background: infringement of iron metabolism is one of fibrosis progressing factors during diffuse liver diseases. the interrelation between the syndrome of iron overload (sio) and svr achievement is studied during chronic hcv infection treatment. methods: 68 patients with chronic hcv infection (genotyping: 1-34; 1+3-5; 3-22; 2-6; 4-2) are investigated. sio criteria: iron increase-more than 37 mkmol/l, ferritin -more than 200 mkmol/l, percent of transferriny saturation with iron (%tf) -more than 50%. results: sio revealed in 10 patients (14.7%): 5 patients -1 genotype (2 assotiative with a diabetes) and 5 patients-genotype 3 (3 -in combination with liver steatosis and obesity). venipuncture series were done up to getting ferritin referential parameter values before therapy beginning. rvr: sio -5 patients, normal metabolism -48; evr: 6 and 54, svr: 4 and 54 relatively. 5 nonresponding patients (sio) had steatosis and diabetes, hereditary hemochromatosis (c282y/h63d) is verified in 1 case. increase of ferritini values and %tf during therapy and positive hla-a3 and hla-b7 is registered in nonresponding patients. conclusions: sio in hla-a3, hla-b7 and c282y/h63d positive patients is independent predictor of nonresponse during peginterferon alpha-2a (40 kd)" ribavirin treatment. a.p. srivastava 1 , g. dogra 2 , s. sachdeva 1 , n. nigam 1 , a. chakravarty 2 1 dr. rml hospital, new delhi (india)-110001, 2 maulana azad medical college & associated hospitals, new delhi, india background: hepatitis c virus (hcv) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. genotyping and assessment of viral load in hcv patients are vital for designing therapeutic strategies. we aimed to determine the pattern of hcv genotypes and its association with viral load and biochemical profile. methods: 71 hcv rna positive patients were included in the present study attending the medical-opd and wards of dr rml hospital, a tertiary care hospital in new delhi during 2006-2008. hcv genotyping was carried out by restriction fragment length polymorphism (buoro et al 1999) followed by the type specific primers from the core region (ohno et al 1997) . viral load estimation was carried out by taqman real time pcr system using previously described method (martell et al 2000) . result: 67.6% of cases were having genotype 3 (3a, 3b, 3f & 3i) followed by genotyping 1 (1a & 1b) in 26.8% and genotype 2 in 5.6%. there was no statistical significant difference seen in the biochemical profile between the three groups of genotypes. genotype one was associated with a significantly higher viral load as compared to the genotypes three and two. parentral mode of transmission was accounted for the 68% of all the infected cases. conclusion: hcv genotypes 3 and 1 accounted for 94% of our cases. the genotype 1 is associated with higher degree of disease severity as assessed by viral load. also two unusual subtypes 3i and 3f were identified from this geographical region. a.p. srivastava 1 , g. dogra 2 , s sachdeva 1 , n. nigam 1 background: the development and resolution of an inflammatory process is regulated by a complex interplay among cytokines that have pro and anti-inflammatory effects. regulatory mechanisms that control the production of cytokines include genetic polymorphism in particular promoter/leader region. polymorphisms may directly or indirectly affect the binding of transcriptional factors, consequently increasing or decreasing the production of mrna, thus regulating cytokine production. we aimed to determine the polymorphism of tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) genes in chronic hepatitis c patients. methods: 40 hcv rna positive patients were included in the present study conducted during 2006-2008. 25 healthy controls were also included. genomic dna was extracted by using q1a amp dna blood kit protocol according to manufacture's instruction and desired fragment was amplified by using the primer's of vidigal et al 2002. result: genotyping of -308-promoter variant of tnf-alpha was performed by pcr. polymorphism in the tnf-alpha (g/g, g/a and a/a allele) was different between hcv patients and healthy controls. il-10 variants (c/t, c/c) were more frequent among hcv patients as compared to healthy controls. conclusion: genetic polymorphism analysis on il-10 promoter have indicated that distribution pattern of il-10 polymorphism was significantly different between controls and hcv patients. furthermore, polymorphism in promoter region of tnf-alpha (-308) was found, though the difference was not significant. since this is a preliminary study, we believe that our findings may stimulate further research on larger number of patients. introduction: the assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. although liver biopsy is the gold standard for fibrosis assessment, it is invasive and may have some risks, this has led to the development of non-invasive biochemical markers of liver fibrosis. fibro-test which have five parameters used for the quantitative assessment of liver fibrosis. our aim is to validate the performance of fibro-test in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 50 patients with chronic hepatitis c genotype 4. all biopsies were scored using metavir system by two independent pathologists. fibro-test was done with (biopredictive, houilles, france) for the assessment of liver fibrosis. sensitivity, specificity, ppv and npv were measured for distinguishing between different degrees of severity of fibrosis. results: patients (45 male and 4 female) age ranged 21-56 years, liver biopsy showed 4% (f0), 40% (f1), 20 % (f2), 28% (f3), 8% (f4). the efficacy of fibrotest is 63.26%, sensitivity 83.3%, specificity 53%, positive predictive value 50% and negative predictive value 85%. conclusion: fibrofast has a low performance in assessment in fibrosis in chronic hepatitis c genotype 4. introduction: liver biopsy is the reference method for assessing liver fibrosis. however, it is invasive, costly and has some limitations. european liver fibrosis (elf) markers have shown to be accurate in assessing liver fibrosis in a range of chronic liver disorders. our aim is to test the performance of elf markers in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 199 patients with chronic hepatitis c genotype 4. all biopsies were staged for fibrosis using metavir system by two independent pathologist. elf markers were done by (diagnostic & operations, england) and fibrosis scores were derived using the published elf algorithm. the area under the curve (auc) for receiver operator characteristic curves was measured along with sensitivity and specificity, positive (ppv) and negative (npv) predictive values for distinguishing between different stages. results: patients (179 male and 20 female), age was ranged 25-51 years, liver biopsy showed 2% (f0), 27% (f1), 34% (f2), 26% (f3) and 11% (f4). elf markers had no correlation with fibrosis score where r = -0.003, p = 0.963, aucs: 0.469, specificity 87.9 %, sensitivity only 9.3 %, ppv: only 6.03 %, npv: 61.5 % and efficacy 58.2%. conclusion: the performance of elf marker is low and can not be used for assessment of fibrosis in chronic hepatitis c genotype 4. background: the prove2 trial is a randomized, placebo-controlled study that assessed the safety and efficacy of 750mg q8h telaprevir (tvr) combined with 180 g/week peg -ifn alfa-2a (p) ± 1000-1200mg/day ribavirin (r) in chronic hcv genotype 1-infected treatment-naïve patients without cirrhosis. methods: overall, 323 patients received tvr + pr for 12 weeks (t12/pr12; n=82), tvr + pr for 12 weeks then pr for 12 weeks (t12/pr24; n=81), tvr + p for 12 weeks (t12/p12; n=78), or to pr for 48 weeks (pr48; n=82). primary endpoint: sustained virologic response (svr, undetectable hcv-rna 24 weeks post-treatment). results: baseline characteristics were well balanced across groups. numerically higher svr rates were observed in patients receiving t12/pr24 (69%; p=0.004 for difference vs. pr48) than t12/pr12 (60%), t12/p12 (36%) or pr48 (46%). relapse rates were lower in the t12/pr24 group (14%) than the t12/pr12 (30%), t12/p12 (48%) and pr48 (22%) groups. the relapse rate in patients receiving t12/pr24 with 4-week and 12-week undetectable hcv-rna was 7% (3/45). the aes occurring more frequently with the t/pr regimen were pruritus, rash, asthenia, nausea and anemia. in the t/pr arms, 12 patients discontinued due to rash, 2 discontinued due to pruritus, and 2 patients due to anemia. conclusion: these results showed that a telaprevir-based regimen led to significantly higher svr rates than pr, and indicate that this regimen could shorten the overall treatment duration from 48 weeks to 24 weeks for most patients infected with hcv genotype 1. a.c. cardoso 1 , c. stern 1 , r. moucari 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: this study evaluated the effect of the response (svr) to therapy on fibrosis stage, as assessed by ls, in patients with advanced fibrosis (f3) or cirrhosis (f4). methods: hcv patients with f3 or f4 who received interferon-based treatment were studded. ls was assessed after treatment (median delay of 36 months, 2-206) in patients with or without svr. correlations between ls and clinical and treatment characteristics were analyzed. results: 114 patients were included: male gender (72%), mean age (54±9 years), diabetes (26%), mean bmi (26±6 kg/m2), genotype 1 (59%). 33% had svr. ls was performed 0-3, 3-6, >6 years following treatment. by linear regression, the median of the ls was independently associated with svr (p=0.005) and diabetes (p=0.008). svr patients had lower ls (8.4 kpa; range 3.3-45) than non svr patients (15.7 kpa; range 5.3-75) (p<0.001). among the svr patients the median ls was lower when the delay between ls and the end of treatment was longer (10.9,8.8,6. 3) (p=0.02). on the opposite, among the non-svr patients the median ls was not significantly different (p=0.57). the median of liver stiffness was higher in patients with diabetes (p=0.006). bmi and dyslipidemia did not influence the median of the ls. conclusion: in patients with advanced fibrosis or cirrhosis, ls was lower in patients with svr and decreased with time while it was higher and did not decrease in non-svr patients. ls could be important for assessment of fibrosis stage during the post-treatment follow-up. to study peripheral blood and intrahepatic natural killer (nk) cells in patients with chronic hepatitis c in relation to disease activity and severity of hepatic fibrosis. patients & methods: fifteen untreated patients with histologically-proven chronic hepatitis c, and 12 matched healthy subjects. the nk cells and natural killer t (nkt) cells were identified in fresh whole blood samples using two-color flow cytometric assay as cd3 -cd56 + and cd3 + cd56 + positive cells. immunohistochemical staining of liver biopsies taken from all patients was done using monoclonal antibody against cd56 for detection of nk cells and rabbit polyclonal antibody against smooth muscle actin (sma) for identification of activated hepatic stellate cells (hscs). results: patients with chronic hepatitis c showed significant decreases in the percentages of nk cells and nkt cells in peripheral blood. a negative correlation was found between serum hcv rna levels and the percentages of peripheral blood nk cells and the intensity of intrahepatic nk cells. the percentages of circulating nk cells and nkt cells and the intensity of intrahepatic nk cells were inversely correlated with the metavir fibrosis stage and the steatosis grade, and also with the intensity of intrahepatic activated hscs. conclusion: patients with chronic hepatitis c had significant deficiency in circulating nk and nkt cells as well as in intrahepatic nk cells. this may provide a possible mechanism for the suppression of innate immunity against hcv. background: hcv infection is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. the virus is classified in six genotypes and more 50 subtypes, which are related distinct with antiviral therapies reply. in brazilian amazon, epidemiologist's studies in blood donors had pointed high frequency of genotype 1 (74%) followed by genotypes 3 (25%) and 2 (1%). however, epidemiological research in populations of risk to the infection still is scarce. aim: to determine hcv genotypic frequency in 116 blood donors, 55 patients with blood transfusions multiples, 57 patients in hemodialysis and 52 drugs users in the state of pará, brazilian amazon. methods: using real time pcr and nucleotide sequencing followed phylogenetic analysis had been gotten viral diagnosis and genotyping. results: in blood donors, hcv distribution was constituted by genotypes 1 (93.1%) and 3 (6.9%). in multitransfunded patients occurs maximum prevalence of genotype 1 (100%), probably reflect of genotype 1 specific transmission of blood donors population. on the other hand, in hemodialysis patients had been detected genotypes 1 (85.7%), 2 (3.6%) and 3 (10.7%), result of a bigger diversity of transmission routes (transfusional, interfamilial, nosocomial, etc) . in drug users occurs the biggest frequency of genotype 3 (38.1%) with prevalence of genotype 1 (61.9%), suggesting that the sharing of abuse machinery is allowing strains diffusion of genotype 3. conclusions: the genotype 1 possesses the biggest frequency in different population. moreover, through hcv genotypic frequency if it detached the contribution of transmission distinct routes indicated by previous epidemiologists researches. virol. 2007 ). we established real-time polymerase chain reaction (pcr) assays for the easy detection of these hcv mutations. methods: plasmids p-core-w, including wild type hcv core coding region (70r and 91l), and p-core-m, including mutant type hcv core (70q/h and 91m), were constructed by cloning and pcr-based mutagenesis for control vector of wild type core and that of mutant core, respectively. using serially diluted forms of these vectors, sybr green-based real-time pcr detections with mutation-specific primers were performed. results: analysis of known scalar concentrations of references indicated that the detection limits of these methods were at least 10 copies, 10 copies, 1000 copies, and 10 copies of 70-wild, 70-mutant, 91-wild, and 91-mutant, respectively. each primer could clearly distinguish the difference between p-core-w and p-core-m at the same copy numbers. concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-w versus p-core-m could be distinguished. on the other hand, for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. conclusions: this method could represent a useful alternative for the detection of genotype 1b hcv core amino acid substitutions 70 and 91 and be reliably applied for rapid screening. efficacy and tolerability of hcv treatment in asian patients according to age and genotype at a tertiary centre in western australia n. saroj 1 , n. kontorinis 1 , t. lorenzo 1 , m. marion 1 , s.l. chen 1 , w. cheng 1,2 1 royal perth hospital, 2 centre for international health, curtin introduction: race and ethnicity can influence efficacy and tolerability to treatment in hcv. the higher response rate in asians is thought to be associated with better adherence and tolerability. objectives: (1) to evaluate the adherence according to age and genotype (2) to assess the effect of age on treatment efficacy (3) background: hepatitis c virus (hcv) infection is a major health problem. there is huge regional variation in its prevalence and genotypic distribution. voluntary blood donors are thought to have somewhat lesser prevalence than the rest of the community. reliable statistics are not available for the entire country, particularly for the rural areas. it is important to know local situation and rationalize use of limited resources. methods: retrospective study of the records of patients attending the free liver clinic (flc) of our hospital located in a rural area of pakistan, and those screened for hcv infection prior to voluntary blood donation. results: patients at flc (324 out of 1638 [20%; males 65%] were found to have higher chances of being reactive for hcv antibodies as compared to voluntary blood donors (121/804 [14%]; p = 0.004; or 1.39 -95% ci = 1.11 -1.75). out of a total of 1022 hcv reactive patients, 904 (88%) were found to be positive on hcv rna testing. out of a total of 166 typeable genotypes, 125 (75%; 95% ci = 68.7 -81.9, estimated odds = 3.05) were infected with a single genotype, and only 7 patients (4%) were infected with genotype 1, either alone (n=4) or in combination with 3a. conclusions: one out of every 5 people tested in our flc is seropositive for hcv, and 14% of "healthy" voluntary blood donors have the same results. genotype 1 is very rare in our region. s.a. batool 1 , s.z. abbas 1 1 department of gastroenterology, muhammad hospital, mirpurkhas, pakistan background: hepatitis c viraus (hcv) infection is common in our region. data is not available on success rates of conventional interferon (inf) based products here. we attempted to find out the dominant genotype, and to determine the success rate of conventional inf-based treatment in eradicating hcv. methods: retrospective case series study of hcv infected patients' records treated with 14 different brands of inf. results: 320/1858 (17%) of all patients tested were positive for hcv antibodies. hcv-rna was tested by pcr for 1022 patients, of which 904 (88%) turned out to be positive. genotype type 3 was the dominant genotype -found in 118/168 (70%) patients. 101 men and 57 women were treated with various brands of inf with the same manufacturer's brand of ribavirin. the overall etr achieved was 100/142 (70%) -58/85 (68%) men and 42/57 (74%) women. 41/62 (66%) of genotype 3 achieved etr. there was no significant difference in average ages for those who achieved good etr and those who did not (39 years each). the etr achieved by different brands ranged from 48% to 91%. svr was achieved by 17/30 patients. conclusions: 17% of all people tested positive for hcv antibodies, of which about 88% had evidence of active hcv infection. etr achieved by different brands averaged 70%. this was 74% in female sex, although age did not appear to be a factor in determining a favourable etr. patients & methods: a total of 439 consecutive diabetic patients of either sex were evaluated for hcv and hbv infection by using enzyme linked immunosorbant assay (eliza-3) along with serum alt levels. on the basis of this test, the patients were divided into two groups, sero +ve and sero -ve. different variables were: age, sex, bmi, area of residence (rural or urban), type and duration of dm, smoking, literacy and alt. results: males 50.3% and females 49.7%. age ranged from 18 to 95. majority were married (98.4%), from rural area (70.4%), had type-2 dm (99.8%), normal weight (39.2%), normal alt(60.1%) and non-smokers (78.6%). seroprevalence for hcv, hbv and both were 25.1%, 1.8% and 1.6%. two groups were made, sero +ve and sero -ve. raised alt (59.2%) was significant (p<0.05) factor while all others variables were insignificant (p>0.05). conclusion: hbv and hcv infections are more prevalent in dm with increased alt levels. while hcv infection is more common than hbv in patients with dm. hepatitis c virus (hcv) envelope proteins (e1 and e2) mediate the entry of virus into host cells by binding to its cellular receptors and resulting in the fusion of the viral membrane with host cell membrane. the expression and secretion of biologically active envelope proteins in vitro have proven to be a difficult task due to the high degree of glycosylation and the existence of hydrophobic domains within these sequences. in order to obtain glycosylated, correctly-folded hcv envelop proteins in large quantities, we optimized the dna sequences of hcv envelop proteins by substituting the encoded sequence with human preferable codons and expressed them in human embryonic kidney (hek) 293 cells. both proteins were detected intracellularly, with a small portion secreted into supernatant. in order to enhance secretion, truncated forms of envelop proteins including e2 tm, e2384-661, e2484-661 were also expressed. both full-length and truncated forms of envelop proteins were glycosylated and expressed at high level. in addition, we also expressed the codon-optimized hcv receptors cd81 and claudin-1 in 293 cells. by comparing the expression level of codon-optimized sequences and the sequences that were obtained from cdna library by pcr, we found that codon-optimization enhance protein expression significantly in 293 cells. these results not only lay solid foundation for further research concerning the mechanism of hcv entry, including the optimal ph and right protein conformation for fusion, cell types that permit viral entry; but also potentiate a useful cell model for testing antiviral agents. background: prolactin (prl) is an immunoregulatory hormone secreted from lymphocytes, however, prl induction in relation to hepatitis c virus (hcv) infection has not been elucidated. methods: serum prl levels were measured in both 232 subjects of our hcv cohort study and 31 male patients of the hospital, who were chronically infected with hcv. furthermore, serum prl levels were compared in 27 male patients before and after interferon therapy. we measured expression of prl mrna level in pbmcs in 12 male patients, and also investigated prl mrna of pbmcs collected from 5 healthy men that stimulated by hcv produced by huh7.5 cells in vitro. result: serum prl levels were significantly higher in the hcv-infected subjects than in the controls (p< 0.01). they were significantly higher in hcv-infected male subjects than in the controls (p< 0.001). serum prl levels were significantly higher in male patients than in the controls (p<0.01). serum prl levels decreased significantly after interferon therapy in patients with sustained virological response to therapy (p<0.05). the levels of prl mrna in pbmcs derived from hcv-infected patients were significantly higher in 12 male patients than in the controls (p<0.001). conclusion: the high levels of prl expression are associated with hcv infection in carriers. background and objectives: hepatitis c virus (hcv) is a major cause of chronic liver hepatitis, cirrhosis, and hepatocellular carcinoma.current clinic standard therapy is interferon alpha (ifn-) combination with ribavirin, but this treatment is associated with adverse effects and often fails to induce a sustained response. until recently, development of a hcv cell culture system (hcvcc) provides a suitable tissue culture system to study the complete hcv life cycle. in this study, we tested the effect of ifn omega (ifn-)-a member of type interferon on hcv compared with ifnbased on hcv 1b replicon and hcvcc. methods: we compared ifn-and ifn-2a effects on hcv rna replication and protein expression, as measured by ribozyme protection assay and western blot. we also compared the intracellular protein level of phosphorylated signal transducer and activator of transcription 1 (p-stat1) treated with different interferon type and concentration with western blot analysis. results: hcv rna and protein level were inversely related with ifnconcentration and compared with ifn-2a, at the same concentration, the hcv rna and protein levels treated with ifn-were lower than that treated with ifn-2a p 0.05 .also based on the hcv rna analysis, ec50 of ifn-was 10 folds lower than ifn-2a. ifns increased intracellular p-stat1 level at a dose dependent manner and compared the same concentration of ifn-and ifn-2a, p-stat1 protein level was higher in ifn-treated group p 0.05 . conclusions: these results demonstrate distinct antiviral effect of ifncompared with ifn-2a and this difference maybe partly caused by the stronger stimulation of ifn receptor . outstanding antiviral activity of ifnmay be useful for developing new hcv treatment strategies. background & aim: hepatitis b virus (hbv) infection with undetectable levels of hepatitis b surface antigen (hbsag) is called an occult infection, which although has been described among subjects with chronic hepatitis c liver disease in the western world, it's prevalence and clinical significance are still ambiguous in the indian subcontinent. materials and methods: we investigated hbv-dna pcr in serum samples of 260 hbsag negative subjects with chronic hcv-related liver disease, and 70 apparently healthy volunteers negative for hbsag and anti-hcv as control. results: serum samples found positive by at least two independent pcr assays were considered hbv dna positive. hbv-dna was detected among 19 hcv-related chronic liver disease (cld) patients (7.3%), which was higher (p = 0.2) as compared with the control volunteers (4.3%). it was more frequent (37.5%) in 24 anti-hbs negative/anti-hbc positive patients than in 180 anti-hbs/anti-hbc positive (5 %, p < 0.05). hcv rna by qualitative pcr was significantly (p <0.001) higher in occult hbv compare to non-occult. hcv genotype 1b was predominantly associated with occult hbv (73%), especially among subjects with hepatocellular carcinoma (hcc) (p<0.05) as compared to non-occult hbv cases. though not significant, frequency of occult hbv infection was higher than healthy controls and hcv 1b genotype was significantly associated in patients with hcc. conclusion: this study suggests that in all hbv-endemic areas, the possibility of occult hbv in patients with hcv should be considered and hbv-dna should be performed. j. zhao 1,2 , w.d. cai 2 , l. chen 2 , y.x. gan 2 , m.l. he 1 , x.r. wang 1 1 background: besides hiv and syphilis, hepatitis c virus (hcv) is also rapidly spread among men who have sex with men (msm). this study was designed to identify the prevalence of these 3 sexual transmitted diseases in msms in shenzhen, china. methods: a cross sectional study was conducted by using time location sampling method from april to july, 2008. 831 msm participants (including 454 male sex workers) were recruited and finished guided self-administered questionnaires (or interviews if they have difficulty in reading or understanding) in 37 venue-date-time randomly selected from 48 active venues. results: results were analyzed using spss. 736 blood samples were collected for hiv, syphilis and hcv test. participated msms were between the age of 18 to 51 years (25.5±5.9) with a majority of 20-29 years (73.7%). most of them finished junior high school education (74.9%). 84.2% had high level of knowledge on modes of transmission and prevention. likewise, 56.0% msms have ever sold sex to men, 38.3% of them were self identified as gay, 35.1% as bisexual. 78.3% msms had multiple male sexual partners and 50.3% msms always used condom. 48.6% of them had sex with women in the past 6 month, and the condom use rate decline to 35.1% during both male and female sex. hiv positive rate is of 6.7% and syphilis for 18.3%, hcv is only found in 3 cases (0.4%). conclusions: a greater number of the participants have both male and female sex partners. this survey shows that hcv infection rate is still low among msms in shenzhen, although the hiv and syphilis rate is high and continuing increased in the past few years. the change of insulin sensitivity in hepatitis c patients with normal insulin sensitivity s.g. park 1 , y.k. cho 1 , j.w. lee 1 , j.w. yun 1 , h.j. kim 1 , w.k. jeon 1 , b.i. background: hepatitis c virus (hcv) infection is associated with a high prevalence of diabetes mellitus (dm). insulin resistance (ir) is known to play a crucial role in the development of dm in chronic hepatitis c (chc) patients. we prospectively investigated the change of insulin sensitivity in chc patients during 5-year period, and analyzed factors significantly associated with ir. methods: subjects consisted of 62 non-cirrhotic chc patients with normal alanine aminotransferase (alt) and normal insulin sensitivity (chc group), and healthy control group of 172 subjects matched by age, sex, body mass index and life styles. we compared initial baseline insulin sensitivity, metabolic parameters and incidence rate of ir at the end of follow up period in both groups. the change of insulin sensitivity and metabolic parameters and development of ir was analyzed, and factors associated with development of ir were evaluated. results: ir developed in 22.5% of 62 chc patients and 5.2% of 172 normal individuals (p<0.001). hcv infection per se and genotype 1 were independent risk factors of ir. initial fasting glucose 90-100 mg/dl, fasting insulin 10 uiu/ml, homa-ir 2.3-2.7 were significantly associated with development of ir in chc group. conclusions: hcv infection is independent risk factor of ir. even if chc patients with normal insulin sensitivity, careful monitoring for ir is necessary. prevalence of viral hepatitis c in latvia i. tolmane 1,2 , b. rozentale 1,2 , j. keiss 1 , f. arsa 1 1 sa infectology center of latvia, 2 riga stradin's university background and aim: viral hepatitis c (vhc) because of its prevalence and clinical course has become one of the most actual infectious diseases in the world. to date chronic hepatitis c affects over 170 million individuals worldwide. chronic vhc is a leading cause of cirrhosis and hepatocellular carcinoma. the aim of this study was to investigate how many residents of latvia, that are over 18 years of age have been exposed to vhc (anti-hcv prevalence) and how many are infected at the moment (hcv-rna prevalence). until now such research has not been performed in latvia. methods: from the register of general practitioners there were randomly selected 26 gp's from different regions of latvia, 60 persons over 18 years of age were selected out of each gp register and tested for anti-hcv with screening test (elisa). in case of positive result antibodies were confirmed with western-blot reaction and person was tested for hcv-rna (pcr). results: in total 1591 person was invited by general practitioners for the test and 1442 persons responded (response rate 90.6%). confirming test (western-blot) was positive in 34 participants and out of which hcv rna test was positive in 25 patients. conclusions: there are 2.4% of people exposed to hepatitis c virus in latvia and 1.73% are infected with hepatitis c virus, respectively, 1734 infected persons per 100 thousand individuals. genetic variation in the ikk/nf-b pathway and the live fibrosis progression in chronic hepatitis c r. sho 1 , k. ishii 1 , r. ishii 2 , h. watanabe 2 , k. sugahara 2 , y. nishise 2 , k. okumoto 2 , t. saito 2 , s. kawata 2 , a. fukao 1 1 department of public health, 2 department of gastroenterology, yamagata university faculty of medicine background/aims: i b kinase/nf-b (ikk/nf-b) signaling pathway is thought to play critical roles in liver inflammation and fibrogenesis. we carried out a haplotype-based association study to examine the contribution of common genetic variations in the genes encoding nf b inhibitor kinase alpha and beta (ikbka and ikbkb; the major components of ikk/nf-b pathway) to the progression of live fibrosis in chronic hepatitis c. methods: based upon the common single nucleotide polymorphisms (snps; minor allele frequency(maf) 0.05) and linkage disequilibrium (ld) information derived from the hapmap, we selected 5 and 3 tag snps from ikbka, and ikbkb, respectively, for genotyping. by using melting curve analysis, snps were genotyped in 217 chronic hepatitis c patients, including 80 patients with hepatocellular carcinoma. association between common genetic variations in ikbka/ikbka and platelet count (plt) was tested by both genotype-and haplotype-based approaches. results: we succeeded in genotyping a total of 8 tag snps that efficiently capture common variation across the 32 kb-block of ikbka and the 25 kb-block of ikbkb. for each of genes tested, 5 haplotypes were found in population studied. all snps were in hardy-weinberg equilibrium, but no significant association was observed between any single tag snp or haplotype and decreased plt in patients analyzed. conclusions: our data suggest that it is unlikely that polymorphisms within the ikbka and ikbkb genes are involved in the progression of live fibrosis in chronic hepatitis c. further studies on genetic variations in other nf-b-related genes in chronic hepatitis c are needed. background: hepatitis c virus infection is a major burden after liver transplantation. the effective treatment for patients who underwent liver transplantation has not been well established. management of these patients is the most challenging task. cyclophilins are essential host factors for hcv replication. we report here the efficacy of divided administration of ifn plus cyclosporine a in the treatment of chronic hepatitis c patients who failed peg-ifn or ifn combined ribavirin. patients and method: we prospectively included 59 patients (median age, 63) with genotype 1b and, failures to combination ifn plus ribavirin or combination pegylated ifn plus ribavirin. the present treatments consisted of an induction therapy, an intensified therapy and a maintenance therapy. the induction therapy comprised intravenous 1 mu ifn every 4 hours for the first 3 days, 1.5 mu ifn every 6 hours for the next 4 days and 2 mu ifn every 8 hours for the following 3 weeks, totaling 168 mu of ifn . the intensified therapy was induction therapy shortened to 2 weeks. the maintenance therapy comprised of pegylated ifn 2b and ribavirin. csa was given 4 times daily during the induction and the intensified therapies. ribavirin was given twice daily during the maintenance therapy. results: the end treatment response and sustained virological response rate of the present study were 73 % (43/59) and 59% (35/59), respectively. the relapse rate was 19 %(8/43). non-responders was 16 % (3/19). all adverse effects were completely reversible. the treatment protocol was well tolerable. conclusion: we concluded that our protocol should be effective in failures to the previous combination therapies. host factor targeting treatment will become a promising treatment option. cyclophilin targeting treatment is a promising new anti-hcv treatment k. inoue 1 , t. watanabe 1 , s. yoshiba 1 1 background: hepatitis c virus (hcv) is the most common cause of chronic liver disease. however, the efficacy of currently available treatments is limited. we recently reported the effects of combined interferon-/cyclosporin a treatment. cyclophilins are associated with hcv replication and bind cyclosporin a. which cyclophilins are closely associated with hcv replication remains controversial. in this study, several cyclophilins were found to be essential host factors for hcv replication and hcv replication was rescued by overexpression of cyclophilin a in the presence of cyclosporin a. methods: we evaluated the effect of cyclosporin a and its analogues on the replication of hcv in vitro using several types of hcv replicon. the gene expression of representative cyclophilins and pin-1 was knocked down using small interfering rna2 (sirna) to identify cyclophilins associated with hcv replication. the specificity of the effect of sirna was confirmed by western blot analysis. the effect of overexpression of cyclophilins on hcv replication in the presence of cycloporin a was also studied. results: cyclosporin a and its analogues suppressed hcv replication in a dose dependent manner. cyclophilin f, cyclophilin lc1 and cyclophilin lc2 as host factors which are closely associated with hcv replication, in addition to the previously reported cyclophilin a. knockdown of chclophilin b showed little effect on hcv rna replication. cyclophiln-dependent hcv replication varied among the three hcv replicon cell-lines used. overexpression of cyclophilin a rescued hcv replication in the presence of cyclosporin a. conclusions: these findings suggest several cyclophilins are essential host factors for hcv rna replication. thus potent cyclophilin inhibitors have the potential to be anti-hcv drugs. background/aims: hepatitis c virus (hcv) genotypes 1-6 have a worldwide distribution. types 1a and 1b are predominant in northern europe and north america, and in southern and eastern europe and japan, respectively. type 3 is endemic in south asia and is variably distributed in different countries. genotype 4 in egypt, genotype 5 in central and south america and genotype 6 is common in china, japan and south east asia. in pakistan 3a is the commonest genotype, which is associated with the most favorable outcome regarding end treatment response and sustained virological response after 24 weeks of therapy. the aim of this study is to find out hcv genotypes in newly diagnosed chronic hepatitis c patients. methods: this observational study was conducted in chronic hepatitis c patients. all patients had raised alt levels for last 06 months, had positive polymerase chain reaction (pcr) for hcv rna by real time method and liver biopsy was done in all patients under national program for prevention and control of hepatitis during year 2006 -2007. genotyping was done on roche genotyping kit. data was analyzed by spss 13.0 results: out of 164 patients, 85.9% (n=141) were genotype 3a. 6.1% (n=10) were genotype 3b. 3.0% (n=5) were genotype 1a. n= 01 had genotype 1b. 4.2% (n= 7) had mixed genotype (3a,3b/1a,1b,3a,3b). conclusion: majority (85.9%) of chronic hepatitis c patients were genotype 3a which is associated with favorable outcome after 24 weeks of interferon and ribavirin therapy and only 3.0% had genotype 1a in this cohort. s.t. zhou 1 , y. zhao 1 , f.j. zhang 1 background/aims: as human immunodeficiency virus (hiv) infected children who are receiving antiretroviral therapy (art) are living longer in china, comorbidities of hepatitis b virus (hbv) and hepatitis c virus (hcv) coinfection should be carefully considered when making management decisions. however, the coinfection rate of either hbv or hcv is unknown in hiv-infected children in china. we evaluated the seroprevalence of hbv and hcv in the china national pediatric art cohort of hiv-infected patients. methods: patients were selected from hiv infected children medically eligible for art who were enrolled into the china national pediatric art cohort since 2004. interviews, medical assessment, serology for hbsag, anti-hcv antibody, transaminase levels, and hiv serostatus and cd4 counts at baseline of patients were obtained. results: 42 of 763 hiv-infected children were hbsag seropositive (5.50%; 95%ci: 3.88%-7.12%), and 69 of 662 children were anti-hcv antibody seropositive (10.42%; 95%ci: 8.09%-12.75%). only age was associated with hbv coinfection. multivariate analysis revealed that children infected with hiv through contaminated blood or transfusion of blood products were 6.35 times more likely to be anti-hcv antibody positive than those infected with hiv through other routes. and children from central china provinces, henan, anhui, shanxi, and hubei were 2.5 times more likely to be hcv seropositive. conclusion: the high seroprevalence of hbv and hcv coinfection in hiv-infected children attending china national pediatric art cohort calls for routine screening for hepatitis viral coinfection and modification of the management of hiv-infected children in china. background: bms-790052 is a first-in class and highly selective hepatitis c virus (hcv) ns5a inhibitor with picomolar in vitro potency against genotypes 1a and 1b. in a sad study with healthy subjects, bms-790052 was safe, well-tolerated, and had a pharmacokinetic profile suggestive of once-daily dosing. methods: the objectives of this randomized, double blind, placebo-controlled, sad study were to evaluate the safety, tolerability, antiviral effect and pharmacokinetics of bms-790052 in patients with genotype 1 chronic hepatitis c (chc). treatment naïve or experienced patients were randomized to receive 1, 10, or 100 mg of bms-790052 or placebo. results: all bms-790052 single doses were well tolerated and had a safety profile similar to that of placebo. following oral administration, bms-790052 was readily absorbed with dose proportional exposures over the studied dose range. the mean terminal half-life of bms-790052 was approximately 12 hours. mean decline in hcv rna 24 hours after a single 1, 10 and 100 mg dose of bms-790052 was 1.8 log10 (range 0.18 to 3.0 log10), 3.2 log10 (range 2.9 to 4.0 log10) and 3.3 log10 (range 2.7 to 3.6 log10), respectively. the 100 mg dose resulted in a mean decline of 3.6 log10 (range 3.0 to 4.1 log10) 48 hours after dosing, which was maintained at 144 hours. conclusions: single doses of up to 100 mg of bms-790052 were safe and well tolerated in patients chronically infected with hcv genotype 1. bms-790052 produced a robust decline in hcv rna and has a pharmacokinetic profile that potentially supports once-daily dosing. background: the global infection rate of hcv is approximately 3%, and nearly 3.2% in china. only 42%-46% of patients with genotype 1b can achieve sustained virological response (svr) after antivirus therapy, nearly half of them experienced treatment failure. the study aimed to determine hcv-1b sequence evolution in patients experienced treatment failure during and after therapy, and further analyze relations between the mutations and treatment outcome. methods: 19 patients with genotype 1b accepted antiviral treatment of ifn plus ribavirin for 48 weeks, and long-term follow-up after therapy. 2 patients experienced treatment failure were further analyzed (one for relapser, another for nonresponder). sera were reserved at baseline, 12w, 48w and 4-year after therapy. hcv-rna was extracted. hcv full-length orf was amplified by rt-nested-pcr and sequencing. result: 9 of the 19 patients achieved svr (47.4%). from sequence alignments of relapser at baseline and 48w, we find that p7, ns5a and ns4a have higher mutation rate both in nucleotide and amino acid level (7.41% and 6.35%, 4.08% and 5.63%, 4.32% and 5.56%, respectively). but there is no significant difference in the alignments of 48w and 4-year after therapy, the mutation rate is lower. mutation rates of the non-responder among baseline, 12w, 48w and 4-year after therapy are very low. conclusion: antivirus effect is correlated with specific hcv sequences in chronic hepatitis c, mutations in hcv non-structure protein p7, ns5a and ns4a have important impacts on treatment outcome in ifn-based therapy. background: the results of antiviral therapy for hepatitis c (hcv) have improved recently with the use of peg-interferon (peg-ifn)/ribavirin therapy. however, age of patients are concerned because of side effects and safety. as we known, a few studies have targeted therapy in elder with chronic hcv. aim: we reviewed the results of interferon based antiviral therapy in the elderly with chronic hcv at our institution. methods: patients were defined as elderly if they were 65 years and elder who received therapy for hcv. the prescribed treatment duration, end of treatment response were mention. the data recorded included laboratory tests, adverse events (ae), dose modification, and withdrawal rate of therapy. results: 304 of chronic hcv patients treated with peg-ifn/ribavirin between nov 2004 and feb 2008. 50 patients were older than 65 years old. the mean age of the elder patients was 70.3 ± 4.8 years old. 21 were male and 29 were female. histological studies showed 18 with cirrhosis. almost all patients had experienced ae/side effects. the most common abnormalities were anemia and neutropenia. therapy was discontinued in 22% (11/50). the rate of dose modification was 46% (18/39) patients who received 24 weeks therapy. transaminases were normalized in 66% (33/50) after 24 weeks treatment and sustained in 66% (29/44) one year later. conclusion: the elder patients are more at risk of developing ae while on treatment. most patients should be discontinued or decreased dosage of medication. however, the elder patients with chronic hcv can be treated successfully. background: in the general population the incidence of interstitial lung disease is estimated to be 0.03% and has also been reported with the use of interferons. the higher reporting rate of ip in japan has created interest and warrants further investigation. methods: using both data from 12 randomized clinical trials (ex-japan) and the roche world-wide safety database (advent), the frequency of ip was estimated in patients treated with peginterferon alfa-2a ± ribavirin. ip was defined as: interstitial lung disease, alveolitis, pulmonary fibrosis, pneumonitis and pulmonary toxicity. results: one case of ip was reported among the 6180 patients included in the clinical trials (0.02%). in the advent database considering the estimated 926,000 patients with cumulative exposure to peginterferon alfa-2a (42,600 in japan and 883,400 us/row) the 228 reported cases of ip represent a rate of 0.02% with a proportional reporting ratio (prr) of 1.7 (p<0.0001). of these cases, 140 were reported in japan (prr 2.9; p<0.0001), 34 in the usa (prr 0.7; p=0.06) and 54 row (prr 1.2; p=0.11) representing reporting rates of 0.3% in japan and 0.01% in the usa and row. japanese patients with reported ip were older (66 versus 50-55 years) and were more likely to have been treated with peginterferon alfa-2a monotherapy (81% versus 21-39%). furthermore, the yearly incidence rate has remained unchanged. conclusions: the apparently higher rate of ip reported in japan may result from differences in patient demography, diagnostic criteria and treatment patterns. the overall incidence of ip remains low. background: hepatitis c virus (hcv) infection carries a significant risk for development of insulin resistance (ir) and/or diabetes (dm). recently, retinol-binding protein 4 (rbp4) has been reported as a protein contributing to ir. this study aimed to assess the different expression of serum rbp4 between chronic hcv infection (chc) patients and non-chc controls. methods: serum rbp4 was measured in 105 treatment-naïve chc patients and its correlation with the homeostasis model assessment of insulin resistance index (homa-ir), liver histology, virology and metabolic factors was investigated. patients were stratified into different stages of glucose tolerance by oral glucose tolerance test. another 100 sex-and age-matched non-chc adults served as the controls. results: the mean rbp4 level of controls tended to be higher than that of chc patients (32.46 ± 20.92 vs 25.48 ± 13.13 g/ml, p=0.07). the mean rbp4 level of 34 igt control-group subjects was 43.7 ± 24.0 g/ml, which was significantly higher than that of 34 ngt (24.4 ± 13.0 g/ml, p<0.001) and 32 dm controls (29.0 ± 19.5 g/ml, p<0.01). in contrast, the mean rbp4 level (22.2 ± 10.3 g/ml) of 32 dm/chc patients was not significantly different from that of ngt/chc (24.9 ± 10.5 g/ml, n=28) and igt /chc (28.1 ± 15.8 g/ml, n=45) patients. amongst chc patients, there was a significant decreasing linear trend of rbp4 dependent of both histological grading and staging progression, whilst a significant increment of homa-ir was found. conclusion: serum rbp4 is dysregulated in chc patients. introduction: sustained viral response (svr) in hepatitis c treatment with interferon alfa and ribavirin is affected by adherence and compliance due to severe myalgia, fatigue-anxiety and disturbed sleep. pregabalin, an orally effective gabasergic drug is not metabolized via cytochrome p450 and is used in fibromyalgia and fatigue-anxiety syndromes without hepatic toxicity.this study evaluates the addition of pregablin to standard agents in achieving svr by reducing side events. methods: thirty patients with chronic hepatitis c {mean age -46 years, male: female -2:1,genotype(g)1(n=29), g6 (n=1), fibrotic score f2-3 (n=24) and f4 (n=6), mean bmi > 29 kg/m 2 , initial viral load > 800,000 iu/ml} were randomized to pregablin100mg (n=15) or duloxetine 20mg (n=15) both orally daily with interferon alfa 2a 180mcg sq once a week and ribavirin 1200mg daily for 48 weeks. myalgia anxiety scale, modified quality of life score -evaluated at entry and tri-monthly. all were tested for rapid viral response, early viral response and end treatment viral response and svr. results: at the end of 48 weeks, in the pregablin arm, 14(97.3%) completed the therapy without interruption, one stopped due to excessive somnolence. duloxetine arm -10(66.6%) completed with interruptions, 4(22.2%) withdrew from the trial due to side events, one left the country. 9(62.2%) achieved svr in pregablin arm and 5(33.3%) with duloxetine. conclusions: pregablin may be considered with ifn and rbv for better adherence and compliance in achieving svr in treatment of chronic hepatitis c. larger randomized studies are needed to confirm the findings. in this study we extended this treatment approach to on treatment nonresponders (defined as having detectable hcv-rna after at least 24 weeks of soc). methods: so far, 5 pts. hcv-rna pos. after 24 weeks of soc (3 male, 2 female, genotype 1:4; genotype 3a:1, 3 with cirrhosis) participated in this protocol; 4 were treatment naïve pts, 1 relapser to two previous therapies (24 and 48 weeks). 20 mg/kg/d sil was given for 14 days, soc was continued. hcv-rna was quantified by taqman (roche diagnostics, usa) at monthly intervals on standard treatment and weekly after starting sil. results: all patients received at least 24 weeks of soc, at week 24 3 had a log drop < 3, two patients had detectable but unquantifiable hcv-rna (< 15 iu/ml). after 14 days of sil all 5 had undetectable hcv-rna, in one hcv-rna increased to 100 iu/ml and recived after a second course of sil. .all 5 patients are still on soc and are hcv-rna negative. conclusion: sil iv. is an effective "rescue treatment" for on treatment nonresponders to full dose of peginterferon/ribavirin combination therapy. poster exhibition -imaging modalities poster session, hall 5b background: levovist-enhanced ultrasonography using subtractions makes it possible to depict the perfusion of hyperechogenic nodules. our institution performs sonazoid-enhanced ultrasonography using a toshiba aplio80 that is set to a ps low images, as generally recommended. the resulting images, however, are difficult to evaluate the kind of staining image that is obtained from a hyperechogenic nodule. these staining images were then compared to advanced dynamic flow (adf) images of a hyperechogenic nodule recorded using levovist-enhanced ultrasonography. methods: the subjects were five nodules who had undergone sonazoid-enhanced ultrasonography. two patients had experienced a recurrence of hcc after tace, while three patients had a hyperechogenic nodule of hcc that had never been treated. one patient with hcc after tace was imaged at a ps low. the second patient with hcc after tace and the three patients with hcc showing a high echoic nodule, were imaged using adf. results: in the patients with hcc after tace, the remaining tumor was difficult to observe in both the vascular phase and the kupffer phase taken at a ps low. in the other patients, however, images taken using adf clearly showed the residual tumor. also, with regard to the findings from the perfused images obtained from the three patients with hyperechogenic nodules of hcc, the hcc was more easily detectable in the adf images than in those taken at a ps low. conclusion: hyperechogenic perfused nodules are easier to identify in images taken using adf than in images taken using ps low. y. komorizono 1 , t. shibatou 1 , k. sako 1 1 nanpuh hospital background: this study aimed to evaluate the usefulness of sonazoid enhanced radiofrequency ablation under real-time virtual sonography (rvs) guidance in a series of patients with hepatocellular carcinoma (hcc). method: twenty-five patients with a solitary hcc tumor measuring < = 2.5 cm in greatest dimension were enrolled in this study. eight patients received an initial treatment, seven also received an additional treatment for local recurrent tumors, and the remaining ten had distant recurrent tumors. all patients were easy to scan by multiple detector ct (mdct), but not by conventional ultrasound ( conclusions: the combination of the rvs system with sonazoid-enhanced us appears to have a high potential for use on patients that are difficult-to-scan by us examinations for percutaneous radiofrequency ablation. background & aims: contrast enhanced ultrasonography (ceus) with sonazoid can be expected to be useful not only for detection of tumor but also for us guided ablation therapy because kupffer imaging lasts for long time. the aim of this study is to investigate the usefulness of sonazoid in rfa for hcc. material & methods: a total of 716 hcc nodules in 316 patients admitted to receive rfa were studied. the detection ability of hcc was compared between ceus and conventional us using dynamic ct as reference standard. the effectiveness in the treatment was assessed by comparing the mean numbers of treatment session of rfa in patient treated with ceus assistance and that in historical controls matched for tumor and background conditions. results: the detection rate was 83.5% in conventional us and 93.2% in ceus (p=0.04). sixty-nine nodules in 52 patients were not detected by conventional us and detected after injection of sonazoid. the mean increase in detected tumor number with contrast enhanced us were well correlated with serum albumin level (p=0.016). ceus was not superior to conventional us in patients with low albumin level. the mean number of session was 1.33±0.45 as compared to 1.49±0.76 in the historical controls (p=0.0019). conclusions: ceus with sonazoid is useful for detection of tumor in patients with well-conserved hepatic reservoir. the decrease in the mean number of sessions compared to historical controls suggested that sonazoid is an excellent supportive agent in rfa treatment of hcc. direct measurement of peri-operative change in portal blood flow and pressure is difficult in human. in the present study, computational simulation of pre-and post-operative portal blood flow and pressure was performed using computational flow dynamic (cfd) software in patients with primary liver cancers. methods: patients with fibrotic or non-fibrotic livers were analyzed. according to preoperative md-ct, mesh models of portal branches were constructed. cfd software (fluent 6.2, fluent inc.) was employed for flow simulation. on the fluent 6.2, changes in flow dynamics in the remnant portal branches were simulated by virtual cutting of an interested portal branch. the simulation was also performed 14 days after the operation using dicom data obtained at that time. results: relative increase in blood flow in each remnant portal branch was not uniform throughout the liver in each patient. the sudden increase in portal pressure just after the virtual cutting of interested portal branch was almost normalized by day 14 in non-fibrotic liver according to the flow simulation, while the increase in fibrotic liver did not return to the pre-operative values by day 14. these results suggest that responsive dilatation of remnant portal branches and subsequent regional regeneration could normalize the sudden increase in portal pressure after surgery in non-fibrotic livers, while the mechanism is impaired in fibrotic livers. discussion: computational flow dynamic simulation is useful to analyze the differences in the peri-operative portal flow dynamics and liver regeneration between non-fibrotic and fibrotic livers. aim: to determine if roi analysis can characterize washout in hepatocellular carcinoma (hcc) better than visual analysis. methods: surgically proven hccs from a single institution were studied. the patients' gender, age, date of scan, date of surgery were recorded. 94 patients with pre-operative triphasic (n=67) and quadriphasic ct scans (n=27) were included. a representative section containing the lesion was selected for each case. the hu change between the precontrast and arterial (huabsolute hypervascularity) and the hu change between the peak attenuation and late portovenous phases (huabsolute washout) were recorded. cases were deemed positive if the hu change was more than the standard deviation (11 hu). this was compared against visual analysis to determine if our method would increase sensitivity of ct for hcc. results: the mean patient age was 63.7 years (range 19 to 84 years); there were 77 males and 17 females. the mean duration between surgery and the scan was 39.5 days (range 1 to 348 days). peak enhancement was seen in the early portal venous phase in 76.6% cases. the mean huabsolute washout was 23.8 hu (range -5 -54). roi analysis detected 86/94 cases (91.5%). this was 12.8% more than visual assessment, which detected 74/94 cases. this was statistically significant (p=0.014). conclusion: visual assessment of lesion density is subjective. quantitative measurement of lesion attenuation changes between scan phases is a simple and objective method that is more sensitive than visual assessment in determining lesion washout. background: abdominal ultrasonogram(usg) is a common available diagnostic tool to screen and follow up for hepatocellular carcinoma(hcc). but it has been reported that the specificity of ultrasonogram is high but the sensitivity of it is insufficient. we investigated the characteristics of hccs that was missed in the usg but was detected in the ct. methods: total 122 patients who were diagnosed with hcc between december, 2003 and february, 2008 , were enrolled and analysed retrospectively. all patients were performed with a usg prior to a spiral ct. the period between usg and spiral ct was limited within 1 month. we investigated age, gender, cause(hbv, hcv, alcohol), the size of hcc(the length of long diameter), stage(modified uicc), child-pugh grade, cirrhosis, tumor number, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns), and the tumor location at segement 8 as the possible related factors. results: the mean period between usg and spiral ct was 3.04±5.70 days. the diagnostic accuracy rate to hcc was 84.4%(103/122). there was no interobserver variation. in analysis of associated factors, there was no statistical significance in age, gender, cause(hbv, hcv, alcohol), stage(modified uicc), child-pugh grade, cirrhosis, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns) (p > 0.05). there was statistical correlation in the tumor size less than 2 cm, the solitary tumor and location at segement 8. (p > 0.05). conclusion: tumor size less than 2 cm, solitary lesion and location at segment 8 are significant factors to miss hccs in usg diagnosis. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 background: histopathological examination is required in the evaluation of various liver diseases for both diagnosis and prognosis. earlier blinded percutaneous liver biopsy was done commonly but now there are various studies suggesting that sonographic guided percutaneous liver biopsy could be more precise and safer. our aim was to compare the safety and diagnostic utility of sonographic guided versus blind percutaneous liver biopsy. methods: it was a retrospective single center study done between june 2003 and may 2007. trucut liver biopsy needle was used in all patients. demographic, clinical and histological characteristics between the two groups were evaluated. insufficient biopsy was defined as a sample with less than 6 portal spaces. we reviewed the type of complications and if hospitalization was required, or any mortality related to the procedure. results: out of 256 liver biopsies done in this period after excluding 16 patients we included 240 patients, 144 in group a(60%, blind approach) and 96 in group b (40%, sonographic guided approach). mean age was 38±12.4 years and male: female ratio was 1.6:1. biopsy was sufficient in 76% in group a and 94% in group b (p < 0.05). minor complications occurred in 58% in group a and 49% in group b which was not significant. major complications occurred in 2.8 % in group a and 1.2% in group b which was statistically significant. mortality was 1.2% in group a and 0.4% in group b which was statistically significant. conclusion: our study suggest that sonographic guided percutaneous liver biopsy is superior in the diagnosis of liver diseases in all aspects when compared to blind approach as it is more safe, has more diagnostic utility with significantly less complications and mortality. poster exhibition -liver fibrosis poster session, hall 5b background/aims: hmg-coa reductase inhibitors have been shown to reduce hepatic stellate cell proliferation and collagen production and decrease oxidative stress and hepatic vascular tone in cirrhotic patients. therfore, the aim of the present study was to examine whether the lipid lowering agents atorvastatin (ato) or rosuvastatin (ros) would prevent experimentally-induced acute or chronic hepatic damage in rats. methods: liver cirrhosis was induced by thioacetamide (taa, 200 mg/kg, i.p.) twice a week, for 12 weeks. acute damage was induced by two consecutive taa injections (200 mg/kg in a 24 h interval). rats were treated concurrently with taa only or taa and either ato or ros daily by nasogastric gavage. another group was treated with taa+pentoxifyline (ptx), an agent with known antifibrotic effect through a different mechanism and served as positive control. results: presented in the conclusions: the lipid lowering agents used in our study had no effect on the development of acute or chronic hepatic damage in rats or on oxidative stress induced by taa. purpose: the development of hepatic fibrosis in patients with chronic liver disease increases the risk of liver cancer. the present study was conducted to determine whether an easily performed myocardial examination technique can be applied to the assessment of hepatic fibrosis. strain rate imaging is a new method based on tissue doppler imaging (tdi). the usefulness of strain rate imaging in assessing the degree of hepatic fibrosis was evaluated. this time, it mede comparative study with fibroscan in 11 cases. methods: strain rate imaging was performed using a diagnostic ultrasound system (aplio tm , toshiba medical systems corporation, tochigi, japan) in a total of 47 subjects:25 in the chronic hepatitis group, 12 in the cirrhosis group, and 10 in the normal control group. tdi-q, the tissue doppler analysis software installed in the aplio system, was used for analysis. measurement was performed five times from the epigastrium, with the roi size set to 10 mm and the derivative pitch to 3 mm. results: (i): both scores were largely reproducible among the different laboratories. however, compared to the histological findings, the error ratio was 77% for all results calculated by fibrotest and actitest. (ii): calculated scores varied among f2 (9%), f3 (31%), f3-f4 (6%), and f4 (54%) (fibrotest), as well as a1/a2 (48%), a2 (9%), a2-a3 (5%), and a3 (38%) (actitest). results: the mean strain value was 0.156 in the chronic hepatitis group, 0.055 in the cirrhosis group, and 0.26 in the normal control group.the correlation was not thought to be fibroscan. conclusion: the results of the present study suggest that this noninvasive method permits quantitative assessment of the degree of hepatic fibrosis to be performed easily and in a short time. it is expected that the accuracy of the strain rate imaging method in determining the degree of hepatic fibrosis will be improved when it is used in combination with histological examination. conclusion: despite reproducibility of fibro-and actitest results among the six laboratories, large scale investigation (n=64) displayed increasing variability of the results depending on interlaboratory differences that were still in a quality controlled, analytically acceptable range. furthermore, calculated scores coincided with histological findings only in less than 25% of all cases. thus, the diagnostic accuracy of these tests seems low, if histology is accepted as gold standard. background: current knowledge attributes connective tissue growth factor (ctgf/ccn2) a crucial role in enhancing tgf-actions during hepatic fibrogenesis. recently, we demonstrated that caffeine leads to an upregulation of ppar in hepatocytes, thus sensitizing these cells to the well known inhibitory effect of 15-deoxy-12,14 -prostaglandin j2 (15-d-pgj2) on ctgf expression. however, upregulation of the receptor alone is not sufficient per se, its physiological ligand 15-d-pgj2 is required for exerting its inhibitory effect on ctgf synthesis. aim and methods: this study compares serum concentrations of 15-d-pgj2 in caucasian patients with fibrotic liver diseases (n=289), caucasian controls (n=136) and caucasian non-liver disease sick (n=307), as well as of chinese patients with hepatocellular carcinoma (n= 43) and chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with ppar inducing (i.e. ctgf inhibitory) drugs such as caffeine. results: presented data show that caucasian patients with ongoing hepatic fibrogenesis (mean 6.2 ± 5.9 µg/l) display impressingly higher serum concentrations of 15-d-pgj2 than healthy probands (mean 2.3 ± 1.0) and caucasian patients with non-liver disease (mean 2.7 ± 1.4 µg/l). similar results are found in chinese patients with fully developed hcc (mean 1.3 ± 0.7 µg/l) compared to chinese healthy controls (mean 0.4 ± 0.2 µg/l). we identified the predictors of tumor recurrence using cox-regression model. introduction: non-invasive, i.e. serum-based multiparametric panels of biomarkers have been proposed for the diagnostic assessment of liver fibrosis. aims/methods: (i) haptoglobin, alt, ggt, alpha 2-macroglobulin, apolipoprotein a1 and bilirubin in sera of 4 patients with histological proven fibrosis (f1-f4, a1-a3) were determined in 6 different quality-controlled laboratories. interlaboratory variations of the calculated fibrotest score for staging and actitest score for grading (both biopredictive tm ), and their error ratios compared to biopsy results were calculated. (ii) the variability of obtained fibrotest/actitest scores depending on 64 differential combinations of the allowed analyt-specific maximum/minimum permissible values as determined by the external quality control of the german association of laboratory medicine was determined and the frequency distribution of the results calculated. results: a total of 51 patients (mean age, 54.3±9.8 years; male, 78.4%) were included. median follow-up duration was 14.2 months (range, 5.6-37.3) and 20 patients (39.2%) experienced local tumor recurrence during the observational period. multivariable analyses showed that low p2/ms level (relative risk, 0.98; 95% confidence interval [ci], 0.96-0.99; p=0.035) and serum alpha-fetoprotein level >100 ng/ml (relative risk, 5.41; 95% ci, 1.59-18.18; p=0.007) were independent risk factors for tumor recurrence. patients with p2/ms level <45.0 revealed 3.79-fold (95% ci, 1.05-13.76; p=0.042) increase in the risk of recurrence after adjustment for serum alpha-fetoprotein level, as compared to those with p2/ms level >45.0. however, tumor size, child-pugh score, and hepatitis b virus dna level failed to significantly affect the time-to-recurrence. conclusion: our study suggests that lower p2/ms value, which means more severe liver fibrosis, is an independent predictor for hcc recurrence after rfa. background/aims: despite of its high prevalence, osteoporosis is an underestimated complication of liver cirrhosis. the aims of this study is to prove the prevalence of osteoporosis and osteopenia in patients with liver cirrhosis and to identify the principal risk factors associated. methods: the prevalence of osteoporosis and osteopenia was studied in patients with alcoholic or viral liver cirrhosis who were admitted to the institute of gastroenterology and hepatology, cnuh between march 2008 and september 2008. osteoporosis and osteopenia was evaluated by measuring their bone density using dual energy x-ray absorptiometry (dexa) at lumbar spine and femoral head. the variables taken into consideration were: sex, body mass index (bmi), presence of cholestasis, severity and duration of liver disease. results: total 45 patients (male 32 and female 13, respectively) were estimated for association of liver disease and osteoporosis. of these, 39 patients were estimated for bone density of lumbar spine and neck of femur by dual x-ray absorptiometry (dexa). morning blood samples were taken for hormonal and biochemical analysis from all patients. among 39 patients, 25 patients (64%) were found to have osteopenia or osteoporosis. there was no statistically significant correlation between age, bmi, severity and duration of liver disease, pth, vitamin d, alp and igf-1. conclusion: there is high prevalence rate of osteopenia or osteoporosis in liver cirrhosis. although the causes of osteopathy are heterogeneous, the early diagnosis and treatment of osteopathy in patients with liver cirrhosis is important. background: to build and to evaluate mathematical models for predicting liver fibrosis progression by using conventional laboratory indicators in chronic hepatitis b. methods: liver biopsy and routine laboratory tests were performed in 391 patients with chronic hepatitis b. using multiple logistic regression to analyze evidently relevant indicators, then the predicting models were built and analyzed by roc curve. results: after spearman analysis, factors such as age, platelet count(plt), international rate(inr), total bilirubin(tbil), albumin(alb), aspartate aminotransferase (ast), gamma glutamyltranspeptidase (ggt), total bile acid(tba) and cholinesterase(che) were found to be correlated with liver fibrosis p 0.01 . three models (s 2, s 3, s=4, respectively) were built by plt, inr, alb, ggt and che, which were independent predictors after multiple logistic regression analysis.finally, fibrosis score (fs) was calculated to predict different liver fibrosis stages. roc curve analysis revealed that the auc of fs was 0.784 in model1 (s 2), 0.768 in model2 (s 3) and 0.806 in model3(s=4) fig1 .the cut-off fs in model1 was at 7.09 with 67.4% sensitive, 79.3% specificity and the accuracy was 71.1%. the cut-off fs in model2 was at 5.67 with 75.0% sensitive, 67.7% specificity and the accuracy was 72.9%. the cut-off fs in model3 was at 3.65 with 71.4% sensitive, 78.5% specificity and the accuracy was 73.7%. conclusions: the predicting models, built by using conventional laboratory indicators, have fairly well value for diagnosing hepatic fibrosis or hepatocirrhosis in chronic hepatitis b. background: to investigate the effect of liver cirrhosis on the development of atherosclerosis in the rabbits chronically fed with high fat diet. methods: normal male new zealand white rabbits were randomly divided into four groups: a control group, a high fat diet group, a carbon tetrachloride (ccl4) group and a complex group. pathologic changes in ascending aortas and livers were observed. the levels of serum alanine aminotransferase alt , lipid, c-reactive protein (crp) were also determined. results: significant hepatic steatosis, inflammation and fibrosis could be observed in the three treatment groups; while atherosclerosis and typical arteriosclerotic plaques in ascending aortas could only be observed in the two high fat diet groups. compared with the control group, serum alt and lipid levels in ccl4 group were increased significantly (p<0.05), but no difference of arterial intima-media thickness (imt) and i/m ratio between these two groups. the levels of serum alt, lipid, crp and imt in two high fat diet groups were significantly increased compared with the control group (p<0.05). the level of serum alt in the complex group was significant higer than that in the high fat diet group, but the i/m ratio was just opposite (all p<0.05), and there was no difference of imt between the two groups. conclusions rabbits treated with ccl4 can elevate serum lipid levels, but can not induce atherosclerosis. though the activity of liver inflammation was aggravated in the complex model group, it has no effect on atherosclerosis possibly partly because of malnutrition. higher values of liver stiffness in males with mild chronic hepatitis c c. stern 1 , a.c. cardoso 1 , r. moucari 1 , a.d. pumpo 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: liver stiffness (ls) measured by fibroscan (echosens) is a noninvasive method to assess liver fibrosis in patients with chronic liver diseases. we evaluated the impact of factors on ls results in mild chronic hepatitis c (chc). methods: chc patients with metavir fibrosis stage 1 at liver biopsy and a reliable ls exam were eligible. all patients had no prior antiviral treatment. the ls values were compared to clinical and biochemical data. results: 93 patients were included with the following characteristics: mean age 50 11, male gender (46%), mean bmi 23 2.7, median ls 5.8 kpa (3.2-21.8), diabetes (7%), genotype 1 (61%), metavir activity a1 (86%), a2 (10%), steatosis at biopsy 30% (92%), mean glucose 4.9 1, abnormal alt (78%), abnormal ggt (47%), homa (2 1.2). the ls values were associated wtih male gender (median 6.1 in males vs 5.2 in females) (p=0.04), bmi (p=0.03), alt (p=0.006), ggt (p=0.02) and glucose levels (p=0.04). no association was found between ls and activity stage (p=0.34) or steatosis (p=0.14). in the linear regression, the only factor independently associated with higher ls was gender (p=0.038). in men, higher ls was related to levels of alt (p=0.005), but not to necro-inflammation grade (p=0.4). in women, ls was not associated with alt levels, but with bmi (p=0.045) and ggt levels (p=0.035). conclusion: in patients with mild chc, liver stiffness values are higher in males. these results suggest that different cut-off for fibrosis stage 1 should be proposed according to gender. aims: to investigate the effects of shuanghu qinggan granule(sqg) on prevention and treatment of hepatic fibrosis induced by carbon tetrachloride in rats. methods: 60 sd rats were divided into 6 groups, normal control groupamodel groupb, sqg largec1, middlec2small dose groupsc3 and silymarin positive contrast groupd. the rats of bc1c2c3d were injected with carbon tetrachloride for 8 weeks. the rats of c1c2c3 were then administered with sqg for 8 weeks. the rats of d were then administered with silymarin for 8 weeks. results the liver structure of rats of b was severely damagedlarge amount of liver cells became obviously degeneratedand hepatic veins were clearly congested. the hepatic cells fatty degeneration and infiltration of inflammatory cells in rats of c1c2c3d reduced significantly. there was no fiber hyperplasia in liver tissues of rats of c1c2c3d. blood serum ha cp p levels in rats of b were significantly higher than those in ac1c2c3d. conclusion: sqg has remarkable therapeutic effects on rats with hepatic fibrosis induced by carbon tetrachloride, the higher the dosage of sqg was, the more effective the results would be. conclusions: none of sophisticated biomarkers had value in addition to readily available laboratory data for the prediction of significant fibrosis in hbeag positive patients. two markers out of 7 sophisticated biomarkers provide additional diagnostic information in hbeag negative patients. before new biomarkers are accepted, their superiority to routine laboratory data should be meticulously appraised. objective: to evaluate the efficiency and safety of "tinmax" hb-3 herbal compound (cpd) in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. methods: a double-blind randomized method was employed. 60 patients of hepatofibrosis or cirrhosis post hepatitis b were separated into study group ("tinmax" hb-3 group) and control group (natural vitamin group) by randomized method. the course was 52 weeks. patients visited once every 12 weeks and the last visit at 12 weeks after the cessation of treatment. part of patients had liver biopsy before and after treatment. before, during the course and at the end of therapy, clinical symptoms and physical signs were evaluated, hepatic function, and serum markers of hepatofibrosis (such as hyaluronate acid, laminin, serum type iii procollagen and collagen iv) were tested, and ultrasound evaluation was performed. results: 60 patients enrolled in the evaluation. 58 patients completed the evaluation according to the protocol. 20 patients had liver biopsy twice, 10 from the study group and 10 from the other one. at the end of therapy, the total effective rate of hepatofibrosis in histopathology is 74.13% in the study group, much higher than that of 21.95% in the control group (p<0.05). the total effective rate of serum markers of hepatofibrosis at the end of therapy in the study group was 70.10%, much higher than that of 30.24% in the control group (p<0.05). the total effective rate of non-invasion markers of hepatofibrosis at the end of therapy in the study group was 76.08%, much higher than that of 19.41% in the control group (p<0.05). the drugs of adverse event had not happened in both groups. conclusion: "tinmax" hb-3 herbal compound (cpd) is effective and safe in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. w.h. sha 1 , xiaohui zeng 1 , yuyuan li 1 1 gi department, first municipal hospital of guangzhou, guangzhou aim: to investigate the clinical value of serum indices for hepatic fibrosis in chronic liver diseases. methods: competitive radioimmunoassay was used to determine the serum level of collagen type ( c), laminin (ln) and hyaluronic acid(ha) in 193 patients with different severity degree of chronic liver diseases, and in 30 healthy subjects. results: the serum levels of c, ln, and ha in the patients with liver diseases increased to different extent, compared with those in the healthy subjects. of which the highest of c, ln, and ha were found in the patients with primary carcinoma of liver or hepatocirrhosis and the serum level of ha is highlight. the combination detection of serum c, ln, and ha is more valuable than single index. conclusion: joint detection of serum c , ln, and ha is of higher significance in clinical diagnosis and prognosis of hepatocirrhosis, and is also available for successive observation on the development of liver diseases. aims: to investigate the mechanism of fuzheng huayu decoction (fzhy) on hepatic stellate cells (hscs) activation relating to tgf-1 signal transduction pathway. methods: hscs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. at day 4 after isolation, cells were stimulated with 100pm tgf-1 for 24h, then incubated with 10% fzhy pharmacological serum or 10 m t r -i inhibitor (sb-431542) for 24h. protein expression of -sma, smad3 was assayed by immunofluorescent stain; total protein expression of -sma, t r -i, smad2/3 and nuclear expression of smad3 was analyzed by western blotting. results: fzhy pharmacological serum significantly decreased expression of -sma, t r -i, and inhibited smad3 nuclear expression and translocation in tgf-1 stimulated hscs. conclusions: fuzheng huayu decoction can prevent hscs activation through tgf-1 signaling transduction pathway in hscs, which may be the important molecular pharmacological mechanism of fuzheng huayu decoction action against liver fibrosis. background: fatty liver disease has become a health problem related to metabolic syndrome worldwide although its molecular pathogenesis has remained further studied and it is unclear whether advanced fibrosis induced by steatohepatitis will regress when diet is controlled. aim of this study is 1) to study the involvement of endoplasmic reticulum stress (er stress) in the occurrence of seatohepatitis and 2) to obtain the evidence of resolution of fibrosis by changing the diet. methods: non-alcoholic steatohepatitis with advanced fibrosis was produced in rats by giving methionine-choline-deficient diet (mcdd) for 10 weeks. methionine-choline-control diet (mccd) instead of mcdd was given for the last 2 weeks in an experimental group. fibrosis and inflammation was determined by several tissue stainings. gene expression related to fibrosis and inflammation was determined by immunoblotting and real-time pcr. expression of caspase-12, caspase-7, and glucose-regulated protein 78 was evaluated to clarify the presence of er stress aim against liver fibrosis relating to hypoxia and angiogenesis regulation. methods: the rats were divided into normal, model, sa-b and perin control group. rats in sa-b and perindopril group were administrated with sa-b and perindopril respectively. liver fibrosis was induced by ip dimethylnitrosamine (dmn) for 4w. fibrosis degree was observed by sirius red staining. col-i protein expression was analyzed by western blot; col-i , vcam-1, icam-1, hif-1 and vwf expression in liver tissue was checked by immunohistochemistry; gelatinase activities in liver tissue were detected by gelatin zymography and in situ flourescent zymography. result: compared to normal group, col-i, hif-1 , icam-1, results: 1) changing the diet from mcdd to mccd triggered the reduction in fat in hepatocytes, the decrease of inflammatory gene expression and oxidative stress, and the regression of fibrosis accompanied by the disappearance of activated stellate cells and macrophages. 2) immunohistochemistry, immunoblotting, and rt-pcr analysis all indicated the occurrence of er stress in steatohepatitis while it recovered immediately after changing the diet from mccd to mcdd. vwf protein expression and gelatinase activity in liver tissue were increased obviously in model group, while sa-b and perindopril treatment significantly decreased these protein expressions and gelatinase activity. conclusions: this simple experiment clearly shows that the changing diet from steatohepatitis-causing mcdd to mccd triggers the resolution of inflammatory and fibrotic reaction in the liver, suggesting that food intake is a very important factor for controlling the state of fat and pathology of the liver. er stress is involved in the process. background: liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver disease. under injury conditions, hepatic stellate cells (hscs) are activated to transdifferentiate into myofibroblasts, which are capable of secretion of many connective tissue elements, especially collagens i, iii, and iv. gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in asia. gypenosides are the major saponins derived from g. pentaphyllum. in previous study, gypenosides have hepatoprotective and anti-fibrotic activities in rat chronic liver injury induced by ccl4, and anti-proliferative effect in rat isolated hscs. methods: in cultured hscs model, we detected type1 procollagen protein and mrna by western blot and rt-pcr. result: we found that g. pentaphyllum inhibited type1 procollagen protein expression in 66% at 48 hours. furthermore, g. pentaphyllum also inhibited type1 procollagen 1 and 2 mrna expression in 39% and 11% respectively. in addition to transcriptional inhibition, we found that g. pentaphyllum also enhanced the degradation rate of type1 procollagen protein. base on the effect of enhancing protein degradation, we used some protease inhibitors like ca-074 me, z-fa-fmk, aebsf, tpck and tlck to identify the potential target of g. pentaphyllum. on the other hand, in the ubiquitin-proteasome system analysis, we quantified the change of some target proteins of proteasome in the presence or absence of g. pentaphyllum. conclusion: g. pentaphyllum reduced type1 procollagen protein by inhibiting transcription and enhancing protein degradation. aim: excessive oxidative stress in diabetic patients has been implicated in the pathology and complication of liver. the present study was designed to examine whether ginger has a direct hepatoprotective effect in diabetic cases. methods: wistar strain albino rats were selected for this study. the rats were divided into 4 groups: (i) control, (ii) ginger treated (200mg/kg b.w. orally, 30 days) (iii) diabetic (50 mg/kg b.w., i.p.) and (iv) diabetic + ginger treatment. the lipid metabolic profiles such as total cholesterol, triglycerides, phospholipids and lipid peroxidation as stress markers and histopathological studies were carried out to assess the damage in hepatic tissue. results: ginger treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals. diabetic rats have shown increased total cholesterol, triglycerides, phospholipids and lipid peroxidation content in hepatic tissue compared to control, indicate prevailing of oxidative stress and alterations in fatty acid metabolism in these rats. further, degenerative changes of hepatic cells in diabetic group are minimized to nearness in structure by administration of ginger as evinced by histopathological examination. conclusion: we summarize that the hypolipidemic and antioxidant compounds present in ginger may be useful in delaying the complicated effects of diabetes. this results also reveal that ginger possess hepatoprotective properties in diabetic cases. anti-fibrotic action m. naime 1 , s. ali 1 1 hamdard university rhizomes of valeriana jatamansi (family, valerianaceae) have long been used in indian subcontinent by the traditional healers for the treatment of various diseases. this study provides experimental evidence suggesting the therapeutic effect of the crude extract of rhizomes on rat liver fibrosis, and demonstrates its antiproliferative role. crude extract (50% ethanolic) at a dose level of 800 mg/kg body weight was administered to rats to study the effect on biochemical and other markers of liver fibrosis. administration of the extract for 9 weeks could bring down elevated the levels of biochemical markers of liver injury, and modulate several other biochemical responses. morphology and hisopathological examination cooroborated with the biochemical changes, and indicated partial reversal of fibrosis. dpph assay confirmed the antioxidant property of the extract, which is suggested to be due to -ionone, -sitosterol and other chemical constituents. further, treatment could restore depleted glutathione level, inhibit lipid peroxidation, and inhibited elevated xanthine oxidase activity in fibrosis. the study also reports anti-tumour promotion activity of the extract as evident by a significant decrease in [ 3 h]-thymidine incorporation by hepatic dna in extract treated rats. results suggest that v. jatamansi extract has curative effect and can partially reverse biochemical and histological changes associated with liver fibrosis. with chronic hepatitis c c. wongjitrat 1 , s. chainuvati 1 , a. manuyakorn 1 , s. aroonparkmongkol 2 , t. tanwandee 1 1 mahidol university, 2 background: leptin is a peptide hormone that mainly regulates food intake, energy expenditure and reproductive function. leptin also releases from activated hepatic stellate cells and may have a role in regulation of fibrogenesis and inflammation. in human chronically infected by hcv, the role of leptin-associated fibrosis of the liver is still unclear. there is no data in thai patients chronically infected by hcv regarding leptin level and its correlation with hepatic histology and fibrosis.the purpose of this study was to evaluate the relationship between leptin level and severity of liver fibrosis in thai patients chronically infected by hcv. methods: sixty-six patients (31 men, 35 women) with chronic hcv infection and liver biopsy was done within 3 months were enrolled. fasting blood samples were obtained and serum leptin levels were measured by elisa. bmi, blood sugar, liver function test, lipid profile, hcv rna viral load and hcv genotype were also measured and related to histological findings. results: mean serum leptin levels were significantly higher in women than in male. there was a significantly correlation between serum leptin and bmi (r = 0.469, p < 0.001). leptin levels were not associated with hepatic fibrosis (r = 0.166, p = 0.183) and necroinflammation (r = 0.203, p = 0.102). steatosis was significantly associated with severe necroinflammation (r = 0.261, p = 0.034), but not fibrosis (r = 0.22, p = 0.076). conclusions: these findings failed to demonstrate correlation of serum leptin and hepatic fibrosis in thai patients chronically infected with hcv. background and aim: liver cirrhosis is one of the leading causes of mortality in our country as well as in our region. even though deterioration of glucose metabolism and existence of insulin resistance in liver cirrhhosis has been well documented in many studies, it is still unclear how insulin resistance mechanism develops. the aim of the present study is to assess insulin resistance, cytokines and crp levels in patients with liver cirrhosis and control subjects. in additon, we aimed to investigate the relation of insulin resistance in liver cirrhosis with such parameters as age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp. material and method: a total of 79 patients with cirrhosis of different etilogy (49 male, 30 female) were included into the study. as controls, 50 (23 male and 27 female) subjects were taken. the two groups were compared with each other in terms of glucose, insulin, c-peptid, homa-ir, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. in the second part of our study, the liver cirrhosis group was divided into two subgroups: patients with homa-ir value >2.7 as insulin resistance positive, and those with homa-ir value >2.7 as insulin resistance negative. these two groups, i.e. , homa-ir positive and homa-ir negative, were compared in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. results: in liver cirrhosis group, glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp levels were determined to be significantly higher than controls. between patients with homa-ir positive and negative, however, statistically no significant difference was found in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels, but il-8 level was seen to be significantly low in patient homa-ir positive. conclusion: in patients with liver cirrhosis, the levels of glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp increase with respect to normal population. determination of increased homa-ir level in liver cirrhosis supports the view that insulin resistance develops in liver cirrhosis as reported in related studies. in the study, it was also determined that the mechanism of insulin resistance development occurs independent of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels. the determination of statistically lower level of il-8 in patients with homa-ir positive with respect to those with homa-ir negative does not indicate similarity with the studies carried out earlier. ) in patients (48.08%) than in controls(14%)in group i hla-b5 significantly increased in patients (60%)as compared to controls (14%) . in group ii hla -b5 significantly higher in patients (45.46%)than controls (14%) also hla-aw19 significantly higher (40.91%) in patients than controls (12.67%).in group iii hla-aw19 significantly increased in patients (46.67%) compared to controls.no significant association between hla antigens and cases with hbv or hcv infection. conclusion: the significantly high association of hla-aw19 and hla-b5 in patients with hepatic schistosomiasis as compared to normal controlstogether with the lack of any association with active intestinal schisto . antigens predispose to liver affection.individuals possessing hla-aw19 appear to be more prone to severeform of liver disease background: atp8b1 mutation is one of the factors that result in cholestasis and progress to chronic liver disease, but has never been reported in the mainland china before. the aim of this study was to elucidate the role of atp8b1 mutation in mainland chinese patients with progressive intrahepatic cholostasis and low ggt. methods: 24 children who presented with progressive intrahepatic cholostasis and low ggt were admitted in a tertiary pediatric hospital in eastern china. abcb11 gene was analyzed firstly to exclude bsep deficiency. afterwards, all the encoding exons and their flanking areas of atp8b1 gene were sequenced in the remaining 19 patients in whom only one or no mutations of abcb11 were found. results: 10 mutations of atp8b1 gene were found in 9 patients. i694n had been reported in taiwanese patients with pfic1, and the others were novel. p209t and ivs6+5t g were linkage and found in 4 of 9 patients, including 2 homozygote and 2 heterozygote. liver biopsy had been performed in 6 patients with atp8b1 mutations and 5 with abcb11 pe408 mutations. variety portal fibrosis was showed in 2 patients with atp8b1 mutations and 4 patients with abcb11 mutations. giant cell transformation was detected in one patient with atp8b1 mutations and 4 patients with abcb11 mutations. 1 laboratory of exercise biochemistry, taipei physical education college, jhongcheng rd., no. 101, sec.2, taipei city-11153, taiwan, roc background/aim: generation of reactive oxygen metabolites are depends on the consumption of oxygen and their cumulative effects may be different from lean to obese population. this study was designed to investigate the deleterious effects of oxidants on hepatic antioxidant defence system in lean and obese rats under hypoxic condition. methods: zucker rats lean (200±10gms) and obese (450±15gms) were divided into control and acute hypoxia groups. the acute hypoxia treatment was performed in a hypoxic chamber at 14% oxygen consumption. objectives: to compare the diagnostic value of morning urine copper to zinc (copper/zinc) ratio and 24 hour urinary copper excretion in wilson's disease (wd) children. results: in the results, acute hypoxia caused a significant (p<0.05) decrease in major antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px), glutathione reductase (gr) and glutathione (gsh) content in lean groups when compare to their controls. the decrease in the activities of all antioxidant enzymes was also noticed in obese group with hypoxia treatment. however, this decrease was not significant in case of cat, gsh-px activities and gsh content. the mda levels (lipid peroxidation marker) were higher in obese rats compare to lean rats. methods: morning urine and 24 hour urine were collected from 96 patients over three years age who were hospitalized in a tertiary pediatric liver service. each patient was re-evaluated according to wd scoring system, and was assigned to one of the three groups: wd, suspecting wd, and non-wd. 24, 6, and 64 cases were assigned to wd, suspecting wd, and non-wd respectively. urine copper and zinc concentration was determined simultaneously by using inductively coupled plasma mass spectrometry. conclusions: the higher hepatic mda values observed in obese rats indicate that accumulation of free radicals may be more in obese rats thus leads to promote the lipids oxidation. from this study it is concluded that decrease of antioxidant enzymes (except gr) with acute hypoxia treatment were more in lean group compared to with that of obese group. results: the morning urine copper/zinc ratio and 24hr urinary copper excretion correlated well (r=0.758, p < 0.001). the median of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, 24h copper excretion, and 24h zinc urinary excretion were 0.370, 0.394, 87.1 and 398.7 in wd group, and 0.051, 0.061, 24.2 and 358.9 in the non-wd group respectively. the differences of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, and 24h copper excretion were significant (z-value -6.502, -6.020 and -6.208 respectively, all p values < 0.000 chd1l is a recently discovered oncogene localized at 1q21, one of the most frequently amplified chromosomal regions in hcc. herein, by yeast-two hybrid assay, we demonstrate that the anti-apoptotic ability of chd1l is associated with its interaction with nur77, a critical member of a p53-independent apoptotic pathway. as the first cellular protein identified to bind nur77, chd1l inhibits the nucleus-to-mitochondria translocation of nur77, and subsequently hinders the release of cytochrome c and the initiation of apoptosis ( figure 1 ). further study found that c-terminal macro domain of chd1l is responsible for the interaction with nur77, and a chd1l mutant lacking residues 600-897 failed to interact with nur77 and prevent nur77-mediated apoptosis. we also find that chd1l confers cellular chemoresistance to drugs that induce apoptosis via the nur77-mediated pathway, which may lead to the identification of new therapeutic targets for hcc treatment. background/aim: accumulation of oxidative damage to proteins, lipids and mitochondria could increase with advancing of age. the current study was aimed to test the hypothesis that swimming exercise training could revert the age dependent oxidative damages in liver. methods: sprague-dawley rats of young (3 months) and old (12 months) were divided into four groups; young control (n=5), young exercise (n=5), old control (n=5) and old exercise (n=5). 90 minutes of swimming exercise was given to the exercise group for a period of two weeks. results: the estimated antioxidant enzyme activities including, superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px) and glutathione reductase (gr) were decreased with age and significantly (p<0.05) increased with exercise training. however, elevated protein carbonyls and mda levels were noticed in old animals, which indicate that old liver had greater accumulated oxidative damages. the significant drop in protein carbonyl content and increase in mitochondrial succinate dehydrogenase (sdh) activity was observed with response to swim training in old rats. conclusions: this data implied that swim exercise training could revert the oxidative damages in liver. this was also proven by enhanced antioxidant enzyme status with response to exercise training in old rats. to sum-up these results it is cleared that age induced detrimental effects to the liver might be reversed by regular swimming exercise training in old rats. results: peg10 was expressed in l02/peg10 (fig.1) . peg10 accelerated the growth of l02. after treatment with 400mm h2o2 for 24 h, the inhibitory rate of l02/peg10 cells was 32.5%; the chromosomal condensation and ladder-like dna fragmentation were not observed (fig.2) . methods: hepg2 or hepg2.2.15 were co-cultured with jurkat cells, with blocking test by adding anti-pd-1 antibody. the pd-1 expression was detected by flow cytometry (fcm); cytokines in culture supernatant in blocking groups and controls were measured by enzyme-labeled immunosorbent assay (elisa); cytotoxic test of t cells were measured by methyl thiazolyl tetrazolium (mtt). conclusions: over-expression of peg10 can significantly promot l02 proliferation and ameliorate apoptosis-inducing effects of h2o2 on l02. results: the pd-1 expression on jurkat cells was induced by hepatoma cells, the expression rate were 16.17±6.5% (by hepg2) and 17.43±6.8% (by hepg2 2.2.15), respectively. the cytokines il-2 level (202.9±53.0 pg/ml), inf-level (88.6±4.6 pg/ml) and il-10 level (63.7±13.4 pg/ml) in culture supernatant of blocking groups were significant higher than that of controls (il-2, 102.9±53 pg/ml, inf-, 39.3±4.2 pg/ml and il-10, 34.6±13.7 pg/ml, respectively. p < 0.05). the cytotoxic test (od value) was markedly higher in blocking group (0.29±0.06) than that of control group (19±0.09 p<0.05) . conclusion: the pd-1 expression on lymphocytes can be induced by hepatoma cells, and cytokines expression and cytotoxic test were recovered by blocking pd-1/pd-l1 interaction. background: hedgehog (hh) pathway is well known as a positive regulator for tissue construction( during development) and reconstruction (in adults). our aim to observe the expression change of hh pathway on rat hepatic regeneration . materials and methods: adult male sprague-dawley rats underwent approximately 70% partial hepatectomy (ph) or sham operation (so). liver specimens were collected at 2, 6, 12, 24, 36, 48, 72 , and 168h after ph or so. hedgehog expression was determined in mrna level by rt-qpcr as well as in protein levels via immunohistochemical staining and western-blotting. results: so treatment did not induce remarkable changes in hedgehog expression; however, the level of transcript for hedgehog was significantly upregulated after ph. we found sonic hedgehog(shh )and glioblastoma (gli1-3) mrna expression in the regenerating liver arrive at its peak at as early as 24 h and returned to its physiologyical level 168 h later. it is similar to the change of proteins (shh and gli1) .as seen from immunohistochemistry experiments; shh protein was expressed uniquely in regenerating hepatocytes. similarly, ph induced over expression for shh protein occurred from 12 h with a peak level at 36 h after surgery. but gli protein mainly located in nucleus and no significantly changes in the phrase of liver regeneration. conclusion: hedgehog pathway may play a role in the activation of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as ph. background: to investigate whether peg10, an imprinted gene with an active paternal but silent maternal allele, was involved in hydrogen peroxide (h2o2) induced cellular apoptosis. methods: peg10 gene was stable transfected into l02. cellular gene expression was determined by rt-pcr, western blot and immunocytochemistry. cell proliferation was analyzed by mtt. after treatment with different concentrations (50-400 mm) of h2o2, cell proliferation inhibition rate was measured by mtt. morphological changes of apoptotic cells were determined by hoechst33342 staining, dna fragmentation was observed by agarose gel electrophoresis. hua tang 1 , xiao-yan tang 1 , min liu 1 , xin li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china we determined how afp modulates the proliferation of hepatoma cells. a recombinant adenovirus expressing sirna against afp (adv-afpsirna) was created and found that it reduced expression of afp specifically in hepatoma cells, and markedly inhibited the proliferation of hepatoma cells in vitro. local treatment using adv-afpsirna caused significant repression of the growth of hepatoma derived hepg2 cells in xenograft in nude mice. knockdown of afp resulted in an obvious delay in the g1/s transition of cell cycle, but did not affect apoptosis in hepg2 cells, as analyzed by flow cytometry and tunel assay. also, differential expressions of some genes related to the cell cycle, including skp2, cyclin d1, csk and ebag9 were identified by microarray and rt-pcr in hepg2 cells and hepg2 cells with knocked down afp. these results suggest that endogenous afp is a critical determinant of the growth of hepatoma cells. hematopoietic stem cell (cd 34+) therapy can improve liver function in patients with cirrhosis. these cells can be mobilized into peripheral blood using granulocyte colony stimulating factor (gcsf). this study was undertaken to assess feasibility and safety and of gcsf induced cd 34+ cell mobilization and its impact on liver function in patients with cirrhosis. patients with liver cirrhosis (cryptogenic or alcoholic with 6m abstinence) with cpt > 7 and < 12, and splenic diameter < 17 cm were included. gcsf injection was given for 5 days (5mcg/kg/dose). baseline & day-6 cd 34+ counts in peripheral blood were done by flow cytometry. follow up was weekly for 4 weeks and then monthly. cpt was compared at baseline and 6 months. 9 patients (median age 51 y, range 33-64 y, 8 males; etiology: 6 alcohol, 3 cryptogenic; median cpt 10, range 8-12) were included. cd 34 + cell counts at baseline and day 6 were 2(0-3) and 15 (13-41) respectively (median, range). side effects were fever in 8, allergic reaction in 1 and increase in splenic size in 1 (excluded). in follow up, 3 patients died (1, 3 & 4m after therapy, 1 after olt), 1 lost to follow-up. 4 patients showed improvement in 10 -7) at 6-month follow-up. gcsf treatment is safe and yields adequate cd 34+ cells in peripheral blood. in short term it results in improvement in liver function in patients with cirrhosis pe415 molecular cloning and transcriptional analysis of kctd9 gene promoter b. pi 1 , j.s. wang 1 , m.f. han 1 , y.y. zhou 1 , x.j. liu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science and technology aim: our previous work has shown that high expression of kctd9, a potassium channel associated gene, correlated with the disease severity of patients with severe chronic hepatitis b(schb). to further understand the gene transcription and regulation, kctd9 promoter was cloned and gene transcription was studied. methods: a full length of isolated promoter and series of 5' truncated promoter of kctd9 gene was subcloned into the luciferase report vector pgl2-basic to form the promoter-report constructs. the kctd9 promoter-report construct upstream of the luciferase report gene was cotransfected with constructs expressing hbv x,c and s protein respectively or stimulated with cytokines (il-2, ifn and tnf ) in 293 t cells to investigate kctd9 gene regulation upon both viral factors and host cytokines. result: a 759bp kctd9 segment upstream of atg translation start site was evidenced to contain potential regulative domains. an important regulation site located between -268bp and -81bp upstream of atg translation start site. based upon the luciferase activity assay, il-2 was able to upregulate the transcription of kctd9 whereas there was no effect from neither hbv viral proteins nor ifn and tnf . conclusion: here we first successfully cloned the full length promoter of kctd9. il-2 significantly enhanced the transcription of kctd9, a gene which has been shown to be involved in t cell activation and disease severity of schb from our group. this work was supported by nsfc(30571643, 30672380, 30700702) 1 1 istanbul university, cerrahpasa medical school, 2 marmara university, 3 okmeydani teaching hospital, 4 background: the treatment in chronic hepatitis c virus (hcv) is not highly effective, and cost, duration, and side effects are challenging. predicting favorable factors of response to treatment would make it possible to give it only responsive patients. recent studies report more conclusive results about the role of apoptosis in inflammation and fibrosis seen in chronic viral hepatitis. hepatocyte damage in hcv is mediated by cytotoxic t-cells. apoptosis primarily developed by the interaction between fas antigen on hepatocyte and fas ligand on t-cell corresponds to a main mechanism for hepatocyte damage. methods: in this study, we aimed to detect any relationship between apoptotic markers (fas, fas ligand, fas-associated death domain, caspases 3,8, and 9, insitu apoptosis) in liver biopsy taken before the treatment and response to the treatment of interferon+ribavirin. additionally, any relationship between these parameters and the other ones predicting the response to therapy including alt level, viral load, genotype, and gender were studied. results: the study includes the patients in 4 centers managing chronic hcv infection. all parameters were studied in 180 patients. study results revealed that histological activity index is correlated with cd95 staining density, caspase 8 intensiveness, and portal and parenchymal fas ligand scores. fibrosis is also seen to be correlated with the same parameters. apoptotic parameters of the responsive cases were not significantly different from unresponsive ones. conclusion: apoptotic parameters studied in the liver tissue is associated with inflammation and fibrosis, however these parameters may not predict the response to the treatment. s. gao 1 , d. xi 1 , j.w. guo 1 , c.l. zhu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science objective: this study was designed to explore the opportunity of microrna interference technique in the inhibitory application of human fgl2, human fas and tnfr1 expression. methods: the eukaryotic expression plasmids of human fgl2, fas and tnfri genes were constructed and have successfully expressed hfgl2, hfas and htnfri protein. mirna expression plasmids of hfgl2, hfas and htnfri complimentary to the sequence responsible for hfgl2, hfas and htnfri respectively were also constructed, meanwhile an irrelevant mirna plasmid was used as control. by respective cotransfection of p-hfgl2mirna and pcdna3.1-hfgl2, p-hfasmirna and pcdna3.0-hfas, p-htnfri mirna and pcdna3.0-htnfri expression construct into 293t cells, the inhibition of hfgl2, hfas and htnfri expression were analyzed by quatitative real time pcr and western blot. results: the experiments showed the significantly inhibitory effect of p-hfgl2mirna on hfgl2, p-hfasmirna on hfas and p-htnfri mirna on htnfri expression at 48h post-transfection both at rna level and at protein level, as well in 293t cell lines the inhibitory efficiency reached as high as 89.3% for hfgl2, 87.5% for hfas and 80% for htnfri, respectively. conclusions: the study demonstrated the constructs of p-hfgl2mirna, p-hfasmirna and p-htnfri mirna successfully interfered their target genes expression in vitro, which provides the foundation for further investigation of these constructs' application in vivo and further more as a therapeutic strategy for a targeting intervention in the diseases which the gene fgl2, fas and tnfri contribute to. this work was supported by nsfc30571643, 30672380, 30700702; 2005cb522901, 2007cb512900 pe418 influence of the id2 on the anti-tumor activity of histone deacetylase inhibitor in hepatocellular carcinoma cells r. tsunedomi 1,2 , s. harada 1 , n. iizuka 1 , m. oka 1 1 dept. of digestive surgery and surgical oncol., yamaguchi univ. , 2 research fellow of the japan society for the promotion of science for young scientists background: our recent study revealed that levels of the inhibitor of dna binding/differentiation 2 (id2) were associated with the progression of hcv-related hepatocellular carcinoma (hcc) and can affect susceptibility of hcc cells to histone deacetylase (hdac) inhibitors. we here aimed to investigate how and whether id2 expression affected on the anti-tumor activity of sodium butyrate (nab), one of hdac inhibitors. methods: two hcc cell lines, hle and huh-7, were used for gene targeting experiments. the id2 over-expressing and knockdown cells were subjected to mts assay to evaluate the susceptibility to nab. time-course of the expressional change of bcl-2 and bcl-xl genes after nab administration was measured by real-time rt-pcr. result: upregulation and downregulation of id2 levels in hcc cells resulted in decreased and increased susceptibility to nab, respectively. we observed that after nab administration, the id2 expression was induced gently, the bcl-2 expression was greatly increased immediately, and the bcl-xl expression was decreased to less than half once and then recovered. these increase and recovery of the expression of anti-apoptotic genes were inhibited in the id2 knockdown cells. in the id2 overexpressing cells, the bcl-2 expression was more upregulated than mock-transfected cells. conclusion: in hcc cells, id2 influences the susceptibility to the hdac inhibitor by regulating the expression of anti-apoptotic genes caused by the hdac inhibitor stimulus. we suggest that id2 could serve as a fascinating marker predictive of response to hdac inhibitors. the role of zinc finger protein 146 in liver cancer m.m.y. waye 1,2 , t.l. yeung 1,2 , j.l. zhu 1,2 1 the chinese university of hong kong, 2 croucher laboratory for human genomics background/aims: the aim of this research project is the characterization of a krüppel zinc finger protein, zinc finger protein 146 (znf146) using hepatocellular carcinoma as a disease model. zinc finger protein 146 (znf146), also named as only zinc fingers protein (ozf), is a 33kda nuclear zinc finger protein consisting solely of ten c2h2 zinc finger motifs of the krüppel type. like most of krüppel proteins, it is assumed to be the transcription factor and involved in the regulation of gene expression. the znf146 gene is amplified in 15-25% of pancreatic carcinomas and overexpressed in more than half of the tumors including liver cancer. it is thus proposed that overexpression of the znf146 may contribute to the development or progression of hepatocellular carcinoma. methods: we used flow cytometry, microarray, green fluorescent recombinant protein, rt-pcr site-directed mutagenesis, and transfection to study the effect of expression of znf146. results: our results shown that znf146 was over-expressed in two human hcc cell lines hepg2 and hep3b. expression profiles of znf146 over-expressing shown that genes related to the p53 tumor suppressor activity or dna damage, repair response and control were deregulated upon overexpression of znf146. conclusions: znf146 is possibly involved in liver carcinogenesis by affecting dna repair and cell cycle control upon induced dna damage. background: in the present study, we reported the establishment of a real-time monitor system for directly observing the catalytic, kinetic characteristics of dnazyme 10-23 in vitro cleavage on the target rna molecules as well as for rapid, accurate, high-throughout evaluation of varied dnazymes on their counterpart rna molecules. methods: dnazyme named dz-hcv-9 specific to hepatitis c virus (hcv) orf aug were designed and synthesized. dz-hcv-mis-9 with mismatched substrate-recognition domains, dz-hcv-mut-9 with mutant catalytic domains, antisense oligonucleotide ason and nonsense oligonucleotide nson were synthesized respectively as controls. a chimeric oligonucleotide of 29nt containing both rna and dna bases was designed and synthesized as the substrate: 5' fam-gt agaccgugcaccaugagcacgaaucct-bhq 3', corresponding to the330-354 nt (underline) of hcv genome(gi: 329873) the reporter fam/bhq was incorporated at the 5' and 3' end, respectively. under simulated physiological conditions (37 ), kinetic characterization of rna-cleaving dnazyme was analyzed in a real-time way. factors that influencing dnazyme cleavage were analyzed. results: dz-hcv-9 specific to hcv orf aug could cleave target rna at a•u site, a continuous change of fluorescence intensity was monitored. while the control oligonucleotides couldn't cleave rna, there were no change of fluorescence intensity. factors that influencing dnazyme cleavage concluded different substrate-recognition domain, mg 2+ concentration and ph. conclusion: a real-time monitoring system for kinetic characterization of rna-cleaving dnazyme was successfully established in the first time. the study on the apoptosis of hepatoma cells synergeticly induced by plasmid-mediated anti-angiogenesis and immunopotentiation therapy p.y. li 1 , q. zhang 1 , y. chang 1 , j.s. lin 1 , d.a. tian 1 1 background: angiogenesis is improtant to hepatoma and decreasing of host immunity promotes the development of tumor. we want to study the effect and mechanism of apoptosis of mice implanted hepatoma cells induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. methods: mouse endostatin eukaryotic plasmid (pseces) and mouse il-12 (interleukin 12) eukaryotic plasmid (pmil-12) were extracted and purified from e. coli. h22 hepatoma cells were inoculated into the leg muscle of mice, which was divided into four groups and injected with pseces, pmil-12, pseces+pmil-12 or pcdna3.1 naked plasmid dna respectively into implantation sites repeatedly. tumor formation and its weight was evaluated. tumor microvessel density, tumor infiltrating lymphocytes and apoptosis of tumor cells were assayed by cd31 staining, he staining and tunel assay respectively. results: inoculated mice received pseces, pmil-12 injection formed tumor slowly with less microvessel density, more tumor infiltrating lymphocytes in the latter and more tumor apoptosis cells in both groups compared with pcdna3.1 injection. there were much more tumor apoptosis cells in pseces+pmil-12 group (19.9±5.5 per 400× microscope field p<0.05) than any other single plasmid injection group (400× microscope field: pseces 11.3±4.1, pmil-12 14.6±3.2, pcdna3.1 1.4±1.3). conclusion: tumor cells of implanted hepatoma in mice could be synergeticly induced to apoptosis by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocytes to infiltrate, by which mice implanted hepatoma was inhibited. (20, 40, 80, 160, 200 ng/ml) in a serum-free medium for 24 h. cell proliferation was measured by brdu incorporation analysis, untreated wb-f344 cells were taken as controls. after treatment with wnt3a (160ng/ml) for 24 h, subcellular localization and protein expression of -catenin in wb-f344 cells treated and untreated with wnt3a were examined by immunofluorescence staining and western-blot analysis. cyclind1 mrna expression was determined by semi-quantitative reverse-transcript polymerase chain reaction (rt-pcr). mrna levels of some phenotypic markers (afp, ck-19, alb) and two hepatic nuclear factors were measured by rt-pcr. expressions of ck-19 and afp protein were detected by western-blot analysis. results: wnt3a promoted proliferation of wb-f344 cells. stimulation of wb-f344 cells with recombinant wnt3a resulted in accumulation of the transcriptional activator -catenin, together with its translocation into the nuclei, and up-regulated typical wnt target gene cyclind1. after 3 d of wnt3a treatment in the absence of serum, wb-f344 cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as afp, ck-19 following activation of the canonical wnt signaling pathway. conclusion: the canonical wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells. the expression level of bid and other pro-and anti-apoptotic proteins were detected by immunoblotting. results: hbx/51 showed the most sensitive towards dox treatment, and truncated bid (tbid) was also only detected in this cell line. the level of bax was also increased in hbx/51 cells. conclusions: the carboxy-terminal of hbx may enhance the processing of bid into tbid, which may contribute to increased sensitivity of the cell towards the dox treatment. cell homeostasis were performed with concentrations of oxysterol (3x10 -5 -10 -4 m) faraway from the physiological and/or pathological one (0.2 and 2x10 -7 m). in our study, we asked the effects of oxysterols (7k and 5'6s) on hepatoma cell lines homeostasis. to this purpose we used concentrations similar to those described in physiological or pathological conditions. sub-physiological (10 -9 m) to pathological (10 -7 m) oxysterol (7k and 5'6s) concentrations were used to stimulate hepg2 cells. a surprising pro-proliferative effect of 5'6s at sub-physiological (10 -9 m) concentration was observed. this behaviour was confirmed by the synergic increase of erk1/2 levels. facs analysis revealed an early progression of cells in s phase at the lowest concentration of 5'6s, while all the remaining concentrations of the two studied oxysterols induced a weakly accumulation of cells in g2/m phase. apoptosis was absent at all concentration used, except for the highest one (10 -5 m). at this point we asked if cells didn't undergo apoptosis but acquired a senescent profile. effectively, both 7k and 5'6s, at all concentration used (except for 10 -9 m), induced cell senescence (revealed by sa-ß-gal staining and sirt1 and p21 over-expression). in conclusion the two oxysterols analyzed have different and in same case opposite effects on hepatocellular line. the main effect is surely the senescence induction, but it is important to highlight the proproliferative effects of 5'6 secosterol at low concentration. mortalin, a member of hsp70 family protein, has been shown to play an important role in hepatocellular carcinoma (hcc). it has been reported that mortalin is binding to the c-terminal of p53, which acts as a safety guard and is a commonly mutated gene in hcc. in this study mortalin was silenced by specific shrna in plc/prf/5, a hcc cell line constitutively expressing p53ser249, and normal liver cells miha, and we found that suppression of mortalin can selectively trigger the mitochondria mediated apoptosis pathway by p53 dependent way in plc cells. tunel staining positive cells were only found in the plc cells mortalin knockdown group, and apoptosis associated protein, such as p53, bax, bcl-xl, cleaved-caspase 3, have been screened by western blot after transfection. quantitative-pcr data also showed that p53 mrna level are upregulated about 2 folds in mortalin knockdown group compared with the control groups in liver tumor cells. two p53 inhibitors, pft-and pft-, which can reverse this apoptosis was applied to demonstrate p53 dependent way. in summary, knockdown mortalin can selectively kill liver cancer cells through reactive apoptosis by sensitizing mutant p53 in plc cells, but had no effect on normal cells. the clinical application of this study suggested that motalin specific shrna might be a potential anti-cancer drug for hcc. background: nafld can proceed to nash and are at risk of cirrhosis and hcc. aim was to study profile of bangladeshi nafld patients. methods: 52 patients with nafld were included. of them 59.6% were males and 40.4% females. patients were between 12-60 years of age. they presented with dull right upper abdominal ache and/or incidental detection of raised alt/ast and/or fatty liver on ultrasonography. all tested negative for hepatitis b and c. none had history of alcohol. all underwent per-cutaneous liver biopsy for histopathology. they were also tested for dm, dyslipidaemia, insulin resistance, hypothyroidism and hepatitis c. their bmi and bp were recorded. results: 88.5% had nash. 63.0% of them were males and rest 37.0% females. 11.5% had nafl. of them 50% each were males and females. majority had nash. 47.8% were obese and 41.3% had dyslipidaemia. 28.3% had hypertension, 28.3% insulin resistance and 13% were diabetics. 6.5% had hypothyroidism. none had hepatitis c. alt was raised in 72% and ast in 40%. although all patients with nash did not have elevated alt, it was raised in majority, contrary to ast, which was normal in most. conclusion: majority nash patients in bangladesh are obese. other leading causes of nash include dyslipidaemia, hypertension and insulin resistance. some nash also had diabetes and hypothyroidism. this study also reveals that elevated alt in patients with nafld is suggestive of fibrosis, although normal serum alt does not exclude nash. the study further suggests that alt is superior to ast in predicting nash. background: non-alcoholic fatty liver disease is prevalent in obese patients. liver biopsy remains the best diagnostic tool for confirmation. we tried to find out the correlations of laparoscopic parameters with histology and laboratory data. besides, we also evaluated the effectiveness of laparoscopy in liver disease diagnosis. methods: in the period of one year and five months,126 morbidly obese patients submitted to laparoscopic bariatric surgeries at our institutions were prospectively studied. results: laparoscopic parameters of significant correlations with histologic steatosis, inflammation and fibrosis were summarized in table 1 . besides, important parameters with relationships to laboratory data were summarized in table 2 department of internal medicine, seoul national university hospital gangnam healthcare center, seoul, south korea, 2 department of internal medicine and liver research institute, seoul national university college of medicine, seoul, south korea background/aims: hepatic fibrosis is associated with poor prognosis in non-alcoholic fatty liver disease (nafld). recently, many non-invasive fibrosis markers have been studied to overcome the limitations of liver biopsy. among them, bard score and guha's simple panel are easy to use in clinical practice. in this study, we evaluated the efficacy of bard score and guha's simple panel as a noninvasive fibrosis marker in korean nafld patients. methods: data from 79 patients with biopsy-proven nafld in seoul national university hospital from 2000 to 2007 were used. bard score and guha's simple panel were calculated by using clinical and biochemical data and were compared with the histological fibrosis stages. results: stage 0 fibrosis were found in 67 patients, stage 1 in 4, stage 2 in 2, stage 3 in 1 and stage 4 in 5. the relationship between fibrosis stage and bard score ( = 0.43, p < 0.001) was statistically significant. all patients with advanced fibrosis (stage 3-4) had bard score greater than 2. mean values from original guha's simple panel for no fibrosis were not different between the patients with and without fibrosis. however, after adjusting coefficients by logistic regression analysis, the differences in mean values became statistical significant (p < 0.001). conclusions: our data suggest that bard score may be effective for detecting high risk patients for advanced fibrosis, and modification of coefficients within the guha's simple panel may be needed to use as a fibrosis marker in asian nafld patients. s.k. mohan 1 , s. subramaniam 2 , s. subramaniam 3 1 assistant professor, department of biochemistry, saveetha medical college & hospital, saveetha university, t.n, india., 2 consultant, department of biochemistry, apollo hospitals, chennai, t.n, india. , 3 department of biochemistry, apollo first med hospitals, chennai, t.n, india. background: non-alcoholic fatty liver disease (nafld) covers a spectrum of liver diseases from simple fatty infiltration to progressive fibrosis. non-alcoholic steato hepatitis (nash) is a severe form of nafld and progresses to the end stage of liver disease. it is becoming the leading cause for referral to liver clinics in most areas. the prevalence of nafld in indian population is estimated around 7 -13%. the nafld has the potential to progress to hepatocellular carcinoma or liver failure, both events that ultimately lead to early death. aim: to evaluate the combination of inter cellular adhesion molecule -1 (icam -1), adiponectin and type-iv collagen, a new biomarker profile for nash in patients with nafld. methods: 76 patients with nafld and age & sex matched 68 normal healthy individuals as controls were selected for this study. levels of serum icam -1, adiponectin, type-iv collagen, lipid profile and liver function test parameters were estimated in patients and compared with controls. results: serum icam -1 & type -iv collagen levels were significantly increased in patients with nash among the nafld patients compared to controls. the serum adiponectin levels were significantly reduced in patients with nash among the nafld patients compared to controls. compared to liver function test parameters and lipid profile levels, nash profile has got positive negative predictive value among the nafld patients. conclusion: in patients with nafld, nash profile test -a simple, noninvasive and reliable to predict the presence or absence of nash. background/aim: oxidative stress and cytokines plays an important role in the pathogenesis of nonalcoholic fatty liver disease (nafld). aim of study was to assess lipid peroxidation, serum levels of transforming growth factor-( tgf-) and tumor necrosis factor-( tnf-) in patients with nafld and compare it with patients of chronic viral hepatitis (cvh) and healthy controls (hc). methods: lipid peroxidation was studied by estimating plasma malondialdehyde (mda) levels as per the methodology described by buege and aust and tgf-& tnf-levels were measured by elisa kits (ray biotech, usa, & diaclone, uk) in the stored sera in 10 biopsy proven patients with nafld (m: 4, f: 6, mean age: 41.7 ± 11.0 yrs), 15 patients with cvh ( m:14, f:1, mean age: 33.7 ± 11.4 yrs) and 5 hc (m:5, mean age: 25.2 ± 2.7 yrs). results: there was no difference in mean plasma mda levels amongst patients with nafld (17.28 ± 3.6 mol/l), cvh (15.29 ± 2.3 mol/l) and hc (16.79 ± 1.2 mol/l). serum tgf-levels between nafld (0.56 ± 0.41 ng/ml) and cvh (0.52 ± 0.25 ng/ml) patients and hc (0.58 ± 0.57 ng/ml) were also comparable. though patients with cvh (17.2 ± 27.0 pg/ml) and nafld (7.5 ± 6.3 pg/ml) had higher levels of tnf-than hc (5.5 ± 1.1 pg/ml), the difference was not significant statistically. conclusion: lipid peroxidation, tgf-and tnf-need to be studied in a larger number of patients with nafld. background/aim: burnt out nonalcoholic fatty liver disease (nafld) may be responsible for cirrhosis and hepatocellular carcinoma (hcc) in the absence of other causes. aim of this study was to evaluate the surrogate markers of nafld in patients with cryptogenic cirrhosis (cc) and cryptogenic hcc (chcc). methods: sixty five patients with cc and 31 patients with chcc were analyzed for the presence of abnormal body mass index (bmi) and type 2 diabetes mellitus (dm aim: to investigate the relation of phosphatidylethanolamine n-methyltransferase pemt gene g175a single nucleotide polymorphism (snp) with the susceptibility to nonalcoholic fatty liver disease nafld . methods the genotypes and allele frequencies of pemt exon 8 snp g175a were analyzed by using pcr-rflp in 51 nafld patients and 50 controls. results: the g to a variation of the pemt gene g175a snp was significantly higher in nafld group compared with controls. the frequencies of gg ga and aa genotypes were 58.8 39.2 and 2.0 in nafld and 78.0 22.0 and 0.0% in controls (p=0.038 . the a allele of the pemt gene was significantly more frequent in nafld group (21.6%) than that (1.0%) in controls p=0.042 .there were significant differences in serum levels of cholesterol, triglyceride, hdl-c and ldl-c between gg and ga/aa genotypes p <0.05 . n. assy 1,2 , g. lipez 3 , s. korem 3 , m. grozovski 3 1 sieff hospital, safed, israel, 2 2technion institute, faculty of medicine, haifa, israel, 3 ort braude college, karmiel, israel background: previous studies reported increase in serum protein c and decrease in serum paraoxonase levels in patients with non alcoholic fatty liver diseases (nafld). conclusion people with pemt gene g175a snp were more susceptible to develop nafld aim: 1) determine whether there is a relationship between nafld, protein c and paraoxonase levels in quiescent and in regenerating rats fatty liver 2) determine the effect of isa on hepatic "protein c" and paraoxonase mrna. pe439 methods: forty-eight sd rats were treated with fructose enriched diet (fed), or fed with metformin (2 mg/kg/d), fed with rosiglitazone (3 mg/kg/d), or the combination of both drugs for 5 wks. 30% phx was performed at wk 5. protein c, paraoxonase mrna expressions, lipids, mda were measured before and 24 hours after phx. results: hepatic "protein c" mrna was higher in rats with fatty liver than control rats (+105%, p<0.01) whereas hepatic paraoxonase mrna was lower in rats with fatty liver than control rats (-28%, p<0.005). hepatic protein c and paraoxonase mrna increased in rats with fatty liver in regeneration (+116%, p< 0.01, and +15%, p<0.01 respectively). the combination of metformin and rosiglitazone decreased hepatic protein c expression at 24 hours after phx by -170% (p<0.001) and increase paraoxonase mrna by +50% (p<0.01). serum paraoxonase correlates with serum protein c (r=-0.2), mda (r=0.4), background: non-alcoholic steatohepatitis (nash) is a type of non-alcoholic fatty liver disease (nafld), and may progress to hepatic fibrosis and cirrhosis. the pathogenesis of nash remains unclear. the aim of this study was to explore the arginase change in the progress of steatohepatitis in rats. methods: male sd rats weighing 70-80g were obtained. twenty animals were randomly divided into two groups. in the model group, five animals were fed with high lipid forage that includes 3% cholesterol and 20% lard for 6 weeks, five were fed for 12 weeks, while the control group ate normal foods. the animals were sacrificed after 6 weeks. the animals were sacrificed after 6 weeks and 12 weeks. liver and blood serum were collected while the serum levels of alt, ast, tg and tc were measured. the pathology of liver was observed by he staining. western blot was used to investigate the expression of arginase in control and model group. tg (r=-0.23). conclusion: hepatic "protein c" mrna levels are high at baseline, up regulated during liver regeneration and decrease after treatment with (isa) whereas hepatic paraoxonase mrna levels are low at baseline, up regulated during liver regeneration and increase after treatment with isa. results: vacuolization were observed extensively in hepatic cells in the model group after 6 weeks and 12 weeks of high-fat diet. it is demonstrated that rats fed with high-cholesterol food are indeed fatty liver models. western blot showed that the level of arginase ii increase in the liver of model group rats as compared to the control group. furthermore, the level of arginase was higher in liver samples obtained from model rats that were 12 weeks on a fat diet as compared to rats that were only 6 weeks on the same diets. conclusion: the level of arginase ii was altered in the progress of non-alcoholic steatohepatitis in rats suggesting that arginase ii is putative biomarkers and may represent new targets in the development of therapeutic strategies against fatty liver disease hepatic fibrosis and cirrhosis. methods: c57bl6/j mice were fed with mcd diet to induce hepatic fibrosis and rosiglitazone was given in treated group. effect of rosiglitazone was assessed by comparison of the severity of hepatic fibrosis in liver sections, expression of mmp-2/9, timp-1/2 mrna and protein detected by rt-pcr and western blot respectively. the ethanolic extract of fructus schisandrae chinensis decreased hepatic triglyceride level in mice fed with a high fat/cholesterol diet results at week 8, fibrosing nash models showed severe hepatic steatosis, infiltration of inflammation and fibrosis, which is associated with down-regulated mmp-2/9 mrna and protein, up-regulated timp-1/2 mrna and protein. rosiglitazone significantly reduced mcd-induced fibrosis by induced mmp-2/9 expression and reduced timp-1/2 expression by activating ppar . s.y. pan 1 , z.l. yu 2 1 beijing university of chinese medicine, 2 hong kong baptist university effects of the ethanolic extract of fructus schisandrae chinensis (etfsc) on serum and liver lipid contents were investigated in mice fed with normal diet or high fat/cholesterol diet for 8 or 15 days. single dose of etfsc (1 or 5 g/kg/day, i.g.) increased the serum triglyceride (tg) level (40 and 142%, respectively), but decreased hepatic total cholesterol (tc) level (15 and 16%, respectively) in normal mice. the hypertriglyceridemia produced by etfsc was suppressed by the co-administration of fenofibrate. the induction of hypercholesterolemia by high fat/cholesterol diet caused significant increases in serum and hepatic tc levels (up to 165%) and hepatic tg levels (up to 528%) in mice. etfsc treatment (1 or 5 g/kg/day for 7 days, i.g.) significantly decreased the mouse hepatic tg level (by 35%) and slightly increased the hepatic index (by 8%). whereas fenofibrate treatment (0.1 g/kg/day for 7 days, i.g.) significantly lowered the hepatic tg level (by 61%), it significantly elevated the hepatic index (by 77%) in hypercholesterolemic mice. the results indicate that etfsc treatment can invariably decrease hepatic tg in hypercholesterolemic mice, suggesting its potential use for fatty liver treatment. aim: to investigate the influence of multiple gene polymorphisms in the susceptibility of nafld. methods: the data of single nucleotide polymorphisms (snps) in 201 nafld patients who had at least one of the genetic variations at the sites of tnf--238, adiponectin -45 and leptin-2548 were analyzed. the genotypes were determined by using pcr-rflp. our previous studies showed that the variations of these sites increased the susceptibility of nafld. results: the prevalence of nafld in adiponectin variation alone group (n=45) was 35.6%; in tnf-alone group (n=33) 42.4%; in leptin alone group (n=54) 35.2% (p>0.05). in comparison with the above groups with single snp, the prevalence of the groups with two gene variations of tnf-plus adiponectin (57.9%, n=19) increased significantly (p<0.05). however the prevalence of other two groups i.e. adiponectin plus leptin (34.6%, n=26) and tnf-plus leptin (40.0%, n=15) did not differed significantly from those of groups with single snp (p>0.05). the prevalence in the group with three gene variations (55.6%) differed significantly from all (p<0.05) except that of tnf-plus adiponectin group (p>0.05). the metabolic features of the nafld patients in the 7 groups mentioned above were not different significantly (p>0.05). conclusion: nafld is a polygenic disease. multiple gene polymorphisms may, but not always, increase the susceptibility of nafld. chronic hepatitis b patients with nonalcoholic fatty liver disease r.d. zheng 1 , c.r. xu 1 , j. chen 1 , b.f. chen 1 1 southeast hospital background: to investigate clinical pathological characteristic in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). methods: we measured fasting blood glucose, insulin, triglyceride, cholesterol, alanine aminotransferase (alt), aspartate aminotransferase (ast) in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). and we detected hepatitis b virus marker, hbv-dna, counted body mass index, insulin resistance index and observed pathological characteristic. all these patients with diagnosis were confirmed by clinical and pathological evidence. result : the body mass index, homeostatic model assessment (homa) of insulin resistance, fasting blood glucose, insulin, triglycerides, cholesterol, were significantly higher in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld) than hbeag negative chronic hepatitis b patients. but the alanine aminotransferase (alt), aspartate aminotransferase (ast), hbv dna levels were significantly lower in hbeag negative chb patients with nafld than in hbeag negative chronic hepatitis b patients. histologic features in hbeag negative chronic hepatitis b(chb) patients with nonalcoholic fatty liver disease (nafld) are in zone 3 predominate macrovesicular steatosis and mild inflammatory infiltrate in portal region. conclusion: the hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease, whose hepatic steatosis changes are mainly caused by the metabolic factors. to carry out liver biopsy selectively for the patients with hbeag negative chronic hepatitis b having metabolic factors, which is helpful for early diagnosis in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). aims: to investigate the preventive effect of cordyceps sinensis and its possible mechanism on apoptosis of nafld. methods: rats were randomly divided into basic diet group (b group), pathologic group (nash group) and cordyceps sinensis group(cs group).the latter two groups were administered with high-fat diet to establish nafld animal models. cs group were treated with cs at the 9th week after high fat diet. rats were sacrificed at the end of the 18th week. biochemical examination were used to detect superoxide dismutase (sod) of liver tissue. hepatocyte apoptosis was assessed in each group using the tunel assay and immunohistochemistry for activated bax bcl-2 caspase-3 and nf-kb p65. results: (1) compated with the b group, severe hepatosteatosis, inflammative necrosis and local fibrigenisis were showed in liver of nfsh group. sod lever was significantly decreased (p<0.01) and tunel-positive cells were significantly increased (p<0.01). immuunohistochemistry test demonstrated active bax caspase-3was increased (p<0.01) while no apparent change was observed in bcl-2. (2) in cs group, only diffusive steatosis but not inflammation or fibrosis was found. sod lever was increased than that of nash group (p<0.05). tunel-positive cells and active bax caspase-3 were significantly decreased (p<0.05 p<0.01) that those of nash group. bcl-2 and nf-kb p65 were increased (p<0.01) than those of nash group. conclusions: hepatocyte apoptosis is a prominent feature of nafld. cordyceps sinensis may be useful as an antiapoptosis theraphy in this syndrome through increasing activity of sod, decreasing express of bax and increasing express of bcl-2 and nf-kb p65. background: non-alcoholic steatohepatitis (nash) is a leading cause of chronic liver disease. insulin-sensitizing , anti-inflammatory and anti-fibrotic effect of thiazolidinediones support their use in the treatment of nash. we aimed to evaluate the efficacy of thiazolidinediones in the treatment of nash. methods: we have identified randomised clinical trials, evaluating the efficacy of thiazolidinediones versus placebo in nash, through medline, embase, ami, cochrane central register of controlled trials. data were abstracted from each study and disagreements were resolved by consensus. dichotomous outcomes were reported as relative risk with 95% confidence interval based on fixed-effects model. results: we included three trials, two evaluating pioglitazone and another rosiglitazone. a total of 171 patients were involved in the analysis. thiazolidinediones was noted to improve liver function tests. it was effective in the reduction of steatosis among patients with nash (rr 0.67, 95% ci 0.52-0.87). it was found to be beneficial in improving ballooning necrosis (rr 0.79, 95% ci 0.66-0.95). it was also found to improve lobular inflammation (rr 0.72, background: it is well known that the weight reduction is effective for alt normalization in patients with non-alcoholic fatty liver disease (nafld). the necessary condition for alt normalization is still unclear. to clarify the necessary and sufficient condition for alt normalization, we investigated the effects of body fat decrease in nafld patients by body composition analyzer. methods: forty-six nafld patients (23 male, 23 female, mean age 49.8±12.9 years old) with abnormal alt levels were evaluated. the volume of skeletal muscle, body fat and bmr were examined by using the body composition analyzer (in body 720; biospace co. ltd., tokyo japan). all patients were received an individualized diet consultation by dietician every 4 weeks for 6 months. daily energy was bmr (basal metabolic rate) x1.2 kcal and protein was 1.0-1.2g per ideal body weight. result: twenty-eight of 46 patients (60.9%) were achieved normal alt level. in alt normalized group, the body weight and fat loss were 3.6±2.3 kg, 3.0±1.5kg (2.8±1.7 %body fat) respectively. on the other hand, in cases with alt remained abnormal level, the body weight and fat loss were 0.5±1.5kg, 0.4±1.6kg (0.5±1.6 %body fat). conclusion: our results demonstrate that the fat loss of 3 kilograms or more was necessary to normalize alt level in nafld patients. a. somani 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background : nafld is often clustered within families and the causes include both genetic and environmental factors. family studies done thus far have been limited by small sample size. to examine the familial patterns , we performed a prospective study to see (a) whether nafld is more common in first degree relatives (b) genetically determined risk factors associated for clustering. methods: first degree relatives of histologically confirmed nafld patients and spouses (controls) were included after excluding other causes of fatty liver. those having raised transaminases >3 months or sonographic examination consistent with fatty liver, had undergone liver biopsy for histological confirmation. they were divided into three groups. group i patients group ii first degree relatives group iii spouses results: nafld was more prevalent among first degree relatives then spouses (37% and 17%, p<0.01). anthropometric measurements, systolic and diastolic blood pressure, lipid profile and liver function tests were comparable in three groups. homa-r was similar in group i and ii (p=0.073), but was significantly different in group i and iii (p= 0.0001) and group ii and iii (p=0.0001) respectively. metabolic syndrome was present in >70% of patients and were comparable in three groups except for fasting glucose > 110, which was present in 76%, 77% and 57% of patients in group i, ii and iii respectively. majority (>50%) of our patients among groups i, ii and iii were having only steatosis while nash was present in 20%, 13% and 14% of patients. a. somani 1 , v. dixit 2 , a. jain 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha background: normal levels of alanine aminotransaminase (alt) have been demonstrated in nafld patients. alt levels are also modulated by age, gender, bmi, fasting glucose, and serum triglyceride levels. we performed a prospective study of patients with histologically confirmed nafld and having alt < 1.5 times and compared them with those having raised alt to determine (a) clinico-pathologic features of nafld patients with normal alt (b) to observe any differences between them. methods: patients with fatty liver on sonography had under gone biopsy for histological confirmation after excluding other causes of fatty liver. participants were divided into two groups (a) those having alt > 1.5 times normal (n=97) (b) those having normal alt (n=47) results: mean age was comparable with slight male predominance. there were significant differences in anthropometric measurements like bmi (p=0.0001) and whr (0.90±3.57 and 0.88±3.23, p=0.0071). mean bp, lipid profile, fasting glucose, insulin, and homa r were comparable. there were significant differences in both mean ast (50.2±5 and 37.4±3.21, p=0.0001) and alt (104±7.29 and 69.9±11.5, p=0.0001) levels. metabolic syndrome was present in >75% of patients and individual components were comparable except for increased waist circumference which was significantly more in those with raised alt (78.35% and 44.68%, p<0.001). majority of our patients were having only steatosis, while nash was present in (27.82% and 8.5%, p<0.05) of patients. conclusion: nafld can exist in patients with normal alt values. although more work is needed to determine who should be screened for nafld and how such individuals should be evaluated, this study is a step toward the identification and characterization of nafld patients with normal alt. we can suggest that patients having metabolic syndrome or insulin resistance, despite having normal alt, should be screened for nafld. also alt values should be adjusted for variables like bmi to appropriately screen nafld patients. background: scientific evidence has demonstrated that traditional chinese medical (tcm) approaches and products can be beneficial for managing non-alcoholic fatty disease (nafld), but few rigorous criteria of patterns of tcm therapy are available to guide practitioners in deciding the cam interventions. objectives: to evaluate criteria of patterns of tcm therapy for the management of nafld identified by biomedicine. methods: literature research, clinical epidemiological investigation and mathematical statistics were employed to make information collecting tables and to establish database. descriptive analysis, factor analysis, and cluster analysis were involved. results: (1) background/aim: serum uric acid level has been suggested to be associated with factors that contribute to the metabolic syndrome. the aim of this study was to investigate the association of serum uric acid level with nonalcoholic fatty liver disease (nafld). methods: a cross-sectional study was performed among the employees of zhenhai refining & chemical company ltd., ningbo, china. results: the study included 8925 subjects (6008 men) with a mean age of 43 years. the prevalence rate of nafld and hyperuricemia was 11.78% and 14.71%, respectively. nafld patients had significantly higher level of serum uric acid than controls (370.3 ± 86.6 vs. 321.1 ± 82.6 mol/l; p < 0.001). the prevalence rate of nafld was significantly higher in the subjects with hyperuricemia than those without hyperuricemia (24.75% vs. 9.54%; p < 0.001), and the prevalence rate increased along with serum uric acid levels (p value for trend < 0.001). multiple regression analysis showed that hyperuricemia was associated with increased risk for nafld (odds ratio [or]: 1.291, 95% confidence interval [ci]: 1.067 -1.564; p < 0.001). conclusion: serum uric acid level is significantly associated with nafld, and increased serum uric acid level is an independent risk factor for nafld. background: development of fatty liver is believed to be an early and reversible consequence of excessive alcohol consumption. however, the cellular and molecular events in the early development of alcoholic liver diseases (ald) and the contributory effects of a high fat diet are not fully understood. methods: this study was designed to quantify specific enzymatic and cytokinetic activity as well as the development of hepatic steatosis in a rat model of alchohol-induced liver injury without high fat diet. results: ethanol-fed rats exhibited high blood ethanol levels (0.85+0.31%) and significant increases in serum alt (67.7+ 13.4 unit/l), ast (136.3+ 60.2 unit/l), and alp (400+108.9 unit/l) when compared with control rats (p<0.01, respectively). histopathological examination found unevenly raised knodell scores (5.33+1.15 in the ethanol-fed livers vs. 0.33+0.58 in control), which were characterized by scattered hepatocyte ballooning, portal inflammation and collagen fiber deposition. however, typical steatosis lesions were absent. qpcr demonstrated up-regulation of genes in the ethanol-fed livers, including hepatocyte metabolism enzymes/receptor (adh1, p<0.05; cytochrome p450 2e1, cyp2e1, p<0.05; gsta2, p<0.01; ppar , p<0.05), and genes coding for pro-inflammatory cytokines (il-1 , p<0.01 vs. control livers; tnf-p<0.05; tgf -, p<0.05; rantes p<0.05), ecm components and proteinases (collagen-1, p<0.01; sma, p<0.01; mmp -9, p<0.05 and timp-1, p<0.05). conclusion: chronic administration of ethanol to rats without high fat diet productively induces alcohol hepatitis in the absence of fatty liver, suggesting that alcohol hepatitis may precede steatosis in the development of ald. the aim of the present study was to evaluate the changes of several cytokines associated with inflammatory liver disease and liver regeneration by molecular adsorbent recirculating system (mars) in 15 aclf patients versus 15 patients treated with medical standard therapy (smt) that presented alcoholic liver disease etiology and similar model end-stage liver disease (meld). methods: mars group: fifteen (10 male and 5 female) patients were treated with mars® (gambro). five patients were excluded by study.the number of mars applications was about 9, the length of applications was about 10h. smt group: fifteen patients (10 male and 5 female) were treated medical standard therapy such as prophylaxis against bacterial infections, albumin and fresh plamsa and judicious use of diuretics. three patients were excluded by the study. the patients were valued during 30 days from inclusion with a survival follow up a three months. results: mars group: we observed a significant changes in levels of il-6(p<0.01), il-1(p<0.002), il-8(p<0.04) and tnf-alfa (p<0.03) in association with improvement of hepatic growth factor (p<0.001). the patient's survival at three months was 50%. smt group: we observed only a significant changes in il-1(p<0.01) and tnf-alfa (p<0.2). the patient's survival at three months was 33%. conclusion: the mars liver support device has corrective effects on disturbed pathophysiology of aclf and may be used to enhance spontaneous recovery or as bridge to transplant. a study of protective effect of centella asiatica in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury n. haleagrahara 1 , s. chakravarthi 1 , p. kumar 1 1 international medical university, malaysia background: centella asiatica has been used for centuries as a medicinal herb for wound healing, memory enhancement, cancer, vitality, respiratory ailments, psoriasis and eczema, revitalizing connective tissue, burn and scar treatment, skin infections, arthritis, rheumatism, periodontal disease, varicose veins, hypertension, sedative, anti-stress, anti-anxiety, aphrodisiac, and as immune booster. results: ppc significantly reduced hepatocyte damage, hepatitis, and hepatic fibrosis, but did not affect steatosis. phosphorylation of apoptosis signal-regulating kinase 1, p38 mitogen-activated protein kinase, and protein kinase c, as well as activation of nuclear factor-kappa b, were markedly suppressed by ppc. these effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ros)-generating enzymes, including cytochrome p450 2e1, acyl-coa oxidase, and nadph oxidases, in addition to restoration of ethanol-induced increases in toll-like receptor 4 and cd14. ppc also decreased the pro-apoptotic proteins bax and truncated bid, thus inactivating mitochondrial permeability transition. furthermore, ppc suppressed overexpression of transforming growth factor-1 and hepatic stellate cell activation, which retarded hepatic fibrogenesis. conclusion: ppc exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ald as a result of inhibition of alcohol-induced ros production. background: dysctamnus dasycarpus has used for the promotion of health in south korea. but, there were rare a report concerning the hepatotoxicity. we report cilinical features of liver injury by dysctamnus dasycarpus. method: eighteen patients diagnosed as acute toxic hepatitis by dysctamnus dasycarpus in chungnam national university hospital between january 2005 and arpil 2008 was enrolled. toxic hepatitis was diagnosed by rucam score ( 4). the medical records were reviewed, retrospectively. result: eleven patients (61%) were female and the mean age was 53.5. most common symptom was jaundice. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4 g/dl, platelet 212×10 3 /l,alt 1375iu/l, total bilirubin 11.3 mg/dl, alkaline phosphatase 159 u/l, ggt 244u/l, prothrombin time(inr) 1.1. the mean hospitalization was 21.5days. peak laboratory findings were as follows: alt 1382iu/l, total bilirubin 15mg/dl. recovery time of each biochemical finding was as follows: alt 37days, total bilirubin 39.4 days. recovery rates of alt and total bilirubin were 27.8% and 38.9%, 89.9% and 89.9% at 4 weeks, 8 weeks, respectively. the main biochemical pattern of hepatotoxicity was hepatocellar (72.2%) type. prednisolone was prescribed in six patients. progressive anemia and thrombocytopenia were detected in one patient diagnosed as pure red cell aplasia. other one patient had prolonged jaundice (117 days). but, all patients had recovered without sequelae. conclusion: in south korea, liver injury by dysctamnus dasycarpus was more frequent in women. the main pattern of hepatotoxicity was hepatocelluar type. most patients had prolonged icteric phase and hospitalization. patients were recovered by supportive management after drug cessation or prednisolone therapy. in korea, traditional medicine that is based on the use of herbal medicine developed from a long time ago. however, clinical study of the herbal medicine is not conducted in a structured manner. we report three cases of toxic hepatitis caused by the intake of dictamnus albus. the first patient, a 44 year old woman was admitted due to nausea after ingestion of liquor containing dictamnus albus for 2 months. total bilirubin was 2.55 mg/dl ast/alt 774/1,424 iu/l on admission. liver biopsy observed hepatocyte necrosis and cholestasis. the elevated bilirubin and transaminase returned to normal 2 weeks later after cessation of dictamnus albus. the second patient, a 64 year old man was admitted due to jaundice after ingestion of boiling dictamnus albus for 3 months. total bilirubin was 11.37 mg/dl ast/alt 2,010/2,522 iu/l on admission. liver biopsy observed pericellular fibrosis and necrosis. the bilirubin decreased slowly compared to the transaminase and normalized 3 months later after cessation of dictamnus albus. the third patient, a 48 year old man was admitted due to jaundice after ingestion of liquor containing dictamnus ablus for 1 month. total bilirubin was 12.8 mg/dl ast/alt 1,097/1,869 iu/l the hepatocyte necrosis was observed by liver biopsy. the elevated bilirubin and transaminase levels normalized 1 month later after cessation of dictamnus albus. all patients had negative viral markers and non-specific ultrasonographic findings. the above mentioned three cases demonstrate that liver may have been damaged by dictamnus albus, which indicated clinical characteristics. background/aims: cmili poses a diagnostic challenge as no tests are available to confirm the causality. the aims of this study were 1) to evaluate clinical features and patterns of cmili and 2) to assess the likelihood of causality among patients with liver impairment and exposure to chinese medicine (cm) by a multidisciplinary approach. method: between 5/2005 and 8/2007, patients who had liver derangement and cm or proprietary cm exposure within six months managed in the united christian hospital were studied. clinical features and the cm were reviewed by a multidisciplinary team involving a hepatologist, a toxicologist and cm experts. literature search of relevant herbs in chinese and western journals were performed. cm samples or residue were sent to toxicology laboratory for analysis to look for any toxic constituents, adulterant or contaminant. the likelihood of causality was ranked by various experts independently and disagreements were settled by a consensus meeting. results: there were forty-six cases of suspected cmili, nineteen cases with alternative causes of liver diseases were excluded. twenty-seven cases of cmili proceeded to detailed analysis. median age of patients was 51 (21-76) with female predominance. the median duration of cm exposure to presentation was 20 (1-150) days. majority of them (82%) had hepatocellular liver injury pattern. one case of adulteration with nsaid and erroneous substitution of herb was identified respectively causality were classified as unlikely, possible, probable and highly probable in 3, 12, 8 and 3 patients respectively. conclusion: a multidisciplinary approach allows systemic evaluation of suspected cmili. mouse model i. nassar 1 , t. pasupati 1 , i. segarra 1 , j.p. judson 1 1 international medical university, kuala lumpur, malaysia background: imatinib, a selective tyrosine kinase inhibitor, exhibits drug interactions with other drugs that are metabolised via the cytochrome p450 pathway. acetaminophen, a widely used analgesic and anti-pyretic drug is also metabolised via p450 pathway. this study aimed to evaluate the nature of hepatotoxicity after co-administration of imatinib and acetaminophen in a preclinical mouse model. methods: four groups of male icr mice (30-35g) were used. the mice were administered either saline solution orally, imatinib 100 mg/kg orally (control), acetaminophen 700 mg/kg intraperitoneally (positive control) or co-administered imatinib 100 mg/kg and ip acetaminophen 700 mg/kg (study group). the mice (n=4 per group) were fasted overnight, dosed respectively and sacrificed at pre-determined time intervals of 15, 30 minutes, 1, 2, 4, 9 and 12 hours and liver samples obtained by dissection. h&e stained liver sections (3µm thick) were histopathologically analysed. results: the liver samples showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, with both imatinib and acetaminophen, administered separately. the damage increased gradually with time, peaked at 2 hours and then resolved completely by 6 hours. liver samples showed irreversible damage (cytolysis, karyolysis and karyorrhexis) when both drugs were administered concurrently, the damage increased with time and had not resolved after 12 hours duration. conclusion: co-administration of acetaminophen and imatinib increased the hepatoxicity caused by acetaminophen and imatinib to become irreversible. this may be due to the fact that both drugs are metabolised by the cytochrome p450 pathway in the liver. background: a higher risk of antituberculosis drug (att) induced hepatotoxicity has been reported in indian subcontinent compared to the western counterparts. slow acetylator genotype of n-acetyltransferase2 (nat2) and ci genotype of cytochrome p4502e1 (cyp2e1) gene are two known risk factors associated with this disease. cyp2e1 gene encodes a rifampicin inducible enzyme which increases hepatotoxicity. therefore slow acetylation of isoniazid and simultaneous use of rifampicin may augment the toxicity of isoniazid. objectives: to analyze the allelic distribution of nat2 and cyp2e1 gene in patients of pulmonary tuberculosis who developed att induced hepatitis materials and methods: the study included cases of pulmonary tuberculosis (190) and att induced hepatitis (95). polymorphism of nat2 and cyp2e1 gene was studied by pcr-rflp method in both these groups. results: occurrence of att hepatotoxicity was 17.89%. there was a higher prevalence of slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 in patients with hepatotoxicity compared to patients without hepatotoxicity (72.09% vs 44.2%, p value < 0.05). no association of cyp2e1 rsai polymorphism could be considered with att hepatotoxicity. however, drai c/d genotype of cyp2e1 appears as a risk factor for predicting the occurrence of antituberculosis drug induced hepatitis (or 5.06, p value < 0.05). conclusion: the study demonstrates that patients with slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 and heterozygous mutant c/d genotype of cyp2e1 gene are predisposed to develop antituberculosis drug induced hepatotoxicity. regular monitoring of clinical and biochemical profile may be considered in these patients when they receive antituberculosis treatment. background: drug-induced liver injury is the most common adverse drug reaction. we often use two kinds of diagnostic scales to evaluate suspected patients. however, we still can't diagnose accurately without the direct drugs history and the pathological evidence. methods: twenty-seven drug-induced liver injury cases with liver biopsies from 2001 to 2008 were reviewed retrospectively by maria and japanese scale. result: there were 11.1% of cases with increasing eosinophils. herbs (29.63%) were the most common suspected drug and unknown drugs intake history (14.81%) were described in these cases. the high possibility and possibility were 25.93%, 29.63% by maria scale and 85.19%, 3.7% by japanese scale, respectively (p=0.015). conclusions: japanese scale seems more sensitive than maria scale in these cases. however, there are still some definite cases ignored as low possibility due to absence of obvious drug using history. early treatment and suspected drugs prohibition interferes the outcomes of the two diagnosis systems and lead to a false result. it is still a clinical challenge without strong drug using history or pathological evidence of liver biopsies to diagnose the drug-induced liver injury quickly and accurately. background: previous study suggested that oxidative stress may be an important mediator of methamphetamine-mediated tissue injury. the study was to examine the mechanism of antioxidant activity and methamphetamine-mediated liver injury. materials and methods: the 25 days old male sprague-dawley rats were subcutaneous injected daily with methamphetamine (10 mg/kg body weight) for 15, 30, 60 and 120 days. control group received equal volumes of vehicle. the liver tissues were extracted to measure the activities of sod, catalase, glutathion reductase (gr), and glutathione peroxidase (gpx), and the level of glutathione. western blot were used to measure the expression of rho and phosphor-ezrin-radixin-moesin (p-erm). results: compared with vehicle group, treated with methamphetamine for 15 and 30 days, the activities of liver sod, gpx, and catalase were significantly decreased. in 60 and 120 days group, the activities of antioxidant enzymes of methamphetamine-treated liver was not different from that of vehicle group. the levels of glutathione production also had the same trend. the activities of gpx and catalase on vehicle group gradually reduced following the days of treatment. however, administration of methamphetamine resulted to a lower activity of catalase through the treated days. there was no difference on the activity of gr between vehicle and methamphetamine group. the expression of rho and p-erm were also increased by methamphetamine treated for 30 days. conclusion: these results suggested the methamphetamine lead to liver remodeling via decreased antioxidant activity. finally, the situation of mechanism needs taking in advantage discussion. background: to observe intervening effects of preventive and theraptical treatment of radix sophorae tonkinensis's polysaccharides(rstp) on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: kunming mice intoxicated with anit 50mg/kg orally and treated with rstp 50mg/kg for 7 days before anit exposure and for 2 days after anit exposure respectively, the general condition,mortality rate and serum alt activity are obeverated. result: it was found that by preventive treatment the general condition and mortality rate were improved, serum alt activity reduced.by therapeutic treatment,the general condition deteriorated,mortality rate and serum alt activity increased. conclusion: the preventive treatment of rstp reduce the liver damage due to increasing the anti-stress ability such as the antioxidant capacity,its therapeutic treatment increase the injuried liver damage due to increasing the non-specific immune response and aggregating the preexisting liver inflammation. background: the product's instruction pointed out that in some patients polyphenolic acids' salt from salvia miltiorrhiza(ppas-sm) may lead to a temporary increase in serum alt activity.so we observe effects of ppas-sm on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: 24 hours after intoxicated with anit 50mg/kg orally, kunming mice were treated with ppas-sm 50, 25,10mg/kg/days for 2 days orally, then serum alt activity was measured. result: all doses of ppas-sm led to rise of serum alt activity in mice, most obvious in group of high dose.but the general situation and mortality rate did not increase significantly. conclusion: ppas-sm lead to rise of serum alt activity in mice with damaged liver.the auther suggests as a double-edged sword,the antioxidant ppas-sm may have a prooxidative effect in some condition too. *this project was supported by grants from shanghai municipal education commission under high school high-tech characteristic development programme (no smec finance (2005) 81) pe467 a. somani 1 , a. jain 2 , v. dixit 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha introduction: hepatic encephalopathy, a complex neuropsychiatric syndrome secondary to acute liver failure, chronic parenchymal liver disease or portal-systemic shunting, may possibly develop through mediators of endotoxin and tumor necrosis factor-alpha (tnf-). several studies have shown that serum levels of (tnf-) are significantly elevated in patients with acute and chronic liver diseases, where these elevations are independent of the etiology of the underlying disease. it has been shown that plasma levels of tnf-correlate with the severity of hepatic encephalopathy (he) in fulminant hepatic failure. however, still there is very few published data regarding the relationship between serum levels of tnf-and the presence or severity of he in patients with chronic liver failure. methods: the aim of this study is to determine the relationship between serum levels of tnf-and clinical grades of he in patients with chronic liver failure. this prospective study included 149 consecutive male patients with alcoholic cirrhosis in various clinical grades of he (according to west haven criterion). detailed clinical, biochemical and sonographic examination was done in all patients. circulating levels of tnf-was measured using solid-phase elisa. results: the mean±sem values of serum tnf-at presentation in patients with mhe (n=37), grade 1 (n=17), grade 2 (n=41), grade 3 (n=44), and grade 4 (n=10) were 6.2±0.4, 9.5±0.6, 15±0.7, 26.3±1.7, and 46±5 .9 pg/ml, respectively. significant positive correlation was found between serum levels of tnf-and severity of he (correlation coefficient = 0.7). conclusion: from the present study we can suggest that there is significant relationship between tnf-and he in patients with alcoholic cirrhosis and it could be involved in its pathogenesis. background: acute hepatitis a (aha) is one of the most common infectious diseases and usually a self-limiting disease. although extrahepatic manifestations are not common, a few cases associated with acute renal failure (arf) have been reported. methods: we reviewed clinical features of aha patients complicated with arf (group a) and compared with non-complicated aha patients (group b). medical records of 191 patients with aha were reviewed between january 2003 and december 2007. we experienced 11 patients (5.8%) with arf associated aha. result: there were no differences between group a and group b in sex ratio and age. the peak value of alt (median: 6133 iu/l vs 1685 iu/l, p<0.001), alkaline phosphatase (median: 235 iu/l vs 201 iu/l, p=0.03), prothrombin time (inr, median 1.72 vs 1.09, p<0.001) was significantly higher in group a than b. nine patients (81.8%) recovered completely with hemodialysis (6 patients, 66.7%) and only conservative management (3 patients, 33.3%), while 1 patient underwent liver transplantation and 1 patient died due to fulminant hepatic failure. there were 4 patients who underwent kidney biopsy. two patients were diagnosed as acute tubular necrosis and 2 patient as acute interstitial nephritis and iga nephropathy. conclusion: aha patients with arf had higher alt and more prolonged prothrombin time. the prognoses were poorer than those without arf. however, arf patients with nonfulminant aha had a good prognosis with a proper treatment and should not be confused with hepatorenal syndrome. background/aims: to investigate the hev infection among different animals and people with special profession, and to analyse the genotype of hev isolated in this study. methods: serum and fecal samples were collected from various animals and people with special profession in the south suburbs of beijing. hev antigen and anti-hev antibody were detected by das-elisa. hev rna was extracted from fecal samples and amplified by rt-npcr. the nucleotide sequence homology and phylogenetics of hev strains isolated from swine were analysed. results: the anti-hev antibody positive rate of adult swine, cow, sheep and younger swine were 98.23% (222/226), 29.35% (54/184), 9.80% (20/204) and 60.73% (99/164), respectively. the hev antigen positive rates of adult swine, cow, sheep and younger swine were 2.65% (6/226), 4.35% (8/184), 1.45% (3/207) and 9.75% (16/164), respectively. the hev antigen and anti-hev antibody positive rate of professional group was 0.40% (1/247) and 42.51% (105/247) respectively. the hev rna positive rate of fecal samples from younger swine was 22.89 %(19/83). 16 of 19 samples were hev rna positive by pcr with primers of hev orf1 and orf2. the sequence analysis of the 16 samples showed that there were 2 groups designated as bj-1 (11/16) and bj-2 (5/16). the nucleotide homology of bj-1 and bj-2 was 99%. phylogenetic analysis of hev orf2 indicated that both of them belonged to genotype 4d. conclusion: phylogenetic analysis of hev orf2 indicated that hev isolated in the south suburbs of beijing belonged to genotype 4d. 6 bracops hospital brussels , 7 st. jan hospital, bruges , 8 chu brugmann, 9 chu sart tilman, liège , 10 gent university hospital , 11 zna middelheim, antwerp hepatitis delta virus is a subviral satellite requiring hepatitis b virus to propagate, usually leading to severe, chronic liver disease. as data on epidemiology and management practice of hdv infection in belgium are lacking, a retrospective and prospective, multi-centric questionnaire-based registry is performed in 2008. results of 32 patients are reported. background/ aims: hepatitis a is an acute infectious disease that is transmitted by fecal-oral root. because the incidence of hepatitis a has been increased in gwangju and chonnam province of korea recently, hepatitis a patients in chonnam national university hospital employees had been increased. so we investigated the seroprevalence of igg anti-hav in hospital empolyees less than 40 years old. methods: we analysed seroprevalence of anti-hav igg from 1,002 hospital employees (men: 190, women: 812) . serum alt and bilirubin at admission were 2,979 1,711 iu/l and 3.9 2.4 mg/dl, respectively. these levels were elevated up to 3,397 1,789 iu/l and 6.8 3.7 mg/dl, respectively. ana was positive in 81 patients (64.3%). age, duration from peak-alt day, duration from peak-bilirubin day, alt level, and peak-bilirubin level were not different between ana(-) patients and ana(+) patients. in the while, sex, duration from symptom-onset day, and bilirubin level, and peak-alt level were significantly different. in 51 (62%) of 81 patients with positive ana, ana was followed after 1 month and ana became negative in 38 patients (74.5%). among 13 patients with positive ana after 1 month, titer decreased from the baseline in 7 patients, showed no interval change in 4, and increased in 2. conclusions: positive ana result is not rare in patients with acute hepatitis a. it is considered that ana transiently appear during the course of acute hepatitis a and then, disappear with the improvement of acute hepatitis. (89), 2007(107). the clinical data such as sex, admission period, ast, alt, total bilirubin, prothrombin time, crp, alt normalization time did not show difference. just wbc and gtp were higher on 2008 group. the older age patients were more on 2008 group. the patients admitted mainly on april, may, june, july (84%) on 2008 while admitted even on past years. conclusion: acute hepatitis a ptients is increasing. it is occurring in older age people and mainly on specific period. the more concern to prevention should be needed. background: we analyzed the 5' non-translated region (5'ntr), non-structural proteins 2b and 2c of hepatitis a virus (hav) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all of our data and examine whether genomic differences in hav are responsible for the range of clinical severities. methods: our accumulated hav strains of 5'ntr (nt 200 and 500), entire 2b and 2c from 25 japanese patients with sporadic hepatitis a, consisting of 7 patients with fulminant hepatitis (fh), 5 with severe acute hepatitis (ahs), and 13 with self-limited acute hepatitis (ah), in whom the sequences of all 3 regions were available, were subjected to phylogenetic analysis. results: fh patients had fewer nucleotide substitutions in 5'ntr, had a tendency to have more amino acid (aa) substitutions in 2b, and had fewer aa substitutions in 2c, than ah patients. four fh and 2 ahs with higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis a and genomic variations in 5'ntr, 2b and 2c of hav. conclusions: our study suggests that genetic variations in some parts of hav might cooperatively influence replication of the virus, and thereby affect virulence. viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis a. aims: the incidence of acute viral hepatitis a in adults is increasing very much in south korea, 2008. the aim of this study was to the clinical features and course in daejoen and its surrounding area. methods: forty seven patients admitted as acute viral hepatitis a in chungnam national university hospital between january 2008 and june 2008 were enrolled. the medical records were reviewed, retrospectively. results: the mean age was 30.5. common occupations were company employee and studuents. most common symptom was jaundice. presumptive infection sources were raw fish or shellfish and raw meat. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4g/dl, platelet 212×10 3 /l, ast 2268iu/l, alt 2755iu/l, total bilirubin 5.2mg/dl, alkaline phosphatase 207u/l, ggt 379u/l, prothrombin time(inr) 1.2. hospitalization was 21.5days. peak laboratory findings were as follows: alt 3181iu/l, total bilirubin 8.8mg/dl. leukopenia (<4000/ul) and thrombocjtopenia (<10×10 5 /l) were ocurred in sixteen and six patients, respectively. recovery time of each biochemical finding was as follows: alt 20.2 days, total bilirubin 26 days. recovery rates of altand total bilirubin were 74.4% and 74%, 88.5% and 92% at 4 weeks, 8 weeks after diagnosis, respectively. prolonged jaundice (115days) was detected in one patient. all patients were recovered by supportive management. conclusions: in south korea, acute viral hepatitis a was more prevalent in young adults, recenlty. presumptive infectious sources were raw fish or 1 guangxi center for disease prevention and control shellfsh and raw meat. if it can not change the food style that many korean enjoy raw seafood, vaccination for adults must be considered to prevent it. objective: to assess the safety and immunogenicity of a new inactivated hepatitis a vaccine (vero cell). pe479 methods: 1507 subjects were selected in gongcheng city of guangxi zhuang autonomous region, and the clinical trail was carried out according to the random, double-blind and parallel principle from january to august, 2005. after vaccination by 0, 6 schedule, adverse events of the subjects were observed, the seroeonversion rate and geometric mean titer (gmt) were tested by the competitive inhibition elisa. results: after immunization, the systemic and local reaction rates of adults were 8.80% and2.67%, which was no significantly statistical difference compared with control group, 12.41%and 4.41%; while the rates of children were 10.60and 2.28%%, and no significant statistical difference compared with control group, 10.71%and2.86%. one month after first dose of vaccination, the seroconversion rates of children and adults were 88.2% and 93.8%, and one month after second dose of vaccination, the rates were all 100%, the gmts of children and adults were 16447miu/m1 and 8555miu/ml, which was significant statistical difference in children compared with control group, 1946 miu/ml and 5881 miu/ml, respectively. methods: igg anti-hav was measured in a total of 3905 subjects under the age of 50, who visited hanyang university seoul and guri hospitals between january 2001 and may 2008. results: fig. 1 shows the relatively low positive rates of the antibody in ages of 11 to 30 and the lowest rates of 9.9% and 9.2% in the age group of 16 to 20, following the ages of 21 to 25 with rates of 12 background: some viruses encode proteins that affect their cap-independent internal ribosomal entry site (ires)-mediated translation and their replication. it was recently reported that hepatitis a virus (hav) proteases interact with intracellular dsrna-induced retinoic acid-inducible gene (rig-i)-mediated signaling, but it remained unknown whether hav proteins have any effects on hav ires-independent translation. in this study, we investigated the effects of 7 hav non-structural proteins on their ires-mediated translation using a reporter assay. pe480 methods: the bicistronic reporter constructs, termed psv40-hm175-ires, psv40-a1-ires, psv40-a2-ires, psv40-f1-ires, and psv40-f2-ires, contain the sv40 promoter that controls the expression of a bicistronic message coding for renilla and firefly luciferases separated by hav ires, and are derived from strain hm175, acute convalescent hepatitis clones a1, a2, fulminant hepatitis clones f1, f2, respectively. human hepatoma cell lines were co-transfected with psv40-hav-ires and each hav protein-expression vector. luciferase activity was determined 48 h after transfection. were from other countries within asia, africa, middle east, and eastern europe. patients of a wide age range were affected by hepatitis delta (mean age 46.0, median 47.0, range 19-79). 18 (40%) of 45 were co-infected with hcv. hepatitis b virus (hbv) dna was detectable in 43 (78%) patients and negative in 8 (15%) patients. all hepatitis delta patients were extracted from a prior study conducted by this collaboration. there were 1,191 chronic hbv carriers. 18 (1.51%) were hbv/ hcv/hdv infected. 32 (58%) of 55 patients carried a diagnosis of cirrhosis compared to 262 (22%) of 1191 chronic hbv patients. 13 (72%) hcv co-infected patients had evidence of cirrhosis while 4 (22%) patients did not. conclusion: individuals with hbv/hdv co-infection have higher rates of cirrhosis. individuals with hbv/hcv/hdv infection have rates of cirrhosis significantly higher than individuals with either chronic hbv infection or hbv/hdv co-infection. testing for hdv should be performed in all patients, especially those with advanced liver disease or high risk behavior. clinical characteristics were compared between the patients with significant endoscopic findings (group a) and without such findings (group b). peak ast and alt level were higher in group a (p<0.01). there were no statistical differences in age, gender, comorbidity, and etiology of acute hepatitis between group a and group b conclusion: significant endoscopic findings were found in considerable proportion of patients with acute hepatitis. severity of acute liver injury was associated with significant upper gastrointestinal endoscopic findings. in patients with severe acute hepatitis who complain of upper gastrointestinal symptom, esophago-gastro-duoenoscopy should be performed. background: in japan, hepatitis e virus (hev) testing is not allowed as routine one. to study the role of hev testing, we checked 22 sera of the patients diagnosed as etiology-obscure acute liver injury. methods: we have seen 54 cases of acute liver injury from january 2004 through december 2007 in our hospital and 25 cases of them were etiology-obscure. in 25 cases, 22 were retrospectively tested for hev-igm, hev-iga and hev-rna (rt-pcr) by direct sequence method on stored sera taken at the time of presentation. result: two of 22 cases (9.1 ) were positive for both hev-igm and hev-iga and one case was positive for hev-rna. in 54 cases of acute liver injury, the cause of virus was 31 cases (57.4%) and unknown was 8 cases (14.8%). hev was occupied in 3.7% in all cases and 6.5% in the cases caused by virus. one of the two cases had been misdiagnosed as "drug induced hepatitis". hev of genotype 3 was detected in one case and its nucleotide sequences of hev showed quite a high degree of similarity to the reported one at closed city in the same year. conclusion: hev is not rare in japan and the hev testing can reverse the diagnosis of acute liver injury. hev testing sould be used as routine one for acute liver injury. association of progesterone receptor gene with hepatitis e disease severity in pregnancy p.d. bose 1 , b. das 2 , a. kumar 1 , p. kar 1 1 maulana azad medical college, 2 background/aims: incidence of fulminant hepatic failure (fhf) in hepatitis e is high in pregnancy particularly during 3 rd trimester when there is an altered status of hormone and immunity. progesterone receptor (pr) up regulation provides fetal protection via immunosuppression but lower immune status in pregnancy may add to the disease severity. till now, no data is available whether pr can play any role in hepatitis e disease severity during pregnancy. progins, a haplotype of pr consisting of 320-bp insertion in intron g together with point mutations in exons 4 and 5 is associated with increased stability and higher transcriptional activity. the aim of the study is to analyze pr mutation (progins) and m rna expression in hepatitis e virus infected pregnant women with avh and fhf. methods: a total of 68 avh and 32 fhf cases were studied. blood and placental tissue were collected from the medicine and gynecology wards of lnjp hospital, new delhi. cases were screened for acute viral markers by commercially available elisa kit. extraction of dna from blood and rna from placental tissue was done by qiagen kit. mutation in pr was detected by pcr-rflp. semiquantitative rt-pcr for pr expression was performed in placental tissue using beta-actin as internal control. results: pr mutation (progins) was significantly more in fhf compared to avh (28.1% vs 10.3%, p value<0.05). protein expression was found higher in progins carriers. conclusion: progesterone receptor mutation (progins) may have a role in the hepatitis e disease severity in pregnant women. results: the hepatitis e was predominantly sporadic, some patients superinfected with other viral hepatitis, especially hepatitis b. in the old patients, jaundice lasted longer and the length of stay was longer, the incidence of complication was higher than the young men. the incidence of complication in the superinfected group was higher than the simple infection. the transaminase in the simple infection group was obviously raise than superinfected with liver cirrohsis. methods: liver sample were paraform-glutaral fixed, paraffin-embedded, sectioned and immunohistochemical stained, and positive samples were selected for histological analysis and rt-pcr detection. result: positive rate of hev immunohistochemistry ranged from 90% to 100% (fig. 1) . hepatocyte degeneration, scattered singled karyopyknosis, lymphocytic infiltrate, hyperplasia of bile canaliculus at the portal area and fibrous connective tissue hyperplasia been observed during histological analysis (fig. 2) , and two genotype 4 hev which closely related to many strain isolated from patients with sporadic acute hepatitis been detected. conclusion: the patients infected with hepatitis e of young men were frequently. jaundice lasted long in the old patients, the incidence of complication was higher in the superinfected men and the old men. conclusion: additional public-health concerns might be placed on pork safety and the risk of hev infection via the consumption of undercooked pork products. poster session, hall 5b aim: esophageal varices (ev) recurs frequently after endoscopic variceal ligation (evl) or endoscopic injection sclerotherapy (eis). we retrospectively investigated risk factors for early recurrence of ev after endoscopic treatment. methods: we treated 110 patients with ev, who had no past history of ev, at ehime prefectural central hospital from october 2005 to june 2008. of those, 78 (71%) were observed for at least 2 months after treatment and enrolled. we divided them into rupture cases at initial endoscopic treatment [(bleeding group; n=25 (32%)], and cases with preventive evl or eis performed [preventive group; n=53 (68%)]. all received periodic upper endoscopy examinations to confirm recurrence or no recurrence of ev. results: recurrence of ev occurred in 18 of all subjects and the average period after treatment was 6.5±3.0 months. the recurrence rate was significantly higher in the bleeding group (11/25) as compared to the preventive group (7/53) (p=0.0026). there was a significant relationship between recurrence of ev and hepatic reserve function (child-pugh a+b, c; 11/64, 7/14 respectively; p=0.0083). in logistic multi-variant analysis, ev rupture at initial treatment and child-pugh c were risk factors for recurrence. in contrast, age, sex, hepatocellular carcinoma, portal tumor thrombosis, continuous alcohol consumption, therapeutic modality (evl or eis), number of treatment sessions, and operator experience did not have a significant relationship with recurrence. conclusion: in cases with ev rupture at initial treatment or child-pugh c, the risk for early recurrence must be considered and patients carefully observed in follow-up examinations. endoscopic cyanoacrylate injection: less oil for less ectopic embolism c.z. li 1 , l.f. cheng 1 , z.q. wang 1 , f.c. cai 1 , q.y. huang 1 , e.q. linghu 1 1 general hospital of chinese pla background and aim: endoscopic injection sclerotherapy with n-butyl-2cyanoacrylate (nbca, histoacryl) has been reported to be effective for hemostasis of bleeding gastric varices, but occasionally the gel flows to other organs and causes ectopic embolism. the present study aimed to determine whether less amount of iodized oil preload in nbca injection helps in decreasing ectopic embolism. methods: from january 1997 to april 2006, 2 different methods of endoscopic nbca injection, "sandwich method" and "modified sandwich method" (in which iodized oil preload was minimized), were applied on 635 gv cases, to evaluate if decrease of iodized oil preload resulted in less ectopic embolism. results: altogether 5 cases of ectopic embolism occurred in the whole group (0.8%), including 3 cases of splenic infarction, 1 case of transient paralysis and 1 case of minor infarction of the lung. the modified sandwich method showed some superiority over original method in decreasing ectopic embolism (0/276 vs. 5/359, p=0.049). less cough during procedure was also found with the modified method (3/276 vs. 18/359, p=0.006). conclusions: less amount of iodized oil preload in endoscopic nbca injection is beneficial to decrease ectopic embolism. background: portal hypertension is closely associated with serious complications of liver cirrhosis which contribute to bad prognosis. hepatocellular carcinoma (hcc) and low serum sodium (sna) are manifestations of end-stage liver disease (esld) and are associated with poor survival in decompensated cirrhosis patients. therefore, we aimed to determine the relationship between hepatic venous pressure gradient (hvpg) and the development of hcc or low sna in decompensated alcoholic cirrhosis patients. methods: child-pugh scores, meld scores, and hvpg at baseline, and the development of low sna (sna <130 meq/l) or hcc during follow-up were analyzed prospectively in 170 patients with decompensated alcoholic liver cirrhosis. the predictive values of different risk factors for the progression to the esld were investigated by multivariate analysis and the kaplan-meier method results: twenty-four patients developed hcc during the follow-up period. in the multivariate analysis, only baseline hvpg>15mmhg was an independent predictive factor for the development of hcc (relative risk (rr)=1.128, p<0.05). those with hvpg >15 mmhg showed a significantly shorter time for the development of hcc on kaplan-meier analysis. twenty patients developed low sna during follow-up. initial hvpg was also an independent predictive value for the development of low sna in the multivariate analysis (rr=1.169, p<0.05). those with hvpg>15 mmhg also showed significantly shorter times for the development of low sna on kaplan-meier analysis. conclusions: in decompensated alcoholic cirrhosis, hvpg may be a useful predictive factor for the development of hcc and low sna, both of which are characteristic of esld and poor prognosis. the effectiveness of the treatment of octreotide on chylous ascites after liver cirrhosis d.x. zhou 1,2 , h.p. hu 1,2 background: octreotide is a crucial drug used for treating patients with chylous ascites; however, there have been few reports related to octreotide that are being used in cirrhotic patients. thus, this thesis is designed to determine the effects of octreotide on patients with chylous ascites after liver cirrhosis. methods: eight patients were diagnosed with chylous ascites, on the basis of laboratory findings on ascites samples, between january 2003 and may 2008. octreotide was given to the six patients, while the remaining two were treated as a control. all patients had persistent peritonea drainage with the quantity and quality of the drainage fluid observed once every other day. all the necessary care was individually given to the patients during the therapy results: all patients properly received combined therapy including low fat and sodium diet, and diuretic and peritoneal drainage. the volume of the peritoneal drainage was reduced to zero in one of the six patients who received octreotide therapy, while the other five had the drainage volumes decreased from 2000 ml to 50 ml with a clear appearance and negative qualitative analysis of chyle for those two patients who did not receive octreotide therapy, the conditions of peritoneal drainage seldom changed both from the qualitative and quantitative aspects. conclusion: octreotide, along with combined therapy, can rapidly relieve portal hypertension and reduce fat absorption from intestinal mucosa. it appears to be an effective therapy available for the treatment of chylous ascites caused by liver cirrhosis. albumin <38g/l were the best predictors of large varices. a model using these predictors in a validation cohort study is planned. background-aim: cirrhosis is associated with raised acute phase proteins (app), irrespective of infection. it is, however, unclear whether their values differ significantly or whether a particular app might be more indicative of infection, and these questions were addressed in our study. methods: we measured serum crp, fibrinogen, ferritin, haptoglobin, 2-microglobulin, c3, c4, and c1 inhibitor in 88 consecutive, cirrhotic patients, on admission. all patients were investigated according to a standard protocol for infection. child-pugh scores (cps) were calculated. results of app were expressed as means sem and compared with the mann-whitney test. results: 19 (21,6%) patients, median age 60 years, (cps: a=0; b=7; c=12), were diagnosed with infection (spontaneous bacterial peritonitis=7; pneumonia=5; septic shock=4; extensive cellulitis=1; listeria monocytogenes meningitis=1; viral infection=1), while 69 (78%) patients, median age 59 years, (cps: a=25; b=28; c=16), showed no infection. although most app values were raised, there was no statistically significant difference between patients with or without infection, or among different cps groups, except for crp, which was significantly more raised in patients with infection (p<0.01). this difference remained even after cps a cases in the non-infection group were excluded from analysis. interpretation: a significantly raised crp in cirrhosis would seem to be independent of cps staging and should prompt a thorough work up to exclude infection. by contrast, the discriminating power of all other app in the face of possible infection is negligible. the predictive value of crp towards infection is under investigation prospectively. although bleeding from ectopic varices such as duodenal, jejunal, ileal, colonic, and rectal varices is less common, it can also cause life-threatening problem, which is often difficult to diagnose and treat successfully. here we present a novel endoscopic approach for hemorrhagic rectal varices using endoscopic injection sclerotherapy with ligation (eisl). patients and methods: in 2000-2008, we performed endoscopic treatment in 215 patients with portal hypertensive varices. among those, four cases of hemorrhagic rectal varices were treated with the combined evl and sclerosing technique. the etiology of portal hypertension included oen idiopathic portal hypertension and three hcv cirrhosis. all patients had a history of prior abdominal surgery or endoscopic treatment for gastro-esophageal varices. results: hemostasis was obtained easily by the evl initially. furthermore, to avoid recurrent bleeding, the patients underwent endoscopic varicerography injection sclerotherapy (evis) using 5% ethanolamine oleate with iopamidol and the feeding vein was sclerosed successfully with no major complication occurred during the entire course of the treatment. conclusions: it is important to recognize the possibility of ectopic varices as a cause of gastro-intestinal haemorrhage especially in patients with a history of variceal therapy or abdominal surgery. the eisl technique is useful to control the initial and recurrent bleeding from rectal varices. t. hirano 1 , t. okada 1 , j. yamanaka 1 , y. iimuro 1 , n. kuroda 1 , k. oh 1 , y. yoshida 1 , j. fujimoto 1 1 aim: interferon (ifn) therapy is a powerful treatment for hcv-related hepatitis and is known to decrease the incidence of progression of hepatocellular carcinoma (hcc). however, thrombocytopenia is a common side effect of ifn treatment, often leads to discontinuance without insufficient therapeutic effect. in this study, we investigated the efficacy and safety of laparoscopic splenectomy ( ) in reversing thrombocytopenia in patients with hepatitis c cirrhosis and portal hypertension. patients and methods: out of 41 patients who underwent ls in our department during aug 2003 and december 2007, 13 patients associated with portal hypertension. among these patients, three patients had hcc, and they were simultaneously underwent partial hepatectomy after splenectomy. platelet count, operative time, blood loss, complications and length of stay were calculated. results: thirteen patients underwent laparoscopic splenectomy; their mean age was 59 years (range 20 to 68 years). six patients were child's class a and seven patients were class b. mean operative time was 178 minutes (range 97 to 255 minutes). blood loss was little, and none required transfusion with packed red cells. a hand-assisted laparoscopic technique was used in four cases (30.8%). average length of stay was 12.7 days. there have been no major complications during follow-up. platelet counts improved from a preoperative mean of 62000/ul (44000 to 108000) to 273000/ul (126000 to 469000) postoperatively. six patients are ongoing ifn treatment without remarkable thrombocytopenia. conclusion: laparoscopic splenectomy is safe and in patients with portal hypertension and thrombocytopenia. it may allows these patients by reversing thrombocytopenia. background: hepatic encephalopathy (he) is a significant cause of mortality in advanced cirrhosis patients. l-acyl-carnitine has been suggested as an alternative treatment for patients with he patients. to assess the clinical efficacy of acetyl-l-carnitine in the treatment of hepatic encephalopathy in cirrhotic patient, especially in diminishing the recurrence and reduction serum ammonia level. methods: we performed a randomized placebo-controlled, cross-over study. we administered acetyl-l-carnitine to group 1 during 3 months first then placebo during later 3 months, and administering acetyl-l-carnitine to group 2 alternatively. results: between january 2008 and february 2008, thirty two selected cirrhotic patients were enrolled in this study. following randomization, the patients were divided into two groups (group 1=14, group 2=18). during administering acetyl-l-carnitine period, serum ammonia level was decreased significantly in both groups significantly (p=0.005, vs. p=0.001 respectively). however, during administering placebo period, serum ammonia level changes were not significant. in group 1, the first recurrence cases of hepatic encephalopathy were more than group 2(group 1=5, group 2=2), and the first recurrences were occurred during first 3 months in all groups. conclusion: our study demonstrates that acetyl-l-carnitine administration reduced serum ammonia level, but not definitely diminishing the recurrence of hepatic encephalopathy. sodium (na + ) and water retention are the most common abnormalities in cirrhotic patients and the magnitude varies from patients to patients. aim: to assess the relationship between the meld score and urinary excretion of na+ in non-azotemic cirrhotic patients. methods: fifty four cirrhotic patients with ascites and normal serum creatinine (<1.5 mg/ml) were admitted and placed on a low sodium diet (4g/day), while all diuretics were withdrawn for 3 days. the electrolytes (na + , k + , na + / k + ) were measured in a random urine and both the volume and na + concentration of urine collected for 8 h after administration of furosemide 80 mg i.v. were determined. results: table. conclusions: the meld score was significantly correlated with the degree of impairment of urinary na+ excretion. the ratio of na + /k + in a random urine specimen and furosemide-induced na+ excretion reflect the degree of impaired natriuresis in non-azotemic cirrhotic patients with ascites. background: portal hypertensive gastropathy (phg) is common finding in patients with liver cirrhosis and portal hypertension. despite portal hypertension remains the crucial trigger for the development of phg, the relationship between portal hypertension and phg has not been widely investigated. methods: fifty-three cirrhotic patients (48 males, mean age 51 years) who were performed hepatic vein catheterization between november 2006 and august 2008 were prospectively included in this study. the degree of phg was assessed according to the third baveno international consensus workshop, and classified three degrees as no, mild and severe. the hepatic venous pressure gradient (hvpg=whvp-fhvp) measurements were performed by triplicate in each case, and results were given as arithmetic means of the three determinations. result: hvpg values did not differ between the patients without phg (13.28±4.72 mmhg) and those with phg (14.91±3.84, p=0.187), nor between those with mild (15.31±3.69 mmhg) or severe phg (14.27±4.12mmhg, p=0.323). the degree of phg and hvpg did not differ regarding the etiology of the cirrhosis(p=1.0, p=0.085) nor regarding the child pugh classification(p=0.085, p=0.738). no correlations were found between the degree of phg and child pugh score, age, with or without ascites, albumin, bilirubin, creatinine, meld score and the degree of gastroesophageal varices. conclusions: our data show that the presence and the severity of phg does not correlate with the degree of hvpg, and that correlate with esophageal varices in patients with liver cirrhosis. introduction: phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the colonic wall due to fibrous degeneration of the submucosal layer and fibrotic sclerosis of the venous wall. there are a few reports those this entity might be related to portal hypertension with disturbed venous return from the colon and mesentery. case description: a 61-year old man with alcoholic liver cirrhosis presented with right lower abdominal pain/tenderness and bloody diarrhea. a colonoscopy revealed multiple circumferential ulcerations in the transverse colon and the scope could not get through the ascending colon due to luminal stenosis, showing histologic finding of ulcerative inflammation with inflammed granulation tissue. abdominal computed tomography demonstrated liver cirrhosis with splenomegaly, multiple portosystemic venous collaterals, diffuse vascular engorgement and the wall thickening of right proximal to mid ascending colon with increased density in the surrounding fatty tissue. a follow-up colonoscopy performed one month later showed still remained multiple ulcerations in the transverse colon and could not further advance to ascending colon. superior mesenteric angiography revealed no main branch occlusion but pooling at the venous phase on ascending colon. a right hemicolectomy was performed because of the colonic obstruction. gross findings on operation showed thickening of the cecum and ascending colon. microscopic examination showed fibrous thickening in the submucosa, abundant neurovascular bundles in the mesentery and several intravascular hyaline thrombi of the mesenteric vessels. here we report the first case of early stage of phlebosclerotic colitis in a cirrhotic patient in korea. spontaneous bacterial peritonitis (sbp) is one of the severe complications in advanced cirrhotic patients with a high mortality rate. although a more rapid diagnosis should lead to the better survival, it takes several days to detect the causal bacteria from ascitic fluid cultures. furthermore, despite the use of sensitive methods, ascitic fluid cultures were negative in more than 50% of patients with suggestive clinical manifestations of sbp. therefore, diagnosis of sbp is based on the polymorphonuclear leucocytes (pmn) cell count in the ascitic fluid. the hybrizep kit (fuso pharmaceutical industries, osaka, japan) detects the dna of bacteria that have been phagocytized in neutrophils and macrophages, using in-situ hybridization method within one day. here we present a case of the patient for whom the hybrizep kit was used to detect the causal pathogen of sbp. a 76-year-old man had been admitted for the treatment of ascites and esophageal varices. one week after the admission, he complained abdominal pain and fever. because the pmn cell count in ascites fulfilled the criteria of sbp (1141/mm 3 ), we started an empirical antibiotic therapy without waiting for a result of the culture, and his symptoms improved within a few days. on the following day of the onset, in situ hybridization showed the positive signals by the ek probe, which detected the genomeic dna of e.coli species. however, the ascitic fluid culture was negative. this case suggested that the hybrizep kit was useful for the rapid diagnosis of sbp with high sensitivity. background: it has not been known that the hemodynamic effect of a portal hypertension for splenomegaly or esophageal and gastric variceal formation. this study was performed to access the parameters of doppler ultrasonography associated with splenomegaly or varices in patients with cirrhosis. patients and methods: from may 2007 to may 2008, 144 cirrhotic patients were performed the doppler ultrasonography. 91 of these patients were accessed the severity of varices endoscopically. the three dimensional volume of spleen was measured from a length, width and thickness on sonography. results: the splenic volume (415.2ml vs 505.6ml, p=0.048) and blood flow of main portal vein (12.6cm/s vs 15.1cm/s, p=0.012) were statistically significant different in alcoholic (38/144) and non-alcoholic (105/144) cirrhosis groups. the splenic volume (600.1ml vs 384.4ml, p=0.001), damping index (0.52 vs 0.38, p=0.024), and blood flow of main portal vein(12.5cm/s vs 15.7cm/s, p=0.006) were statistically significant different in esophageal variceal groups (55/91) and non-esophageal variceal groups(36/91). the only splenic volume (669.4 ml vs 461.7 ml, p=0.004) were statistically significant different in gastric variceal groups (24/90) and non-gastric variceal groups (66/90). the hemodynamic parameters venous ammonia and cff at baseline and after one month of treatment with lactulose. mhe diagnosed by abnormal psychometry and/or p300erp.response defined by normalization of abnormal test parameters. results: mhe diagnosed in 35(58%) patients. of 35 patients 26 (74%) had both abnormal psychometry and p300erp whereas30 (86%) alone had abnormal psychometry, 31 (89%) had abnormal p300erp.cff was <39hz in 28(80%) patients. mhe recovered in 55% with treatment and cff >39hz was seen in 22(69%) of 32 patients. cff sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy before and after treatment is shown in table. conclusions: critical flicker frequency is a simple and accurate test without any age or literacy dependence for the diagnosis and recovery of patients with mhe. background/aims: endoscopic injection of n-butyl-2-cyanoacrylate (histoacryl) is an effective treatment of varix bleeding. but nontarget embolizations and septicemia are unwanted complications. we evaluate the risk factors for complications. methods: thirty-three patients with esophageal or gastric varix bleeding received endoscopic histoacryl therapies (54 procedures). baseline varix size, ctp score were checked. serum leukocyte, blood culture and body temperatures were repeated checked within one week after procedure. average volume of histoacryl per each session was 1.6 ml, and dilution volume ratio of histoacryl/lipiodol was 1/1 or 1/2. results: average of ctp score was 8.0 ± 1.7. three cases of septicemia were correlated with ctp score rather than session frequency or injection volume. two cases of systemic embolizations (pulmonary and splenic arterial embolism) were correlated with high lipiodol dilution ratio (1/2) and lipiodol volume rather than histoacryl volume or ctp score. conclusion: ctp score, lipiodol volume and dilution ratio of histoacryl/lipiodol were significant risk factors for complications. detection of circulating toll-like receptor 2 and 4 and cd4+cd25+ regulatory t cells in patients with hbv-related liver cirrhosis x.q. wang 1 , y. zhang 1 , x.f. bai 1 , j.q. lian 1 1 background : to detect circulating cd4 + cd25 + regulatory t cells and toll-like receptor(tlr)2 and tlr4 expression on the peripheral blood mononuclear cells (pbmcs) of patients with hbv-related liver cirrhosis (lc), and to explore the correlation between them. methods: pbmcs isolated from 14 lc patients , 21 chronic hepatitis b (chb) patients and 16 normal controls(nc) were stained with fluorescent labeling anti-tlr2-pe, anti--tlr4-apc, anti--cd14-fitc monoclonal antibodies and anti-cd4-percp anti-cd25-fitc anti-cd127-pe. samples were collected and detected of three-color immunofluo rescence by flow cytometry. results: the expression of tlr2 and tlr4 were significantly up-regulated in patients with lc than those in the controls.the expression of tlr2 was significantly increased in patients with lc than those in patients with chb, but there were no differences of tlr4 expression between lc and chb.treg/cd4 + t cells were significantly increased in patients with chb than those in patients with nc and lc, but there were no differences between lc and nc. there were no correlation between the expression of tlr2,tlr4 and treg in patients with lc . the expression of tlr2 and tlr4 on pbmcs in patients with lc were positive correlation.the expression tlr4 and hbv dna level were negative correlation in patients with lc. conclusion: the expression of tlr2 and tlr4 were up-regulated on pbmcs in patients with lc. it seems to be expression of tlr2 and tlr4 invovlved in the pathogenesis of lc. evaluation of 13c-phenylalanine breath test for the measurement of hepatocyte function in patients with chronic liver disease z.j. bao 1 , d.k. qiu 2,3 , x. ma 2,3 , g.s. zhang 1 , y.q. huang 1 , z.p. fan 2,3 , s.m. yin 1 1 huadong hospital, fudan university, 2 renji hospital, shanghai jiao tong university school of medicine, 3 background: the objective is to investigate whether the 13c-phenylalanine breath test(pbt) would be useful for the evaluation of hepatic function in patients with chronic hepatitis b, liver cirrhosis and minimal hepatic encephalopathy (mhe). methods: l[1-13c] phenylalanine was administered orally in a dose of 100 mg to 80 patients with liver cirrhosis, 20 with chronic hepatitis b and 20 healthy subjects. the pbt was measured at 8 different time points (0, 10, 20, 30, 45, 60, 90, 120 min) to obtain the values of delta over baseline, percentage 13co2 exhalation rate and cumulative excretion (cum). the relationships of the cumulative excretion with the 13c-%dose/h and blood biochemical parameters were investigated. results: the 13c dose h -1 at 20 and 30 min combined with the cumulative excretion at 60 and 120 min showed correlations with the chronic liver diseases, especially child-pugh score and mhe or not. and the data showed correlations with serum albumin hemoglobin platelet and child-pugh score. prothrombin time, total and direct bilirubin were significantly increased, while serum albumin, hemoglobin and platelet, the cumulative excretion at 60 and 120 min values decreased by degrees in healthy controls, child-pugh a, b, and c patients (p<0.01). similar results of pbt were in the patients with and without mhe, while only prothrombin time prolonged and total bilirubin increased (p<0.05). conclusions: the pbt can be used as a non-invasive assay to evaluate hepatic function in patients with liver cirrhosis and mhe. the %13c dose h-1 at 20 min, %13c dose h-1 at 30 min and cumulative excretion at 60 min may be the key value for determination at a single time-point. branched chain amino acids in improving survival and decreasing risk of liver failure among cirrhotic patients: a meta-analysis h. flores 1 , e.l. ang 1 , n. iv estanislao 1 1 philippine general hospital background: the state of a patient's nutritional status greatly affects disease outcome. among cirrhotic patients, approximately 60-90% are in a state of protein-energy malnutrition. hence, adequate nutritional support is essential to improve their general medical condition and long term prognosis. several studies have shown than branched chain amino acids (bcaa) may be of benefit for this purpose. it is the aim of this study to evaluate the effectiveness of diet plus bcaa compared to diet alone in improving survival and in decreasing liver failure among cirrhotic patients. methods: pubmed, cochrane, and embase search was done for articles which compared the clinical effects of bcaa supplementation versus diet alone among patients with liver cirrhosis. the following free-text terms and mesh words were used -"branched chain amino acids", "amino acids, branched chain", "bcaa", "liver cirrhosis", "cirrhosis", "randomized controlled trials'" and "meta-analysis". after critical appraisal of the included studies, a random effects model using odds ratio was used to synthesize the results (revman 4.2). results: 3 rcts were included for analysis with a total study population of 836. combination of the studies showed a significant decrease in the risk of liver failure (or 0.45, 95% ci 0.25-0.82, p=0.009) and a trend towards benefit in improving survival (or 0.57, 95% ci 0.27-1.17, p=0.12). conclusions: the overall trend appears to show benefit in the use of branched chain amino acids for patients with cirrhosis with respect to liver failure and survival. background: the proxisome prolifrator-activated receptor gamma (ppar ) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation, carcinogenesis and gastric ulcer. on the other hand, the frequency of gastrointestinal ulceration is higher in cirrhotic patients compared with the normal population. the present study was designed to investigate the effect of specific ppar ligand, pioglitazone, on the mucosal lesions induced by ethanol in cirrhotic rats and the possible involvement of nitric oxide in the pioglitazone effect. methods: cirrhosis was induced by surgical ligation of bile duct and sham-operated rats served as controls. both cirrhotic and sham rats were kept for 28 days after the operation. different groups of sham and cirrhotic animals received saline, or 5, 10 or 15 mg/kg pioglitazone, daily during last 5 days of the fourth week after the surgery. another 2 groups of bdl or 2 groups of sham rats received l-name, a non selective inhibitor of nitric oxide synthase, alone or along with 5 mg/kg pioglitazone for 5 days. on day 28, rats were killed 1 hour after ethanol administration and the area of gastric lesions was measured. results: the ethanol-induced gastric mucosal damage was significantly more sever in cirrhotic rats than sham-operated ones (p < 0.001). pretreatment with pioglitazone dose dependently attenuated gastric lesions induced by ethanol in both sham and cirrhotic rats, but this effect was more significant in cirrhotic ones. concurrent treatment of l-name and pioglitazone decreased the ulcer index in bdl rats more than the groups that received l-name or pioglitazone alone. conclusion: we conclude that chronic treatment with pioglitazone exerts a potent gastroprotective effect on the stomach ulcers of cirrhotic rats probably due to inhibition of nitric oxide synthase. inhibition of phosphodiesterase 5 -a novel therapeutic strategy for portal hypertension l. halverscheid 1 , p. deibert 2 , b. pannen 1 , r. schmidt 2 , m. roessle 2 , w. kreisel 2 1 university hospital duesseldorf, germany, 2 university hospital freiburg, germany introduction: the no-cyclic gmp system is a key factor in the regulation of splanchnic and hepatic blood flow and may be a target for medical treatment of portal hypertension. clinical data have shown that inhibitors of phosphodiesterase 5 (pde5) lower portal pressure in cirrhotics. methods: we monitored in rats the effects of the pde5 inhibitors vardenafil and sildenafil on systemic and hepatic hemodynamic parameters up to 60 minutes after the drug. the drugs were administered intravenously into the tail vein at 1 (group a), 10 (group b), and 100 g/kg body weight (group c). 0.9% nacl was the control. n = 7 for each group. results: the most prominent changes were observed in the vardenafil b group: mean arterial and portal venous pressure decreased (-9%, -8%), as well as portal venous, hepatic arterial, and systemic vascular resistance (-31%, -30%, -12%). portal venous and sinusoidal flow increased (+31%, +13%). in the vardenafil c and sildenafil b and c groups there was an increase of portal venous flow by 20-30%, an increase of sinusoidal flow by 12-30%, and a decrease of portal venous resistance by about 25%. there was a trend for reduction of portal venous pressure. conclusions: vardenafil and sildenafil influence portal hemodynamics in the rat. portal venous flow increases by 20-30%, portal venous resistance decreases by >25%. dependent on the dose, portal venous pressure decreases significantly. these data yield further evidence that pde5 inhibitors may be a novel therapeutic option for portal hypertension. groups of liver cirrhosis e. havrilyuk 1 1 lviv national medical university introduction: rupture of esophageal varicose resulting in posthemorrhagic anemia is a common life-threatening complication of liver cirrhosis. but it is not clear, why the other patients, having the same degree of sclerosis and histologic activity index, die from hepatocellular failure or other reasons. aims & methods: 3713 autopsy cases performed in lviv regional hospital in 2004-2007 were analyzed. screening of slides with liver tissue allow to select 580 cases (15,6%) with cirrhosis (complete and incomplete), which are examined in order to evaluate the frequency of lethal portal hypertension complications in the different etiologic groups of liver cirrhosis. results: according to the etiologic factor the following groups of liver cirrhosis were examined: alcoholic disease (49,8%), viral hepatitis (7,8%), nonalcoholic steatohepatitis (14,8%), secondary biliary cirrhosis (2,9%), cardiac sclerosis (0,9%), combined lesions (11,9%) and cryptogenic cirrhosis (11,9%). analysis shows that in 287 cases (49,5%) patients die from cirrhotic complications (hepatocellular failure, jaundice, portal hypertension) and only in 105 cases (18,3%) -from posthemorrhagic anemia caused by rupture of esophageal varicose. in the latter cases correlation between the etiologic types of cirrhosis is almost the same, as in the main group and only alcoholic lesions (60%) and biliary cirrhosis (6,7%) are more frequent. conclusion: analysis shows that development of lethal complications of portal hypertension can not be explained only by etiologic factor. probably additional stimuli are more important for morphogenetical variants of cirrhotic transformation. patients with viral cirrhosis k. mumtaz 1 , s. ahmed 1 , h. ali shah 1 , s. hamid 1 , w. jafri 1 background and aims: increased nitric oxide (no) production is incriminated in the pathogenesis of arterial vasodilation and hyperdynamic circulatory state in non cirrhotic models of portal hypertension (pht). we investigated the relative roles of constitutive nos (enos) and inducible nos (inos) isoforms in the development of rabbit models of endotoxemia induced portal hypertension (eipht) methods: eipht was induced by chronic injection of lipopolysaccharide via an indwelling cannula placed in the gastrosplenic vein of rabbit and maintained for 6 months. the concentration of no, expression of nos (enos and inos) mrna and protein was measured in eipht and sham operated control animals. results: rabbits with eipht compared with controls had raised portal pressure (in mmhg-14.34±1.78 vs 6.30±0.60;p<0.05;1mo; 14.91±0.56 vs 7.04±0.42; p<0.05, 3mo; 19.8±3.10, vs 10.2±4.80;p<0.05), arterial hypotension (in mmhg-65.40±3.2 vs 80.04±1.40, p<0.05, 1mo; 62.90±5.40 vs 76.05±2.60,p<0.05, 3mo; 65.85±2.50 vs 79.1±5.10, p<0.05,6mo), splenomegaly (in g-0.92±0.14 vs 0.60±0.13,1mo; 0.90±0.16 vs 0.62±0.04, 3mo; 0.97±0.12 vs 0.67±0.07, 6mo), normal liver functions and preserved hepatic architecture at 1,3 and 6 mo. serum levels of no2 as well as the no3 were significantly elevated in eipht rabbits as compared to the controls. the expression of enos, at the level of mrna, was significantly increased in eipht rabbits consistent with increased levels of expression of inos as compared to the controls. the enos but not inos protein expression was elevated in eipht than control rabbits. conclusion: vascular dysfunction in the splanchnic circulation during the development of endotoxemia induced portal hypertension is predominantly characterized by enos and partly by inos gene up-regulation. liver cirrhosis contributed to the immunocompromised status by shedding the membranous tnfrii t.n. lin 1 , c.h. chao 1 , i.s. sheen 1 , y.p. ho 1 , w.t. chen 1 , c.j. lin 1 , c.t. yeh 1 , c.y. lin 1 1 liver research unit, linkou medical center, chang gung memorial hospital, chang gung university, taoyuan, taiwan background & aim: patients with decompensated liver cirrhosis (dlc) were regarded as immunocompromised, reflected by high incidence of bacterial infection. paradoxically, the proinflammatory cytokine like tnfincreased significantly in patients with dlc even in the face of this immunocompromised status. on the other hand, regulatory t cell (treg cell) is believed to play an important role in inhibiting immune responses, including innate immune responses like blockade of tnf-effect through soluble tnfrii. here, we studied the role of treg cells and tnfrii in patients with decompensated liver cirrhosis. patients and methods: 33 healthy volunteers and 78 cirrhotic patients were enrolled. the percentage of treg cells were enumerated by flow and serum levels of il-10, tgf-and tnf-by elisa. results: the percentage of treg cells increased significantly in patients with dlc associated with increased serum levels of il-10 and tgf-. in addition, these treg cells were mainly memory type reflected as high cd45ro. furthermore, the tnfrii expression increased significantly on these treg cells of dlc. interestingly, these membranous tnfrii on treg cells could be shed-off. lastly, we found the serum soluble tnfrii concentration increased significantly in patients with dlc when compared with normal volunteers. conclusion: our results demonstrated memory treg cells with high tnfrii expression increased significantly in patients with decompensated liver cirrhosis that could possibly blocked the biological effect of tnf-by shedding membranous tnfrii and contributed to the immunocompromized status of dlc. background: portal pressure measured as hepatic venous pressure gradient (hvpg) correlates with severity of portal hypertension and the development of complications. hvpg measurement is invasive. recently, liver stiffness measurement has been shown to correlate with liver biopsy and helps predict outcome in chronic liver disease patients. this study was conducted with the aim to study the correlation between portal pressure as measured by hvpg and liver stiffness as measured by fibroscan among patients with portal hypertension due to various causes. methods: between august and september 2008, consecutive patients with portal hypertension were included and were subjected to hvpg measurement and fibroscan (echosens, france). results: of the 18 patients with portal hypertension, both hvpg and liver stiffness were measurable in 12[9 (75%) males; mean age 37.5(10.6) years]. the etiological distribution was hbv related cirrhosis in 3 patients, hcv cirrhosis in 3, cryptogenic cirrhosis in 2, alcoholic cirrhosis in 2, hbv and alcoholic cirrhosis in 1and primary extra-hepatic portal vein obstruction in1. the mean hvpg and liver stiffness of this group were 13.9 (5.1) mm hg and 26.6 (16.5) kpa respectively. there was a strong positive correlation between hvpg and liver stiffness [r = 0.708; p = 0.01]. conclusions: non-invasive measurement of liver stiffness correlates well with invasive measurement of portal pressure. liver stiffness measurement could be used as a prognostic indicator to predict the severity of portal hypertension. endpoints were rate of rebleeding and mortality till day 30 after inclusion and to see for any adverse events. results: the bleeding was stopped in all 15 patients (100%). rebleeding till day 30 was observed in 4 (26%) patients (2 each in group a and b). total 2 patients (13%) died (1 each in both groups) due to rebleeding. transfusion needs were higher in group a (4.2±2.8 versus 2.3±2.1, p<.05). serious adverse effects leading to treatment discontinuation were not seen in any patients in both groups. conclusion: prolonging terlipressin treatment did not confer any significant decrease of mortality or bleeding recurrence. however transfusion requirements were significantly decreased in patients receiving prolonged treatment. serious adverse effects leading to treatment discontinuation are rare. poster exhibition -miscellaneous poster session, hall 5b background: it is important to know the prevalence of atp7b gene mutations of different geographical areas to justify the local screening strategies for wilson disease (wd). materials: eleven unrelated lithuanian families, including 13 wd patients were tested. genomic dna was extracted from whole venous blood using a salt precipitation method. firstly, semi-nested pcr technique was used to detect the c.3207c>a (p.h1069q) mutation. patients not homozygous for c.3207c>a (p.h1069q) mutation were further analyzed. the 21 exons of the wd gene were amplified in a thermal cycler. direct sequencing of the amplified pcr products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer. results: total of 13 wd patients (mean age 26.4 years; range 17-40; male/female, 3/10) presented with hepatic disorders and 16 their first degree relatives were studied. some of wd patients in addition to hepatic symptoms have had extrahepatic disorders (haemolytic anaemia 3; fanconi syndrome 1; neurophsychiatric and behavioural disorder 2). twelve of 13 (92.3%) wd patients have had c.3207c>a (p.h1069q) mutation, 6 of them in both chromosomes, 5 were presented as compound heterozygotes with additional c.3472 -82delggtttaaccat, c.3402delc or c.3122g>a (p.r1041q) mutation. for one patient with liver cirrhosis and psychiatric disorder no mutations were found. out of 16 first degree wd relatives 11 (68.7%) were heterozygous for c.3207c>a (p.h1069q) mutation. conclusion: c.3207c>a (p.h1069q) missense mutation is characteristic for lithuanian wd patients. even 92.3% of wd patients with hepatic presentation of the disease are homozygous or compound heterozygote for this mutation. background: diabetic dyslipidemia is a crucial problem of diabetic patients with inadequate control. we investigated the relationship between glutamic-pyruvic transaminase (gpt) and high-density lipoprotein cholesterol (hdl-c) in diabetic patients. methods: with informed consents, we recruited outpatients with diabetes at a hospital in rural area in taiwan in [2004] [2005] [2006] [2007] . anthropometric measures, blood tests and urine screening were examined in diabetic patients. results: overall, there were 1241 diabetic patients aged 19-91 years enrolled in this study and 660 (53.2%) of them had low hdl-c. diabetic patients with the highest quintile of gpt had higher average of body mass index (p<0.0001), diastolic blood pressure (p = 0.003), but lower average of hdl-c (p<0.0001) compared with diabetic patients with the lowest quintile of gpt. the prevalence of obesity (45.7% vs. 24.8%, p<0.0001) and low hdl-c (64.6% vs. 48.3%, p<0.0001) were higher in diabetic patients with highest quintile of gpt than in diabetic patients with lowest quintile of gpt. in the multivariate logistic regression, diabetic patients with highest quintile of gpt had higher odds ratio (or) of low hdl-c compared with diabetic patients with lowest quintile of gpt (or = 1.88, 95% confidence interval [ci] = 1.21-2.92). the corresponding or of low hdl-c in patients aged 70 years and older was 3.30 (95% ci = 1.15-9.47). conclusion: high gpt is one of factors associated with low hdl-c in diabetic patients. the effect of desferrioxamine as supplement to cefotaxime in the treatment of spontaneous bacterial peritonitis na. seda 1 , m. el-hamamsy 2 , r. el-wakil 3 , m. al azizi 4 1 ain shams specialized hospital, 2 faculty of pharmacy,ain shams university, 3 faculty of medicine,ain shams university, 4 faculty of pharmacy ,ain shams university background: oxidative damage lead to cell damage, organ dysfunction and death in sepsis. desferrioxamine (dfx), an antioxidant iron chelators. the aim was to assess the efficacy of desferrioxamine supplemented to cefotaxime in the treatment of spontaneous bacterial peritonitis (sbp) in cirrhotic patients. methods: thirty patients divided into two groups: group i (n=15) with sbp and receiving cefotaxime (1g iv every12 hours) alone and group ii (n=15) with sbp receiving cefotaxime (1g iv every 12 hours) with desferrioxamine (500mg im twice daily).all patient were monitored for seven days, their vital organs were screened and their ascitic fluid was assessed completely including microbiological investigations. results: the concomitant administration of desferrioxamine with cefotaxime significantly at (p<0.001)and(p<0.01) improved the therapeutic outcome and the cure rate after 5 days of treatment as compared to patients using cefotaxime only. conclusions: desferrioxamine can improve the therapeutic outcome by preventing iron-induced organ damage and inhibiting bacterial growth. oligella ureolytica is a gram-negative, nonfermenting rod that is infrequently recovered from clinical specimens and is most commonly isolated from the urine of patients with chronic indwelling urinary catheters or other urinary drainage systems. bacteremia due to this organism is an extraordinary finding. we describe here a case of oligella ureolytica being detected in the blood of a patient with decompensated cirrhosis. a 51-year-old male man was admitted to hospital with 9-month duration of debility, poor appetite and abdominal distension. decompensated cirrhosis was diagnosed based on clinical findings such as hepatic face, ascites, edema of lower limbs and icteric sclera. laboratory results showed positive serum anti-hcv and high serum hcv rna level. the patient received a therapeutic regimen of pegylated interferon alpha 2a plus ribavirin after being admitted to hospital. during hospital stay, a fever of 2-day duration with shivering occurred to the patient. three blood cultures were drawn, which all grew oligella ureolytica in pure culture. the organism was identified by the viteck 2 compact (biomerieux, france). additional tests for identification resulted positive for nitrate reduction and urea hydrolysis, strongly positive for phenylalaninedeaminase activity and showed no growth at 42.7c. tests for nitrite reduction and motility resulted negative. the organism was resistant to amikacin, cefoperazone, levofloxacin, piperacillin/tazobactam, trimethoprimsulfamethoxazole, aztreonam, cefotaxime, piperacillin and was susceptible to gentamicin, imipenem, meropenem, and netilmicin. a 14-day of combined therapeutic regimen with cefminox and isepamicin was administered to the patient. within 2 days, the patient became afebrile. background: endoscopic ultrasound (eus) is often performed in patients with unexplained liver tests to assess the gallbladder, bile ducts and pancreas. an unremarkable eus exam and negative hepatology workup often leads to a liver biopsy. eus may provide histopathologic evaluation of the liver in these cases under direct, real-time visualization. aim: to assess the feasibility and efficacy of eus guided core biopsy of the liver in a porcine model. methods: female pigs were used and live procedures were performed under general anesthesia. a linear echoendoscope was used and the liver identified endosonographically. transgastric core biopsies of the liver were obtained with a 19 gauge quick-core ultrasound biopsy needle (wilson-cook) and sent for histopathologic evaluation. live animals were euthanized at the end of the procedure and necropsy performed. results: core biopsies of the liver biopsy were obtained in 4 animals (1 cadaver and 3 live anesthetized). a total of thirteen needle passes were made (mean 3.25; range 2 -4 needle passes per animal) and a visible core of tissue obtained. the maximum length of liver tissue obtained was 10 mm and considered adequate for assessment as more than one such specimen could be obtained. microscopic evaluation confirmed liver tissue. no complications were noted. necropsy did not show any evidence of bleeding, perforation or damage to surrounding structures. conclusion: eus-guided liver biopsy is feasible and can be performed at the time of routine echoendoscopic exam in select patients undergoing eus examination for abnormal liver tests. background: as the common indexes, alt, ast and plt play an important role in disease diagnosis, treatment and prognosis. many researchers suggested that there was inflammatory changes and fibrosis in chronic hepatitis b and c patients whose alt level was persistently normal. a large sample investigation showed that the serum level of alt in healthy persons is lower than the normal reference value. this study re-evaluated the normal serum level of alt, ast and plt. methods: 3815 people were enrolled in the study between sep. and oct. 2007. the platelet count and serum alt and ast levels were measured. frequencies, one-sample kolmogorov-smirnov test and nonparametric tests were used to analyze the difference between age groups, male and female, glucose groups, cholesterol groups and triglyceride groups. result: in the five groups, there is significant difference in alt and ast levels between male and female. in group 1, the alt and ast levels showed a significant difference between different age groups, between different glucose groups and triglycide groups. in the three groups the plt level is significantly different between male and female, and the serum level in male is higher than female. there is significant difference between different age groups conclusion: the serum levels of alt, ast and plt are all significantly different between male and femal. there is significant difference between different genders and age groups for plt. the serum level of plt is higher than the reference value. background/aims: myeloproliferative disorders (mpd) (like polycythemia vera, essential thrombocythemia and primary myelofibrosis) are responsible for 50% cases of hepatic venous thrombosis (hvt) and 35% of portal venous thrombosis (pvt) in western series. latent form of mpd lacks the characteristic blood picture and may be classified as idiopathic thrombotic disorder. a point mutation at val617phe of janus kinase 2 tyrosine kinase gene (jak2 v617f mutation) occurs in high proportion of the patients with mpd. this non-invasive test with high positive predictive value is now considered to be essential for diagnosis of various mpd. this test may be useful in diagnosing latent form of mpd in splanchnic venous thrombosis methods: 232 patients with confirmed pyogenic liver abscesses admitted from 1995 to 2007 in our institution were included. there were 145 men and 87 women ranging in age from 19 to 91 years. the medical records were reviewed for clinical, laboratory and radiographic characteristics. results: among 232 patients, 81 (34.9%) experienced at least one complication. there were 67 pulmonary (pleural effusion, pneumonia, empyema) complications, 16 septic shock, 12 acute renal failure, 2 abscess rupture, 2 pseudomembranous colitis, and 2 pericardial effusion. the predictive factors for its complications were: systemic inflammatory response syndrome (sirs, 2 factors), thrombocytopenia ( 80,000/ml), hypoalbuminemia ( 3.0g/dl), elevated ast or alt (>200 iu/l), hyperbilirubinemia ( > 2.0 mg/dl), k. pneumonia, air within abscess cavity (p<0.05). conclusions: the incidence of complications in the pyogenic liver abscess was 34.9%. the various predictive factors of complication should be monitored carefully. further large scaled study should be warranted. background/aims: hepatic iron deposition is a common feature in chronic hepatitis c (ch-c), however, whether it could enhance the progression of fibrosis or not is controversial. the aim of this study was to evaluate the status and significance of hepatic iron deposition in the korean patients with chronic hepatitis c. methods: untreated, 78 ch-c patients who underwent liver biopsy were included. the hepatic iron was assessed by scheuer's scoring system, and activity, fibrosis, and steatosis were scored by a pathologist in a blind manner to the clinical features. clinical and laboratory data including serum iron indices, virological, biochemical results were analyzed to search for significant factors associated with hepatic iron deposition. results: hepatic iron staining was positive in 26(33%). among 26 patients with hepatic iron deposition, serum levels of ferritin (p=0.005) and -fetoprotein (p=0.002), and body mass index(bmi) (p=0.039) were significantly elevated. there was no significant association between the degree of hepatic iron deposition and fibrosis stage (p=0.321), although elevated levels of serum hyaluronic acid (p=0.049), -glutamyl transpeptidase (p=0.028), and prothrombin time (p=0.012) were associated with advanced fibrosis. conclusions: hepatic iron deposition in asian-pacific ch-c patients seemed to be neither frequent nor related to hepatic fibrosis, but related to obesity. therefore, phlebotomy might not commonly applicable to this area. further studies on the pathogenic role of iron in ch-c in asian-pacific countries are warranted. a late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture, necrosis of hepatocytes and the formation of regenerative nodules contributes to cirrhosis. limitations like organ donors shortage, high cost, absence of proliferation in cultured hepatocytes, inherent risks of infection, rejection in xenogenic cells and other socio-economical complications emerges advanced regenerative human hepatic stem cells(hhpscs) transplantation. hhpscs are located in the ductal plates in fetal and canals of hering in adult livers [schmelzer et al.(2006) ]. hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [schmelzere etal(2006) ].hhpscs express cd326(epcam)marker. scjelzer etal demonstrated that during embryogenesis 90% of the epcam positive cells had hepatoblast phenotype. in animal study, on transplantation of freshly isolated hhpscs in scid mice results in mature liver tissue expressing human-specific proteins. recently, we(aleem etal 2008)have shown clinical improvement in study in patients with crigler-najjar syndrome, biliary atresia using hhpscs infusion. in the present study we transplanted hepatic progenitors to five subjects of end stage liver cirrhosis with meld score >30. hhpscs were sorted using macs with cd326 antibody microbeads and infused through hepatic artery via femoral artery catheterization, a safe procedure provided portal pressure to monitor cell infusion route in order to prevent vascular thrombosis. all the patients showed improvement clinical and functional biochemical parameters after first month of cell infusion. ascites was decreased and changed encephalopathy grade into normal level was observed. meld score system falling to normal level from >30 to <22 after infusion. 1 the aga khan university patients. the apri of 1.5 in combination with a cut-off ha of 300 ng/ml can best detect patients with moderate to severe fibrosis (stages 2-4). it has a ppv of 93.7%. also, for patients without moderate to severe fibrosis, the test is hardly ever positive (specificity of 98.9%). but the apri of 1.5 in combination with different ha as cut-off points is not possible to detect patients with no or mild fibrosis. objectives: terlipressin is used in esophageal variceal bleed (evb) along with endoscopic band ligation (ebl) for 3days (uc). due to its high cost, it was stopped <3days (sc) who could not afford & were stable after achieving hemostasis with ebl. we retrospectively assessed the efficacy of sc vs uc of terlipressin for control of evb and length of stay. conclusion: the apri of 1.5 in combination with ha 300 ng/ml as cut-off points to predict patients with moderate to severe fibrosis (stages 2-4) is an easy and accurate method. methods: patients with evb who had achieved hemostatsis with ebl from jan 2004-dec 2005 were included. all were managed on standard protocol on hospital variceal bleeding pathway. the course of terlipressin as sc or uc was based on patient's inability to afford the cost of hospitalization and terlipressin. the efficacy of terlipressin in the control of evb was defined based on baveno iii criteria. results: total of 117 patients were admitted during the study period. out of them, 66 received uc & 51 sc of terlipressin. the base line characteristics were comparable except younger age in sc. there were 2 re-bleed (3%) in uc and 1 (2%) in sc terlipressin group. the length of stay was shorter in sc group. (2.47±0.57 vs 6.15±2.92 days). conclusions: sc seems as effective as uc terlipressin in the control of evb after initial control of hemostatsis with ebl and may reduces the length of hospital stay. rcts are needed to assess this as all stable patients may not need to continue terlipressin for 72 hours. background/aims: to analyze the relationship between conventional laboratory results and death risk in patients with esophageal varices bleeding due to cirrhosis (cevb), and establish a simple model for timely predicting death rsik of the patients. outcome of patients with gastro-oesophageal bleeding in a tertiary center j. wat, w.h. li, m.t. cheung methods: the medical documents of cevb patients were reviewed retrospectively and the data were collected. univariate and multivariate logistic regressions were performed, in which the discharged results (survival or death) as dependent variable and the results of liver function, kidney function, serum electrolytes and blood cell analysis as independent variables. the multivariate regression equation was as the model for the prediction of patient outcome and its predictive performance was evaluated. objectives: to determine the rebleeding rate, mortality and long term survival in cirrhotic patients presented with acute gastro-oesophageal variceal bleeding. method: this is a retrospective review of adult patients who were admitted to our hospital with the diagnosis of acute gastro-oesophageal variceal bleeding for the first time regardless of their underlying causes for cirrhosis. the study period was from january 2000-october 2008. data were collected from our hospital computer system and records. results: in univariate regression, the significant positive variables for death outcome were dbil, akp, k, wbc and plt, and the significant negative variables were tp, ap, a/g, na, cland ca 2+ . the variables entered the multivariate regression are alt, tbil, dbil, gp, a/g, cr, na + , cl -, ca 2+ , wbc, hb, plt. the sensitivity, specificity and accuracy of the regression model for predicting death of cevb patients were 97.1%, 95.1% and 95.8%. results: a total of 172 patients were included in this study, with 122 male and 55 female. their mean age was 61.9. the initial failure rate in endoscopic haemostasis was 15.1%. the 5-day and 6-week mortality rates were 11.8% and 21.9 %, respectively. poor child's grading, multiple columns of oesophageal varices, high grade of varices, failed initial endoscopic haemostasis, presence of inoperable hcc, low platelet count on admission, and short duration from index bleed to rebleed were factors associating with increased risk of 6-week mortality (p < 0.05). mean duration from index bleed to first rebleed was 15.1 months. poor child's grading and presence of inoperable hcc were associated with both early or multiple rebleed (p <0.05). overall, 37.2% of our patients developed rebleed before their variceal eradication. 5-year survival in patients with child's a, b and c were 65%, 22%, and 10%, respectively (log rank test p 0.000). conclusions: the liver function, kidney function, serum electrolytes and blood cell analysis are generally independent factors for cevb patient death risk, especially dbil, a/g and ca 2+ . the established model shows a excellent predictive performance. an imbalance in plasma amino acids of advaced cirrhotic patients impairs the maturation of dendritic cells via mtor/s6k signaling pathway e. kakazu 1 , y. ueno 1 , y. kondo 1 , k. fukushima 1 , m. shina 1 , j. inoue 1 , k. tamai 1 , m. ninomiya 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university hospital conclusion: although endoscopic haemostasis is an effective treatment modality; rebleeding is still commonly seen among patients with poor child's grading and inoperable hcc. this will result in significant bleeding-related death and poor overall survival. further advancement in treatment strategies for this group of patients are required to improve their outcome and prognosis. background: we have demonstrated that extracellular branched-chain amino acids (bcaas), especially valine, regulate the maturation and function of monocyte-derived dendritic cells (j immunol. 179: 2007) . however, it is not clear whether an imbalance in plasma amino acids of advaced cirrhotic patients influence the function of dendritic cells (dcs). methods: we used human pbmcs and cd1c+dcs in this study. we made two mediums: a serum free culture medium consistent with the average concentration of the plasma amino acids from a healthy volunteer (n=100) was defined as the healthy control medium (hcm); whereas that from advanced cirrhotic patients (n=50) was defined as the advanced cirrhotic active hepatitis b replication is defined as hbeag + or hbv dna > fibrosis was scored according to the metavir system. alt levels were characterized as being normal, < 2 x normal, and > 2 x normal. conclusions: pe494 frequency of portal hypertensive gastropathy and its non-invasive predictors in patients with viral methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent for screening egd for varices in last 2 years was reviewed. phg was defined endoscopically by using mccormack classification. noninvasive markers such as spleen/platelets ratio, meld score and child score of all the patients who underwent for egd were recorded. results: out of 360 patients 226(62.77%) were males. out of 300(83.3%) patients who had phg, 136(45.3%), 93 (31%) and 71(23.7%) had mild, intermediate and severe phg respectively. higher proportion of esophageal varices (89.7%) was present among those who have phg (p<0.001).196(65.3%) with phg has child score of 7. meld score >15 and 15 were seen in 32.3% and 67.7% of patients with phg, respectively. platelet/spleen ratio was 916.08± 400 in patients with phg as compared to 1476 methods: retrospective analysis of cirrhotics undergoing surveillance endoscopy was undertaken assessing for oesophageal varices. clinical, biochemical and radiological indices were analysed. results: 81 cirrhotics underwent surveillance endoscopy during the study. childs pugh scoring (cps) to assess prognosis of liver disease showed cps a(58%), cps b(27%) and cps c(14%). 68% were male albumin (42g/l vs 35g/l significant factors on multivariate analysis were albumin (p=0.003) and platelet count (p=0.026) conclusion: in our cohort there were significant biechemical and radiological differences in differentiating patients with large varices (grade 2-4) on surveillance endoscopy aims and objectives: to study the frequency of ev in patients with cirrhosis due to viral etiology and its correlation with different non-invasive markers. methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent screening egd for varices in last 2 years was reviewed. ev were divided in two grades (small and large) as proposed in consensus development workshop. noninvasive markers such as spleen/platelets ratio, meld and child turcotte pugh (ctp) scores of all patients were recorded. results: out of 360 patients, 226 (62.77%) were males on multivariate analysis ctp score of 7 (or 2.06, p<0.01), meld score >15 (or1.63, p<0.05) and platelet/spleen ratio 900(or 2.35, p=0.005) were found as significant predictors of large ev. conclusion: the frequency of ev is high in viral cirrhosis patients on screening egd. meld score>15, ctp score 7 and spleen/platelets ratio 900 can be used as non-invasive predictors of large ev. pe519 treatment outcome and prognostic factors of spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with hepatitis b virus-related thirty-seven (28.5%) patients had sbp while 91 (71.5%) had cnna. except the higher proportion of renal failure at admission in patients with sbp than cnna (32.4% vs. 7.5%, p=0.001), no significant difference in the clinical and laboratory data related to liver and renal function was observed. overall mortality during hospitalization was higher in patient with sbp than that of cnna evl was repeated every 2-weeks till varicial eradication.bb dose was titrated to achieve a resting heart-rate of 55bpm or a maximum dose 320mg/d or when side-effects began to appear.primary end-points were rebleed and death.secondary end-points were complications as a result of evlor beta-blocker,variceal recurrence afterevl,and decrease in variceal grade inbb limb. results: 71 patients (median age 14[range2-68] yrs, males65%) were included (evl arm[n=37]and bb arm[n=34]. median grade of varices was iii(range ii to iv) nitric oxide synthase isoforms play distinct roles in the evolution of hyperdynamic state in endotoxemia induced portal hypertension m.r. rizvi 1 , m. shahid institute of genomics and integrative biology, mall road, delhi 110 007, 4 department of physiology a seconderc was performed after two or more years to assess the progression of the disease. results: 79 ehpvo patients(median age 20[range 5-62]yr, males 67%) were studied.history of present or previous jaundice was present in 24%,ascites 18% and pain 9%. on erc,92% had portal biliopathy.the type of bile duct involvement was categorized as: type 3 b(51%) and type 1(42%).the pattern of involvement included indentations(49%) and dilatation and strictures(36%).20%of the patients had bile duct stone and 9% had history of cholangitis.the mdian bilirubin was1.1(range0.3-15.9) mg/dl and median serum alkaline phosphatase 171(range 42-617) iu/l.all patients were treated endoscopically by endoscopic stone extraction, dilations with/without stenting. 24(30%) patients underwent second erc after a median interval of 21(range 1-111)months.the type of involvement progressed,75% patients developed type-3 involvement compared to54% at the baseline (p=ns).indentations progressed to develop strictures, from46% to71%.the frequency of new bile duct stones per year was 4%(p= ns). conclusions: portal biliopathy is very common in ehpvo, often remaining asymptomatic.however,it is slowly progressive leading to development of biliary strictures objective: to investigate the mechanisms of angiotonin ii (angii)-induced ca (2+)-independent pathways mediated by rho kinase in hepatic stellate cells (hscs) various vasoactive drugs that reduce portal pressure are used in treatment of esophageal variceal bleeding alongwith endoscopic treatment. terlipressin use decreases both, recurrent bleeding and mortality. it is given usually for 3-5 days, nevertheless there is very little data comparing different time periods. our aim was to compare the efficacy and safety of 5-days versus 10 days of terlipressin treatment in bleeding esophageal varices. methods: out of 15 patients who presented with variceal bleeding, 8 were randomized to receive terlipressin 2 mg 8 hrly, i.v. daily for first 5 days and placebo for next 5 days (group a) and 7 to receive terlipressin 2 mg 8 hrly, i.v. daily for 10 days (group b). both groups were both age and sex matched. (svt) {consisting of hvt and pvt}. there is no such data from india a comparative study of male vs background: mucosal lesions are frequently observed.gender differences are expected due to food habits, nature of job, mobility related to work, consumption of alcohol, tobacco etc. material and methods: procedure done in 1395 m & 19(3.4%) f& duo 48(5.73%) m & 73%)f.varices (48) eso varices 31 (4.3%) m &10(1.8%) f & gastric varices 1 (.11%) m & 1(.11%) f .growth (17)eso growth 5 (.59%) m &1(.17%) f & stomach growth 9(1.07%)m & 1(.17%) f & laryngeal 1(.11%)m. eso monoliasis (7) 3(.35%) m & 4(.71%) f.polyp (8) eso polyp 4(.47%) m & 1(.17%) f & gastric polyp 1(.11%)m & 1(.17%) f & moniliasis and ulcers are more common in female lee 1 1 department of internal medicine, gyeongsang national university school of medicine background/aims: although the pyogenic liver abscess is a common intraabdominal inflammatory disease, this complications are not rare. however, reports dealing with this complications are not good enough and results are often variable. the aim of this study was to identify the predictive factors of complication in the pyogenic liver abscess. pe538 effects of saikosaponinsd (ssd) on expression of c-myc and pcna in experimental hepatocarcinoma of all rats were killed in the 18th week, then general conditions of rats were recorded, the serum alt akp ggt afu was detected and pathological examination was made. the expression of pcna and c-myc were tested by immunohistochemistry. results: he staining showed that rats were induced to hepatocellular carcinoma,the results of liver function in the 18th week displayed that alt akp ggt and afu of all groups were increased than that of normal control group conclusion: ssd can inhibit development of hepatoma induced by den, possibly by down-regulating the expression of pcna and c-myc protein lethal endotoxic shock was induced by single endotoxin (e.coli) 6mg/kg injection into abdominal cavity. ppc in 5% gs was given via tail vein at 1ml/100g (232.5mg/kg) 24h and 6h before endotoxin. rats' behavior and 24h survival were recorded, venous blood taken for ast and alt, liver preserved for he staining and liver/body weight ratio l/b and liver wet/dry weight ratio (w/d) calculated. intercellular adhesion molecule-1 (icam-1) expression in liver tissue was observed with 2-step immunohistochemistry assay. results: rats of ns and ppc group demonstrated similar normal activity, histology and other characters (p>0.05), while lps group showed sag and less water-intak and severe inflammation in liver including inflammatory cell accumulation, parenchymal cells edema and tissue exudation. p+l group turned tired but could drink water the role of insulin resistance, adipokine and cytokine pro-inflammatory in non-alcoholic fatty liver disease n. ratnasari 1,2 , s. anam 2 , p. bayupurnama 1,2 , s. maduseno 1,2 , s. nurdjanah 1, 2 1 dr sardjito general hospital yogyakarta indonesia, 2 background: non-alcoholic fatty liver disease (nafld) is a benign disease during 5-10 years period, with 67%-59% survival. nafld can progress to fibrosis, cirrhotic and cancer of the liver. the etiopathogenesis of nafld is still unknown, however genetic and environment factors are predicted. objective: to know the role of insulin resistance, adipokine and cytokine pro-inflammatory on nafld. methods: the cross sectional study was performed on general check-up population at dr. sardjito general hospital yogyakarta, indonesia. the study was begun from january 2007 until november 2007 at internal medicine outpatient department. inclusion criteria: adult, alcohol consumtion 20g/day, metabolic syndrome patients, and healthy subjects. exclusion criteria: the diseases with increasing liver enzymes (hbv, hcv, ischemic hepatitis, congestive liver), a "bright liver" on ultrasound examination (malnutrition, rapid weight decreased, post gut surgery on obesity patients, and drug induced). based on liver ultrasound subjects were devided into steatosis group and non-steatosis group. data were analyzed by t-test and non-parametrical test. results: 101 subjects that were enrolled the study 61 steatosis (60.4%) and 40 non-steatosis (39.6%) and the subjects who completed cytokine and adipokine examination were 48 steatosis and 30 non-steatosis. there were significantly different on homa -ir and adiponectin level in steatosis group (homa-ir 3.367±4.901 vs. 1.430±1.889, p=0.019; adiponectin 4.049±1.929 vs. 7.034±3.777, p=0.000). there were not significantly different on tnf-, il-6, leptin and visfatin level (p>0.05). conlusions: there were significantly different on homa-ir and adiponectin level in nafld patients compared non-nafld patients. background: to optimize management of nonalcoholic fatty liver disease (nafld), a simple screening tool is necessary. in this study, we aimed to devise a simple index that reflects the presence of nafld in the korean population. methods: a cross-sectional study was conducted on 10,724 health check-up subjects at a healthcare center (5,362 cases with nafld versus age-and sex-matched controls). study subjects were randomly assigned to a derivation cohort or a validation cohort. an index reflecting the presence of nafld was derived in the derivation cohort and validated in the validation cohort. results: multivariable analysis indicated that body-mass index (bmi), serum alanine aminotransferase (alt) to serum aspartate aminotransferase (ast) ratio, sex, and the presence of diabetes mellitus were independent predictors of nafld. using these variables, a formula was derived using a linear regression model: nafld index (nafldi) = 8×alt/ast ratio +bmi (+3, if female; +2, if diabetes mellitus).nafldi had an area under receiver-operating curve of 0.812 (95% confidence interval, 0.801-0.824). at a value <31.0, nafldi ruled out nafld with a sensitivity of 89.0% and a negative likelihood ratio of 0.22, and at a value >36.0, nafldi detected nafld with a specificity of 91.2% and a positive likelihood ratio of 5.42. in the validation cohort, the predictive power of nafldi was maintained at similar levels.aim: since nash could progress to liver cirrhosis and hepatocellular carcinoma, it is important to correctly diagnose between nash and simple steatosis (ss). the aim of this study was to determine the prevalence of nash among nafld patients and to clarify differences in clinical features between nash and ss. subjects and methods: thirty-one patients with nafld showing abnormalities in serum transaminase (ast and /or alt >40 iu/l) were enrolled (sex: male 11, female 20; mean age: 49.5 yrs, mean body mass index: 29.3), after obtaining informed consent. differential diagnosis between nash and ss was performed histologically according to the matteoni classification and clinical features were compared. results: among the patients with nafld, 81% and 19% were diagnosed with ss and nash, respectively. no significant differences in the sex, mean age and bmi were seen between nash and ss groups. the levels of ast, alt, homeostasis model assessment-insulin resistance (homa-ir) and hyaluronic acid were significantly elevated in nash patients compared to ss patients. no significant differences in serum levels of adiponectin, as well as the rates of occurrence of diabetes, hypertension and hyperlipidemia were observed between the two groups. conclusion: the prevalence of nash in nafld patients was about 20%. nash patients showed higher levels of serum transaminase, homa-ir and hyaluronic acid, compared to ss patients. a large-scale biochemical study is required to accurately diagnose nash patients and confirm these results. conclusion: nafldi was a simple, efficient screening tool for nafld that could be utilized for selecting individuals for liver ultrasonography and for determining the need for lifestyle modifications.pe432 aim: this study was conducted to evaluate the hepatoprotective effects of the centella asiatica extract in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury in rats. methods: sprague dawley rats were treated with alcohol extract of centella asiatica orally in two doses (20 and 40 mg/kg/day) for 3 months along with intraperitoneal injection of mptp (1 ml/kg). biochemical parameters such as serum total protein, albumin and marker enzymes were estimated. histopathological studies of liver were also carried out to confirm the biochemical changes.results & discussion: mptp -induced hepatotoxic effects were evident by a significant (p < 0.05) increase in the serum marker enzymes and a decrease in the total serum protein and albumin. administration of extract of centella asiatica effectively inhibited these changes in a dose-dependent manner; maximum effect was with 40 mg/kg. histopathological examination of liver tissue corroborated well with the biochemical changes. hepatic steatosis, hydropic degeneration and necrosis were observed in mptp-treated group, while there was a significant reduction in these changes in the treatment group. conclusion: centella asiatica extract exhibited hepatoprotective action against mptp induced liver injury. further optimization of tuberculosis chemotherapy requires a comprehensive evaluation of the effects of antitubercular drugs on metabolic processes in organism.wistar albino male rats, body weight (b.w.) of 160-200 g, were divided into three groups: group i received pyrazinamide per os at a dose of 1000 mg/kg b.w./day, whereas group ii received a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; the control group was composed of intact animals. the contents of free amino acids were determined using an amino acid analyzer -881 (czech republic). the study of the effects of pyrazinamide administered in different doses on the liver contents of free amino acids showed the largest number of changes at a dose of 1000 mg/kg b.w./day. the content of free amino acids at the level of 16 amino acids and total sum of amino acids significantly differed from controls. part of these changes could be regarded as compensatory answer of organism to this drug action. further pyrazinamide dose increasing caused exhaustion of liver adaptive possibilities. the study of the influence of pyrazinamide on liver contents of free amino acids allows to fully estimate the effects of this substance on metabolic processes in this organ. moreover, the effect of pyrazinamide on the majority of free amino acids in the liver is dose-dependent. background: taiwan is an endemic area of hbv and hcv infection, chemotherapy for lymphoma patients who has been hbv infection, may induce serious clinical sequela due to reactivation of hbv. this study want to clarify the difference of the chemotherapy induced hepatitis between hbv and hcv carrier in lymphoma patients. methods: from july, 2003 to july 2006, 537 non-hepatocellular carcinoma patients were enrolled, 49 (12.5%) cases were lymphoma, in these lymphoma patients, 24(48.9%) cases have been hbv infected, and 22 (44.9%) cases have no hbv or hcv infected. 3 (0.6%) cases have been infected with hcv. all patients received chemotherapy with the regimen of chop or r-chop. liver function , viral markers, hbv dna, hcv rna were checked before and after chemotherapy. results: hepatitis happened in 16 (32.7%) lymphoma patients, 11 (45.8%) cases were in hbv infected patients, 3 (12%) cases were in non-hbv infected patients, 2 cases of hcv infected patients suffered from hepatitis. hbv infected hepatitis patients hbv dna elevated more than 2 log as before chemotherapy. non of the hcv infected patient has elevated of hcv rna after chemotherapy. the mortality rate in hbv infected patient is 33%. no mortality in hcv infected patients after chemotherapy. conclusions: 1.high rate of hbv reactivation and mortality in chemotherapeutic lymphoma patients who has been hbv infected. screening of hbv viral markers among candidates for cancer chemotherapy is mandatory, especially in lymphoma patients. large number and prospect study for chemotherapy induced hepatitis in hcv carrier are needed. background: excessive drinking leads to social, psychological, physical and other problems. this study investigated the epidemiology of ald and analyses the associated risk factors. methods: from 6,043 residents 3,815 blood samples were collected. alcohol consumption and the impact of alcohol on liver function, blood lipids, blood pressure and bmi and mcv have been evaluated. results: the drinking rate and average daily alcohol intake was 35.0% and 36.97±48.76g respectively. the total alcohol intake was 297.90±506.52kg and the average drinking age was 19.21±11.34 years. the average -gt, ast, alt, mcv, chol, tg, ldl-c, hdl-c and bp increased gradually with increase in alcohol intake. the population ald prevalence was 3.98%. the prevalence of ald among the drinking population and the alcoholic population was 11.76% and 44.17% respectively. conclusion: chol, -gt, ast, alt, and mcv were highly correlated with daily alcohol intake which closely related to the occurrence of ald. n. tanaka 1 , w. okiyama 1 , t. aoyama 1 background: alcoholic liver disease (ald) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. polyenephosphatidylcholine (ppc), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons. however, its precise mechanism remains uncertain. we examined the effects of ppc on ald using peroxisome proliferator-activated receptor (ppara)-null mice treated with an ethanol-containing diet, which showed pathological features similar to human ald. methods: male ppara-null mice were pair-fed a lieber-decarli control or 4% ethanol-containing diet with or without ppc at a clinically comparable dose (30 mg/kg/day) for 6 months. o. parkash 1 , a. almas 1 , s.h. ali shah 1 , w. jafri 1 , s. hamid 1 , j. akhtar 1 1 aga khan university hospital karachi 1.hdv-hbv co-infection presents mostly as moderate to advanced liver disease. background: liver injury due to dengue infection is not uncommon. acute liver injury is a severe complicating factor in dengue, predisposing to life-threatening hemorrhage, dic and encephalopathy. results: there were no significant differences of demographic features and laboratory parameters such as peak serum alt, total bilirubin and creatinine between 2 groups. however, peak ast was higher in superinfected group than control group (median: 2,000 iu/l vs 731 iu/l, p=0.035,). additionally, the peak serum albumin levels, prothrombin time and platelet counts were lower in superinfected group than control group (median: 3.0 mg/dl vs 3.3 mg/dl, p=0.02, 51.8 % vs 87.2 %, p=0.027 and 103 x 10 3 /mm 3 vs 165 x 10 3 /mm 3 , p<0.001, respectively). of superinfected group, 9 patients were followed over 6 months after resolution of aha. interestingly, serum hbv-dna levels decreased significantly over 3 months following resolution of aha, then rebounded subsequently (median: -1.89, -1.85, -0.38, 0.58 and 1.10 log10 copies/ml at 1, 3, 6, 12 and 24 months, respectively).methods: the overlapping fragments of hev isolate swgx40 were amplified with reverse-transcription nested polymerase chain reaction (rt-npcr) and the 5' and 3' ends of viral genome were amplified with rapid amplification of cdna ends (race). the pcr products were cloned and sequenced. the phylogenetic analysis of swgx40 was performed.result: the genome of swgx40 consisted of 7,233 nucleotides, excluding the poly (a) tail of 36 residues. the genome contained three open-reading frames (orfs), orf-1 encoding 1705 amino acids, orf-2 encoding 674 amino acids and orf-3 encoding 114 amino acids. the full-length genomic sequencing showed that swgx40 strain shared similarity with all known hev genotype 1, 2 and 3 isolates by 73.4% to 76.5%, and with an identity of 83.1% to 91.2% among genotype 4 hev isolates, and a high nucleotide identity as 94% with chinese guangxi human strain lz-105.conclusions: acute hav super-infection may suppress hbv-dna replication in chronic hbv carriers and chronic hepatitis b, although the suppressive effect did not seem to sustain longer than 3 months. conclusion: the swine hev strain swgx40 was phylogenetically close to the human hev strain lz-105, both from the same region in south china. therefore it was concluded that hev sub-genotype 4b might have existed in south china at least for 6 years and now it was prevalent both in local human and swine, which also strongly supported the zoonosis hypothesis of hepatitis e. associated with splenic volume were damping index (r=0.213, p=0.022) and blood flow of portal vein (r=-0.314, p=0.001). conclusions: the splenomegaly in portal hypertension was more frequent in non-alcoholic groups, and associated with a damping index and a blood flow of portal vein. the measurement of blood flow of portal vein, damping index, and splenic volume by a doppler sonography can be helpful to predict the varices.z.q. zhang 1 , j. cao 1 , w. lu 1 , l.g. shi 1 1 objective: to appraise the clinical efficacy of simple non-invasive models of ast-to-alt ratio (aar), ast-to-platelet ratio index (apri), spleen-to-platelet ratio index (spri), age-platelet index (api), age-spleen-to-platelet ratio index (aspri) for predicting hepatitis b associated cirrhosis. methods: 138 patients and 32 patients were diagnosed pathologically as non-cirrhosis and cirrhosis, respectively. the simple non-invasive models were calculated as described originally. spss 13.0 was used for statistical analyses.background: to reveal the microrna (mirna) expression profile of the hepatic fibrosis inducing cells, rat hepatic stellate cells (hscs), during in vitro activation. results: the areas under roc curve of aar, apri, spri, api, aspri for predicting the cirrhosis were 0.81, 0.62, 0.71, 0.73, 0.74, respectively which were larger than those under the diagnosis reference line (p 0.000, 0.033, 0.000, 0.000, 0.000, respectively).methods: the hscs were isolated from male sd rats by in situ perfusion and density-gradient centrifugation. the quiescent and activated hscs, which were harvested at day 2 and 14, respectively, were then subjected to immunocytochemical staining (desmin and -sma), oil red o staining and quantitative rt-pcr (desmin, -sma, albumin, cd31, cd68 and cytokeratin-19). after extraction and labeling, the hy3-labeled cellular rna samples and hy5-labeled reference pool rna samples were mixed pair-wise and hybridized to the lna mercury microarray. differentially expressed mirnas were filtered and randomly verified by stem-loop rt followed by quantitative pcr.conclusion: all of the simple non-invasive models of aar, apri, spri, api, aspri can be used for predicting hepatitis b associated cirrhosis; and aar has the most practical efficacy for predicting hepatitis b associated cirrhosis. results: both the purity and the total activation of hscs were validated. global analysis of the mirna expression profile based on quiescent and activated hscs demonstrated 21 differentially expressed mirnas. among these, 12 mirnas were up-regulated more than 2-fold in activated hscs as compared to that in quiescent hscs, while 9 mirnas were less than the threshold level (0.5-fold) during the hsc activation. furthermore, the expression of mir-16, 15b, 122, 138, 143 and 140 had been proved. background/aims: only limited patients with chronic hepatitis b virus (hbv) infection will develop liver cirrhosis, and no effective methods to precisely predict ones who will develop cirrhosis. we try to establish a model to predict the patients with the risk of cirrhosis development basing on a clinical epidemiological factor survey. z.q. zhang 1 , w.y. bao 1 , w. lu 1 , l.g. shi 1 methods: cirrhosis patients with hbv markers (case group) and asymptomatic hbsag carriers (control group) were recruited and inquired by researchers with a specific designed questionnaire including 98 items. a multivariate logistic regression analysis were conducted to establish a predictive model, in which two third patients selected randomly as model sample and another 1/3 patients as validating sample, key factors screened out as variables. the predictive performance of the model was evaluated. objective: to explore the practical significance of the peripheral blood corpuscle counts for prediction of hepatitis b associated cirrhosis. methods: 122 and 31 male patients with chronic hepatitis b were pathologically diagnosed as non-cirrhosis and cirrhosis. peripheral blood corpuscle counts were measured by coulter ac•t diff hematology analyzer. results: red blood cell (rbc), platelet (plt), neutrophil (n) counts in cirrhosis were significantly lower than those in non-cirrhosis; and lymphocyte, mid-cell counts were similar to those in non-cirrhosis. the areas under the roc curves of rbc, plt, n counts for prediction of cirrhosis were 0.24, 0.32, 0.34 respectively; according the optimal cut-off determined by the roc curves, the sensitivity, specificity, positive predictive value, negative predictive value, accuracy of rbc, plt, n counts for prediction of cirrhosis were 0.55-0. method: pbmcs of 8 active chb patients under pyg-interferon treatment were analyzed for their th17, treg and pdc by flow cytometry. they were determined by cd4/il-17 for th-17, cd4/cd25/foxp3 for treg and cd123/cd86/cd14-for pdc. pbmc were collected every 4 weekly during the treatment until end of therapy (week 48) and every 12 weekly until the end of follow-up (week 72). alt was quantified at every time of the pbmc collection. ifn-gamma release cells were analyzed by elispot to hbv-core and s ag.background: alpha fetoprotein (afp) is a well-recognized tumor marker for hcc; elevated level of afp is found in at least 70% of hcc. other liver diseases such as cirrhosis and chronic hepatitis are also related with an elevated level of afp. the regulation of afp gene expression has been relatively less studied although the gene has been suggested to play a role in hcc development. in this study, we tried to identify genetic variations in afp gene and analyze its effect on serum afp level and possible hcc progression.results: in parallel with decline in alt for the first 12 weeks, we found decline in both pdc (r=0.71, p=0.03) and elispot (r=0.65, p=0.03 for hbv-core ag; r=0.48, p=0.05 for hbv-s ag). although there is trend that th-17 decline with treg decline, but they are not statistically significant differences in the same period. there is no significant difference between the svp and non-svp patients.methods: direct dna sequencing was carried out to sequence afp promoter and 500 bp upstream and downstream of afp coding regions in dna samples isolated from 99 hcc subjects and 105 controls respectively. for each samples serum afp levels were determined using commercially available elisa kits. conclusions: under pegyintron treatment, pdc, ifn-gamma change in the same trend of alt during 12 weeks of treatment. it implied that pdc take regulatory effects on ifn-gamma releasing cells and be very tightly related to alt. there wasn't a significant difference in both treg and th-17, which implies that treg and th17 might be of important cells in keeping the stability of the immune system.results: a total of 30 snps were detected in the afp genomic region analyzed, including 29 known snps and one novel snp. among the identified snps, the c>g nucleotide change in the position -250 bp upstream of afp transcriptional start site showed a significant association with hcc (p < 0.05) and a decrease in afp gene expression level. conclusion: our preliminary results indicated a possible association between serum afp expression and -205g allele. the identified snp is located in afp promoter region with possible binding sites for known transcription factors, such as tfiid, coup, apf and nfiii. -30°tail-suspension (ts) rats were used as the model to simulate the physiological effects of weightlessness. thirty-two wistar male rats were randomly divided into 4 groups: control for 7d (7d con), 14d con, ts for 7 d (ts7d) and ts14d. histopathological changes of testicle of the rat were observed by he stain. localization and expression of ar and hsp70 in testicle of rat were observed comparatively by means of immunohistochemistry, and the density of ar and hsp70 immunoreactivety in four groups were compared. results: signal molecules mrna level are shown in the table 1 (** p<0.01, * p<0.05). tnfa, il1, il6 and il8 were higher in group 1, 2 and 4 than those in group 5. ifna was higher in group 5 than that in other groups. there are no significant difference in infc, il2, and il4. there was a positive correlation between tnf and myd88 in group 2, tnf and nfkb in group1 and 2. results: obvious pathological lesions presented in testicle of ts7d and ts14d rat. germinal epithelium irregularity and malformed spermatozoa were found in seminiferous tubules. degeneration and necrosis of germinal epithelium appeared in testicle of ts7d and ts14d rat. ar immunoreactive cell density in the ts7d and ts14d groups were significantly decreased compared with the in-phase normal control groups ( p < 0. 01). while hsp70 immunoreactive cell density in the ts7d and ts14d rats were significantly increased than those of control rats( p < 0. 01), and in testicular interstice or extracellular there were very strong ehsp70 immunoreactive positive staining signals. the results indicate that ground simulated weightlessness induced by 7d-14d tail-suspension in rats can lead to the serious injury , depressed expression of ar and enhanced expression of hsp70 in testicle. despite the absence of any serologic marker of hbv recurrence, however, it remains unknown whether there is occult reinfection in the liver graft. we aimed to detect and quantify the presence of intrahepatic hbv dna in the liver grafts of patients who remain seronegative for hbsag for more than 1 year after liver transplantation. materials and methods: liver biopsy and blood samples were obtained from 31 patients who had been receiving nucleoside analogue prophylaxis alone and remained persistently seronegative for hbsag for at least 1 year (median 44.5 months, range 13.6 to 126.4 months) after liver transplantation for chronic hepatitis b. quantitative polymerase chain reaction was performed to detect and quantify total and covalently-closed circular (ccc) hbv dna in the liver (lowest detection limit, 10 copies/ml), serum and pbmc. direct sequencing was used for hbv quasispecies screening. results: liver biopsy was performed and intrahepatic hbv dna as measured by quantitative real-time pcr was detectable in 26 of 31 recipients. donors anti-hbc status before liver transplant was significantly related to the presence of intrahepatic hbv dna in the recipient's study biopsy (p=0.038). donor intrahepatic hbv cccdna levels correlated with recipient post-liver transplant intrahepatic hbv cccdna levels (p=0.004). hepatitis b virus sequencing results and phylogenetic analysis revealed that hbv reinfection in two recipients were of donor origin, four recipients were of recipient origin and four recipients were of both donor and recipient origins. conclusions: our findings demonstrate the presence of occult hbv reinfection with persistence of hbv dna in liver allografts despite long term nucleoside analogue prophylaxis after liver transplantation, suggesting the need to continue indefinite antiviral therapy. the use of liver grafts from anti-hbc-positive donors might increase the risk of occult hbv reinfection. both donor and recipient hbv dna could contribute to occult hbv reinfection in liver transplant recipients. aim: to construct one noninvasive assessment model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with chronic hepatitis b(chb). methods: we have retrospective analyzed 137 consecutive patients with chronic hepatitis b who underwent percutaneous liver biopsy. we calculated sensitivity, specificity, positive predictive value(ppv), and negative predictive value(npv) of an apri 1.5 in combination with different hyaluronic acid(ha) cut-off points medium (acm). we stimulated pbmcs or dcs under hcm and acm, and evaluated the function. results: after adding the stimulants under hcm, the cd83 and cd86 expression of dcs from cirrhotic patients (lc) were lower than those from healthy contorols (hc). in both hc and lc, the cd83 and cd86 expression of dcs stimulated under acm was lower than that under hcm. the il-12 production in acm was lower than that in hcm. the expression of cd98, which is related to amino acid transport, was not different between hcm and acm. however, dcs cultured in acm expressed lower levels of phospho-p70 s6k than those cultured in hcm. finally, we ascertained that the ifn gamma production by pbmcs was significantly decreased under acm.conclusions: an imbalance in plasma amino acids of advanced cirrhotic patients suppresses the maturation of dcs via mtor/s6k signaling pathway. key: cord-023364-ut56gczm authors: nan title: education day monday: plenary session 1 monday: parallel sessions date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00651.x sha: doc_id: 23364 cord_uid: ut56gczm nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023346-8sqbqjm1 authors: nan title: monday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00652.x sha: doc_id: 23346 cord_uid: 8sqbqjm1 nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-023354-f2ciho6o authors: nan title: tuesday plenary session 3 tuesday: posters date: 2005-06-08 journal: vox sang doi: 10.1111/j.1423-0410.2005.00654.x sha: doc_id: 23354 cord_uid: f2ciho6o nan from the perspective of causal inference, there is a hierarchy of evidence, ranging from case-series to large randomized controlled trials (rcts). in addressing a particular clinical or policy problem, clinicians or policy-makers can base their decisions on the types of clinical reports that have been published, along with an assessment of the strengths and weaknesses of each study. rcts are controlled clinical experiments in which patients are randomly allocated by the investigators to receive a treatment under study. if randomization is used, all participants are equally likely to be allocated to either the treatment or the control arm of the study. rcts should be distinguished from observational studies in which investigators passively observe patients who happen to receive a treatment under study. because the allocation of subjects to the treatment and control arms of an rct is random, the play of chance should distribute all confounding factors equally between these two arms. thus, the treatment and control arms of an rct should be equivalent with respect to all confounding factors except for the treatment under study. randomization removes selection bias, because neither the investigator nor the participant knows what the treatment allocation will be before a patient enters a study. moreover, if an rct is double-blind, observation bias is also removed, because preconceived notions about benefit from the treatment cannot color the reporting of symptoms by a patient or the assessment of disease activity by an investigator. when the undertaking of rcts is deemed unlikely, meta-analyses of individual patient data, that is, of the data recorded previously on subjects enrolled in published, small rcts may allow investigators to address issues of possible biases 2ed-01-01 standardizing blood components would allow better monitoring of the effectiveness of transfusion d davenport university of michigan, ann arbor, mi, usa in routine transfusion of red blood cells (rbc) or platelet concentrates (pc), we have only a very rough idea of the dose we administer. the minimum expected hemoglobin content of rbc should be 50 g (fda criteria). however, actual measurements have found a mean hemoglobin content of 46.6 ± 11.7 g per unit, with variability between manufacturers. 25 percent of units may contain less than 34.9 g of hemoglobin while 25 percent may contain more than 56.3 g. the effect of storage senescence can magnify these differences. the resulting hematocrit increase, depending on size of recipient and age of a unit, may be 1.5 to 9 percent. apheresis collection of rbc can help standardized unit contents, but this technology is relatively expensive and time consuming. knowledge of actual hemoglobin content can be used to optimize red cell transfusion. one trial, using a simple formula incorporating the desired hemoglobin and estimated blood volume, showed that nearly 60 percent of transfusion orders could be met with one unit while achieving a mean target hemoglobin of 9.3 g/dl. for pc transfusion, about 66 percent of transfusions achieve the targeted dose of 3-6 ¥ 10 11 platelets in actual practice, with considerable variability between institutions. without knowledge of the actual content of pc, determination of refractoriness and response to matched pc is problematic. standardization and labeling of rbc and pc units for content will permit accurate dosage and quantification of transfusion practice. are transfusion guidelines evidence-based? jp aubuchon dartmouth-hitchcock medical center, lebanon, nh, usa application of the results of randomized, controlled clinical trials to similar clinical situations should increase the predictability of the outcomes of transfusions. unfortunately, too few such trials have been conducted to investigate all the potential situations where transfusion might be applied. however, the ones that have been reported indicate that, in general, transfusion is unlikely to provide substantial benefit in many of the situations where it is routinely used. in the intensive care setting, transfusing red cells at a trigger point of 7 g/dl rather than 10 g/dl not only did not lead to increased anemic morbidity but was actually associated with a reduction in overall mortality. subgroup analyses indicated transfusion at the higher trigger point did not decrease morbidity in patients with cardiac disease nor decrease the time until weaning from a ventilator. following cardiac surgery, a transfusion trigger of a hematocrit of 25% yielded the same outcomes as one of 32%. an observational study suggested that patients with peripheral vascular disease and a post-operative hematocrit below 28% were more likely to suffer a morbid cardiac event, but this group of patients had more evidence of anemia and cardiac ischemia preoperatively, illustrating the pitfalls of observational studies. most would agree that transfusion is necessary below a hb of 7 g/dl and unlikely to be necessary at a hb above 10 g/dl, but most patients fall between these limits, and there are insufficient data in the and residual confounding factors, and may permit them to make a judgment of a causal relationship. sackett proposed that the evidence generated from meta-analyses of individual patient data be regarded as being equivalent in strength to that generated from rcts. meta-analysis is the structured and systematic integration of information from different studies of a given problem. when the results of individual studies are discrepant, the purpose of an overview is to investigate reasons for disagreements among studies. when the results are concordant, the goal of meta-analysis is to derive, through application of a number of quantitative techniques, a measure of the effect of the intervention across combined investigations. this measure is referred to as the 'summary' effect of the treatment under study. meta-analysis differs from the traditional, narrative reviews of the literature, in that: (1) all completed investigations of the efficacy of an intervention that meet specific, eligibility criteria are retrieved and included in the overview; (2) the quality of retrieved studies is assessed systematically; (3) the degree of agreement among studies is evaluated, both conceptually and based on statistical criteria, and the synthesis of the findings proceeds only if the variation in reported results is sufficiently modest to be attributed to chance; and (4) quantitative methods are used to calculate the summary effect of the intervention and to test that effect for statistical significance. 2ed-02-04 biology of stem cell mobilization th papayannopoulou university of washington, seattle, wa, usa at baseline hematopoiesis, the majority of developing hematopoietic cells (precursors, progenitors and stem cells) is actively retained in bone marrow (bm), whereas fully mature cells emigrate to peripheral circulation. a small number of stem/progenitor cells are also present in blood, serving presumed physiologic roles for the repopulation of remote damaged areas. however, in several hematopoietic perturbations, i.e. post irradiation, post chemotherapy or by using empiric treatments, a heightened emigration (mobilization) of progenitor/stem cells was noted over 30 years ago. the introduction of g-csf as an efficient mobilizing agent not only had a major clinical impact, but has triggered a flurry of studies exploring the mechanisms of mobilization. from these studies, significant insight has been gained about the molecular pathways leading to mobilization, especially by studying the altered environment within bm post mobilization, and less so by studying cells mobilized in blood. several attractive scenarios have been proposed and their importance was further bolstered by studying genetic mouse models. a prominent role of the sdf-1/cxcr4 pathway has been emphasized, either by down regulation of cxcr4, changes in sdf-1 gradients, or by disruption of sdf-1/cxcr4 signaling. whether this pathway is disrupted by a proteolytic mechanism prevailing within bm post g-csf or by other protease-independent mechanisms has not yet been settled. in addition to sdf-1/cxcr4, disruption of other pathways responsible for the retention of primitive cells in bm can lead to mobilization. for example, disengagement of the vla4/vcam-1 pathway by anti-functional antibodies or its genetic deficiency in mice results in egress of stem/progenitor cells. mobilization is also seen following the use of other cytokines (i.e. kit-l, flt-3 l, il-3) or chemokines (i.e. il-8, gro?), complement activation, etc., but the detailed mechanisms or the interdependence of these other pathways with the ones already proposed have not been worked out. finally, efforts to improve mobilization efficiency in man has led to the use of combination treatments with g-csf, either by adding another cytokine (i.e. growth hormone), agonistic sdf-1 molecules (i.e. ctce0021), or cxcr4 antagonists (i.e. amd3100) which have yielded synergistic increments, and these will be discussed. a full understanding of the principles of mobilization, as well as the bm homing characteristics of mobilized cells after their i.v. infusion in transplantation, should lead to targeted, more efficient experimental protocols in the future. the possibility of deriving all kinds of mature cell types from embryonic stem (es) cells for the purpose of cell replacement therapies will probably face a major obstacle; a limited supply of available hla types for an ever growing population in demand. one possible solution is to use adult sources of stem cells such as the haematopoietic stem cells (hsc) that can repopulate the entire haematopoietic system after transplantation in a myeloablated host. however cells with the repopulating ability of hscs are still elusive for tissues such as muscle, heart, cns and pancreas. it was therefore exciting news when it was shown that hscs could repopulate other non-haematopoietic tissues arguing for a general role of bmderived cells in tissue regeneration. the term plasticity was thus coined to describe the phenomenon whereby cells of one lineage could trans-differentiate into cells of another tissue. this in turn implied that a certain degree of developmental plasticity was still available in the adult hsc, or their derived progeny, that allowed them to present with novel phenotypes. how exactly this was accomplished was a matter of speculation. in order to address the mechanism we used an animal model of liver failure where bmt with normal (wild type, wt) hsc was shown to rescue the liver failure; the repopulating hepatocytes apparently differentiated from the sole source of wt cells, the bm compartment. by studying the genetic composition of the regenerating liver nodules we observed that both wt and mutant dna was present in the nodules, a finding that was consistent with bm-derived cells fusing with host hepatocytes. the mechanism of plasticity was therefore directly related to the exposure of the donor bm-derived wt nucleus to the transcriptional environment of the hepatocyte and the expression of hepatocyte-specific genes. this fusion mechanism was also shown to underlie perceived cases of haematopoietic cell transdifferentiation to purkinje cells in the cerebellum and to cardiomyocytes. however, there are carefully executed studies that strongly support the alternative transdifferentiation mechanism in tissues such as epithelia or the epidermis. what these observations may imply is that in tissues with high rates of cell turnover, there may be a potent stem cell niche that can reprogram incoming cells to follow relevant cell lineages. if this hypothesis is correct, then the major research effort should focus on what makes the niche and whether it can be recreated faithfully in vitro so as to educate hscs to diverse lineages opening the way for realistic cell replacement therapies. collection and distribution of blood and its products to meet the medical needs of industrialized countries are typically managed through large-scale national or nationally-supported blood programs. the development, maintenance, and ultimate success of such programs are all components of a process that involves the delicate balance of continuously competing pressures, e.g. social, economic, ethical, political, regulatory/legislative. as with all endeavors that directly affect human life, safety is a central, unifying theme of this process. because transactions of blood inherently involve risks at both donation and reception/transfusion ends, efforts to enhance and maintain an acceptable level of safety generally rely on a focused program of risk management (rm). rm involves three equally important elements: identification/evaluation of risk factors in a process, control of exposure to them, and continuous monitoring to assess the effectiveness of countermeasures and the emergence of new risk factors. specific approaches to rm and quality assurance in transfusion medicine will be presented. examples from blood programs currently in effect in selected countries will be discussed. rm efforts within the corresponding blood program in greece will then be examined. the lack of data to adequately assess the status of this program suggests that at least one of the elements of rm -monitoring -can still be improved. a preliminary research effort aims to survey blood donors, transfusion recipients, and physicians in order to build an understanding of the specific factors that drive the perception of risk in the greek population. the findings form important stepping points upon which specific recommendations can be made for the development and maintenance of a comprehensive, effective rm program in greece. 2ed-03-08 the use of haemovigilance data to increase safety in transfusion medicine haemovigilance is a surveillance of all the procedures in the transfusion chain. the intension is to collect and assess information on unexpected or undesirable effects in bleeding of donors and transfusion of blood components. in europe haemovigilance was introduced as a concept in the middle of the nineties. the first national reports on haemovigilance appeared in the late nineties, and were only dealing with complications related to transfusion. later on other kind of events like 'near miss' events were added. nowadays national reports are dealing with all steps in the transfusion line, and to some extend also with complications in blood donors. the state of the art is haemovigilance with retrospective registration of unwanted events that has happened, but also a more active prospective part with an early warning in a rapid alert system about new threats in the transfusion world. the aim of the different national haemovigilance systems has been to improve safety in the transfusion line. after the first 10 years with haemovigilance in the european countries, what has been the outcome of this big effort? data from national reports do not signify major improvements, but safety is suggested to have improved due to many minor changes of procedures and awareness of dangerous situations. however, in most countries nothing really effective has been done to avoid failures, the most important cause of serious complications in transfusion of patients. systems which can prevent most of the failures are on the market. the cost of these equals the cost of nat screening for virus introduced in the same period of time. the common perception of transfusion risks is still much more focused on the hypothetical risk of virus transmission than to the demonstrated magnitude of severe complications related to bleeding of donors and transfusion of patients. therefore, to increase safety in transfusion medicine the nature and occurrence of the risks should be revealed by haemovigilance and not less important the results should be published with the aim to get a realistic common perception of the transfusion risks. the role of diagnostic testing to identify congenital vs acquired disorders of hemostasis and to optimize the management of perioperative bleeding g despotis washington university school of medicine, st louis, mo, usa excessive bleeding with trauma or after surgery can result in hypoperfusion and anemia related end-organ dysfunction and mortality as well as transfusion-related complications. several large studies have demonstrated that if bleeding is excessive after cardiac surgery to the point that reexploration is required, that overall mortality increases by 3-4 fold. transfusion related complications include the following potentially lethal complications: disease transmission of pathogens, acute hemolytic reactions, allergic reactions, transfusion associated acute lung injury, allo-immunization related disease (e.g. post-transfusion purpura, platelet refractoriness, transfusion-associated graft-vs-host disease) and other potential complications (e.g. increased perioperative infection or multi-organ system failure) related to transfusion associated immune modulation. in addition, blood shortages related to increasing consumption (i.e. expanding geriatric population) and/or a shrinking supply (i.e. related to exclusion of donors related to donor exclusion criteria designed to prevent disease transmission) may limit our ability to adequately manage our anemic and bleeding patients. this highlights the relative importance of accurate diagnosis and optimal management of excessive bleeding to minimize bleeding related complications and conserve our blood supply. patients at risk for excessive bleeding include those with an established hereditary disorder (e.g. vwd, hemophilia, connective tissue disorders), end-stage hepatic or renal disease as well as patients with acquired defects of the hemostatic system. in specific, patients with platelet (i.e. platelet refractoriness) or coagulation factor inhibitors at increased risk, extracorporeal circulation related defects or as related to certain pharmacologic agents). patients who require longer periods of extracorporeal circulation for cardiac surgical procedures (e.g. repeat, combined procedures or use of deep hypothermic circulatory arrest) are at a greater risk of developing excessive bleeding (e.g. cpb-related abnormalities). in addition, patients receiving one or more longacting anti-thrombotic medications in the immediate preoperative period (e.g. plavix, reopro, low molecular weight heparin etc) are at increased risk for bleeding. clinicians in the past have resorted to empiric and 'shot gun' approaches when managing excessive bleeding due lack of immediate availability of results from laboratory-based coagulation tests. on this basis, the use of point-of-care (poc) tests of hemostatic function to facilitate the optimal management of excessive bleeding, help differentiate between microvascular vs surgical bleeding and reduce transfusion have been investigated. five of six recently published studies have demonstrated that implementation of a standardized approach to manage bleeding (e.g. algorithm) which when coupled with either point-ofcare or laboratory tests of hemostatic function can optimize the management of bleeding and reduce total donor exposures by 50%. ddavp has bee shown to be beneficial with uremia-induced platelet dysfunction and with type i von willbrand's disease. although previous studies have not been able to conclusively show that ddavp can reduce bleeding and transfusion when administered prophylactically, more recent evidence indicates that this agent may be useful in preventing excessive bleeding when a test (point-of-care) reveals platelet dysfunction. recombinant activated factor vii (rfviia) is licensed for use in bleeding episodes in hemophiliac patients with inhibitors. although, only anecdotal reports and results from small clinical studies have shown that this agent can reverse life-threatening bleeding after major surgery, other reports indicate that there is variability in the effectiveness of rfviia as well as highlight lifethreatening thrombotic complications in a subset of high risk patients (i.e. patients with congenital or acquired thrombotic disorders or systemic activation of the hemostatic system such as with dic or after cardiac surgery). therefore, large clinical trials evaluating the efficacy and safety of rfviia are needed before any widespread use can be recommended. in recent years, molecular methods of blood grouping have become routine diagnostic procedures in many laboratories throughout the world. they have proved especially valuable for the determination of fetal blood groups. the ability to detect rhd in pregnancies where the fetus is at risk from haemolytic disease of the newborn represents a significant advance in obstetric care. furthermore, the occurrence of fetal dna in the mothers' peripheral blood has allowed this diagnostic procedure to be carried out without the risks that accompany amniocentesis (lo ymd. (1999) ann med 31:308-312). determination of the coding sequence of all the genes giving rise to antigens within the 29 blood group systems currently recognised is virtually complete. by correlating the coding sequence of these genes with the phenotype of red cells from individuals with different blood groups it has been possible to infer the molecular bases of most of the antigens comprising each blood group system (daniels gl (2002) human blood groups, 2nd ed. blackwell science). the polymorphic antigens of most systems other than abo and rh, are defined by single nucleotide substitutions (snps) which effect a single amino acid sequence change in the gene product. numerous methods, both manual and automated, are available to determine snps and these have been applied to the determination of blood groups. useful applications of snps detection methods include, determination of the blood group phenotype of patients who have been transfused recently and still have donor blood in their circulation (rozman p, dove t, gassner c et al. (2000) transfusion 40:936-942), and donor screening to find compatible units for patients with multiple blood group antibodies or for the management of patients with diseases like the haemoglobinopathies who are going to be transfusion-dependent over many years (castilho l, rios m, bianco c et al. (2002) transfusion 42:232-238). other applications of molecular methods include zygosity determination for rhd, determination of the blood group phenotype of patients with a positive direct antiglobulin test, selection of donors with rare blood group phenotypes, and as aids to the solution of complex blood grouping problems in the reference laboratory. the molecular bases of abo and rh antigens are complex and cannot be determined reliably by a single snp. nevertheless, methodologies are available that allow the comprehensive sequence analysis of individual genes and could be applied to abo and rh typing. whether or not this will ever be a cost-effective procedure is a moot point. it is clear that molecular methods are a useful addition to the range of diagnostic procedures available to transfusion medicine practitioners but it is important to remember that methods based on dna analysis determine phenotype by inference and not by direct measurement. the results of molecular tests should be interpreted with this caveat in mind. 2ed-06-18 the hla system g stavropoulos general hospital 'g. gennimatas', athens, greece since its discovery in the mouse in 1936 the major histocompatibility complex (mhc) has become one of the most important region in the vertebrate genome with respect to infection, autoimmunity and transplantation. mhc primary function is to provide protection against pathogens. this is achieved through sophisticated pathways in which mhc class i molecules present endogenous antiges to cd8+ t cells and class ii molecules present exogenous antigens to cd4+ t cells. an increasing number of other proteins are being found that support these two pathways; many of these proteins, together with the class iii complement proteins, also map to the mhc. the mhc molecules were originally studied for their ability to cxonfer tolerance (histocompatibility) following tissue grafts or later, organ transplants. nowadays, the success of unrelated hematopoetic cell transplantation is influenced by the degree of mhc compatibility between the donor and patient. thus, for patients who lack matched donors, the rules that govern permissibility of mhc mismatching still need to be identified. for patients with high risk disease who lack matched donors, use of donors with a single mhc mismatch may permit early treatment before disease progression. scientific evidence to fully answer the questions 'when are platelet components clinically effective' and 'what do we really know?' is limited. despite this limitation and the level of uncertainty it generates with regard to treatment options and policies, it is reassuring to note that current prophylactic platelet transfusion protocols -mostly derived from empirical observations collected during the 1960's and 1970's -protect oncology patients from clinically relevant bleeding in more than 95% of thrombocytopenic days spent in hospital or at home, even in aggressive conditions such as leukemia, lymphoma and other severe blood diseases. this evidence seems to justify the prevalent policy of transfusing platelets when the patient's platelet count falls below 10 000 per microliter (or 20 000 in 'unstable' patients). this policy is based on positive outcomes of prospective and retrospective studies performed during the 1990's including some hundred non-surgical patients and on the desire of balancing the will of reducing patient exposure to limited, expensive, potentially infectious and immunogenic blood products with the objective of preventing clinically relevant hemorrhage. several national and international organizations endorse the above policy. although the role of platelet support in surgery or in specific clinical situations requiring invasive procedures or in patients affected by multi-organ and system co-morbidity suffers from even more limited evidence, the latter has been carefully collected and summarized in the guidelines for platelet transfusion published in j clin oncol 2001;19:1519-1538. additional data on lumbar puncture are reported in ann hematol 2003;82:570-573. another important topic where there is room for improvement and need for further investigation is platelet refractoriness, a condition developed by a proportion of chronic platelet recipients, which causes high hemorrhage risk if compatible platelets are not provided and in which consensus on the diagnosis and treatment is lacking. with regard to the type of platelet product, it will be necessary to determine the clinical impact of buffy-coat derived platelets, consistently used in europe and current object of increased interest in canada, as compared to the platelet-rich plasma method traditionally used in the us, of viral inactivation procedures and of laboratory methods for detection of bacterial contamination of platelet concentrates. in addition, ongoing studies on the clinical impact of high versus low platelet doses will provide novel elements to determine the relative merits of apheresis versus whole-blood derived platelets. as far as the established procedure of white cell reduction by filtration, which shows high technical efficiency and has been implemented as a standard by a number of institutions, debate is still ongoing on its equivalence with serologic screening for the prevention of cmv transmission, whereas general consensus supports its pre-storage use to prevent febrile, non hemolytic transfusion reactions. finally, although the high cost of filters do not allow a generalized use of this technology in many settings, white cell reduced platelets have been unequivocally shown to reduce alloimmune refractoriness from about 15% to about 5% of patients. 2ed-07-20 ffp: appraisal of the evidence for the clinical use of ffp although the indications for transfusion of plasma (fresh frozen plasma; ffp) are limited, the use of ffp continues to rise in the united kingdom and has risen by over 20% in the past few years. local uk audits continue to document that a significant proportion of ffp transfusions are not consistent with indications reported in guidelines. a systematic review of the evidence base for the effectiveness of ffp was therefore undertaken to identify, select and appraise all relevant randomised controlled trials, as the most robust form of study to assess effectiveness of ffp. in the systematic review of ffp, the criteria for inclusion of full-published studies were: there must have been at least two groups in the study; • allocation to the groups must have been either by formal randomisation or by a quasi random method e.g. alternation; • one of the arms of trial must include ffp or plasma as an intervention; results on the relevant clinical or laboratory outcome must be presented. the main analysis was qualitative, and differentiated between: 1. studies of interventions comparing ffp with no ffp; 2. studies of interventions comparing ffp with a non-blood product e.g. solutions of colloids and/or crystalloids; 3. studies of interventions comparing ffp with a different blood product; 4. studies comparing different formulations of ffp, e.g. solvent detergent and methodine blue treated. an evaluation of studies comparing ffp with no ffp would be expected to provide the clearest direct evidence for a positive effect of ffp. studies comparing ffp with colloids or crystalloids were separately appraised because these latter products may have variable effects on coagulation tests. studies comparing different formulations of ffp do not directly evaluate effectiveness of ffp but were included because there is now a uk national policy to use these products in younger patients and therefore these trials might identify negative outcomes. the search strategy identified a total 57 publications. although it may appear that this number of randomised trials might provide a reasonable evidence base to help inform clinical policy and decision making, the review identified a number of important concerns about the published trials. few of the identified studies included details of the study methodology (method of randomisation, blinding of patients and study personnel). the sample size of many included studies was very small (range 8-78 patients per arm). few studies took adequate account of the extent to which adverse events might negate the clinical benefits of treatment with ffp. many of the identified trials in groups such as cardiac, neonatal, and other clinical conditions, evaluated a prophylactic transfusion strategy. however, when these trials evaluating prophylactic usage were assessed together as a single grouping in the review, it appeared there was no consistent support for a beneficial effect of prophylactic ffp, irrespective of clinical setting. there is a pressing need to develop new trials to determine the effectiveness of ffp, including for those clinical situations in which it has become an accepted part of current transfusion practice. a law decided upon by the riksdag (the swedish parliament) often sets the general standards as a framework and authorises the central government authority concerned to elaborate and decide upon the more detailed regulations. laws and regulations define the level of quality and safety that has to be reached and state what must be done, while establishments and their managers are kept responsible for fulfilling the requirements and how this is done. guidelines (non-binding) present detailed instructions on ways how to fulfil the requirements. making a law is complicated and time-consuming. the ministry draws up a legislative proposal, refers the proposal to relevant bodies for consideration and comments, and then drafts a bill which is referred to the council on legislation for consideration before it is submitted to the riksdag. a parliamentary committee deals with the bill before it is put to the chamber of the riksdag for approval. when adopted, the bill becomes law. regulations elaborated by central government authorities are handled in a principally similar way. however, regulations are more readily adjusted to scientific and technical progress, and when legislation need requirements for technical details, these are preferably presented in a regulation. the ministry of health and social affairs is responsible for elaborating the bill to be submitted to the riksdag on the blood safety law. two central government authorities, designated as competent authorities, are responsible for preparing the appropriate complimentary regulations as well as for inspecting and licensing the blood establishments: • the national board of health and welfare (nbhw), responsible for requirements related to blood components intended for transfusion, and • the medical product agency (mpa), responsible for requirements related to blood components intended for the manufacture of medicinal products. sweden became a member of the eu ten years ago. since then, experts in transfusion medicine have been appointed by the ministry, nbhw and mpa for advice on scientific and technical issues and for representing sweden at expert meetings arranged by the commission. some of these experts are also members of the handbook committee of the national society for transfusion medicine, preparing and revising the national guidelines. consequently, the requirements of the directives are readily implemented in the daily work of the blood establishments before (due to legal and technical reasons) the new blood safety law and the revised nbhw and mpa regulations can be promulgated. to a great extent, requirements of the blood directives are covered by existing swedish legislation. no major problems or obstacles seem to arise when implementing new requirements into law and regulations and into the blood establishments' daily service. a few detailed requirements have been found difficult to follow precisely in the routine work. these minor difficulties, however, will not compromise the high quality and safety of blood components prepared according to the standards set by the blood directives. the landscape for blood collection and distribution includes availability and safety. true international availability of blood has not been reached. the landscape of safety has been mountainous, if viewed by the degree of frustration among the transfusion specialists. the reasons for frustration have varied from serological problems to those of transmissible infections, to which no end is in sight. mistrust in the safety of blood taken by others is one reason for lack of international landscape in blood collection and distribution. increasing demands on safety, availability and economy force the health care providers to reorganise blood services. national systems are winning ground. this may make it easier to achieve international collaboration. the council of europe has pioneered in creation of international recommendations for blood collection and distribution. in 1958 a european agreement on the exchange of therapeutic substances of human origin, including human blood, was published. its purpose was to make blood available to other parties of the agreement in case of urgent need. many countries ratified the agreement rapidly, some did it first in the nineties. in practice little exchange of blood components has taken place. the council of europe recommendations lack legal power. the european union is different, and it has taken on its agenda activities aiming at improving confidence in the safety of the blood transfusion chain in the community (commission communication 1994) . the agenda is based on an agreement reached at a high level eu meeting in adare, ireland in 1994. first in the commission agenda was a recommendation on donor selection criteria, given in 1998. then came the european blood directive 2002/98/ec, the aim of which is to improve the safety of blood and blood components within the union so that enough mutual trust between member countries can be achieved to make the exchange of blood components possible. being a legally binding document the directive is undoubtedly helpful in reaching a harmonised standard in europe. hopefully the european commission has the resources to follow its implementation. there are concerns, however. the epidemiological differences among the eu member countries and frequent appearance of new infectious agents potentially transmitted by blood make it difficult to foresee that even effective viral inactivation of blood components could totally erase the national differences in donor approval. blood collection and preparation of components are already the same in eu member countries and the directive helps in their further standardisation. there has not been much distribution of blood components internationally, with the exception of export of red cell concentrates to the us from switzerland and the netherlands. the european legislation opens new horizons and widens the european landscape as its purpose is to simplify the crossing of borders between the member states, but before true movement of blood components is achieved more work is needed on the eu commission blood agenda. the eu directive implemented into the legal act for polish blood transfusion service blood transfusion service (bts) in poland is an integral part of the public polish health service. in the country of nearly 39 million people, approximately one million units of blood and plasma are collected every year from voluntary, nonremunarated donors, which is statistically over 25 donations per 1000 inhabitants. blood and plasma are collected in strictly appointed centers and no private collection sites are permitted. the legal basis for the activity of polish bts is polish blood transfusion act of 22nd august 1997 which came into force as of january 1st 1999. this act was then updated in november 2003 according to eu directive 2002/98/ec and came into force as of january 13th 2004. this act introduced the principles for organization, collection, processing, storage, transport and quality assurance in bts. it specified -among others -the system of accreditation, haemovigilence, as well as requirements for bts employees. according to this act the polish bts is obliged to monitor and supervise immunohematology testing and transfusion procedures in all hospitals where blood and blood products are transfused. polish bts consists of 21 regional blood transfusion centers (rbtc), one military center and one ministry of internal affairs and administration center as well as the institute of hematology and blood transfusion (ihbt) which acts as supervisor. the 21 rbtcs have a uniform organization structure, uniform quality assurance system and act according to uniform guidelines issued by ihbt. they have two financing sources -the central budget and hospital reimbursement for distributed blood and blood products. the polish blood transfusion act of 22nd august 1997, in force since january 1st 1999, has been supplemented by 8 decrees: 1. procedures for external bts audits; 2. requirements for donor selection; 3. requirements and procedures for organization and safe management of blood transfusion in hospitals; 4. requirements for implementing of national and regional donor registers; 5. employment criteria for bts personnel; 6. training requirements for hospital personnel involved in blood and blood product administration; 7. national, uniform price list for blood and blood products; 8. organization requirements for setting up of a national committee for blood and blood transfusion. introduction: several technical aspects must be considered in pediatric apheresis due to the size of the patient. factors that must be evaluated are extracorporeal circuit volume, blood flow rates, type of anticoagulant and vascular access. adverse events are mainly related either to vascular access or to metabolic or hemodynamic changes. aim of the study: in this study we show our experience using fresenius hemocare com.tec for pbsc collection in children. methods: twelve pediatric patients (median age 8 years, range 2-16; median weight 38 kg, range 12.2-60.5 kg) with solid tumors at onset or on relapse underwent collections with the p1y kit of the fresenius hemocare com.tec blood cell separator. our cd34+ cells target was 10 ¥ 10 e 6 /kg. collections were started if a peak of at least 0.02 10e9/l cd34+ cells (20 per microlitre) were reached in the peripheral blood. in all the patients, leukapheresis were performed through a central venous catheter and temporary peripheral venous access. acd ratio 1 : 14-1 : 16 was combined with heparin 40 u/kg. in 3 children with <15 kg the separator was initialized with a compatible filtered and irradiated red blood cell unit, suspended in 4% albumin, up to the patient's hematocrit, to avoid transient hypovolemia, due to the volume sequestered in the separator. results: twenty-five procedures were performed. a median blood volume of 5400 ml (range 2.100-12.400 ml) was processed in a separation time of 270 min (range 180-330 min). the median product weight was 108 g (range 37-258 g) and yield of cd34+ cells was 3.2 ¥ 10e 6 /kg body weight (range 0.7-12.5 ¥ 10e 6 /kg body weight). three poor mobilizing patients (peripheral blood cd34+ peak of 20-26 cells per microlitre) underwent more than two apheresis to collect the desired transplantation dose (3.5 and 4). all collection procedures were well tolerated. children never required sedation to perform the leukapheresis. only mild hypocalcemia-related symptoms, promptly responding to small i.v. boluses of calcium gluconate, were reported. no circulatory side effects were observed. blood flow alarms occurred in every procedures but no collection had to be terminated due to insufficient flow. conclusion: in summary, leukapheresis in children can be safely and effectively performed with the fresenius hemocare com.tec separator with minimal technical difficulties even in patients under 15 kg. m-pa-007 alternative methods for prevention of infection transmission (pathogen inactivation etc.), cost-benefit considerations of the procedure itself. however, one must also take the loss of plasma into consideration -and more difficult: calculate the effects of changes in product quality, as reduced content of factor viii, protein s and other labile proteins. for methods involving pooling, there are also concerns about the risks due to the pool sizes. the major benefit of the methods will be the potential reduction of infectious transmission, but also possible advantages as reduction of allergic transfusion reactions and trali must be evaluated. the study types involved in cost-benefit considerations are costeffectiveness analysis where the cost and effects of an intervention and an alternative are presented in a ratio of incremental cost to incremental effect and cost-utility analysis, where quality-adjusted life years (qaly) are used as the effectiveness endpoint. qualityadjusted life years is a method that assigns a preference weight to each health state and estimates life-expectancy as the sum of these products of each preference weight and time spent for each state. a complete glossary of terms is found at http://www.hsph.harvard.edu/cearegistry. in literature, there are several publications on cost-benefit considerations within the field of transfusion medicine. concerning plasma products, the costeffectiveness of solvent-detergent plasma has been the major focus. however, the conclusions of different authors have been conflicting. in 1994, aubuchon and birkmeyer published a paper (jama 1994; 272(15) :1210-1214) where they concluded that the cost was usd 340 000 per qaly, which is far above the 'acceptable limit' of usd 50 000. this estimate was adjusted to usd 9.7 mill. per qaly in a 1999 letter to jama (jackson, jama 282). in 2003 , riedler et al. published (vox sang 2003 85:88-95 ) that the discounted cost/life year saved for sd-ffp use in the uk was gbp 22 278 for neonates and gbp 98 465 for patients aged 70. the main reason for the differences between the two papers (and others) was considered to be different calculation of non-infectious complications. the papers cited above underline the difficulties of the cost-benefit considerations. the age of the patient, the outcome of the treatment, the quality of life in an infected patient and the cost of side effects will differ. in addition, how much are we willing to pay for protection against emerging viruses? this paper will not provide the answers, but introduction: the photodynamic treatment of therapeutic plasma using methylene blue (mb) in combination with visible light is a well established procedure for the inactivation of blood borne viruses. aim of the study: evaluation of the quality and stability of mb/light-treated plasma (mb plasma) prepared under worst case conditions for routine processing. methods: single donor units (n = 12) were treated using the macopharma theraflex mb-plasma system which includes plasma membrane filtration (plas4) and addition of mb prior to illumination followed by mb and photoproduct filtration (blueflex). samples were taken before treatment and from the final product. additionally mb-treated plasma was prepared from four different plasma pools and stored for up to 9 months. treatment was done under worst case conditions for the preservation of coagulation factors: maximum mb concentration during illumination (1.15 mmol/l), maximum storage time of whole blood before separation (4°c, 17 h), maximum storage time of mb plasma before freezing (1 h). several plasma parameters and the concentration of mb and its photoproducts (azure a, azure b, azure c and thionine) were determined. results: mb/light treatment had a significant influence on thrombin time (+20.5%), fibrinogen (clauss) (-20 .4%), factor v (-15.6%), factor viii (-22.3%), factor xi (-13.3%) and protein c (-10.3%). no significant changes were detected for at iii, vwf : rco, vwf cleaving protease, plasmin inhibitor and alpha-1-antitrypsin. the entire virus inactivation procedure including the filtration steps for leukocyte depletion and mb and photoproduct depletion had no significant effect on activation markers (prothrombin fragment 1 + 2, thrombin-antithrombin-complex, d-dimers). no further essential loss of coagulation factor activity was observed during storage of the plasma at 30°c for 9 months. mb and its photoproducts (azure a, azure b, azure c) were depleted to a final concentration of <0.1 mmol/l. thionine was undetectable in all samples. conclusion: photodynamic treatment of fresh frozen plasma (ffp) using the theraflex mb-plasma system leads to only moderate decreases in the activities of different coagulation factors even under worst case conditions for routine production. mb and its photoproducts were effectively removed from the plasma by the blue-flex-filter integrated in the theraflex-system. the quality of mb plasma is well preserved during storage. pulmonary complications, particularly transfusion-related acute lung injury and circulatory overload, are the most common causes of transfusion-associated morbidity and mortality in the developed world. trali is a syndrome characterized by acute respiratory distress, hypoxemia, hypotension and pulmonary edema, occurring within 6 hours (usually 1-2 hours) of transfusion of a plasmacontaining blood product. other signs, including hypertension, leucopenia and hypocomplementemia, are less frequent. all blood products, except for albumin and solvent/detergent plasma, have been associated with trali, but red blood cells, platelets and ffp are the most common. the incidence is unknown, but 1 : 5000 plasma-containing transfusions is the most commonly cited figure. in 80% of patients, recovery is well underway within 96 hours, and leads to complete resolution. death occurs in 6-23%. the profile of the at-risk recipient has not been identified. recurrent cases have been infrequently described. there are two prevailing theories of pathogenesis: (1) antibody-mediated and (2) 2-hit hypothesis. evidence supports both concepts and neither is mutually exclusive. more than 60% of reported cases are associated with blood components containing either hla-specific (class i or ii) or hnaspecific antibodies. in 50% these antibodies correspond to at least one epitope in the recipient. most implicated components are donated by multiparous women. five percent of patients have hla or hna antibodies in their pre-transfusion serum. treatment requires prompt, assertive respiratory intervention, frequently necessitating mechanical ventilation. because trali has a much better prognosis than ards, it is an important diagnosis. some blood collectors are diverting plasma from multiparous donors away from ffp production or routinely screening for hla antibodies. the most effective method for identifying 'high risk' components has not been identified. introduction: trali is a life threatening adverse reaction of blood transfusion. it is characterized by noncardiogenic pulmonary edema developed soon after blood transfusion. in japan, we built hemovigilance system in 1993 and have been collecting the voluntary reports of severe adverse reactions of blood transfusion including trali cases since 1997. since the diagnostic criteria of trali have not been established until recently and no specific diagnostic markers for trali have been discovered so far, it is very difficult to make proper diagnosis of trali in each reported case. we have been collecting the cases with respiratory distress and pulmonary edema developed after blood transfusion as suspected case of trali. we reevaluated each report whether it meet the recommended diagnostic criteria for trali published in transfusion journal in dec. 2004 . aim of the study: purpose of this study is to select trali cases which met internationally recognized criteria so that it will reveal the possible causes of trali with laboratory testing for anti-leukocyte antibodies in donors and recipients. methods: the cases with respiratory failure and pulmonary edema are selected for evaluation from the adverse reaction case reports voluntarily reported to japanese red cross. in order to make a proper diagnosis of trali, we have been utilizing the respiratory distress questionnaire which has recently revised. this helps us eliminate other adverse reactions such as circulatory overload, cardiac failure, anaphylaxis and bacterial contamination, which results in selecting out the internationally recognized trali cases properly. for laboratory testing, flowpra and labscreen for hla antibodies and gift-fcm for hna antibodies are performed. the cross-matching test is also performed if possible. results: during past 8 years, 95 cases of trali and 29 cases of possible trali have been confirmed by critical review of each questionnaire. of 95 cases of definite trali, donor specimens were obtained in 84 cases. of 84 cases, anti-leukocyte antibodies were detected in 43 cases (51%) of donors' blood, which was significantly higher than the positive rate of anti-leukocyte antibodies in donors' blood of other adverse transfusion reactions (<10%). of 43 cases of antibody positive donors, anti hla antibodies were detected in 33 cases, anti hna antibodies were detected in 18 cases, and both were detected in 8 cases. of 33 cases of positive anti hla antibodies, class i antibodies were detected in 22 cases, class ii antibodies were detected in 28 cases, and both were detected in 17 cases. on the other hand, the anti-leukocyte antibodies were detected in 38% of trali recipients, and this rate is almost the same with that of positive rate of other adverse reactions of blood transfusion (39%). these results indicate anti-leukocyte antibodies in the blood donors are one of the prerequisites for developing trali from the antibody-hypothesis-oriented point of view. other cases with no detectable antibodies should be investigated in more detail in the future. thus, reevaluating trali cases based on recommended trali criteria will allow us to reveal new information about trali. of 2087 adverse events analysed by the serious hazards of transfusion (shot) scheme (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) , 556 (27%) were haemolytic transfusion reactions (htrs). 303 were due to incorrect blood component transfused (ibct): 226/303 were abo incompatible and 77/303 caused by other red cell antibodies. a further 40 cases were reported as acute htrs (ahtrs; i.e. occurring within 24 hours of transfusion) whilst 213 were recognised more than 24 hours after transfusion and reported as delayed htrs (dhtr). 21/556 (4%) patients died and 58 suffered major morbidity. htrs associated with ibct result from clinical or laboratory errors and are all preventable. it has been assumed that other htrs are unavoidable. closer scrutiny reveals that this may not always be the case, though review is hampered by incomplete investigations. 9/40 ahtrs occurred in group a (8/9) or group b (1/9) patients given group o platelets. of the 31 ahtrs related to red cell transfusion, 4 were due to errors, 9 were in patients with auto-antibodies, in only 4 of whom alloantibodies had been adequately excluded/identified. at least 24/213 dhtrs were potentially avoidable; in 18 cases the antibody was detectable retrospectively in the pre-transfusion sample; in 6 cases the presence of a previous antibody was not communicated to the laboratory. two patient deaths related to dhtr might have been avoided by earlier diagnosis and clinical involvement. 14% htrs reported to shot would have been avoided by compliance with pretransfusion testing guidelines and provision of group a platelets for all group a recipients. htrs can be clinically overlooked and inadequately investigated. national guidelines are needed for the investigation and management of htr, with focus on the identification of underlying causes to guide the choice of future component therapy. reference laboratories can provide valuable support in elucidating complex serological problems. patients treated with high dose chemotherapy and autologous blood progenitor cell (bpc) support may get malignant cells reinfused together with stem cells. we analyzed bpc products collected from patients suffering from germ cell cancer measuring malignant contamination and followed these patients for up to 98 months after transplantation also with the question if transplanted malignant cells influence survival. aliquots of stem cell apheresis products containing one million mononuclear cells were sedimented on glass slides and by immunocytochemistry quantitation of cytokeratin expressing cells was performed manually with light microscopy and by automated image analysis. in 11 of 35 patients (31%) cytokeratin expressing cells were detected in bpc apheresis products of patients treated for germ cell cancer. we followed the activity of the malignant disease of patients for more than eight years in median 60 months after transplantation. no significant difference in survival was demonstrated for our two patient groups. background: the use of adequate number of peripheral blood stem cells (pbsc), collected by mnc apheresis is essential for effective treatment of hematological malignancies, solid tumors, and other disorders by transplantation. aims: the goals of this study were: (a) to obtain an effective apheretic protocol for mnc harvesting, and (b) to compare the hematopoietic reconstitution after hspc transplantations in different clinical settings. methods: in this study, pbsc transplantations -60 allogeneic from matched sibling healthy donors and 60 autologous -were performed in the management of patients with severe aplastic anemia, leukemias (all, anll, cml), multiple myeloma, hodgkin's and non-hodgkin's lymphoma, breast and ovarial cancer, and extragonadal non-seminal germ cell tumor. pbsc mobilization was achieved with rhg-csf (5-16 g/kgbm/day). mnc-apheresis procedures (generally one and occasionally two) were performed using blood cell separator cobe-spectra. the first mnc-apheresis was accomplished when the leukocyte cont was 5-10 ¥ 10e9/l (autologous setting) or on the 5th day 4-5 hour after the last rhg-csf administration (allogeneic setting). the processed blood volume during one mnc-apheresis was 12. 7-22.2 l, and 24 .3 l for one pbsc transplantation in average. results: using a minimal target dose of cd34+ cell count (3 ¥ 10e6/kgbm), performing one mnc-apheresis procedure for 86% recipients sufficient number of pbscs were obtained. the mnc yield was 10.1 ¥ 10e8/kgbm in allogeneic and 8.1 ¥ 10e8/kgbm in autologous setting in average. the mean cd34+ yields for allogeneic and autologous transplantations were 12.1 ¥ 10e6/kgbm and 6.5 ¥ 10e6/kgbm, respectively. hematopoietic reconstitution was achieved on the 9.4th day for leukocytes and the 13.05th day for platelets when pbsc transplantation was applied. summary/conclusion: improved mnc and cd34+ cell yield, as well as rapid hematopoietic reconstitution were observed when: (a) the intention of auditing any clinical practice is, ostensibly, to improve patient care. the term 'audit' implies comparison against a standard. although absolute indications for transfusion have not been defined by clinical trials applicable to most clinical situations, many institutions have established their own transfusion triggers based on their reading of the literature and common practice at that hospital. assuming these represent prudent guidelines, comparison of actual practice to them may allow physicians to re-assess their pattern of hemotherapy and bring it into conformance with the guideline. there are several common ways of performing transfusion audits. retrospective analysis of transfusions allows appreciation of the clinical situation (and its ultimate outcome) but reviews the event through a 'retrospectoscope' that assesses clinical information in a different manner than that available to the clinician making the decision to transfuse. furthermore, the time lapse between the decision to transfusion and feedback about that decision may render the feedback from the reviewing body (e.g., a transfusion committee or blood utilization review committee) of little practical import. to speed the provision of feedback and emphasize educational rather than any punitive outcomes of the audit, some facilities have abolished attempts at determining whether the decision to transfuse was supportable and have used electronic means to feed back to clinicians a non-judgmental informational message summarizing the literature regarding the indications for transfusion. this appears to be at least as effective as the traditional retrospective audit system. prospective review of requests for blood components have the potential to redirect practice in a manner that immediately helps patients. however, such interactions with clinicians may come at inopportune times, may require considerable (unscheduled) time, and are most likely to be fruitful if a knowledgeable transfusion medicine expert can serve as the intermediary. both approaches (or a combination) have been shown to be beneficial in altering practice, but efforts must be diligent and sustained. providing data comparing a physician's practice to colleagues in the same specialty may prompt additional introspection and practice change, particularly if physician leadership of the institution supports the effort as a quality improvement tool. extending the comparison to a group of physicians and contrasting their transfusion habits with benchmark data from other institutions may also be helpful, but one needs to be ready to counter arguments that differences in patient groups are the reason for the differences in practice (studies have shown that, in general, practice patterns are primarily related to training and habit rather than large differences in patient acuity). increased focus on the performance of hospitals as expressed in outcome data may soon extend to transfusion practices as well. the public or governmental institutions may ask to see data illustrating the transfusion practice of an institution and its improvement over time. carefully conducted, diligent, and ongoing transfusion audits are an integral part of an institution's quality improvement program. informed consent: the term informed consent, appeared for the first time in the late 40s but it was only in the 70s that it attracted attention with regard to health care. numerous discussions and publications have attempted to define the meaning and the justification of informed consent in recent years. initially it consisted in the obligation of the physician to disclose information to the patient, regarding the procedure he was to undergo, but more recently, ethicists have emphasized the need to ensure the patient's understanding and his autonomous decision to consent. current institutional rules of ethics demand that the physician must obtain the informed consent of a patient 'prior to any substantial intervention' . what is however the meaning of informed consent? is it a mutual decision making between physician and patient? in 'principles of biomedical ethics' beauchamp and childress claim that' it is critically important to distinguish informational exchanges through which patients elect medical interventions, from acts of approving and authorizing those interventions. the elements of consent include: disclosure, voluntariness, decision and authorization. when applying the concept of informed consent in transfusion medicine one can distinguish it into donors' and patients' consent. donor informed consent: information to blood donors constitutes a sensitive issue. it refers to their protection from side-effects of the donation, as well as to protection of the recipient. with regard to whole blood donors a detailed history and information as to side effects are necessary for first-time donors. for repeat donors one needs mainly an updated history. because of time-pressure, whole blood donors are usually given written information and are asked to answer written questions. donors however differ in literacy and even literate ones do not always understand medical terminilogy; so, during the interview one should probe the degree of understanding of each donor. with first time apheresis donors more time is needed in order to explain the procedure and potential side effects. since granulocyte and stem-cell donors require premedication with growth factors and or corticosteroids, the responsibility for detailed information is even greater. the fact that all donors sign the informed consent does not mean that they are all adequately informed! interviewers must be familiar with side effects, their frequency and sequelae and must pass on this information. misses and near-misses, (serious) adverse events and failures in medical practice seem to be not preventable. in medical interventions, preparing and prescribing of medication, assistance by doctors and nurses, medical treatment and follow-up, unwanted and unexpected events occur (http://www.mederrors.com.). these events happen also in transfusion medicine and focus on safety is not unique. haemovigilance which is defined in the eu blood directive as 'a set of organised surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiological follow-up of donors' (eu directive 2002/98/ec off. j. european union.8.2.2003:l33/30-l33/40) is established to help in trying to identify and minimise the misses and (serious) adverse events in the chain from donor to recipient of blood components. the causes or reasons should be studied in order to prevent re-occurrence. adverse event reporting in blood transfusion and transfusion medicine is complex. it depends on the cooperation between blood establishments with clinicians and hospitals. it implies knowledge of blood banking, transfusion medicine and routine clinical care of all gender and ages, of potential hazards of transfusion, of immune-haematology, of microbiology, and of epidemiology. an adverse event may have its cause in every single part of the blood chain and reference may take place to a proven problem, a potential problem, or to a justified doubt. in almost all blood transfusion centres, a single donation will be processed into a number of different products, and these units might be divided or processed into more products. blood components are produced from whole blood or apheresis donations, and depending of the blood drawing and processing techniques, a high number of products with different specifications is prepared. the products' shelf life is not equal and therefore the moment in time of actual use of each unit prepared from the same donation may differ. in case the unit of platelets harms the recipient, a rapid alert can warn in order not to issue or to transfuse the unit of red cells or the unit of ffp prepared from the same donation because of the potential adverse reaction, which was detected during or after the transfusion of the first unit used. haemovigilance is not only important to blood establishments and to patients and prescribers, but it is also to clinical scientists, and to the public at large. it should provide a basis for minimising adverse reactions on blood components, and it should enable the therapeutic potential of new or established treatments with components to be maximised, since demonstrations of safety during widespread use may lead to extended usage, and wider availability. it is quite worrisome that underreporting is a general problem in medical care. it might be expected that in transfusion medicine the same rates of underreporting can be found, but also that the same mechanisms for improvement are applicable. although medical misses occur and do not seem to be preventable, the handling of these misses is often quite poor. many patients like to hear a detailed explanation, and the majority expects even apologies from the treating physician. it seems desirable to look for new routes in the prevention of unwanted events of transfusion medicine. for problem solving, analysis and improvement of working methods, where needed and possible, are often the most effective methods. attention should be focused on improvement and not on identification of the person who caused the problem ('bad apple'). lack of communication and insufficient insight in each other work are often the causes of problems and unwanted events. it should be recognised that advices given by the haemovigilance officer or the blood transfusion committee about prevention without the input and commitment of the direct responsible persons will lead in most cases to advices which are not effective or which will not be accepted. setting up a haemovigilance system or appointing of haemovigilance officers or installation of blood transfusion committees will not be sufficient. it will be necessary to develop ways of registration, data collection and analysis, but more importantly to support by giving advice and training to prevent reoccurrence of the adverse events or not optimal use of blood components. the confidentiality of the information should be guarded sufficiently. for a physician-patient relation, even after a medical failure, a 'blame-free culture' with a central role for openness and transparency is necessary. for blood establishments and hospitals, there is an important role in the right assistance and help of the physicians concerned both on a practical and on an emotional way. m-pa-032 8 years of shot data 1996-2004: a view of transfusion safety in the uk and reactions. this will require investment in infrastructure, for which there must be a trade-off in improved transfusion safety. transfusion-transmitted infections and serious immunological reactions are rare; shot has highlighted the need for blood services to implement strategies to minimise bacterial contamination and transfusion related acute lung injury and will monitor their effectiveness. from the inception of shot it has been clear that the most frequent transfusion hazard is 'incorrect blood component transfused' i.e. a patient receiving a blood component intended for another person or not meeting appropriate requirements. only a minority of these events results in patient harm and is reportable under the terms of the directive. haemovigilance schemes such as shot, that analyse no-harm errors and near-misses, can reveal clues as to the root causes of 'wrong blood', which contributed to 16 deaths and 85 cases of major morbidity in the uk between 1996 and 2003 . analysis of such events shows that most errors occur in clinical areas, the most frequent being failure of the 'bedside check' . clinical audit data indicates that 10% of patients are transfused without a wristband or other form of identification, whilst anecdotal reports suggest that urgent clinical situations, massive transfusions and nocturnal transfusions are particularly error-prone. strategies aimed at reducing errors include structured education and competency testing, and methodologies, both high and low-tech, to ensure accurate patient identification in all circumstances. onethird of errors occurs in hospital laboratories; denominator data on laboratory workload shows that work done outside of 'core hours' accounts for 20% of all pre-transfusion testing but 40% of errors, suggesting that biomedical scientists 'on-call' or on shift work are working under pressure and beyond their competency. 85% of hospitals reported that they participated in shot in 2003, but only 47% of eligible hospitals reported adverse events suggesting that transfusion hazards remain under-recognised and under-reported. however, benchmarking of 'wrong blood' incidents against transfusion activity shows that the number of observed incidents is roughly proportional to blood use. if haemovigilance data is to contribute to improved transfusion safety, clinicians must be encour-aged to report all events, thus contributing to an evidence base that can be used to effect change and facilitate learning. barriers to reporting include cumbersome systems, lack of time and resource, lack of feedback and fear of blame. transfusion practitioners have a vital role in the recognition and reporting of adverse events, education and clinical audit, but must be adequately resourced and supported by senior clinicians and managers through an active hospital transfusion committee. *provisional. m-pa-033 passing the borders: when, how and where d pirc-tiljak croatian institute of transfusion med., zagreb, croatia there may come that moment in professional life when you have to follow a strong professional and ethical need and confront your evidence-based statements with the leadership, passing the border of your own small society. in order to protect patient's health and respect human right to be informed about all possible consequences of irregular medical therapy, insisting on professional dignity and truth, you feel responsible and follow-up processing of your serious error report. once you pass the border, trying to warn authorities and find ethical resonance and critical confirmation of your professional fears, you are 'persona non grata' . methods/results: reality checking, personal experiences and observations studying the path of serious error report. although the qc system functions, yet omissions happen . . . the possible reasons could be: lack of knowledge, lack of experience, lack of independency, personal confront of interest, immature leadership, political influence, even corruption . . . how strict do the authorities manage a fault, bearing in mind the responsibility toward the patients under the risk. there is a need to create an available, effective international expert's board which will react and give professional counselling support-asylum for endangered professionals who found enough power to blow the whistle. who will hear it? transfusion transmitted infections (tti) are a major source of concern given the repercussions of hiv, hepatitis c, bacteria and vcjd transmission by blood components. large amounts of resource have been expended in making products safer and in maintaining public confidence in the blood supply. identifying emerging infections of concern is a major activity for many transfusion services. of the long list of emerging infections identified by disease control agencies around the world, identifying those responsible for tti requires, amongst other things, that: • the agent is identifiable. • it is present in blood • it causes a disease of concern. • it is transmitted by transfusion. • it is present at relatively low frequency. • if a test is available (nat, serology or immunoassay) what the infection window period is. once an agent has been identified various approaches are possible, including: • donor selection by testing, geography or lifestyle e.g. wnv; • product selection e.g. erythrovirus (b19) antibody or bacterial testing; • product treatment e.g. pathogen inactivation; • patient selection e.g. cmv matching, immune status. against this background where should our attentions focus? some agents of initial concern are now known to be ubiquitous and have minimal disease association (ttv, gbv) although transmitted by transfusion. for others (coronavirus -sars or the possible kawasaki disease agent, dengue, flavivirus encephalopathies, avian flu, etc.) this is less certain, with agents arising from species crossover being of particular concern (avian flu, vcjd, hiv). • enhancement of automation/computerisation; • process control to provide an 'error-free pathway'; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody 'combi' assays (e.g. for hiv, and recently, for hcv); • awareness of hbsag vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or chagas' disease infection (for retrieval of otherwise wasted blood); • european union's in vitro diagnostics directive: this has caused some problems and reduced flexibility. nucleic acid testing (nat): nat continues to increase in blood service usage world wide, although not (as yet) to replace serological methods. trends include: • reduction in sample pool size; • increased automation (and process control); • increased multiplexing to detect 2 or more agents in the same assay; • increased number of agents being tested by nat (varies in different countries); • introduction of rapid and flexible nat to detect west nile virus, in north america. bacterial screening of platelet preparations: several countries have introduced (or will introduce) routine screening of platelet concentrates either with biomerieux, bactalert or pall ebds (02 depletion assessment). other bacterial testing methods are under active assessment, some rapid enough for possible 'point of use' testing. m-pa-036 evaluation of in vivo red blood cell recovery after processing with a new filter designed to reduce prions e nelson*, h taylor † , p whitley † and t lieu* *pall medical, covina, ca, † american red cross and evms, norfolk, va, usa background: a filter, called the leukotrap affinity prion reduction filter (prf1b filter, pall medical), has been developed to reduce the level of infectious prions, associated with several fatal neurodegenerative diseases including variant creuztfeldt-jakob disease (vcjd), from leukocyte-reduced red cell products. aim: the objective of this study was to evaluate the quality of leukocyte-reduced red cells (lr-rbc) processed through this filter and stored for 42 days. red cell quality was determined by measuring the in vivo red blood cell recovery 24 hours after re-infusion of the 42-day stored red cells. storage hemolysis and atp were also determined. methods: units of blood (450 ml) were collected from normal volunteers into the leukotrap wb system containing cp2d/as-3 anticoagulant/preservative solutions (pall medical). units were either processed to lr-rbc within 8 hours at room temperature (rt units), or after 24 hours at 1-6°c (cold units). the prion filter set was sterilely connected to the units on day 1, and the units were filtered and stored for 42 days. samples were taken pre-and post-prion filtration and post-storage for plasma hemoglobin and atp determinations. post-storage samples were taken for labeling with 51-cr radioisotope, re-infusion, and determination of the 24-hour in vivo rbc recovery. a donor sample was also labeled with 99m-tc to allow for red cell mass determination. thus, both single-and double-label 24-hour recovery values were calculated. results: twelve units were collected. in vitro testing was completed on all units. in vivo testing was completed on 11 units. the mean single-label 24-hour recoveries were 84.4% and 85.7% for the rt and cold units, respectively. the mean double-label recoveries were 81.9% and 84.1% for the rt and cold units, respectively. the overall combined mean in vivo and in vitro results are shown in the table. conclusion: the 24-hour in vivo red cell recovery means are well above the fda and council of europe's requirement of achieving a mean post-transfusion survival of no less than 75% of the transfused red cells, and they are comparable to this center's previous results of red cells filtered using the licensed leukotrap rc system with cp2d/as-3 (pall medical introduction: to reduce the risk of platelet transfusion-associated sepsis (tas), methods to routinely screen for bacterial contamination have been implemented. pathogen inactivation treatment of labile blood components provides an alternative means to prevent tas. the intercept blood system for platelets (baxter healthcare) has received the ce mark and has been introduced into clinical practice. aims: this study compared the efficacy of bacterial screening using a culture method (bact/alert system, biomerieux) with pathogen inactivation (intercept blood system) for prevention of transfusion of platelet components contaminated with bacteria. methods: seven strains of bacteria associated with tas, including gram-positive staphylococcus epidermidis, streptococcus agalactiae, and staphylococcus aureus, gram-negative escherichia coli, and klebsiella pneumoniae, and the anaerobes propionibacterium acnes and clostridium perfringens were studied. for each strain, three double-dose platelet concentrates (~6 ¥ 10e 11 platelets in 600 ml of 35% plasma and 65% intersol) were collected using the amicus® cell separator. on day of collection, calibrated stocks of bacteria (20, 200, 2000 cfu) were added to the double units. each double unit was divided into two identical products containing 10, 100, or 1000 cfu of bacteria and stored overnight under conventional blood bank conditions. the control platelet concentrate was not treated. the test platelet concentrate was treated with the intercept process (150 mm amotosalen + 3 j/sq cm uva). both units were cultured using the bact/alert system at the time of bacterial inoculation and on days 1, 2 and 5 of storage. samples (10 ml) were taken for both the aerobic and anaerobic cultures. a platelet sample was considered contaminated with bacteria if a positive signal was registered within 120 hours of culture. results: for control platelet concentrates, cultures failed to detect low-dose inocula. the time to positive culture varied with the bacterial strain, contamination level, and time of sampling. at 10 and 100 cfu per product, 4 strains (s. epidermidis, e. coli, c. perfringens, s. agalactiae) and 2 strains (e. coli, c. perfringens) tested negative after 5 days of platelet storage, respectively. k. pneumoniae tested positive after 7-8 hours of culture when sampled on day 2 of platelet storage for both 10 and 100 cfu per product. at 1000 cfu per product, p. acnes tested negative in aerobic culture and c perfringens tested negative in anaerobic culture after 5 days of platelet storage. the anaerobic cultures of p. acnes became positive after 50 hours of culture when sampled on day 5 of platelet storage. of the 7 strains studied, only s. aureus consistently tested positive after 7-16 hours of culture. in contrast, all test platelet concentrates treated with intercept remained negative by bact/alert cultures throughout the entire 5-day observation period regardless of the strain and the contamination level. conclusions: bacterial detection using cultures may fail to detect low levels of bacteria typically associated with platelet contamination at time of collection and processing. failure to detect bacteria will result in the release of contaminated platelet products with 'test negative-to-date' status. in contrast, inactivation of bacteria is capable of preventing release of contaminated platelet components. background: since nat implementation for hiv-1 and hcv rna in france, the residual risk (rr) of transfusion-transmitted infections (tti) has dramatically decreased. the rr estimates, for a threeyear period from 2001 to 2003 showed a significant decrease from 1/1 700 000 and 1/1 560 000 before nat implementation to 1/3 150 000 and 1/10 000 000 after nat implementation for hiv and hcv respectively. as for hbv, the serological screening is only based on both hbsag and anti-hbc assays. for the same period, the rr estimate for hbv is 1/640 000, five times higher than hiv one and 16 times higher than hcv one. aims: as the overall rr of tti is mainly related to hbv, and given the availability of hbv nat assays, a study was conducted to determine whether hbv nat has the ability to further reduce the hbv rr and then should be implemented in blood donor screening in france. we have estimated the wp reduction by nat in comparison with one of the most sensitive hbsag screening assays, on 10 commercial seroconversion panels (bioclinical partners, franklin, ma, usa). the nat test was the procleix ultrio assay (genprobe/chiron, san diego, usa). the hbs ag test was the prism hbsag (abbott, france). the comparison was performed on both neat samples and diluted samples 1/8, 1/16, and 1/24, in order to simulate minipools of different sizes. then, we have calculated the yield of hbv-infected donations detected by nat relative to prism hbsag assay. results: on the basis of a window period (wp) of 56 days, ultrio assay is projected to close the wp by an average of 15 days on undiluted samples, 5 days in minipools of 8 samples, 4 days in minipools of 16 samples and only 2 days in minipools of 24 samples. the projected yield calculated on the basis of 2.4 million donations collected per year in france, would be 0.65 unit per year for minipool-nat and 2 to 3 units per year for individual donation nat. conclusion: introduction of minipool-nat will offer only a little added benefit to transfusion safety relative to current serological screening strategies based on both hbsag and anti-hbc assays. hbv minipool-nat is then unsuitable for hbv screening in french blood donors. single-sample nat or minipool-nat with smaller pool sizes and/or modified procedures (genome enrichment or test improvement) would be more relevant. automation when technologically and practically feasible is a prerequisite for single-donation nat. therefore, decision has been made not to implement hbv nat in the french transfusion network until fully automated systems will be available. however, as the prevalence of hbv infections is higher in the overseas territories than in continental france, and as nat is performed on individual donations in these sites, hbv-nat has been implemented since december 2004 in these territories. combined detection of hepatitis c virus core antigen and antibody as an alternative to nucleic acid testing in blood screening grating the capillary cytometer with a robotic workstation and a small footprint centrifuge. significantly, there was no decrement in system performance following automation: 474 of 501 clinical samples (94.6%) typed identically with this system and cat, and 25 of the 27 discrepant results were eventually resolved in favor of the automated cytometry method. testing showed high-throughput capabilities (currently 280 samples/day) and was inexpensive. to demonstrate the flexibility of this testing platform, we also developed a method to perform completely automated counting of residual wbcs (rwbc) following leukoreduction of blood components. there were no significant differences in accuracy and precision when rwbc in analytical controls and authentic clinical samples were quantitated by the automated capillary cytometry method or the leucocount method performed manually. given the flexibility of this system, it is very likely that additional blood bank assays could be modified for high performance automated testing on this platform. noninvasive prenatal genotyping on cell free fetal dna in maternal plasma ce van der schoot sanquin research, amsterdam, netherlands in 1997 lo et al. demonstrated that in the maternal circulation small amounts of cell free fetal dna are present, concentrations ranging from on average 25 genome equivalents(geq)/ml early in pregnancy to about 100 geq/ml at the end of pregnancy. most likely this dna is derived fom apoptotic syncitiorophoblasts. the human placenta is hemichorial, which means that the syncitiotrophoblast is in direct contact with the maternal blood flow, and apoptotic nuclei are directly released into the maternal circulation. the cell free dna is very rapidly cleared from the circulation, the t1/2 being only 15 minutes. in 1998 we have shown that this cell free fetal dna could be used for rhd genotyping. in the last 5 years many groups have shown the successful application of different prenatal genotyping assays such as fetal sexing, thalassemia, achondroplasia, duchenne's disease, adrenogenital syndrome etc. on this source of dna. importantly, no false positive result have been described due to the presence of fetal dna from previous pregnancies, the main draw back of prenatal diagnostics on circulating fetal cells. at present prenatal rhd genotyping has been introduced in routine diagnostics in the united kingdom, france and the netherlands. in large scale high throughput studies it has been shown that the diagnostic accuracy of prenatal rhd genotyping is over 99%, and it is to be expected that in the netherlands this screening will soon be introduced to restrict the antenatal anti-d immunoprophylaxis to women carrying rhd-positive fetuses. in a large european project (safe, co-ordinator maj hulten, warwick uk) many researchers collaborate to further explore the possibilities of cell free fetal dna for future diagnostics. standard operating procedures for the isolation of plasma dna have been established. control pcrs for the presence of fetal dna have been developed. recent findings on differences in methylation status of placental genes in fetal dna opens new possibilities. the main technical problem that hampers wide application of prenatal genotyping is the impurity of the fetal dna, only 1-5% of the cell free dna in plasma is from fetal origin. this makes diagnostic assays on numerical chromosomal abnormalities impossible. and also for many single nucleotide polymorphisms (snps) such as almost all blood group antigens, assays are hampered by aspecific amplification from maternal dna. our own preliminary results indicate that this latter problem can be solved by pna clamping. the addition of a pna probe specific for the k-allele partial d feature d antigen alteration, often identified as distinct 'partial' d epitope loss. the clinical impact of partial ds is due to the ability of their carriers to form anti-d antibodies upon confrontation with regular d after transfusion, or pregnancy. this leads to the -naively spoken -contradictory finding of an allo anti-d antibody in a d positive individual in connection with a negative autocontrol. the antibodies themselves include the same fatal clinical potential as anti-d antibodies of d negative individuals, but may be even more hazardous since unexpected in d positive individuals a priori. d categories (ii to vii) represent a nomenclatorily defined subgroup of partial ds. the molecular cause of partial d lies within single (caused by point mutation in the respective rhd gene sequence), or multiple amino acid exchanges (caused by gene conversion events leading to rhd-rhce-rhd hybrid genes) which determines a qualitative d antigen alteration, rendering them distinguishable from regular d by a partial d carriers immune system. nowadays, transfusion specialists and gynaecologists are more or less aware of these facts and are taking them into consideration in the clinical setting. most partial d exhibit decreased d antigen density, enabling principal recognition of them. however, routine serological methods may not properly recognise all partial ds and will identify their carriers after immunisation only, which represents a reactive diagnostic/therapeutic attitude second best to an actively prognostic one. this actively prognostic proceeding with respect to early detection of partial ds became widely feasible by rhd dna typing techniques. currently, routine rhd dna typing techniques offer an affordable, accurate and fast approach to an unambiguous identification of partial ds and their reliable discrimination from weak d types, not at risk for allo anti-d immunisation. a reasonable proactive proceeding could e.g. demand for (once in a lifetime) routine rhd dna typing of all weakly expressed ds as defined by serology, since most partial ds also meet this phenotype. rhd allele frequencies and their geographical and regional prevalence will certainly have an important impact on dna typing strategies and their (mandatory) specificities. fluorescence cytometry for completely automated immunohematology testing d roback*, b barclay † and d hillyer † *emory university school of medicine, atlanta, † transfusion & transplantation technologi, decatur, ga, usa we previously described a methodology for accurate immunohematology testing by fluorescence cytometry [roback, j.d. et al. (2004) transfusion 44 (2), 187]. this system utilized low-speed centrifugation of 96-well filter plates for red cell staining, and a smallfootprint capillary cytometer for data acquisition. when authentic clinical samples from hospitalized patients were tested for abo group, the presence of d antigen, and red cell alloantibodies, the results were well-correlated with those obtained by commerciallyavailable column agglutination technology (cat). this system determined the correct abo group and d type for 98.7% of 520 samples, compared to 98.8% for cat (p > 0.05). when samples were tested for unexpected alloantibodies, fc determined the correct result for 98.6% of 215 samples, as compared to 96.3% for cat (p > 0.05). this novel method was better than cat at detecting weak anti-a (p < 0.0001) and alloantibodies. based on these promising results, we sought to completely automate this method by inte-prevents the aspecific amplification of the k-allele, and makes it possible to detect the fetal k-allele in the presence of excess of maternal k-alleles. furthermore, it has been shown that fetal dna is in the plasma present in shorter fragments (<300 bp) than maternal dna. size separation of cell free fetal dna can therefore be used to increase the relative concentration of fetal dna, which will help the development of new genotyping assays. in conclusion, cell free fetal dna in maternal plasma is nowadays routinely used for prenatal rhd typing and fetal sexing. new technical developments will make it possible to extend these indications to other blood group antigens in the near future. more insight in the characteristics of fetal dna might finally lead to wider applications, including numerical chromosomal aberrations. furthermore, it might become possible to apply genomic dna microarrays for the screening on many different inherited diseases, including hemoglobinopathies. determination of the affinity of anti-d present in the serum of immunized subjects and in anti-d ig preparations by a method using unlabeled antibodies p lambin*, m debbia* and y brossard † *institut national de la transfusion, † chp hopital saint antoine, paris, france introduction: few data are available concerning the affinity of maternal anti-d responsible for the hemolytic disease of the fetus and the newborn (hdn), and the affinity of anti-d immunoglobulin used for the prophylaxis of that disease. we recently described a method to measure the affinity (ka) of untagged anti-d monoclonal antibodies. aims of the study: in this work, a similar method was applied to determine the affinity constant (ka) of polyclonal anti-d present in the serum from d-immunized mothers and donors and from anti-d ig preparations. methods: a constant amount of o r1r rbcs was sensitized with increasing concentrations of anti-d present in the sera from immunized subjects, and in anti-d ig preparations. at equilibrium, the amount of anti-d bound to rbcs was measured by elisa. the scatchard equation (linear regression) and the langmuir equation (hyperbolic regression) were used to determine the ka of anti-d. the experimental data fitted well with the scatchard equation (mean r † = 0.95) but a better correlation was observed with the langmuir equation (mean r † = 0.99). in 11 maternal sera, the mean ka of anti-d was 5.6 ¥ 10 to the 8 m-1 (from 2.8 to 12 ¥ 10 to the 8 m-1). in the sera from 6 immunized donors, the mean ka was 3.9 ¥ 10 to the 8 m-1 (from 1.5 to 6.8 ¥ 10 to the 8 m-1) and in 5 lots of anti-d ig, the mean ka was 3.4 ¥ 10 to the 8 m-1 (from 3.1 to 4.2 ¥ 10 to the 8 m-1). the comparison of anti-d affinity measured in 5 cases of hdn in which infants presented a fetal anemia and in 6 cases of hdn in which infants presented only a postnatal anemia showed no significant difference. the mean value of ka in the cases of fetal anemia was 5.4 ¥ 10 to the 8 m-1 whereas in the cases of postnatal anemia the mean value of ka was 5.8 ¥ 10 to the 8 m-1. conclusion: the method previously described for monoclonal anti-d was applied to polyclonal anti-d present in the serum of d-immunized subjects and in ig preparations. the experimental data fitted well with the langmuir equation, and the affinity of polyclonal of anti-d was measured with accuracy. in addition, no significant difference was observed (at least in the 11 cases of this study) between the affinities of anti-d measured in the most severe cases of hdn (fetal anemia) and in the less severe cases of hdn (post-natal anemia). introduction: cryopreservation of platelets is widely used in platelet immunology to ensure the availability of well characterised panel cells for the detection of hpa antibodies. but recovered platelets do not express the hpa-15 alloantigens. aim of the study: here we describe a method for the successful preservation of platelets by lyophilization. we report the value of this new reagent for the detection of hpa alloantibodies and especially anti hpa-15 alloantibodies. methods: rehydrated lyophilised platelets (lyo p) were tested for their reactivity with monoclonal antibodies against gpiibiiia, gpibix, gpiaiia and cd109 by flow cytometry. the levels of reactivity were comparable with the ones obtained with fresh platelets. the rehydrated platelets were used in the maipa with a panel of hpa antibodies (anti-hpa-1a, 30; anti-hpa-1b, 3; anti-hpa-3a, 3; anti-hpa-5a, 3; anti-hpa-5b, 50; anti-hpa-15a, 2 and anti-hpa-15b, 4). results: all hpa antibodies showed the expected pattern of reactivity and in several cases absorbance reading were well above those obtained with fresh platelets. absorbance values produced by inert sera were comparable with those obtained with fresh platelets (ranges 0.001-0.008). interestingly, we used lyophilized platelet with a high expression of cd 109 bearing the hpa-15 system and we have detected 24 anti hpa 15 antibodies among 1000 sera previously negative with fresh platelets. nineteen sera concerned patients suffering from hematological diseases and 5 from pregnancy women. conclusion: lyophilized platelets are possibly an ideal reagent for the platelet immunologist to be used for the detection of hpa antibodies. moreover, this work bring new insights on the hpa-15 system in platelet transfusion. we are now pursuing more extensive validation studies with a larger number of samples representing all known hpa specificities and several diseases. the diagnosis and treatment of sick infants and children requires a broad knowledge of physiology, biochemistry, genetics and the application of sophisticated testing and treatment options. one of these options is transfusion of blood and blood products. transfusion of the infant, especially the premature infant, and sick child, especially those with major organ dysfunction, requires careful consideration of their unique metabolic, hepatic and renal clearance mechanisms. guidelines that direct the indications for transfusion differ from those in adults. non-invasive measures of oxygen delivery and oxygen offloading may assist in guidelines for red blood transfusion. metabolic complications from massive transfusion and/or the manipulation of blood products must also be considered. evidence from a high quality randomised controlled trial suggests that anaemia is also well tolerated by critically ill patients. a restrictive approach to rbc transfusion that maintained the hb concentration between 70 and 90 g/l was found to be as effective as and possibly superior to a more liberal strategy of maintaining the hb concentration between 100 and 120 g/l. there are concerns that some groups of critically ill patients, such as those with cardiovascular disease and patients who are difficult to wean from mechanical ventilation, may benefit from higher hb levels. rbcs also have a role in primary haemostasis and higher triggers may be appropriate in coagulopathic patients. it is important to realise that blood is not a uniform product and the clinical efficacy of rbc transfusion may vary. one factor that may have a considerable effect on the quality of the rbc product is the storage time. rbcs undergo marked changes during refrigerated storage. the implications of these changes on tissue oxygenation are not known but these concerns have led some clinicians to request 'fresh' blood for critically ill patients. there is insufficient evidence to support such practice. it is of great practical importance to determine if, or when, fresh rbcs could be superior to stored rbcs. background: with a decreasing blood donor base, fully tested, fresh unrefrigerated whole blood (fuwb) has been found to be a more efficient and effective use of a limited resource in place of, or as an adjunct to, traditional blood component therapy in surgical situations associated with massive blood loss. aims: to outline the use of fuwb in situations where there is potential for intractable bleeding associated with major surgery, and evaluate platelet function in fuwb versus platelet components. methods: outcomes of fuwb and traditional blood component use were examined for 20 cases of cardiac bypass surgery. in addition, exclusive use of fuwb for 23 burns debridement cases was analysed. an evaluation of platelet function in whole blood compared to platelet components was also performed by measuring platelet aggregation and activation parameters. results: there was a decreased requirement for blood components following administration of whole blood post cardiac surgery. whole blood usage for burns debridement surgery eliminated the requirement for additional blood components. platelet activation was markedly reduced in whole blood compared to component platelets, and this may be one reason for the increased efficacy of whole blood in these clinical settings. conclusion: fuwb appears to have a role in minimising blood product requirements and consequent donor exposure in situations associated with massive blood loss. m-pa-051 transfusion practice for coronary artery bypass surgery in greece s lakoumenta, m vassili, g hatzidimitriou, t asteri, p stratigi, s kanellas and g palatianos hellenic society of blood transfusion, athens, greece cardiac surgery is associated with a demand for allogenic blood and blood product availability as well as a considerable consumption. the impact of consensus guidelines for allogenic blood transfusion during coronary artery bypass graft surgery (cabg) in us attracted great attention 1. the present study is conducted in order to reveal the transfusion practice in greece on a similar population i.e. patients undergoing cabg operations. methods: five participating centers collected data concerning transfusion of allogenic blood and blood products in patients undergoing elective first time cabg procedures, as well as parameters that may influence blood loss, such as duration of the operation, cardiopulmonary bypass time (cpb) etc. the total estimated blood loss was calculated as the sum of red blood cell volume reduction [(body weight in kg ¥ 60 ml/kg) ¥ (admission haematocrit-discharge haematocrit)]+(red blood cell volume transfused). results: results are shown in table 1 : means, standard deviations, and p-values of the wilcoxon test comparisons between the hospitals. a preliminary analysis of data from 5 centres (268 patients) showed no difference in patient characteristics (age, body weight, male to female ratio). there is a statistically significant difference (p < 0.0001) between the five centers in duration of the operation, cpb, estimated blood loss and volume of transfused plasma. red cell use showed also a variation which however did not reach statistical significance (p < 0.02). the center with the highest figure for blood loss has the lowest volume of allogeneic red cell transfusion because of the use of cell salvage. conclusions: although variations, as those observed in greek cardiac surgery centers, have been documented in other countries, the variation in the use of plasma is striking and we are in the process of trying to identify the reasons. our study is in progress and additional data are being collected and will be presented. introduction: premature infants and at term newborns have an higher circulating blood volume per kilogram than the adults (100 ml/kg in premature; 80 ml/kg in at term), for this reason, in case of neonatal thrombocytopenia, a specific hemocomponent, with a very high platelet concentration, needs for transfusion therapy. the laboratory criteria for platelet transfusion are the following: (a) a plt count < 150 ¥ 10 9 cells/l if bleeding is observed; (b) a plt count < 20 ¥ 10 9 cells/l without bleeding; (c) a plt count < 50 ¥ 10 9 cells/l in newborns showing critical clinical conditions. aim of this study: in this study, we have monitored the plt transfusion therapy in our neonatal intensive care unit (nicu) in the last four years. methods: effects of plt transfusions have been followed in 37 children (31 premature infants and 6 at term newborns). the weight of premature infants ranged from 600-1800 g and at term newborn from 1800-4000 g. gestation age of premature infants ranged from 26-36 weeks and of at term ones, of course, from 37-42 weeks. for every platelet transfusion in these newborns, the volume of platelet concentrate has been of 10-20 ml/kg, with a plt count < 700 ¥ 10 9 cells/l. results : in the study period, 77 plt transfusions have been performed: 23 children have been only transfused one time, while multiple plt transfusions (ranged 2-10) needed for 14 children according to clinical conditions. the observed clinical indications for transfusions have been the sepsis with haemorrhagic syndrome (26 cases) , haemorrhagic syndrome without sepsis (7 cases) and neonatal alloimmune thrombocytopenia without haemorrhagic syndrome (4 cases). after 24 hours from transfusion therapy, the absolute plt count and the correct count increment have increased in all 37 little patients. the highest increase in plt count was 45 ¥ 10 9 cells/l, while the lowest 5 ¥ 10 9 cells/l. no difference in the efficacy of therapy has been detected between premature group and at term group. 94% of children have been discharged from hospital in good general conditions without complications in following controls. in conclusion, we can affirm that plt transfusion in premature infants and in at term newborns is an efficient and safe treatment of severe haemorrhagic conditions. however a collaboration between nicu and transfusion center is necessary to choice the adequate platelet concentrate's volume for transfusion and the best plt donor for the newborn. developing transfusion strategies 27 fusion society of turkey (bbtst) in 2004 with contribution of 253 blood transfusion centers. according to these figures 51% of centers attended operates apheresis procedures. two centers informed us that apheresis in the hospital was carried out at blood bank. there was not enough information from one center, so it was excluded. of the 51 blood banks performing apheresis, 30 were university hospital blood banks. another 38 blood banks were producing both productive and therapeutic, 10 produce only productive and 2 produce only therapeutic procedures. one center did not respond. all centers reported to prepare and separate erythrocyte and plasma. however only 48 centers reported to prepare random platelets as well. each center had apheresis machines between 8-15. a total of 19 centers was carrying out around <500 procedures, 12 around 501-1000, at 9 centers about 1001-2000, at a further 10 centers around 2001-5000 procedures a year (one center was excluded). of the responders to the survey 36, all procedure were done at blood banks, whereas at 6 of them all were carried out by the hematology clinics. at 8 other centers, productive procedures were conducted by the blood bank, and therapeutics were performed by the hematology division. a total of 48 blood banks stated that they have not kept the platelet suspensions produced and used them straightaway. productive apheresis center capacities were as shown: 13 centers < 5000, 16 centers 5000-10000, 13 centers 10001-20000 and 6 centers > 20000 units have donations a year. around 68% of all apheresis procedures were carried out by large well run blood banks. conclusion: as the use and production of random platelets increase, and settle of apheresis devices in big centers will eventually decrease the demand of apheresis procedures and keep the welltrained staff at big centers, decrease the cost thereafter. • planning of resources for the financing of the bts, adoption of a methodology for creating and adjusting the price list of products, adoption of the yearly plan of needs for blood/products and services of health institutions which use blood/products. • achieving recognition of real costs of products and services from the health insurance fund and ministry of health. • harmonization of low level of acclaimed costs and real costs of basic transfusion activities. results: acclaimed costs for activities in transfusion practice (collection, testing, processing, storage, distribution and transport) as a reflection on the price of the products are 60% lower than real costs. the prices of health services in the official price list are much lower than the proportion of costs of material resources needed for the realization of these services. this especially affects the management of independent blood establishments (bti's in serbia) with core blood transfusion activities as their basic field of work, in comparison with the 44 hospital based transfusion services, which are financed within the budget of the whole hospital. the 44 hospitals with hospital based transfusion services involved partly in core transfusion activities are completely financed by the health insurance fund, while independent blood establishments are financed through the price of products and services they provide. conclusion: in order to provide adequate quantities of safe blood/products for the end user -the patient, it is elementary to create stabile and equal financial management conditions for the whole blood transfusion service in serbia. this can be achieved only by continuous cooperation of the health insurance fund, ministry of health and independent blood establishments. sion centers (rbtc). the activities on promotion and organization of voluntary, nonremunerated blood donation, blood collection and patients' services are carried out in the rbtc and in 24 departments of blood transfusion (dbt), part of the district hospitals. the collected units in dbt are transported by special cars to the ncht and the rbtc for processing, testing and control. the same transport is used for the requested by dbt blood components for storage and distribution to hospital departments. thus the issued components are with an equal quality and safety for all patients throughout the country. lbbdbt introduces hemovigilance as a mandatory system, covering the whole chain of the blood transfusion process. it includes as well the creation of registries at a national, regional and district level of blood donors, recipients of blood products and all activities of the blood transfusion service. . seventy hospitals are exclusively users of blood, blood products and services. the current organization of blood transfusion services faces the following problems: fragmented transfusion service, lack of a national blood policy, the blood program is not nationally coordinated, limited knowledge on quality management, inadequately distributed human resources, limited material resources, lack of it system, lack of planned, continuous skill upgrading. as a direct consequence we have: suboptimal blood collection activities, inadequate blood supplies significantly vary between seasons, high percentage of replacement donors, outdated methodologies, old, even obsolete equipment, the quality of blood products is not standard, there is a lack of traceability. aim of study: to reorganize blood collection activities in serbia to increase collection of safe blood up to 4% (304 000 blood units). methods: division of responsibilities between blood establishments and hospital based transfusion services by: • optimizing organizational structure • implementing blood collection standards to enhance blood safety and donor care • gradually replacing family donors with a network of voluntary non-remunerated blood donors from low risk population groups • creating and implementing a training strategy. results: through the eu funded project support to a national blood transfusion service in serbia, we are in the effort of integrating the services and standardizing their work. the blood collection working group began by dividing serbia into 3 blood collection regions: north, central, and south. each region is divided into sub regions covering approximately half a million population (4 in the north, 8 in the central and 4 in the south region). each sub region will have one standard mobile blood collection team to collect 80 blood units daily, i.e. 19 000 annually. the 19 000 blood units per 16 teams provide the 304 000 blood units (4%) to cover hospital needs in serbia. to this effect, the following has been achieved: • blood collection activities in serbia analyzed • performance analysis of bte and hbts mobile teams in place • two model standard mobile teams tested in the field • national blood collection sop's written • national donor questionnaire form prepared • national set of blood collection standards prepared • list of donor deferral criteria prepared • blood collection equipment renewed • regional reorganization plans in progress. the objectives and results can be achieved by the participation and mutual cooperation of all institutions involved in blood transfusion within an integrated, standardized system with clearly delegated responsibilities. p-005 60 years of the national blood transfusion institute in serbia n nedeljkovic national blood transfusion institute, belgrade, yugoslavia nbti was founded in 1944 . since 1953 and unpaid blood donation is mandatory, organized in cooperation with red cross. blood donation is regulated by the law in 1974, 1993/94. codex of voluntary blood donation and health care staff has also been established; 300 000 blood donors donates blood annually. in the past 60 years, there was over 3 million blood donations, performed in accordance with who regulations. over 250 transfusion medicine specialists and 700 technicians specially trained for the work in blood transfusion service (n = 53), perform transfusion medicine doctrine of rational labile blood component and stable blood derivatives therapy, based on the selfsufficiency concept in fr yugoslavia with 10.3 million inhabitants in serbia, montenegro and kosovo. plastic blood containers and tests are imported or given as humanitarian aid gift and from 2003. now, they have been regulated by tender. in 1951, test to lues was introduced, to hbsag in 1971, to a-hiv in 1985 and to a-hcv in 1993. information system was introduced in 1987. nbti includes: national haemovigilance coordinatoin center, center for medical care of haemophiliacs, tissue typing center, center for prenatal and perinatal protection of pregnant women and newborns. activities of nbti are organized through: center for planning, organization and development of blood transfusion service, center for blood collection, preparation and distribution, center for immunology and immunochemistry, plasma fractionation center for plasma in 8 west balkan countries, center for diagnostics means, center for quality control of drugs and medical and diagnostic means, center for education and training and scientific research work. nbti is the third year of gmp, sop, yus iso 9002 implementation. in the current reform of transfusiology system we are aiming for 4 percent of voluntary blood donation. nbti is the publisher of the national bulletin of transfusion medicine and it is included in the education system of the belgrade university medical faculty and the estm in belgarde 2003. the problems of blood service in russia ea selivanov and t danilova russian inst. of hematol. & transfusiol., st. petersburg, russian federation background: the blood transfusion service (bts) development as a platform for providing the hospitals with blood and blood derivatives is an important national problem. aims: russian blood service assessment with international comparison. methods: a study was conducted on the base of the reports from all regions of russia followed by a computer statistical analysis. results: blood and blood components were collected in the russian federation in 2003 in 190 stations of blood transfusion and in 1065 blood transfusion departments at big hospitals. amount of donors in 2003 was equal to 2 202 853, voluntary donors being 87.2% of them. the average number of whole blood donations in relation to the general population is 25 per 1000 inhabitants, and on average 28 percent of the donor base consists of first time donors. the average number of blood collected in relation to the general population and health care system is 11.4 ml per inhabitant and 1240 ml per one bed. an average volume of one blood donation is 396 ml. blood was collected into plastic bags containing domestic or foreign anticoagulants. about 93.2% of collected blood is used for procurement of blood components and preparations, 0.8% of banked blood is used for transfusions. amount of donors and the volume of whole blood have been significantly decreased for the last 15 years. at present in russia all donations are tested for abo blood group, rh(d) type, anti-hiv-1/2, hbsag, anti-hcv and syphilis. the total percentage of blood discarded after testing for transfusion-transmissible infections is 4.5%. 32% of plasma is obtained by plasmapheresis. blood components collected are as ffp, rbc, frozen rbc, 1eukocyte-and platelet-depleted rbc, rbc suspension, and preparations: 10% albumin, immunoglobulins, and cryoprecipitate. as to blood safety measures -implementation of blood components leucodepletion and ffp and rbc quarantine in process. the new national strategy of bts reorganization has been developed. it includes the following: increasing the visibility and resource commitment to blood issues at the national, regional and municipal levels; the national voluntary donor programme promoting; blood safety increasing; blood collection, testing and pro-cessing concentration in federal and regional bts establishments, appropriate blood and blood components usage. calculating the cost of blood in turkey n solaz, s kemahli and s cin ankara university, ankara, turkey background: like other fields of the medicine cost efficacy is gaining importance in blood banking and transfusion medicine since last few years. since last 5 years even the most developed countries started to discuss about the cost of blood. in turkey ministry of health determines the cost of blood annually. aim: to establish a safe, cost effective and reliable prices for blood components. methods: turkish ministry of health (moh) started to determine the cost of blood components as 'all inclusive' principle. this means that cost of a unit of blood component will cover all conventional expenses such as; blood typing, infectious screening, labour, consumables, etc. this system has provided uniformity to blood component costs but if the system is not controlled and followed properly it will cause serious risks. there might be some blood banks which will not respect the safety regulations and may modify the test standards for decreasing the cost of tests. conclusion: current blood product pricing system looks generally reasonable and reliable but moh should establish close follow up systems for avoiding any abuse on the safety of blood. background: a positive direct antiglobulin test occasionally occurs in normal blood donors, and is often discovered when the donor's red cells are found incompatible in a compatibility test. the incidence of a positive dat was expected to increase since more sensitive techniques (gel test) were installed. the aim of our study was to examine whether dat positive otherwise healthy donors presented any clinical or laboratory abnormalities. methods: in the first 19.000 cross-matches last year (in 10 months) 38 were found incompatible due to dat positivity of blood donors' red cells (0.02%). dat positive [(1+)-(3+)] samples were only igg positive in 32 cases, only c3d positive in 5 and igm positive and c3d positive in 1 case. all blood donors were notified and thirty two of them responded to a request for a further sample. a complete blood count, a reticulocyte count, bilirubin (total, direct, indirect), transaminases, serologic immunological tests (ana, anti-dna, anti-ena, rf, anticardiolipin antibodies), quantitative assessment of immunoglobulins, aptt and lupus anticoagulant were performed, as well as serologic tests for markers of viral infections. dat and iat were performed by gel test (id-diamed) according to the manufacturer's instructions. dat were performed with polyvalent and monovalent reagents (anti-igg, -igm, -iga, -c3c, -c3d). the blood donors were also examined clinically. the donors who had positive immunological tests were referred to a rheumatologist for further investigation. results: among the thirty one blood donors eight had received medication the last 24 hours before blood donation, two had been vaccinated for hepatitis b recently, four presented signs of a viral infection soon after blood donation, three had evidence of an allergic condition, five had positive tests for anticardiolipin antibodies and ana, two were positive for anticardiolipin antibodies only and two had a positive ana test only. in six blood donors we did not find any abnormality that might be interrelated to dat positivity. conclusions: all blood donors with positive dat should be requested to undergo further investigation. some of them are possibly candidates to long medical follow-up, especially those with other immunologic abnormalities such as positive ana and/ or anticardiolipin antibodies. the eligibility of such donors for future donation of whole blood, platelets or plasma needs to be elucidated. tions: usefulness, frequency and sincerity in answering questions. donors could choose one of the offered answers and elaborate in writing the answer they have chosen. results: of the 1000 donors that participated in the survey 947 (97.4%) answered the questionnaire, 820 (86.6%) men and 127 (13.4%) women. that the survey was useful thought 91% and 9% that it was not. opinions were elaborated by 61.1%. that the questionnaire should be completed before each blood donation was the opinion of 81.2%, 17% thought it should be filled out only the first time blood is donated and 1.8% that the questionnaire should not be completed at all. the answers given were sincere in 95.4% of blood donors, 2% were not and 2.6% were given automaticallywithout comprehension. conclusion: most donors believe that completing the questionnaire before each blood donation is an effective way to increase safety by preventing potentially infected individuals from donating blood. they are also aware of the importance of answering questions truthfully because the end result is protecting the wellbeing of both blood donors and receivers. analysis of blood donor's deferral in national institute for transfusion medicine -skopje for the last five years (2000) (2001) (2002) (2003) (2004) p blagoevska*, i nikolovska † and r grubovik* *national institute for transfusion medic, skopje, † medical center, prilep, macedonia introduction: safety of blood and blood products depends on many different factors, starting with selection of blood donors. the aim of this study is to analyze the number of deferred blood donors and the reasons for their deferral, as well as the total number of blood donors in nitm and their correlation (voluntary/family donors). materials and methods: this is a retrospective, epidemiological study and data were taken from the blood donor's registry in nitm from 01.01.2000 till 31.12.2004. statistical mass includes blood donors who came to nitm to donate blood in the mentioned period. results: there were total 14 229 donors in nitm and 541 (3.8%) deferrals. 71.5% of deferred ones are male, as well as in the group with donated blood (males are predominant). the most common reason for deferral is low hb level in 232 (42.9%) blood donors, use of drugs -78 (14.4%), low blood pressure -47 (8.7%), high blood pressure -43 (7.9%), infections -30 (5.5%), cardiovascular diseases -21 (3.9%) and others. relation voluntary/family donors is almost equal (49. 6 : 50.4 ). in the last two years the number of voluntary blood donors is increasing (60 : 40), which is good sign. conclusion: percentage of deferred blood donors in first three years is ~2%, which is result of insufficient data and it is increasing in the last two years (>6%). reasons for deferral are predominantly from temporary character (98.9%). permanent deferrals are only 6 (1.1%), which is probably due to good education of the population and self-deferral. we should establish the national registry for deferred donors, as well as for the donors with positive markers for tti. we should design a strategy for returning of temporary deferred donors. regruting blood donors in multiethnical environment p blagoevska*, r grubovik* and k elezi † *national institute for transfusion medic, skopje, † medical center, gostivar, macedonia introduction: population in r.macedonia consists of 75% macedonians, 22% albanians and 3% others (serbs, gypsies, turks). over 80% of blood donors are voluntary non-remunerated and ~20% are family donors. transfusion service and red cross should recognize the values and cultural differences of minors groups and recruiters should developed methods for reaching and motivating them to donate blood. the aim of the study is to present the ethnical structure of our donors and to develop strategy for their regrutation and retention. the study reviews the results from the blood donation actions among the high schools and university students in west part of the country (multiethnical environment) from 2002 till 2004. results: there were 3704 blood donations for the mentioned period. predominant blood donors are employed and high school students in 75%. family blood donors are ~20%; between them 70% are from albanian population. the ratio between blood donors macedonians vs. albanians is 80 : 20. woman blood donors are presented with 14%. first time blood donors are 53%, and regular donors arẽ 13%. conclusion: first step in planning the blood donation in multiethnic society is creation of special teams of important and devoted volunteers, such as religious leaders, teachers, doctors and businessman. for a successful campaign it is necessary to design special promotional material and address personally to the target population on their mother language. background: pursuit of pharmaceutical purity of the blood in the bag has led to a shrinking donor base and a significantly more expensive product. decisions regarding new infectious marker testing and donor deferrals have typically been made emphasizing decreasing one specific risk without considering the effect the intervention will have on the overall safety of blood transfusion. regulations have been formulated by governmental agencies with limited input from the medical community. the decision making process has lacked risk benefit analyses and has not had the robustness associated with spirited discussions. policies made in this manner may result in certain risks being decreased but can also have adverse unintended consequences. discussion: in the u.s., the fda's implementation of donor exclusions to prevent possible transfusion transmitted vcjd has reduced the donor base by more than 2%. given the demographics of the deferred donors, the impact on plateletpheresis donations has been even greater. to compensate for the loss of donors, blood services will have to persuade present donors to donate more frequently, to recruit new donors, or both. one study has indicated that two-thirds of donors have no intention of donating more frequently. new donors have higher rates of infectious disease markers with positivity for hiv and hcv twice as high as repeat donors. despite sensitive testing techniques, window periods still exist and not all potentially infectious donors will be excluded. another area of concern is the aggressive use of inducements to attract new donors. some blood services are offering lavish incentives such as enrolling donors into drawings to win automobiles. most donors entering the lottery will be low risk; however, it is reasonable to worry that such extreme tactics might also attract persons who should not be donatconclusion: (a) blood donors who were patients' relatives were many more than volunteers as well as more were men than women. also people of young ages were more than those from older ages. (b) the frequency of the diseases for which the blood units were tested was found to be in low levels in the population of the area. specifically as concerns hcv, it seems that transmission frequency has been reduced after the obligatory testing of hcv in blood transfusion centres and stations. genotype 1b of hepatitis c virus is the most frequent in blood donors 2d, from 3a to 3f, from 4a to 4k, 5a and 6a. these are differently distributed in the world: types 1 and 3 are the most common in europe and in usa. aims: considering that, in our region, anti-hcv antibody positivity is variable from 0.87 to 4% of general population, aim of this study has been to evaluate the prevalence of hcv genotypes in blood donors. methods: in period from may 2003 to december 2004, 83 362 blood units were analyzed by nat for viral rna research. nat has been performed on single sample by tma technique. on rna-positive samples, the hcv genotype has been identified by reverse hybridisation with line probe assay. results: 143 blood donors have resulted hcv-rna positive with identification of the following genotypes: 1a = 11 cases (7.7%); 1b = 89 (62.3%); 1a + 1b = 3 (2.1%); 2a/2c = 35 (24.4%); 3 = 1 (0.7%); 4 = 4 (2.8%); none was 5a or 6a. we have also analyzed the differences between the two sexes in hcv-genotypes distribution. hcv-1a has showed a double prevalence in men (8 cases, 5.6%) respect in women (3 cases, 2.1%), while genotype 1b is more frequent in women (48 cases, 33.6%) than in men (41 cases, 28.7%), moreover genotypes 3 and 4 do not compare in women. although an accurate pre-donation selection, discharging all subjects with alt >40 iu, our results show that 1.7 : 1000 donors, apparently healthy and without risk factors, have resulted hcv-positive. analyzing our data, the genotype 1b has resulted the most frequent in blood donors' population, followed by type 2, while the others have showed a very low prevalence. the high frequency of genotype 1 in blood donors is explained by the observation that hcv 1 is usually associated with low alt levels, for this reason affected subjects may escape to donor's screening only based on dosage of alt. on the contrary subjects affected by other hcv types, associated with high alt levels, may be deferred increasing the hcv 1b relative prevalence. at the end, the different distribution of hcv genotypes between men and women and between age's classes probably reflects differences in the pathogenic characteristics of the virus, in the transmission way and in the risk factors. in fact, it has been demonstrated that genotype 1 is principally linked to a not transfusion transmission way; genotype 2 is linked to old age, to female sex and to post-transfusion transmission; genotypes 3 and 4 are associated to young age and to an history of drugs abuse, respectively with high and low viral load; genotypes 5 and 6 are still little known because extremely rare in europe. p-023 kell blood group system and rare blood donors v fakitsa*, p karyda*, s giannoulea † , c antoniou*, j flesiopoulou*, e haliou*, m papakonstantinou*, h dessilla † , e katsadorou*, g lyrakos* and k sofroniadou* *general hospital of nikea, pireas, † blood transfusion center, athens, greece background: the kell blood group system is a compound antigen system exclusively of red blood cells. some of the kell antigens are highly immunogenic. the commoner kell antibody is anti-kel1. the kel2 (cellano) antigen is a high frequency antigen and the blood donors lacking this antigen are quite rare. the blood donors who have not factor cellano are classified in the rare blood donors. rare blood by its very nature is required rarely, but when needed that blood has to be ensured to specified patients. there are other blood donors in their family -74 (35.57%) students, but the number of persons that donate blood from their neighborhood and close environment is much bigger -175 (84.13%). motives for their donation are the following: their wish to help the ones that need blood -142 (68.26%), concern that some day everyone can be a potential recipient of blood -38 (18.26%), because of offered benefits -27 (12.98%), for a friend or relative -23 (11.05%), care for their health -16 (7.69%), because of citizen duty -8 (3.84%), because the others donate -6 (2.88%), curiosity -1 (0.48%). they want to be invited every 6 months -68 (%) students, every 12 months -38 (18.26%), every 3 months -16 (7.69%) and 16 ( the mean age of case group was 30/55 ± 9/33 and the mean weight of them was 71/23 ± 9/3, 72/6% was male and the mean number of blood donation was 2/34 ± 2/19. the mean age of control group was 35/27 ± 10/72 and the mean weight of them was 78/61 ± 12/77. 90/3% of them was male and the mean number of blood donation was 6/58 ± 7/08. the blood donors who were female, first time blood donor low wt the rate of vasovegal rx was higher in female, first time, low weight, younger blood donors (p < 0.005). the rate of vasovegal rx was higher in blood donor (p < 0.005) who were fatigue or first time blood donor, low wt blood donation, fatigue of them and starvation of them had higher absolute donation reaction than other donors. when each variable was adjusted for other variable by regression analysis. young age, first time blood donation, anxiety, fatigue, starvation were significant (p < 0.005) and the others were not. conclusion: donation -related vasovegal syncopal reactions are a multi factorial process. these reaction are more prevalent in blood donors who are young, first time donor, anxiety, fatigue, starvation. these reactions might be predicted vasovegal reaction and these some facth donors need more care. with better donation care, syncopal reaction may be decrease this would be improved donor safety, better donor retention, higher donor satisfaction, and reduce cost and increase regular blood donors. to avoid iron deficiency in blood donors, iron compensation is necessary in most females and males who donate more than 1-2 and 3-4 whole blood units per year, respectively. we present 3 studies dealing with different dose and duration of iron compensation. in the first randomized placebo controlled study iron decreased continuously in males and females at donation intervals of two (males) and three months (females) without iron compensation. 40 mg and 20 mg daily combined with 400 mg ascorbic acid over 2 months (males) or 3 months (females) compensated for iron loss or even overcompensated in females. in the second open study we reduced iron dose to 20 mg daily over one month for both genders. this iron dose was sufficient for compensation of iron loss. a further reduction of iron dose to 20 mg daily over half a month led to negative iron balance in the majority of donors. in all three studies donors with exhausted iron stores profit more from iron compensation, whereas donors with high ferritin values (>50 mg/ml) tend to loose storage iron. aim of the study: one of our campaign strategy how to increase blood donation among adolescents are periodical seminars and excursions for students of secondary schools (more than 40 per year). the aim of this study is to analyze impact of our campaign educational system on adolescents in period 2000-2004. methods: the donation of whole blood and aphaeresis platelets from donors of age from 18 to 20 (max. 3 years for each class) were count for the period of five years (2000) (2001) (2002) (2003) (2004) . the percentage of the man´s donation was calculated for each target class (1982) (1983) (1984) (1985) (1986) . results please see tables 1 and 2. in the tables there is shown observed data in relation to the total number of births in the czech republic in reviewed years. the study showed that number of donation from donors of age from 18 to 20 decreased during objected years. unfavourable state of total number of births in the czech republic (153 801 birth in 1980 republic (153 801 birth in , 133 942 birth in 1986 ) and its decreasing tendency (92 786 birth in 2002!) is with high probability a major demographic factor affected number of young donors. despite energy invested in our campaign educational system our recruitment efforts should be intensified to decrease influence of demographic factors. we should find new ways and methods to attract new blood donors and keep the regular ones, too. the aim of the research was to investigate women's attitudes towards blood donation in cyprus. a statistical sample was selected using stratified sampling and consisted of 369 women from the district of limassol (the second largest urban center of cyprus) between the ages of 19 and 60. using linear logistic regression, the analysis of the data collected revealed that there is a greater probability for a woman to be a blood donor if she is of a higher educational level, a member of an organized group or association, or if she is acquainted with other blood donors. the percentage of female blood donors is higher in rural areas than in urban centers. 40% of women do not donate blood and attribute their reluctance to do so to health-related problems, while about 25% of those who have never donated blood claim to fear the blood donation procedure. in addition, more than half of the women who have stated they would never donate blood again have attributed their denial to healthrelated problems. the research revealed that there could be an increase of up to 30% of the percentage of female blood donors if they were given time off work for a few hours or one or two days afterwards. even though very few female blood donors expressed a preference for the blood donation to take place on a particular weekday, half of them prefer the donation to take place on the discussion: it is about small group of students. the impression is that the altruistic behaviour is present at most of the questioned students. the fact about free school days is not underestimated because it is one of the most important motives of blood donoring of the young population. families where the blood donoring is a tradition have a great influence for young children because the children in these families are better informed for blood donoring. conclusion: including the children in the process of education for young children is of particular importance because the altruistic behaviour as a higher feeling is from an early age of the child and it is under the influence of the environment (family and friends). active participation of the department for transfusion medicine in the educational process, especially in the education of young children, is a guarantee to achieve longlasted positive results. adverse reactions in 2 blood donors taking betablocking antihypertensive medications l paesano*, m d'onofrio*, s misso † , g fratellanza* and e d'agostino* *university federico ii, naples, † hospital san sebastiano, caserta, italy one aim of blood donor's selection is to avoid an adverse reaction to phlebotomy (as vasovagal reaction, syncope and/or hypovolemic cardiac insufficiency). blood donation is surely contraindicated in various pharmacologic therapies, but not in all. in fact a certain degree of discretionarily exists about the assumption, or the period of suspension, relative to a numerous pharmaceutical products, as the antihypertensive agents. according to literature, the deferral of donors taking antihypertensive medication is not indicated when blood pressure is normal, symptoms are absent, and diuretics or similar agents are the only drugs used. on the contrary, it is a common opinion that an antihypertensive therapy by betablockers is not compatible with blood donation for its cardiac effects. nevertheless, in our daily activity, the observation of a blood donor taking beta-blocking drugs may occur for various causes. a possible error is a superficial pharmacological anamnesis, as it can occur in donations on autohemotheca, for a too fast medical visit (due to a large number of donors), or for the inexperience of the selector (often a not specialist of transfusion medicine young doctor). another possibility of observation is constitute by patients, undergoing to elective surgery, included in a program of autologous blood donation, suffering hypertension treated with betablockers. in fact, in this last case, the risk/benefit balance justify the blood letting procedure. in the last year we have just observed two severe post-donation reactions in donors suffering hypertension treated with atenolol. the reactions have been similar, in fact both donors showed lypotimia followed by convulsions about past half hour by the end of phlebotomy. no prodromic symptoms have been observer or referred. cardiac frequencies (cf) before donation were respectively 60 and 64 beat per minutes and blood pressures (bp) were both in the normal range (130/80 and 120/80 mmhg). after donation, during adverse reaction, cf showed no substantial variations, while bp have been decreased respectively to 80/45 and 60/30 mmhg. immediate treatment has consisted in putting the donors in the trendeleburg's position and in applying a dolorous stimulation. in the first case this treatment has been sufficient to report the bp to 100/65 mmhg (with disappearing of all symptoms) in only half hour time. in the second one, the marked hypotension showed a very slow remission, for this reason the subministration of a plasma expander needed, with the complete resolution of the symptoms after two hours. these two donors were not deferred from donation because they were periodic donors that had modified their antihypertensive therapy, without referring it neither in the questionnaire nor during anamnesis. our experience confirms that the blood donation don't must be permitted to subjects taking betablocking antihypertensive drugs. in fact these medications act on cardiac pump decreasing the cardiac rhythm and limiting the postdonation cardiac recover. this effect is very dangerous because it appears relatively in retard respect to the end of donation, when donor may have just leaved the transfusion center. introduction and aim of the study: in society under transition privatisation and marketisation probe all areas of life. transition to market economy is extremely important and sensitive issue in health and welfare services in general, and specifically in the case of blood transfusion service. the aim of the study was to analyze effects of confusing publicity which introduced possible ways of transforming blood transfusion service in serbia (ideas about privatization of some parts of national blood transfusion institute, buying blood from blood donors, selling blood from voluntary blood donors to private clinics, exporting blood from vbd, stories about tradition of paid blood donations in some european countries). publicity was restricted to a small number of sporadic outbreaks concerted in a limited period of time. table. conclusion: surveillance of adverse reactions and injuries or accidents during or after blood donation is essential for maintaining the well being of active blood donors, as well as for the safety and quality of the donated blood components. information on other activities and parameters affecting the quality of blood including materials, reagents and equipment should be collected and any serious deviations from standard operating procedures should be notified to the competent authority using haemovigilance infrastructures. skae has built up such procedures working along the lines of the european haemovigilance network. improvement of existing national haemovigilance systems is expected to follow from the implementation of the eu directive. although inevitable, blood donor deferrals lead to losses in donated blood supply and may affect donor-return rates and subsequent blood donations. to estimate the scope of blood donor deferrals and their causes, we analyzed the 2003-2004 data from regional blood centers using standardized criteria for temporary and permanent blood donor deferrals. within this period (2003) (2004) , 14.6 percent of persons who presented for donation were deferred; 85.9% were temporary deferrals (50% due to laboratory test results, among others low hemoglobin, 6.4% due to risk of acquiring a transfusiontransmissible infection) and 14.1% were permanent (20% due to the infectious diseases markers, 8.7% due to cardiovascular diseases). for regional blood centers the temporary deferral rates varied widely (see the table below ). in the case of individual regional centers, the differences as well as the most common causes were often difficult to explain. according to our analysis, some blood centers have a more restrictive approach to donor acceptance than others and this results in increased donated-blood loss. to some extent such losses could be avoided. further studies are recommended to elucidate the problem and eliminate unnecessary deferrals. caption 1: percentage of deferrals aims: from our experience in selecting blood donors, a certain number of issues have been noticed that remain obscure and need to clarification since those seem to 'haunt' the whole process of blood donation. methods: many first time blood donors and especially volunteers think that rejection reasons are permanent and they are completely incapable of donating blood their entire life. this is a 'tragic' misunderstanding since the doctor did not explain that the reason of the rejection is only temporary and in the future this man is capable of donating blood. those potential donors will never even approach again blood donation centre and when in the future they are asked why they do not donate blood, they repeat the cause of the past rejection. results: one of these rejection reasons is for example low blood pressure (12.54% of total causes of rejection). as we all know blood pressure must be determined according to age, sex, weight and from other factors as sleep, emotional status, food and liquid intake. therefore blood pressure is very important but should be evaluated with all the above factors and must not be alone the only reason for rejection. even when one blood donor is rejected it should be made clear to him that this is only temporary and if in the future he is in better physical condition, he could donate blood. in fact 85-90% of those donors rejected for hypotension are readmitted in blood donation after meeting the above mentioned criteria. another matter of equal importance is anemia (25.04% of total causes of rejection), especially concerning young women. since most of those women tend to develop anemia due to depletion of iron stores, they should be advised to donate blood at longer periods than regular, to receive proper medication and diet according to their needs. the doctor must explain the donor the reasons for iron depletion, so blood donation should not be considered as the only cause for this situation from the donor. there are many factors contributing to anemia, menses, specific diets, overwhelming stress and exercise, not to mention other medical reasons. it is the duty of the doctor to correct those factors that resulted in iron depletion or anemia and readmits those donors in blood donation in the future (75-80% of those rejected are readmitted in our centre). summary/conclusions: at our blood centre we have created a program of regular tests (blood tests-physical examination) for all our blood donors. our experienced and well taught personnel offers advice and provides useful information in every aspect of blood donation and more. we have created a friendly environment for all our volunteers with love, understanding and appreciation and believe that this is the only way to keep a constant 'flow' of blood in our region. introduction: an innovative perception for blood donation in a new and evolving environment must focus on specific matters and ideas and adopt in a certain level lifestyles and concerns of society. aims: the purpose of this study is to find methods and ideas that can help blood donation centers throughout our country to create new blood donors, give a motive and inspiration for blood donation by adopting new trends of society and finally accomplish national need. methods: by having a personal interview with many volunteers about their feelings for healthier life, their nutritional habits, daily physical activity, sports, vitamins, smoking, weight, cholesterol levels. we investigated whether they believe that blood donation has, if any role towards a more hygienic life. results: we divided blood donor volunteers according to their age, educational level, and number of blood donations per year. our results indicated that there is a tendency among young educated people to adopt a personal lifestyle that includes consuming healthier food, keeping their weight low close to the ideal, having some kind of personal activity, not smoking, watching cholesterol levels, following doctors advice and concerning seriously about their health. this dynamic group of blood donor volunteers considers blood donation as a contributing factor to well being and donates blood at specific intervals. besides the yearly run lab tests that are done by our blood centre they also seek advice and discuss any matter concerning their health with the blood centre doctor. it appears that they are extremely sensitive in those matters and they seem to appear well informed about issues concerning their health, they also believe that blood donation is part of the plan they have to keep fit and being well. in our blood centre we encourage this belief and we also provide information concerning this new trend towards healthier habits. summary/conclusions: this approach has already shown some positive results in our blood centre as many people especially young educated women have joined our blood donorship program and donate blood at scheduled intervals. in order to achieve our goal which is to raise the percentage of blood donors in the region we have to be flexible, innovative according to new habits and lifestyles. we have to move with society and modernize the way we attract various groups of people. blood donation against prejudice as saltamavros*, s dimitrakopoulos † , v zacharaki*, p giannaros*, s markou* and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: in order to achieve a greater population to be admitted in blood donation we have to provide information concerning any obscure issues that presents in selecting donors. to examine the accuracy of hb measurements obtained by the noninvasive clinical device, as compared to values detected by standard methods, (cell-dyn 1600, abbott laboratories, usa), in a blood donor setting. methods: the nbm-100 device utilizes a finger base sensor using occlusion red/near-infrared spectroscopy (o-rnirs) to detect and analyze the hb/hct levels. the clinical trials were conducted at two blood donor centers (israel and usa). studies were carried out on a group of 159 subjects (86 females, 73 males) aged 19-64. subjects were healthy volunteers who had come to donate whole blood or aphaeresis components. after obtaining informed consent, hb/hct levels of all the study volunteer participants was tested non-invasively, using the nbm-100 device, followed by a venous blood sample. additionally, the usa center tested a capillary blood sample using the hematastat hct measurement device (71 donors). hb levels were considered normal when readings were equal to or >12.5 g/dl. results: venous hb measurements ranged from 9.9-16.2 g/dl. the mean nbm-100 hb level was 13.20 ± 1.35 g/dl, only 0.28 g/dl lower than the mean hb result obtained by venous sampling, which reached 13.48 ± 1.22 g/dl. the standard deviation of the difference between the invasive and noninvasive hb readings was found to be ±1.04 g/dl. the mean absolute error (mae) of their difference was 0.81 g/dl. when checked against the cell-dyn in the usa center, where 21 subjects had hb of 12.5 g/dl or lower, the nbm-100 and hematastat devices showed comparable sensitivity results. the nbm-100 using o-rnirs is a promising noninvasive technique for hb screening in blood donors. the device is easy to use and agreeable for both blood donors and personnel. the technique reduces the need for the invasive finger prick or venous blood sampling, thereby enhancing safety, reducing costs, and improving the experience of blood donation. the effect of short-term, temporary deferral on blood donor return rates and subsequent blood donations background: blood donors are deferred for numerous reasons. some deferrals like intravenous drug use, male homosexual contact or certain positive test results are permanent. the majority of donor deferrals, however, are short-term temporary deferrals (sttds) that are resolved in a matter of days, weeks or months, after which time the person is again an eligible blood donor. the effect of sttds on blood donor return rates and subsequent blood donations is studied. materials and methods: donors given sttds during the 15 december 1999 to 15 march 2000 were computer-matched with non deferred donors on the basis of age, sex, and donation date (case group: 804 donors -control group: 295 donors). computer records were evaluated during the next 3 years (21 march 2000 to 21 march 2003 to determine donor return rates. significance for comparison between the two groups was based on chi-square analysis. results: the most common reasons sttds were elevated blood pressure (52%), deferred for medication (29%) and colds and/or sore throats (4.2). non deferred donors were a little more likely than donors with sttds to return over the next 3 years (36.6% vs. 34.8% pv = 0.0001) and non deferred donors donated more whole blood units. . according to ethnic structure, women -ethnic macedonians donate blood in largest numbers -1096 (14%), while all other ethnic groups are present with only 3%. the most prevalent is the group of adults aged 41-50 (358 -27.5%), with high school education -800 (61.4%) and mostly those who donated blood 1-5 times (719 -55%). conclusion: having in mind that 50% of the population in macedonia is female, the obtained results reveal a significant, yet insufficient participation of women in blood donation with 17% in relation to the total number of blood donors surveyed in the period 1999-2003. this is due to insufficient motivation and education of women from all ethnic groups especially those from the younger population and with elementary education. incorporating them in education and organization would contribute to their more extensive participating in blood donation. comparison of serum beta 2-microglobulin (b2-mg) between hbsag positive donor and healthy control f tarabadi*, m shaeigan*, g babaee † , a talabiean* and m khadir* *iranian blood transfusion center, † tarbiat modarrs university, tehran, iran background: beta 2-microglobulin (b2-mg) is a low molecular weight protein (11 800 daltons) and found in all biological fluids it is light chain of histocompatibility class1-human present on the most membranes of cells. in the hepatitis infection the viral antigen presentation on the hepatocyte in the presence of class 1-hla antigen plays a role in the elimination of the virus. method & samples: beta 2-micro globulin was measured in serum drawn from 45 hbs ag positive blood donors include 5 (11.1%) female and 40 (89.9%) male in age between 17-56 years, and 50 healthy 16 (32%) female and 34 (68%) male in the same age we detected serumic b2-mg by enzyme immunoassay (ela). results: our studies showed b2 mg level increased in 7 (15.6%) hbs ag positive donor that was significant differences with healthy control (p = 0.0001). conclusions: it seems that serum b2mg is a good marker for hbs ag replication. the role of b2mg in monitoring of response therapy needs to be more evaluated. and 1635 (0.8%) were contributed by vd, rd and dd respectively. over the last 5 1 / 2 years, voluntary donations have shown a rising trend from 54.3% to 60.9%, where as rd (44.9% to 38.4%) and dd (0.9% to 0.8%) have shown a declining trend. the percentage of female donors was maximum in voluntary group as compared to rd and dd (11.01% vs. 2.02% vs. 6.17%) respectively. the rates of all tti markers reactivity were significantly higher in rd as compared to others donors. the hbsag and anti hcv reactivity in vd and dd is comparable (0.84% vs. 0.85% and 0.45% vs. 0.49%). hiv antibodies was found more frequently in vd as compared to dd [0.15% vs. 0.06% (p < 0.05)] whereas, vdrl reactivity was lower in formal as compared to latter [0.26% vs. 0.37% (p < 0.05)]. conclusion: voluntary blood donation has shown a rising trend over a last few years, thus highlighting efficient donor motivational strategies. these strategies need to be strengthened to increase the female donor base. the safety of dd is equivalent to vd when the rates of tti are compared. thus, dd should be advised to donate blood regularly as voluntary blood donors. blood safety depends on a number of factors. the chain of safe blood starts with the donor. one of the procedures for obtaining safe blood for transfusion is the medical selection based on the completed questionnaire and the possibility of self-exclusion from the process of blood donation, the medical history of the potential donor and the medical examination. donor selection consists of two sets of information necessary for protection of the blood recipient as well as the donor himself. aim: to present the most frequent reasons for declining volunteer blood donors. material: the materials used for analysis were the questionnaires completed by all the potential blood donors at the transfusion department of the medical center in strumica as well as the record books of the blood donors which contain the results of the analysis we make for the potential donors. these donors donated blood in the period between 2001 and 2003. results: during this period 4129 people volunteered to donate blood, out of which 3940 were allowed to donate blood, while 189 were declined. out of the total number of blood donors 3313 were male and 627 female donors. the reasons for declining potential donors were the following: 42.3% had low levels of hb, 22.36% were taking antibiotics, 9.37% were ill, 7.83% had low blood pressure, 6.75% had high blood pressure, 11.39% for other reasons. conclusion: donor selection and their care on one side and obtaining safe blood for transfusion on the other side entails obligatory organized medical control. the obligatory completion of questionnaires, the medical examination of the potential donor and their self-exclusion as a result of the feeling of personal responsibility as well as the obtained information are very important for the selection of quality blood donors and obtaining safe blood for transfusion. questionnaire on 2700 subjects-students, their knowledge and motivation on blood donation f vladareanu, a bugner and s sirian national institute of heamatology transf, bucharest, romania the research theme of this questionnaire is as follows: 'what is the level of knowledge and of motivation in the non-remunerated and voluntary blood donation at students?' we also tried to see the practical implications that this study will have and how it will influence the knowledge in this area. the purpose of this questionnaire was not dissimulated. the general theme of the knowledge and motivation on blood donation had been studied before through two big questionnaires applied in 1987 and 1992, but the general population was their target. students had never been an investigated lot up to now. the hypothesis referring to this problem is as follows: students are not informed either on the act of donation, or on the crisis of blood. 1. the lack of information is a first cause of the indifference of the studied lot towards the idea of donation. 2. the lack of motivation of the studied lot is another cause. the questionnaire was applied on a 2700 lot of students from seven different cities: bucharest, iasi, constanta, cluj, sibiu, brasov, timisoara. the number of the questions was limited to 22, which we consider best for a questionnaire applied on the street or at college. as a conclusion, we can say that a passive-defensive attitude towards the blood donation was revealed after this questionnaire. not knowing the issue caused by their lack of information sometimes determines indifference at the statement of the subject. on a general dissolution environment of the responsibility of the youth, the donation problem is not in their aria of preoccupations, the general attitude being of non-involvement for the moment, at this idea which is not yet in every individual conscience and which is normally administrated at an institutional level. the donor data and the details of blood application of the north west transdanubian region of hungary k vörös*, c bercsényi † , o petró † , r jáger † and e miskovits ‡ *hungarian national blood transfusion s., györ, † blood bank, tatabánya, ‡ headquarters hungarian n.b.s., budapest, hungary the ongoing fundamental reorganization of the blood service began on the 01.07.1998 in hungary. as the consequence of reorganization till 01.07.2000, 28 blood banks had been established instead of 63 existing before, under direction of the hhnbts. the working profile of the 6 regional blood centers and 22 local blood banks will be changed step by step. virus screening, blood group serology and processing will be made in the 6 regional centers. one of the regions is the 'north west transdanubian region' (nwtr, city györ as the center, with about 930 000 inhabitants and 8000 hospital beds). 3 local blood banks (tatabánya, sopron, and szombathely) are belonging to nwtr. the regional center and the local blood banks provide the labile blood products and high level clinical-transfusion service (cross-matching, antibody screening, outpatient immunhematology investigations, etc.) for the hospitals. annually 56 000 donors donate blood in this region. this donation activity covers about the 6% of all inhabitants. the acceptance ratio of the donors is good (6-8% of the donors were deferred). there are 14 hospitals in our region. the regional demand on rbcc is 30-45.000 u/year, on ffp is 5.500-7.000 u/year and on pc is 8-10.000 u/year. the poster shows the donor data and the details of blood application of this region since 1999. p-054 implementation of 2rbc collection using haemonetics mcs ® +: medical staff training, donor recruitment and acceptance g woimant, c fretz, d puydupin, e pélissier and jl beaumont efs ile de france, paris, france background: single donor 2rbc collection is an approved apheresis technique in france. aims: our goal was to evaluate the implementation of 2rbc collection in our center in terms of donor recruitment and acceptance, as well as medical staff training and adaptation. methods: donors were selected according to the french requirements for 2rbc collection (weight ≥ 65 kg, height ≥ 165 cm, hb ≥ 13.5 g/dl, ferritin ≥ 20 ng/ml for repeat 2rbc donors). all personnel were trained on adequate communication with donors. eligible donors were contacted by mail, by phone or during pre-donation interview. among the 102 recruited donors, all donors were male, 63% were regular whole blood donors, 36% were regular whole blood or apheresis donors and 3% were new donors. the medical staff was trained on 2rbc collection with the sdr protocol and disposable set ln 948pf on the mcs ® +. most of the medical staff was already used to autologous rbc donation with similar apheresis devices. blood samples were taken from donors pre-and post-donation, as well as 2 to 4 months later for those returning for a subsequent donation. donors were asked to fill out a post-donation survey for assessing donor comfort and information. results: donor profile and clinical follow-up are summarized in table 1 . six percent of the donors had a ferritin level below 20 ng/ml; these donors were regular whole blood donors. the collections were well tolerated and no changes in vital signs were noted. four reactions were reported: 2 hematomas and 2 citrate reactions. no reaction was observed post-donation and hemoglobin levels measured before next donation were back to normal. the technique was easily implemented by the medical staff and fitted well in the existing blood center processes. the medical staff as well as the donors found collection duration short (average of 35 min). the results of the survey were very favorable as more than 95% of the donors considered their donation and the information they received as satisfying. most of them agreed to donate again and several actually donated twice during the evaluated period. conclusion: the implementation of 2rbc collection in our center, using haemonetics mcs ® +, was successful in terms of ease of use of the technique, as well as user and donor acceptance. we now plan to evaluate donor loyalty in the longer term. risk from first-time blood donors e zhiburt, s golosova and p reizman federal blood center, moscow, russian federation introduction: each third dose of whole blood in russia is donated by first-time blood donor. there are two reasons for attention to this kind of donors: (1) possible risk of infectious disease in seronegative study; (2) possible risk of donation for person with contraindication. aim of the study: we investigated role of regional deferred donors registry (rddr) in by first-time donor selection. methods: moscow rddr includes parts: hiv, viral hepatitis, syphilis, tuberculoses, malaria, drug users, psychiatry, 60 days after blood donation. rddr was complete and our center began actively work with it since last year. each donor has to be registered in rddr and automatically checked for deferral reason. effectiveness of rddr was investigated. results: first-time donors donate less than 10% blood in our center. about a quarter of them are deferred before possible donation. part of donors deferred by rddr has been significantly increase in 2003 (c2 = 19.6; p < 0.001) at the expense of seropositive people. conclusion: rddr is effective for blood donor selection and decreases necessity in laboratory screening. first-time blood donors have to be examined before blood donation. if they have not contraindications, donation can be performed up to 10 days before examination and screening. the double unite platelet production is important especially if the relatives of patient find the donors. we evaluated the effectiveness two apheresis machine for platelet collection. in our blood bank, one fenvall amicus and one cs3000+ apparatus were used for platelet apheresis. 1531 apheresis were performed between 2/12/2002 and 13/2/2005. including criteria of donors are that estimated process time is smaller than 90 minute and estimated postapheresis platelet count is higher than 100 ¥ 10 9 /l. donors firstly was enrolled to amicus. if amicus was busy, then it was enrolled to cs. the properties of our donor populations were given in blood and plasma cell components are obtained either by traditional manual method from whole blood or by apheresis. modern medical treatment is based on transfusion of deficient components such as erythrocytes, leukocytes or plasma proteins. this involves new solutions to achieve higher yields and better quality of such components. the aim of our study was to estimate the efficacy of blood cell separator cobe trima in obtaining platelet concentrates (pcs) as compared to older-generation cobe spectra blood separator. apheresis procedures were performed on both these blood cell separators. the quality of platelet concentrates was tested during 5 day storage period (see table below ). we have tested the effect of apheresis procedure on donors and estimated the operating comfort of both separators. the tolerance of both separators was satisfactory except for more frequent hypocalcemia when trima separator was used. most donors were more satisfied with trima procedure because of single venipuncture although it involved special donor selection (good vein access). in general we may say that trima is undoubtedly a more modern and more friendly separator. however, cobe spectra may continue to be used with success especially when a more versatile cell separator is necessary (leukocyte concentrates, peripheral blood stem cells or therapeutic apheresis). methods and results: tls (587 procedures on 67 patients) were used successfully in patients with acute or chronic leukemia with hyperleukocytosis (white cell count >100 ¥ 10e9/l or blast count >50 ¥ 10e9/l) when high cell count would promote leukostasis with vascular occlusion in the microcirculation. performed tl procedures were rapidly reduced both the white cell count and the whole blood viscosity. average fall in white cell count after treatment was 73.3%. tp-treatments (186 procedures on 32 patients with symptomatic thrombocythemia and/or platelet count higher than 1000 ¥ 10e9/l) were applied in order to prevent the development of 'thrombotic-hemorrhagic syndrome' . the tps performed resulted with rapid platelet counts reduction (84.3% in average) and with clearly noted clinical improvements, subsequently. tes (429 procedures on 196 patients) were performed using manually technique in patients with 'cellular hyperviscosity syndrome' induced by high red blood cell count. it was shown that te procedures resulted to red blood cell number lowering and decreasing of blood hyperviscosity. average fall in hemoglobin and red blood cell concentrations after te treatments was from 20.5% till 35.7%. rbcx treatment (8 procedures on five patients with malaria and two with severe aiha crysis) was performed on an urgent basis, particularly when clinical symptoms indicate life-threatening situations and resulted with rapid and significant reduction of concentration of unwanted pathogen affected rbcs and summary/conclusion: the effects of tcs depended on the nature and stage of the basic hds, of adequate selection of patients and of timely applied apheresis. rapid cytoreduction is obtained justly in patients with excessively high cell count, and this effect did not associated with bone marrow remission. thus, tc should be looked upon as adjunct to the standard treatment of different cithemias, but not as replacement therapy. the present study indicates that the best therapeutic effects were obtained by rbcx. were carried out with continuous flow blood cell separator cobe spectra and all patients underwent large volume leukapheresis (lvl). in all procedures, a blood warmer was connected to the return line and a continuous calcium infusion was administered preventively. six patients, who were under 25 kg body weight, had the extracorporeal circuit primed with irradiated, filtered packed red cells diluted with 5% albumin solution. seven children had vital signs and ecg continuously monitored during the procedure. results: each patient underwent a median of 2 collections (range 1-6). the inlet blood flow ranged between 16.0 and 95.4 ml/min (median 54.5 ml/min). the median blood volume processed was 11 754 ml (range 2700-22 500). leukapheresis lasted a median of 240 min (range 120-270). the median total nucleated cell yield was 11.86 ¥ 10e8/kg (range 1.94-21.21), mononuclear cell (mnc) yield was 6.01 ¥ 10e8/kg (range 0.97-12.73) and cd34+ cell yield was 3.5 ¥ 10e6/kg (range 0.19-28.01). the median of mnc collection efficiencies was 56.11% (range 12.34-158.09). in 9 (39.1%) patients, in only one apheresis procedure more than 2 ¥ 10e6 cd34+ cell/kg were collected. during 6 (9.09%) procedures patients had experienced apheresis-related side effects. the citrate-induced reactions were most commonly observed. the reactions were mild and cessation of collection was required only in one case, because of catheter related complication. mild sedation was required only in few very small children. post-donation platelet count was less than 30 ¥ 10e9/l in 3 cases and these patients required platelet transfusion before subsequent procedure. our results show that lvl in pediatric patients is relatively safe procedure, well tolerated and with a very low risk of serious adverse events. close monitoring of blood counts, especially platelets, between pbsc collections is necessary. the cessation of procedure was required in only one case and no life threatening side effects occurred. neonatal alloimmune thrombocytopenia (natp) caused by fetomaternal mismatch for human platelet (plt) alloantigens (hpas) worsens approximately 1/2000 pregnancies and can lead to a serious bleeding diathesis, intracranial hemorrhage (ich) and sometimes death of the fetus or newborns. we describe the successful management of a 37-year-old pregnant woman, alloimmunized to the hpa-1a (p1a1, zwa) antigen, with a history of two previously children with severe thrombocytopenia and ich. the pregnant woman was at her terminal pregnancy and was suddenly admitted. to evaluate the risk of ich in the fetus, cordocente was performed to demonstrate fetal thrombocytopenia (plt 4.000/mmc). to ensure a rapid provision of compatible negative-antigen platelets, we decide to collect platelets from the mother using apheresis. plateletapheresis was performed using com.tec separator, fresenius. blood processed was 1.100 ml in a short time procedure (35 minutes). no significant adverse effects were observed in the mother and fetus, during and after the procedure. platelets collected (1.5 ¥ 10e11) were transferred to the preparation set and plasma was removed after centrifugation to resuspend the platelets in octaplas ab. then we separated the platelets into two units containing 0.7 ¥ 10e11 each. the day after the donation, the mother gave birth to a girl by caesarean section. after the transfusion, the plt account increased from 4.000/mmc to 35.000/mmc and after a week the child had plt 75.000/mmc without hemorrhagic complication. according to the literature data and our observations of the patients, there are changes of the hemostasis system indexes in the most patients with the endogenous intoxication syndrome and immune disturbances. in the number of cases medicamentous therapy appears to be not enough to normalize the changes, but it is especially important for pregnant women and women in childbirth, because on the background of these disturbances different complications of pregnancy and postnatal period take place. the aim of our study was the substantiation of plasmapheresis using in complex therapy of purulent inflammatory complications in obstetrics and immunoincompatible pregnancy with hemostasiologic disturbances. 10 patients with hemostasis system disturbances: one woman in childbirth with exacerbation of chronic pyelonephritis, who had in the first 24 hours some signs of hypocoagulation on the background of permissible blood loss (prolonged coagulation time up to 20-30 minutes with episodes of its absence on the background of the normal indexes of general coagulogramm, quantity and function of thrombocytes and the dilute fibrin monomer complex level in 7 times higher than the norm) and nine pregnant women with the perinatal losses in anamnesis severed by the pregnancy (threat of abortion, places of fetal egg detachment). these women were examined, the following was revealed: the high antibody titer to chorionic gonadotropin, parameters of partially activated thrombin time were higher than the norm (47-52 seconds), thrombin time (18-20 seconds), the dilute fibrin monomer complex (8-12 mg%), coagulation time (15-20 min). in all these cases the conservative methods of treatment (antibacterial, hemostatic, hormonal therapy) were effective for a short period of time and they didn't succeed to correct the given parameters of hemostasiogramm. the discrete centrifugation plasmapheresis was included in the complex of medical treatment. the woman in birth 5 operations were done, in the programme of plasma replacement during the first two plasmapheresis procedures donor fresh-frozen plasma was included. six pregnant women on the given stage one course consisting of 3 plasmapheresis procedures for plasma replacement with crystalloids was done, the volume of the removed plasma was 25-30% of the circulating plasma volume. three pregnant women before delivery were required two courses of plasmapheresis more consisting of 3-4 procedures each. the system heparin was not used. in all patients already after the first procedure of plasmapheresis the normalization of hemostasis indexes was marked, that allowed to prolong pregnancy, to prevent the coagulopathy bleeding and the development of disseminated intravascular syndrome. four women are discharged from the hospital, the other patients are observed with progressive pregnancy. thus, the using of discrete centrifugation plasmapheresis is effective at the signs of hypocoagulation in patients with isoimmunisation with fetal antigens and infectious pathology, and is the reserve in prevention and treatment of obstetric complications. extracorporeal photochemotherapy: an alternative therapeutic approach to control graft versus host disease after allotransplant with reduced intensity conditioning regimen c del fante*, c perotti*, gl viarengo*, p bergamaschi*, p pedrazzoli † and l salvaneschi* *irccs policlinico s. matteo, pavia, † ospedale niguarda cà granda, milano, italy background: extracorporeal photochemotherapy (ecp) can be defined as an immunomodulatory therapy that demonstrated to be efficacious in treating patients affected with graft versus host disease (gvhd) after allotransplants for oncohematological diseases. reduced intensity conditioning regimen (ricr) for allotransplant is a relatively new practice in patients (pts) ineligible for a conventional myeloablative conditioning regimen. the use of immunosuppressive therapy (ist) to control gvhd is limited for the high risk of developing infections and disease relapse due to the strong reduction of graft versus tumor (gvt) effect. aims: to evaluate the effectiveness and safety of ecp in treating pts affected with gvhd post rcr and the possibility to taper, at the same time, the ist. methods: 6 pts (5 females, 1 male), median age 47.5 years (25-63), affected with agvhd grade ii (1) and extensive cgvhd (5) gtx with median total granulocyte doses of 55 (21-150) ¥ 10 9 per gtx corresponding to 1.46 ¥ 10 9 granulocytes/kg in children and 0.58 ¥ 10 9 granulocytes/kg in adults. the wbc counts increased from baseline values of 0.2 (0-0.5 ¥ 10 12 ) g/l for both pediatric and adult patients to peak values of 3.8 (0.4-18.2) ¥ 10 12 g/l (children) and 0.9 (0.3-9.4) ¥ 10 12 g/l (adults) at one hour after gtx and to 1.4 (1.1-13.6) ¥ 10 12 g/l (children) and 0.4 (0.3-8.6) ¥ 10 12 g/l (adults) at 24 hours after gtx. in 42 out of 54 patients (80%), the crp levels significantly declined (60 (11-91)%; p£0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and gtx. patients without crp response to gtx (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. background: in recent years, the use of platelet concentrates obtained from single donors by automated apheresis has grown steadily. plateletpheresis donation is considered to be a safe procedure with modern instruments. so far, no studies have identified donor or procedure specific factors that may be associated with serious adverse events. aim: to evaluate the incidence of adverse events during plateletpheresis procedure, over a five-year period in our hospital. materials and methods: eight hundred single-needle plateletpheresis collections were performed by using two automated intermittent-flow cell separators: 700 of them with mcs3p and 100 with mcsplus (haemonetics), according to automatic standard protocols a3p and ldplp, respectively (with the collection of an additional plasma unit). acd-a was used as the anticoagulant in all apheresis procedures (acd-a: blood ratio was 1 : 11). most of the donors (84% men and 16% women) were patient´s relatives. half-hour before the initiation of the procedure, 250 mg of calcium (1 tablet cal-c-vita) were administered to each donor. the mean platelet yield was 3.2e11 /unit. the overall rate of the donor related adverse events was 3.4%. feeling faint was the most frequent event, which was occurred in 1.62% of donations. hypotension and citrate related rates were 0.77% and 0.73%, respectively. all citrate related symptoms were only transient perioral paresthesias, which were relieved by slowing the i.v. rate, without additional administration of oral calcium. donor unconsciousness was the only observed severe event, the rate of which was 0.25%. other adverse events were venipuncture related (2.37%), machine related (2.7%) and miscellaneous complications (1.25%). (1) plateletpheresis using the mcs3p and the mcsplus automated cell separators is a safe procedure, with a low risk of serious adverse effects. (2) with the used acd-a-to-blood ratio (1 : 11) satisfactory platelet concentrates were obtained with very low incidence of citrate-related events. (3) the peros administration of calcium before the initiation of the procedure, probably lowers the rate and the severity of hypocalcemia symptoms. quality assessment of ffp collected as a byproduct of plateletpheresis 1. from the donors immediately with the initiation of the procedure (citrated whole blood) and 2. from the final platelet concentrates after one hour rest at room temperature without agitation. in vitro platelet response to the aggregation-inducing agonists adp, collagen, ristocetin and arachidonic acid was investigated by means of an aggregometer (pap-4c, bio/data). results: there were no significant differences between the groups of donors with respect to age, sex, smoking habits, preapheresis wbc and plt counts and hemoglobin concentration, as well as in the harvesting time between the two cell separators. our findings are shown in the following table. mildly decreased response to all agonists was observed (mainly to adp and arachidonic acid) in the samples taken right after the initiation of the procedure, in both groups. platelets from the final component showed a further slight decrease in response to adp, which was more prominent in the mcs3p device (p = 0.025). on the contrary, an increase in platelet response to the other three agonists was observed in both devices, which, however, was statistically significant upon collagen and ristocetin stimulation. conclusions: reduced response to aggregation stimuli is possibly caused immediately with the initiation of the apheresis process. literature reports regarding further platelet traumatisation due to the procedure, are rather conflicting. in our study, such traumatisation was observed only in the case of adp in the mcs3p obtained collections and this could be correlated with the technological differences between the two devices. recovery of platelet aggregability, as it was expressed by the upregulation in platelet response in the other stimuli, could be attributed to the resting period and seems not to be affected by the timing of the leucodepletion procedure. background: lupus erythematosus often is accomplished with severe symptoms, such as polyarthritis, nephritis, pericarditis or dermal alterations. in pregnancy cytostatic therapy affects gestation. on the other hand the course of disease can be refractory to corticosteroid therapy. elimination of autoantibody and immuncomplexes by plasmapheresis could be an efficient way to amend the severity of symptoms. a 24 year old pregnant woman in the 24th gestation week with systemic lupus erythematosus showed severe symptoms like polyarthritis, nephritis and pericarditis. treatment was initially 1 mg/kg bw prednisolone for 3 weeks and subsequently 8 mg/kg bw for 2 weeks. plasmapheresis was applied daily in the beginning and continued depending on the condition of the patient ( table 1) . the eliminated plasma was substituted by fresh frozen plasma. the medium volume was 2.951 ml per apheresis. after day 11 plasmapheresis treatment was suspended to avoid problems with coagulation and was followed by a cycle of 3 immunoabsorptions to eliminate circulating immuncomplexes. results: prednisolone therapy alone brought no effect even after changing to high-dose treatment. a significant amelioration of all symptoms could be observed after the first plasmapheresis. good condition of the patient remained stable over the period of daily plasmapheresis for 4 days. intermitting apheresis treatment for one day lead to a significant aggravation of symptoms. apheresis no. 6 again lead to a recovery of the patient which held on until day 11. conclusions: treatment of systemic lupus erythematosus in pregnancy especially in combination with resistance to corticosteroid therapy, is an effective therapy to ease severe symptoms such as polyarthritis, pericarditis and nephritis. exposure to cytostatic drugs can be avoided and therefore the impairment of the fetus can be reduced. background: the collection of mnc represents the first step of photopheresis procedures and could be of critical importance in achieving a therapeutic goal. in this work we compare the cell yield of two collection programs on cobe spectra device: the mnc versus the autopbsc program using the ecp procedure modified by andreu. methods: 39 procedures were carried out with mnc program and 19 procedures with autopbsc on 4 patients with cgvhd. both hemoglobin increased from 35.9 ± 5.7 to 204 ± 100 mg/tu, k+ from 1.7 ± 0.7 to 44.8 ± 13.9 mmol/l, level of glucose decreased from 26.65 ± 2.06 to 9.75 ± 2.06 to 9.75 ± 0.91 mmol/l, ldh from 1.81 ± 0.48 to 6.24 ± 1.89 ukat/, lactate from 2.77 ± 0.47 to 22.72 ± 14.02 mmol/l, ph from 7.50 ± 0.14 to 6.74 0.08. the volume of apheresis units was lower than wb-rbc, the leucocyte count was normal in all units. the rbc loss by filtration was 24.9 ± 3.1 ml/tu and was lower than at wb-rbc. in apheresis rbc there were the differences in hb and ht value between the day of storage 0 and 40, in wb-rbc there were no differences. during the storage period we found no differences in k+ increasing value and no change in ph value between apheresis rbc and wb-rbc, the increasing of lactate was higher in wb -rbc, increasing of ldh correlated to hemolysis. the plasma hb value increase was higher at apheresis rbc in contradistinction to literature. hb and ht correlation in apheresis units according to predonate value in donors was lower than at wb -rbc. the method is a useful alternative to conventional whole blood donation, we get rbc units with high standard of quality and low correlation according to predonate hb and ht value in donors. acknowledgement: the study is supported by grant iga ministry of healthy cr n. nr/8015-3. background: in life-threatening exacerbations of sle a satisfying efficient therapy is lacking. despite intensive immunosuppressive therapy some patients are resistant or contraindicated to conventional treatment. in particular circulating antibodies and immune complexes play an important role in the pathogenesis of sle and mctd. an extracorporeal removal of these pathological substances may be effective in the treatment of active disease. methods: five patients with severe therapy-resistant sle/mctd underwent immunoadsorption onto protein a. blood was drawn from patients by using a jugular catheter or a peripheral intravenous catheter. anticoagulation was performed with acd-a and heparine or acd-a and r-hirudine. plasma was separated by centrifugation. the 1.5 to 2-fold total plasma volume was treated in every immunoadsorption. the columns were floated with a maximal plasma flow of 20 ml/min. the procedure was carried out every second day. additionally supplementary intravenous immunglobulin therapy was given only once. results: remission of the disease was achieved in four patients. see table below . conclusion: pa-ia is highly effective regarding the elimination of autoantibodies and circulating immune complexes, might induce a remission in patients with sle/mctd. it is an acceptable alternative treatment option in patients when other therapies are ineffective or contraindicated. background: purification of bone marrow from erythrocytes is used to prevent early hemolysis in major abo incompatible allogeneic hemopoietic cell transplantations. erythrocyte depletion is strongly recommended to reduce product volume and stem cell purification before storing autologous and even allogeneic bone marrow in order to prevent early hemolysis and dmso toxicity that might develop after thawing. centrifugation, sedimentation with hes, and cell separating devices are methods for erythrocytes depletion. aim: in our center, we prefer to use cell separation device, since it is a reliable method and has a high-yield and risk of contamination with erythrocytes is low. success of the process is retrospectively analyzed for high and low volumes. method: erythrocytes depletion of bone marrow harvest was done in hemapharesis unit with cobe spectra device in the last five years in 20 cases with bone marrow volume over 750 ml, and 17 cases with bone marrow volume under 750 ml. fifteen of these cases were allogeneic, and 22 were autologous procedures; a software uploaded with cobe pbsc coll vers 5.1 and (catalog no: 777-006-000) set was used in the procedure, and at the same time, double bag system with intermediate connectors were used to prevent re-circulation (catalog no: 777-006-300). results: the mean volume reduction was 90.48% (83.81-93.54) for volumes over 750 ml, and 89.79% (84.03-94.02) for volumes less than 750 ml. regarding the success of the procedure no statistically significant difference was found between procedures with high and low volumes. no complication developed related to the device or product, and waste bag never had to be re-used. in none of the patients early massive intravascular hemolysis was observed. conclusion: erythrocyte depletion and volume reducing with cell separation device is a reliable method. this process is successfully applied with high volumes (over 750 ml); and in low volumes as well for reducing erythrocytes, and gain of mononuclear cells and cd34+ cells. platelet concentrates obtained by apheresis procedure-correlation between the initial count and the final concentration v srejic*, g bogdanovic*, z garic*, n vavic* and b balint † *national blood transfusion institute, † military medical academy, belgrade, serbia apheresis team of the national blood transfusion institute processed and classified data of 100 donors who donated platelets by apheresis procedure from january till april 2004. procedures were performed in accordance with the ldplp protocol, using haemonetics mcs+. initial donors' platelet count and the absolute platelet concentration in the final preparation were followed, as well as red blood cell and leukocyte contamination and the volume of the processed blood. donors' initial platelet count was not less than 191 ¥ 10 9 /l and the volume of the processed blood was not less than 2300 ml. according to histogram, the most frequent donors' initial platelet value ranged from 210 ¥ 10 9 /l to 308 ¥ 10 9 /l (70%). final concentration of the samples of 100 tested donors ranged from 3.9 ¥ 10 11 to 6.1 ¥ 10 11 in the average volume of 400 ml. regression analysis demonstrated that there was a correlation between the initial donors' platelet count and the obtained final concentrate. student's t test showed p < 0.0011. leukocyte contamination of the final concentrate prepared without the filter ranged from 0.0 ¥ 10 9 /l to 0.4 ¥ 10 9 /l. presence of red blood cells in the final concentrate ranged from 0.01 ¥ 10 12 /l to 0.02 ¥ 10 12 /l. p-077 therapeutic apheresis (ta) in croatian hospitalsadherence to respectable guidelines z zivkovic*, b jeren strujic*, s boras † , i bojanic † , b golubic cepulic † and z ivankovic † *clinical hospital dubrava, † clinical hospital center zagreb, zagreb, croatia introduction: besides considerable resources, ta requires high costs and risk for patients. therefore, indication for ta often considers interests of patient, hospital and requesting physician. the most respectable guidelines for the implementation of ta were defined by aabb and asfa, classifying total of 58 diseases into 4 categories (ctg), ranging from 'standard therapy' to 'lack of efficacy' . the objective of this study was to determine indications for ta performed in 6 croatian hospitals in the period 2001-2004, respecting aabb/asfa guidelines. results: during the observed period in croatia, ta was performed in 777 patients suffering from 26 various diseases. in 514 (66%) patients ta was performed by membrane filtration, while in 263 (34%) separation by centrifugation was used (table 1) . according to the 4 ctg, s of the aabb/asfa guidelines, ta was performed in 573 (74%) diseases from ctg i, 126 (17%) ctg ii, 54 (6%) ctg iii, and 25 (3%) ctg iv of patients. the most frequent indications included in ctg i were: myasthenia gravis (32%), collection of pbpcs (31%), sy. guillain-barré (17%), and plasmacytoma (6%). in ctg ii frequent indications were: poisonings (35%), systemic lupus erythematodes (30%), and rapidly progressing glomerulonephritis (14%), and in ctgs iii and iv: cytoreduction-polycythaemia (52%), thyroid storm (15%), gvhd (10%), and reumatoid arthritis (8%). (1) the time spent for resolving h 52/40, (2) mtp 0/0, (3) discarded blood units 10/4. iii group: wrong data input 5/4, donor replacement 4/1, marking errors 3/10, error in determining blood group at the first blood taking 28/13, errors in input medical consulting 9/12 and disregard of prohibitions 2/13. the consequences are: (1) the time spent for resolving h 35/40, (2) mtp 2.5/3.5, (3) discarded blood units 9/18. conclusion: the analysis of errors has showed that the number of errors can be decreased by implementation of corrective/preventative action based on continual education of the staff, appropriate sop, effective organization, qmp for equipment. the conclusion of our study is that reducing the rate of work errors will decrease the waste of material and time, also that will decrease the number of discarded blood units. an iso standard for blood transfusion? background: in our search for an independent, objective assessment at western province blood transfusion service, we were unable to find one single model that met the specific requirements of blood transfusion. we therefore resorted to developing our own model but ask the question: why not have one international standard for blood transfusion? aims: our aim was to have a standardised system for the independent, objective assessment of our blood transfusion service with audits carried out by an internationally recognised body. we wanted a formalised, professional system of accreditation with inspection checklists, reports, certificates etc. methods: having moved away from accreditation by the american association of blood banks in mid 1990's, we evaluated various other options such as inspection by our government department of health or the world health organisation but neither organisation had trained inspectors or systems in place. we also investigated iso 9000 certification but, although this was acceptable on the quality management side, it did not cover the technical parameters relating to blood transfusion. results: we therefore developed our own model for accreditation that consists of three parts: • a quality management section incorporating iso 9000 principles; • a technical section incorporating specifications from the south african standards of practice (we also consulted the european, american, canadian and australian guides); • a laboratory section incorporating iso 17025 parameters (soon to be updated with iso 15189). we then chose to be accredited by the south african national accreditation system, sanas, an internationally recognised institution. once we had written a national accreditation checklist, sanas submitted this to two international accreditation bodies (iaf and ilac) for approval. the system has been in place for three years now during which time we have had four successful assessments. summary/conclusions: in developing our system, we reviewed what was being done elsewhere in the world and it became evident that, although there are great similarities between countries, there p-078 software for the management of the scansystem bacterial detection method the scansystem tm was developed for bacterial detection in blood products. to be implemented in blood banks, a specific software is now available in compliance with blood bank regulation in order to manage sample traceability and data file transfer. the software is divided in 3 main levels: an administrator level to create an application configuration in compliance with customer needs (product bar code characteristics, frequency of the positive controls, manual or automatic data file transfer . . .), a technical level to manage the operators (password, id) and to validate some specific results, an operator level for routine testing. the software assess sample traceability when testing pools of samples from 1 to more than 20. indeed, bacterial detection is performed for pools of 1 to 3 platelet samples and 1 to 20 red cell samples. each sample in the pool is traced through its barcode until the final result. the system is compatible with most of the barcode standard including isbt 128. in addition, the system checks each barcode protecting the sample against duplicate testing. the software assists and monitors the bacterial detection process from the sampling to the end of the test (final result), each step of the procedure is identified through its barcode and at each time, it is possible to know the test status for each sample. for a pool of samples, 2 results can be obtained: 'negative' or 'on hold' for a positive result. for a 'negative' pool, each sample constituting the pool are determined as 'negative' . for an 'on hold' pool each sample constituting the pool must be tested as a single sample and the final result is 'negative' or 'positive' . data transfer may be manual or automatic. a final technical validation is necessary before the transfer through an active selection of results to download. final results are provided in a compliant format for an easy import into the blood bank database. all necessary information are displayed: 'machine id' 'product/sample barcode id' 'date' 'time of transfer' 'operator id' 'result' ('negative' or 'positive'). the main advantage of this software is a continuous check of each step reducing the risk of error in testing. it makes the scansystem tm test compatible with a routine use in blood banks according to the current regulations and quality assurance programs. is no overall consistency. we feel it would be of benefit to establish an international working group to investigate the feasibility of writing an iso standard for blood transfusion. the standard would harmonise quality management parameters based on iso principles and technical/laboratory parameters specific to blood transfusion. minimum technical specifications would need to be agreed upon based on the various standards and guidelines available around the world. this iso standard could then be used for the purposes of certification/accreditation or government inspections. this would ensure global standardisation of world-class best practices. first world countries would be able to achieve compliance and a subsequent step could be the establishment of an international forum to assist developing countries to work towards compliance in the longer term. residual leukocytes in leukoreduced cellular blood products -evaluation by flow cytometry web-based outcome review: do you know how productive your trima® can be? background: web-based outcome review is a new software tool developed by gambro bct for the management and the interpretation of data from the trima and trima accel tm automated blood collection systems. aim: does the interpretation of reports obtained through outcome review lead to an increase in the number of products per run and the overall productivity of the apheresis center? method: run data files (rdf) from trimaᮀ were collected and transferred onto the outcome review server. these rdf do not contain any donor related data that can lead to possible donor identification. the reports were generated on the outcome review website (gambro bct intranet), interpreted by a gambro bct employee and presented and discussed with the management of blood centers. results: a total of 21 different reports can be generated on the website as a pdf file. for this study, 9 reports were investigated. they are: doses per collection, doses per collection trend, platelet collection trend, platelet procedure performance, platelet procedure performance trend, product distribution, average procedure time, procedure time, machine productivity trend. the results obtained for a center can be compared to word-wide, national, regional or to other individual trima devices in the centre (benchmarks). this benchmarking allows the management of an apheresis center to compare the results, to draw the right conclusions and to develop and implement corrective actions. implementing successfully these corrective action plans will lead over time to productivity results that are more in line with the figures that are generally accepted to be the optimal production capabilities of trima. the reports also help to monitor the effects of the corrective action plan over time and to adjust this plan if the results are not in line with the expectations. reaching and maintaining the optimal production capabilities of trima will also increase the net revenue by procedure or decrease the cost by procedure for the blood centre. conclusion: web-based outcome review allows getting more products from the existing donor base by interpretation of the multiple reports and implementing the required corrective actions until optimal production capabilities of trima are reached and maintained. introduction: to examine the cell vitality of packed rbc's during storage several parameters like atp, free hb or 2,3-dpg are used. less kits for the determination of atp are available and they need either a large sample volume and/or are time consuming. here we present the modification of a commercial testkit for a time-and cost saving detection of atp. methods: samples were analysed with (a) 3-phosphoglyceratekinase reaction according to bergmeyer, h. methods of enymatic analysis 2nd. edition. academic press, new york 1965; (b) detection via hplc and c) using a commercial testkit (r. greiner bio-chemica, germany). the atp-kit were minimized from 3500 ml to a total volume of 200 ml and tests were performed in microtiterplates. results: 245 samples were analysed in hplc and modificated commercial testkits, another 20 samples have been examined in all tests. comparison of the results showed no discrepancies in the above mentioned methods. standard curves have been performed (range 5-1000 mm atp) and statistical analysis demonstrated a given linearity (r = 9.97). variability has been calculated as 1.2% (intraassay; n = 25) and 4.6% (inter-assay; n = 70). the hand-on-time calculated for 96 samples has been decreased from 4.5 hours to 30 minutes. at least the costs of atp-determination have been reduced from €3.45 to €0.22 per sample. conclusion: performance of test kits in microtiter format is a fast and rapid method, reliable for high-throughput determination of atp in packed rbc's. background: in order to preserve both blood safety and availability it is mandatory that a minimal amount of blood units would be discarded due to defects in the materials and supplies used for blood collection, or to deviations in blood processing or storage. aims: (1) to monitor the derangements of different materials and disposables used during blood collection and processing, and to study the suppliers' responses and corrective actions taken. (2) to asses the relative contribution of different defective materials (dm) to the need to discard valuable blood components. materials and methods: about 831 000 whole blood units were collected and processed by mda national blood services in [2002] [2003] [2004] . as part of the routine quality control activities, derangements of the dm used were recorded and analyzed. some 40 different types of dm were defined. out of 68 reports sent to the corresponding manufacturers for investigation, 45 responses (66%) were received and analyzed. about 70% of dm were detected during blood collection. manufacturer defects of different materials were the reason for components discard in nearly 25% of cases. conclusion: defective materials are one of the major causes of the infringements of blood collection and blood component preparation processes. analysis and monitoring of the different defects and of the suppliers' responses and corrective actions are essential to improve products' safety and availability. establishment of a network for the exchange of information among international blood centers would enable the blood banking community to compare between different suppliers and to use the documented cases for training of personnel of both the blood services and the manufacturers. such a system may contribute to the improvement in quality of materials used and might lower the discard rate of valuable blood units. results: table 1 analysis and characteristics of t (1) comprehension of the blood bank's processes and the interaction between them and between the processes of the whole hospital. (2) monitor, measure and analyze these processes, in order to improve their effectiveness continuously. (3) implementation of internal and external quality controls for blood and blood products and implementation of appropriate statistical techniques for monitoring their results. (4) identification of interested parties (doctors, donors, patients) satisfaction and taking up the necessary preventive or corrective actions to improve their satisfaction. the qms of our blood bank was certified by tuv rheinland in 16/11/2004 and the scope of the certification is: 'blood collection, testing of infections markers, production of blood components, compatibility screening for blood transfusion and other immunological tests and implementation of therapeutic schemes in thalassaemia patients' . the implementation of the qms based on iso 9001:2000 standards ensures the improvement of services provided by the blood bank and the increase in customer satisfaction, whether donors or patients are concerned. the former enjoy the respect and recognition of their social contribution, while the latter are assured of very high levels of service and health protection. finally, we shall not underestimate the positive impact of qms in the motivation of blood bank personnel. quality becomes integrated both in their professional and personal attitude and allows for achieving increased satisfaction from their work. equipment management in the national blood transfusion service in serbia introduction: new equipment was urgently needed in three blood transfusion establishments (bte) in serbia. equipment was mostly inadequate for core blood transfusion activities, placed in inappropriate facilities, very old without routine maintenance or calibration. also, technical documentation for most of the equipment did not exist, and procedures for equipment management and responsibilities were not defined. further more, coordination on equipment issues with the qa department was not recognized. service funded by the european agency for reconstruction, provided various equipment for the three blood transfusion establishments. the new equipment includes blood collection equipment, centrifuges, refrigerators, incubators, automated testing equipment, genetic analyzer and it equipment of 1.6 million euros value. before the new equipment is installed the bte's agreed to have the national procedures on installation, validation, calibration and preventative maintenance in place. this will ensure that the equipment can be properly installed and validated before use. the project has provided training on validation to the working group (wg) on quality. the wg has created national procedures related to the equipment, including quite new term validation. the same problems in implementation of procedures were present in all three bte's. significant efforts are made to explain to the staff how the equipment has an impact on quality, how to ensure that the equipment does what it is supposed to do, how to be confident that the results obtained are accurate and how important it is to generate records. qa managers played an important role in the preparation of facilities for equipment installation, making plans for equipment layouts, creating documents (master cards, instruction for use) and designing the validation protocols. the same procedures and records enable an exchange of results, comparability, sharing information on what works and what does not work between bte's. the qa managers also prepared an introduction of equipment requirements to the heads of departments, with special attention to the validation process so that they are able to fully understand what is required and why to validate their equipment. results: national standards in equipment management in serbia are set and are being implemented. qa managers carefully managed that the new, numerous equipment, delivered in the short period of 2 months was correctly installed, validated, obtained with necessary documentation, followed by previously trained staff, so that equipment can be considered as controlled. the additional, positive effect is that validation is performed in one establishment for all bte in the country, allowing a more prompt response to problems and presenting of joint request to suppliers, as well as an easier way of monitoring equipment performance of the three bte's. organized equipment management has affect on every aspect of blood activities and finally to the quality of blood and blood components. background: the vista information system (vista tm ) is used in centers in europe as an apheresis management system. with vista tm it is possible to increase productivity, donor comfort and loyalty and therefore simultaneously improving the overall process in the center. aim: a calculation tool (microsoft excel) was developed to evaluate the added value of vista tm . three blood centers in different european countries completed a questionnaire using their local data. summarizing these figures gives us an idea about the impact of vista tm on the daily work and budget of a blood centre. method: the excel calculation tool that was used investigated 3 major areas where vista tm could show added value: improvement of regulatory compliance, increase the efficiency in operations and improve productivity. results: regulatory compliance: the number of infringements decreased, causing a considerable direct financial gain because these events are very expensive to deal with. the time spent on regulatory reviews decreased with a mean value of 35%. operational efficiency: the number of reports is very site dependent: sometimes a report is made for every procedure together with the printout of a blood loss history form. because vista tm tracks all procedure related data, some sites decided to stop printing these types of reports and to go completely paperless. the percent time reduction in reporting is therefore very variable. however the % of errors related to these reports decreased considerably with a mean value of 30%. efficiency in operations was also obtained because of the number of reports that are available in vista tm . productivity, management and process reports allow verifying and correcting the daily operations of the blood center. increased productivity: depending on the center, also an increase in the number of platelet and plasma products collected was detected. the number of product discards caused by infiltration reduced with a mean value of 50%, mainly due to the possibility to have customized and more donor adapted trima settings. the percentage of whole blood donors targeted for conversion was very site dependent (min 0.5%-max 4%). but because with vista any procedure brings between 2.0 and 3.0 products in general, the financial gain was considerable when donors could be converted from whole blood to apheresis. the use of vista allows the apheresis center to work with a reduced error rate and to increase the operational efficiency and the productivity. the financial impact of this has been estimated by the centers between 4€ and 26€ (mean value 10€) per procedure. establishment of national quality system in blood transfusion service in serbia introduction: the production of blood products is a semiautomated process in which the manual steps may be difficult to control and standardize. aim of the study: we introduced a specialised team for the blood production to test if this improved the control of the quality of the blood products. methods: the blood products tested for statistical process control were red cells in additive solution, buffy coat removed, and leukodepleted (ld) platelet pools prepared from 4 buffy coats. the products were collected in t&b triple opti-pac from baxter and the platelet pools were ld using plx-5 filters from asahi and stored in platelet bags from baxter. using control charts, namely x-mrchart, exponentially weighted moving average ewma chart and for autocorrelated stationary data the ewmast chart, we examined if time series of quality control values were in statistical control. if not we examined if autocorrelation and/or differences between the technologists producing the blood products could explain the lack of control. data included approximately biweekly measurements of volume, haemoglobin (hb) concentration, hb/unit, haematocrit and log leukocyte count (wbc)/unit of 352 units of red cells, measurements of volume, platelet concentration and platelet count/pool of 79 ld platelet pools produced by a team of 13 technologists and of 79 ld platelet pools produced by a specially trained team of four technologists. results: log wbc/unit was out of statistical control due to systematic differences between technologists. apparent lack of control of volume, hb-concentration, hb/unit caused by autocorrelation disappeared when the ewmast chart was used. platelet concentration and volume of the platelet pools produced by the 13 technologists were out of control. in that some technologists systematically produced low values. this could be explained by inappropriate handling of the platelet product between centrifugation and separation. systematic differences between the four specially trained technologists could not be demonstrated and they produced platelet pools with a significantly higher platelet count/pool. however, standard deviations of the four technologists differed significantly causing occasional outlying values. conclusion: training and routine in blood production or process automation, and also importantly, feed back to the technologists based on control chart quality control data, is recommended. background: one important principle of the use of blood and blood products is the ability to trace the units from donor to the recipient. this study set out to establish whether or not there was sufficient reporting on transfusions from the hospitals supplied by fort portal regional blood bank in western uganda as a means of establishing sound haemovigilance and look back systems. were reported with no unit number and could not be traced to the patients. there was sufficient reporting on the data requested by the blood bank. these results suggest that it is possible to establish effective 'look back' and haemovigilance systems. capture of data on outcomes and adverse effects will be necessary to fully establish the system. further efforts are required to educate those involved in transfusing blood about the need for adequate and accurate documentation. external quality assessment of blood grouping were misinterpreted as rhd-positive samples without the use of control reagent. rbc phenotyping was made correctly by 79.7% of participants. the remaining 20.3% of participants carried out the phenotyping incorrectly, while false-positive and false-negative results were derived in 8.91% and 11.39% of cases correspondingly. polyspecific human sera and monoclonal antibodies were used for abo, rh and antigens typing. the reason of errors in antigen detection was low quality of reagents. antibodies identification was carried out in six distributed exercises. 75% of participants detected anti-d-k-c alloantibody correctly. the rest of participants did not found alloantibodies or detected their specificity incorrectly. results of testing depended on quality of screening cells. thus, the participants using homemade pooled screening cells had a significant lower detection rate of antibodies comparing with those using diamed ag cell panel. consequently, the results of the first federal external quality assessment scheme show the necessity of improving the quality of red cell reagents produced in russia. in addition, the more appropriate training of staff is required. the importance of iso 15189-quality system in increasing safety of blood transfusion introduction: hadassah hospital transfusion medicine department received on 3/2004 iso 15189-quality system accreditation. an essential element of this standard is the development of a reliable system to identify, document, analyze and correct actual and near miss events and assess the effectiveness of corrective and preventive actions. aim of the study: assessment of events and corrective actions following implementation of iso 15189 quality system. methods: events in the blood bank were identified by the staff, by internal or external audits and by complaints from the wards. all events were recorded and classified into two categories; quality system and technical. the latter were further classified into preanalytical (sample receipt), analytical (abo rh typing, antibody screen and identification, cross matching and phenotyping) and post-analytical (issue of components to wards). all events were graded into 4 levels; 1-most severe, potentially harmful to patient. 2-severe, damage to process and result. 3-moderatly severe, damage to process only. 4-benign, no harm. events were corrected and effectiveness of corrective actions was assessed by monitoring recurrence of the event. results: during the years 2003-2004, 248 events were detected and recorded in the blood bank, they comprised 0.072% of all tests performed (343 595). most of the events were technical. all events were detected before causing harm to the patients. results are summarized in the table. *the percent analytical value is a summary of rates of events per test types included in this category. events detected in the quality system were mainly of severity level 2&4, whereas technical events were mainly of severity levels 2&1. analysis of event recurrence in the quality system revealed that 97% of events were resolved, whereas only 14%-31% of technical problems were completely solved. the main source of event identification and documentation, in the quality system were audits whereas in the technical system, staff members revealed most events. the implementation of iso 15189 quality system provided a powerful means for recognition, analysis and study of patterns of near misses and actual events. understanding the root causes of events enables to choose the most effective corrective and preventive action to control event recurrence. evaluation of the frequency of events confined to the blood bank revealed a very low rate of 0.072%. these results are in agreement with data in the literature. creating a non-punitive, non-stressing open environment, motivates personnel to identify and document events, which are regarded as opportunities for improvement and serve as important tools for upgrading transfusion safety. the explosive use of information technology and the speed with which it has spread into all life activities has created vulnerabilities for all organizations. those vulnerabilities are compounded by the complexity of information technology, limited time to market, development constraints, and constantly changing relationships between organizations and suppliers. growth in the sophistication of security threats makes it imperative that organizations remain equally competent in identifying vulnerabilities and mitigating security risks. aim: the goal of this document is to explain how isbt intends to provide guidance to the blood banking community on implementation of effective information security policy. when using information for critical activities, blood banks should consider information security as an important aspect of their management policies. evaluation of existing standards, such as iso 17799 and hipaa, allows us to establish a framework for information security without regard to the type of organization. it remains very difficult however, due to the complexities involved, to establish an information security policy without guidance. method: the isbt information task force was created to provide guidelines on information security for blood banking organizations of all sizes. the intent is to help them understand existing standards as well as provide tools for implementing information security policy. these guidelines are based on existing standards that are followed by most worldwide countries: iso 17999 and hipaa. results: information security can be defined as the 'protection of systems, information and services from accidental and deliberate threats to confidentiality, integrity and availability' . understanding existing information security standards was the first step for establishing a structure for the guidelines. the core is organized within an implementation framework and presented under the three following layers: 1. administrative for defining the it security organization, the information security policy, and information security awareness and training. 2. physical for providing solutions that relate to physical environment protection and access, equipment and it infrastructure security, and control for accessing computerized equipment. 3. technical for maintaining confidentiality of electronic information and ensuring that authorized access to information systems is maintained (technology relating to identification and authentication, logical access, operating system, network management, application access, etc.). strategy guidance is also included for senior managers in charge of establishing organization policy, including responsibilities and methods to successfully implement policy. further, the task force is addressing both risk analysis and management including identification of potential dangers to information systems (threat-source) and existing controls (risk description), as well as a plan to address identified vulnerabilities and mitigation of specific risks. conclusions: information security standards are prerequisite to understanding the issues involved when considering information vulnerability. international guidelines for information security, specifically directed to the blood banking community, are equally necessary if we are to identify, plan for, and mitigate risks associated with vulnerabilities to critical blood banking information. the isbt task force is committed to providing such guidelines. introduction: the important part of quality planning and quality assurance within production of blood components is measurement system analysis (msa). measurement system analysis was performed on microscopic counting of blood cells in our study. aim of the study: aim of this study is determination if microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. methods: we practiced measurement system analysis in counting of residual elements (leukocytes and erythrocytes) in whole blood plasma. the counting performed two lab technicians in ten samples of plasma from whole blood. leukocytes and erythrocytes were measured in naggeotte counting chamber. we used the method of mean and range for the determination of reproducibility and repeatability (r&r) and analysis of variance for complex measurement system analysis. regulation diagrams were applied for the graphic statement. we determined the value of repeatability, reproducibility, coefficient r&r, variability among samples of plasma and total variability of measurement system. the important conclusion was to determinate if the microscopic counting of samples of blood components is sufficient with regard to quality parameters specified in guide to the preparation of blood components (erythrocytes: <6.0 ¥ 10 9 /l, leukocytes: <0.1 ¥ 10 9 /l). our results show that microscopy counting of residual elements in whole blood plasma is suitable for quality control of blood components. aim of the study: to provide transfusion services with a tool for proper qms implementation, an international collaborative study on qms applications, employing the 'process approach', has been undertaken by a group of transfusion services of varying sizes and structures with experience of qms, in collaboration with a university institute offering a master in qms implementation in health services, and an expert from a quality association. the 'process approach' serves as a tool to manage transfusion activities as a system based upon a network of processes and their interactions. guide-lines have been produced based upon this principle and will be published (volume and cd-rom) and distributed at no cost to all transfusion services of nations participating in the study. the main contents of the guide-lines' chapters are: through definition/analysis of single processes and the correlation network amongst these, the 'process approach' methodology renders the transfusion centre's functioning units completely interdependent, eliminates process interface barriers, provides personnel with a unified focus on the main transfusion objectives, and lays the basis for improvement of transfusion service quality, organization and performance through efficient control of processes' interactions. the blood screening system 400 automated high-throughput nat system for simultaneous screening of hcv, hbv and hiv nucleic acids: full process surveillance e pfeifer*, b alessandri † , hr bachmann † , t barker † , y ohhashi*, c parkhouse*, j pinsl-ober ‡ , p wenzig ‡ and g ziegler ‡ *roche molecular systems, inc., pleasanton, usa, † roche instrument center ag, rotkreuz, switzerland, ‡ roche diagnostics gmbh, penzberg, germany introduction: the blood screening system 400 combines on one deck both automation of dna/rna extraction from blood/plasma samples and multiplex pcr amplification and detection of nucleic acid targets. the system is designed for high-throughput single unit testing and pooled specimen processing. a data management system supervises and controls the complete process from initial sample pipetting through to result compilation and reporting. the objective of this project was to present the system 400 assay and device built-in quality control measures that guarantee process safety and reliability. the study shows how internal control, external batch controls, pipetting sensors, validation & maintenance procedures, and a controlled development & manufacturing process yield an optimal test method and system stability. method: failure modes and effects criticality analysis (fmeca) was used to evaluate a mathematically derived safety metric for optimizing risk reduction. this method makes use of a system risk objective-function (srof), which provides a multivariate description of sample processing, amplification and detection steps. the method for analyzing system behaviour first employs product classification into risk domains, followed by ranking of process steps that are determined to be linked to known system hazards. this provides objective means for directing system design leading to risk minimization. the srof responses associated with redundant liquid sensing channels were shown to substantially reduce risk during sample or reagent transfer steps. the system 400 liquid flow, airpressure-based (plld) and capacity-coupled liquid (clld) sensors detect aspiration and dispensing inaccuracies. sensor signal tolerance band widths studied for fluid classes representative of system 400 reagents and for plasma samples from lipemic, icteric, and hemolized sample sources were shown to correlate with srof multivariate modelling. the impact of surveillance design elements on the srof response demonstrates that risk is functionally dependent on design elements. additional surveillance examples are presented that describes temperature sensing, robotic positioning and motion control. treatment of residual risk is addressed by introducing external controls that are configurable to specific workflow scenarios. the negative external control (nc) is used to check for contamination of reagents. five low concentration armored external controls, i.e. hiv-1 group m arna, hiv-1 group o arna, hiv-2 arna, hcv arna, and protected hbv dna are run at the beginning of a batch as a run-control measure. in addition to these roche controls the system 400 supports running of user-defined external controls for co-validating the batch. the internal control (ic) is based on armored hiv-1 group m rna that is co-extracted and co-amplified with the external controls and the target nucleic acids potentially present in the sample. lastly, the system 400 resource management monitors the status of samples, ready-to-use reagents and disposables via rack sensors and bar-coded bottles and tubes. the fmeca methodology provides a risk-minimized, comprehensive system design. redundant sensors with internal and external controls are evaluated through comparison of modelled versus actual run results. the system 400 brings a new level of surveillance and throughput for automated pcr testing, and emphasizes roche's commitment to increasing the safety of the global blood supply. technical standards for safe storage and transport of blood components and blood samples objective: to implement standardization of technical specifications for safe storage, transport and distribution of blood samples and blood components intended for transfusion, as part of a quality system in blood transfusion medicine. methods: in the course of implementing a quality system in our blood establishment (distributing 10% of the national blood supply), we have developed standard operating procedures (sops) for temperature and hygienic conditions to maintain and control storage of blood components during their shelf life and to ensure their safe distribution to other blood services in the country and abroad, in compliance with eu directives 2002/98/ec and 2004/33/ec and the recommendations of the council of europe and who. procedures are validated and relevant records are kept. a statistical process control is also in place to monitor deviations from specified temperature and time range throughout the period of transportation. blood components collected and prepared for specific purposes (e.g. directed donations, irradiated units, hla-typed units, anti-cmv negative blood components and blood for neonates) are stored separately, and alarms and warning systems are in place. packing and transport conditions of red cells, platelets and ffp are submitted to the tests and criteria of adr (european agreement concerning the international carriage of dangerous goods by road). in greece, the adr legal framework has applied since 1999. blood samples are classified according to adr in division 6.2 under un 3373 as diagnostic specimens, and the criteria for safe carriage include packaging, specific labelling and vehicle requirements as well as carrier obligations and personnel training. our blood establishment has a contract with biotrans, a private company accredited for packaging, storing and transporting blood, organs, tissues and cells as well as potentially infectious biologic substances and blood samples for diagnostic purposes. assurance system). the approach is specified in the form of requirements of art. 4.1 of the standard: the organization shall: (a) identify the process needed for the quality management system and their application throughout the organization, (b) determine the sequence and interactions of these processes, (c) determine criteria and methods needed to ensure that both the operation and control of these processes are effective. regional centre for transfusion medicine in biaĺystok was the first transfusion service in poland which was certified according to iso 9001:2000 standard. our expected profits from the implementation qms were: possibility to overview organization's pathways of operation and to inspire corrective and improvement actions, emphasis on the role of staff in the system, focus on self-control and responsibility for one's own work as a factor of staff's mentality creation/modification. our one year of experience with iso 9001:2000 standard proved the system to be handy tool of management for the organization collecting, processing, testing blood and releasing of blood products for the hospitals and to be well accepted by the staff. the implementation and certification of the internationally nor-malized quality management system simplify and shorten all accreditation and registration procedures required for legal activities of transfusion service as well as for any supplemental medical activities which may be performed by the centre. the implementation of qms facilitates the implementation of other quality systems and simplifies procedures required for the ce certification for the products. red blood cells stored in blood banks, normally undergo a series of chemical alterations, or storage lesions. the ultimate consequence of these lesions is a decrease in the viability of the red cells following transfusion. the chemical alterations are mainly changes in levels of na+, k+, cl-concentrations, ph and 2,3 dpg levels and they affect the electrical impedance of blood. the electrical impedance, cole-cole parameters, is determined mainly by the resistance of the red cell extracellular fluid (re), the resistance of the intracellular fluid (ri) and the capacitance of the cell membranes (cm). in this study we aimed to investigate the relation between blood parameters and electrical impedance changes, and their further clinical implication. all parameters were measured on 51 erythrocyte suspension (es) samples during 42 days of storage at 4oc on days 0, 10, 21, 35 and 42. for 31 whole blood (wb) samples during 21 days of storage, same parameters were measured on days 0, 10, and 21. the measurement of the complex impedance of blood samples were performed in the frequency range from 100 khz to 1 mhz. by using the 4284a hp lcr meter, the impedance z, and the phase angle a for each sample were read. these values were corrected according to the gain and phase characteristics of the amplifier; and the resistance r and the reactance x were calculated. each data was first fit to the cole-cole model; hence the cole-cole parameters, intracellular resistance ri, extracellular resistance re, characteristic frequency fc and phase angle a were obtained by using lms software. afterwards, cm was calculated by using ri, re, a and fc. whereas ri and cm decreased progressively with time on both wb and es, re changes showed some differences. the electrical impedance alterations were explained by measurements of na+, k+, clconcentrations, ph and 2,3 dpg by indicating days. storage of red cells resulted in a rise in extracellular k+ and a fall in extracellular na+, cl-, ph and 2,3 dpg. anova was used to evaluate differences in blood measures in relation to storage time. the results were presented as the mean ± sd. according to the regression analysis in spss, the intracellular resistance (ri) on both es and wb was affected more efficiently from all blood parameters among all other electrical parameters. although ri and re were correlated with na+, k+, cl-, ph and 2,3 dpg more significantly, cm measurements were failed to show correlations with blood parameters because of the intervening parameters, a and fc. the best relationship between the parameters mentioned above on both es and wb was ri and k+. ph changes were the same for ri and re both for es and wb. the correlation between parameters on es was better than those on wb, because whole blood consists of several particles that may affect our measurements. our study showed that 2,3 dpg has an effect on ri and re as efficiently as other blood parameters and therefore electrical impedance measurements may serve future implications. background: issue of quality blood products and donor safety are the main aims of blood transfusion services. a comprehensive quality system should be in place to fulfill these aims, which can be attained through strict adherence to the established standard operating procedures (sops). the drugs and cosmetics act of india, which controls the licensing of blood transfusion services, does not provide clear guidelines regarding plateletpheresis procedure. aim: we therefore established our own sop and operational flow chart for plateletpheresis that can be easily followed by other centers in india. methods: a total of 100 plateletpheresis procedures performed using two cell separators (cs3000 baxter, usa, mcs3p, hemonetics, usa) were evaluated following our established sop. the mean platelet yield in cs3000 was 2.9 ± 0.84 ¥ 10 11 and in mcs3p, it was 2.88 ± 0.75 ¥ 10 11 per unit, however, only 4-7% of sdps showed wbc levels <5 ¥ 10 6 . six of 100 donors complained of hypocalcemic symptoms. the operational flow chart designed in this study was found to be simple and easy to adapt by blood transfusion services in this country. the first advanced quality management training course for blood transfusion services in the western pacific region mk tan*, jp yu † and d teo* *health sciences authority, singapore, † who regional office for wpr, manila, singapore background: blood transfusion is a key part of modern medicine. a well-organised blood transfusion service (bts) is a prerequisite for the safe and effective use of blood and blood products. in 2000, the world health organization (who) introduced a new initiative of quality management project (qmp) to achieve the goal of safe and adequate global supply of blood. through qmp, regional training centers were identified and quality management training (qmt) courses were established. in 2001, centre for transfusion medicine (ctm) of health sciences authority singapore was appointed as a collaborating center for qmt in the western pacific region (wpr background: spectrophotometric method according to harboe was traditionally used at our institute for determination of free haemoglobin in supernatants of rbc blood components at the end of storage time. in the year 2002 hemocue plasma low hb system (hemocue, sweden) was introduced as a replacement method. aim: the aim of the study was to examine reliability and suitability of the hemocue method for measurement of free haemoglobin in supernatants of rccs. methods: hemocue plasma low hb method was validated and compared with harboe method. supernatants of 36 rbc products were tested by two methods and results compared using regression analysis. additional testing was performed to investigate precision of hemocue method (within-run and between-day variation), trueness using reference material and to define optimal sample handling. results: regression analysis showed high correlation between two methods (r = 0.98), with higher values obtained with hemocue method. immediately after centrifugation one supernatant was measured 6 times in order to determine within-run imprecision. coefficient of variation (cv) calculated from 6 consecutive measurements was 6.1%. to investigate between-day imprecision of the hemocue method one sample was divided in 5 aliquots and frozen. one sample was thawed each day and measured. cv of five measurements was 1.95%. during the period of validation 37 measurements of reference material (low, medium and high) were performed. cv calculated for these measurements were 7.86% (low), 1.88% (medium) and 1.04% (high). in order to investigate possibility of batch testing, supernatants of 5 different rbc products were divided in aliquots and measured periodically during 1-month period. cvs calculated from 7 measurements of each sample were in range 3.1-9.9%. conclusion: hemocue plasma low hb method appears to be an excellent replacement for the harboe method. it is consistent, easy to use, measurements are performed in short time, and errors are minimized by eliminating dilutions and manual calculations. background: determination of haemolysis in red cell concentrates (rccs) at the end of storage time is routine method in quality control of blood components at our institute. for this purpose, haemoglobin concentration in supernatant of rccs is measured using hemocue plasma/low hb system (hemocue, sweden). according to manufacturer recommendation, visually turbid samples should be filtered before analysis with a 0.2 mm filter. because visual estimation of turbidity is highly subjective and unreliable, filtration of all supernatants before analysis using appropriate filters is possible solution for standardisation of the method. aim: the aim of the study was to investigate the effect of filtration of visually non-turbid samples on results of free haemoglobin measurement. methods: haemoglobin concentrations were measured in 42 visually non-turbid supernatants before and after filtration using hemocue plasma/low hb photometer (7 wb negative controls were used for each blood component. in all blood components tested bacterial contamination was detected using both types of bottles. in comparison with sa/sn bottles, nearly all enrolled microorganisms were detected faster in bpa/bpn bottles (see table 1 ). all negative controls were negative (no false positive). the results of the study performed support the use of bact/alert bpa and bpn plastic bottles in quality control testing of different blood components. objective: management of returned blood products is important part of quality assurance activities in transfusion medicine. blood products are returned most frequently because of routine rotation of stock and because of nonconformities discovered after the product has been received. methods: qa data about returned blood products were retrospectively analysed for the 5-year period (2000-2004) . only blood products returned because of nonconformities were taken into consideration. data about the reasons for returns are presented and discussed. the top 4 reasons for returns were positive dat, labelling errors, blood bag defects and visual appearance of blood units (see table 1 ). in all cases positive dat was confirmed in our institute, and blood donors managed accordingly. nonconformities related to visual appearance of blood component and other nonconformities related to the quality of blood products were investigated and corrective actions conducted. in case of blood components returned because of damaged bag, significant problem is to investigate the cause of nonconformity (usually inappropriate transport conditions). conclusion: management of returned blood products is important tool in improving the quality of blood products. introduction: hemovigilance is the systematic monitoring of the blood transfusion chain for side effects and adverse incidents from the moment of blood collection until after administration of the unit to the recipient, and comprises all activities that can lead to a safer and more effective use of blood components. attempts to achieve a more safe and effective use of blood components constitute a huge task given the number of interventions and array of medical and not-medical personnel involved in the transfusion process. registration of the impact of all participants is a tremendous job as such. information management may enhance the chances for a successful hemovigilance. aim: the aim is, to establish a basis for all the information related to the chain, such as standard operating procedures, (inter)national guidelines, local transfusion protocols and forms, and procedures to direct the process. additional factors that contribute to the final results are the profile and role of employees, incidents, points of care, product information, prices, budget, contracts, etc. by clarifying and explaining the basis to all participants by means of an existing infra structure, everyone will gain access to identical, consistent and up-to-date information at all times. the primary activity is to create the basis by an outline of every individual link in the transfusion chain from donor to recipient. secondly, the resources, responsibilities, actions, and internal controls (what task, who is performing, why, which help, where, when) are documented. by creating distinct databases for these 6 'w's' in combination with a separate database for the hemovigilance process itself, it is possible to establish relations, hyperlinks, between the different databases. all the information collected in the foundation, complete with all the resources, responsibilities, actions, and internal controls is made available to target groups by means of the intranet in our hospital as well as in a printed handbook format. collecting and displaying the information on hemovigilance this way, creates a transparent and more easy-to-manage process. it establishes a basis, which incorporates all resources, responsibilities, actions, and internal controls to all participants and enables the organisation to operate both efficiently and effectively. it allows us to show auditors (both internal and external) which processes are related to which guidelines as well as the results in daily. in addition, it reveals which factors determine the genuine application of the guidelines in clinical practice. conclusion: by outlining the process, followed by defining, analysing, securing, on the 'w6' method, the hemovigilance process offers a stable basis and framework for all participants and may stimulate a more safe and effective use of blood components. background: transfusion medicine is a basic post-graduate specialty for medical doctors with a specific national curriculum in bulgaria. in order to improve the post-graduate training of medical doctors in transfusion medicine, a new curriculum was elaborated in 2004. purpose of the new program: independent work of transfusion medicine specialists on all levels of the blood transfusion service (bts) -organization of bts, promotion of voluntary, nonremunerated blood donation, collection, processing, testing, storage and transport of blood and blood components, immunohematologic testing of patients, laboratory hematology, clinical experience, assistance and advice on diagnostic and therapeutic problems of patients, requiring treatment with blood products, teaching transfusion medicine to bts and clinical personnel. admittance and duration to training -medical doctors are admitted to post graduate specialization after a successful state examination. the overall duration of training is 4 years, of which at least 2 are obligatory for training in the national or regional blood transfusion centers. main sections of the curriculum: 1. organization of blood donation and blood transfusion, including promotion of voluntary, nonremunerated blood donation; organization, planning and information systems; organization of blood transfusion; quality management. 2. collection, processing, storage and transport of blood and blood components 3. laboratory hematology and specialized methods (general laboratory methods, immunology, immunohematology, transmissible infections, hemostaseology, nat techniques, flowcytometry) 4. clinical use of blood components and plasma products (treatment with blood products, adverse events and reactions, alternatives to blood transfusion). the new curriculum of transfusion medicine guarantees a better training of medical doctors, thus leading to safe and effective blood products their proper clinical use. introduction: transfusion medicine is integrated medical discipline based on clinical and laboratory practice. it is closely connected with molecular biology, genetics, as well as with apheresis, automatically techniques and transplantation. the aim of studies in transfusion medicine is proper education, skills, attitudes and knowledge of students at the medical faculty and of doctors on residency in transfusion medicine and other specialties about blood and its usage. material and methods: there will be presented how transfusion medicine is implemented in educational and health sector in r. results: there is 3-year specialization in transfusion medicine, established 30 years ago (over 90 specialists finished this specialization till now). we have postgraduate studies in tm started 15 years ago. transfusiology, as subject at the medical faculty, is now included in new curricula of medical student, consists of 15 theoretical and 15 practical lectures. also, medical doctors on specialization in surgery, anesthesiology, obstetrics and gynecology, pediatrics, internal medicine, maxillo-facial surgery and others have obligatory 1-month education in tm. transfusion medicine is integral part in education of nurses and laboratory technicians; all personnel that work in transfusion field in our country finished 6-mounths course in tm and get certificate. conclusion: although we have already done so much in educational field, we will continue with our efforts to improve our collaboration with clinicians and to involve ourselves more in clinical disciplines. introduction: in hospitals of saint petersburg donor blood, its components and preparations, autoblood and its components, hemocorrectors are applied with the medical purpose at 20-30% of hospital patients. the service of blood and the existing organization transfusion therapy in hospitals should provide maximal immunological and infectious safety and also its high medical efficiency. it demands special preparation for all doctors: general physicians on clinical transfusiology and doctors of blood service on all sections of the general, industrial and clinical transfusiology. clinical gemostasiology, pharmacology of hemotransfusion means and hemocorrectors, the organization of the blood service, the donor service; the organization, technique and technics of preparation of blood, its components and preparations, quality assurance, transfusion therapy programs, technique and technics of transfusion medicine, a problem of maintenance of immunological and infectious safety, preventive maintenance, diagnostics and treatment of posttransfusion complications, feature transfusion therapy in pediatrics and medicine of accidents). clinical physicians master all basic questions of clinical transfusiology, thus the special attention is given questions of clinical immunohematology and clinical gemostasiology, programs and a technique of transfusion therapy, the prevention of posttransfusion complications. employment are carried out in departments of city blood bank, hospitals blood departments. final examination under the test program and at interview shows sufficient mastering a theoretical and practical material (right answers of 80-98%). the described system of postgraduate studies provides an opportunity of successful independent work of doctors on the workplaces both regular increase and qualifications not less often than 1 time in 3-5 years. conclusion: the existing system of postgraduate education for doctors on transfusiology provides the blood services and hospital by the qualified staff. introduction: nowadays in hospitals of saint-petersburg more and more it is frequently applied the extracorporal hemacorrection (haemapheresis, haemasorbtion, plasmasorbtion, krhyoplasmasorbtion, ultrafiltration etc.) and photohemotherapy (optical radiation influence on blood) at various diseases and traumas. they are used at treatment almost 6% from the general number of patients of hospitals with positive results at 85% from them. for this purpose in hospitals there are specialized branches or cabinets with staff of the doctors -transfusiologists. therefore an actual problem is organization of postgraduate special education for them. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: even the history of public blood banking in turkey has dates back to 1957, whole blood was comprising the majority of transfused units (more then 96%) in turkey until the last few years. aim: the main target was to decrease the whole blood use in a short time and establish countrywide effective blood transfusion practice. methods: there were no specific blood banking and transfusion education either at undergraduate and postgraduate medical education in turkey until the last few years. blood banks and transfusion society of turkey (bbtst) was established at 1997 with the main aim of education of the related people about blood banking and transfusion medicine. bbts has organized 35 symposiums, 13 national courses, 1 national and 1 international congresses since 1997. due to increased knowledge about the blood components and improved infrastructure of blood banks whole blood consumption has decreased to national average to 60%. in most of the university hospitals and training hospitals this is around 19%. conclusion: like most of the public based behaviours dedicated efforts of civil initiative has changed the traditional habit of blood consumption in turkey by the direct affect of well organized educational activities. training of general practitioners in blood banking and transfusion medicine n solaz*, b keskinkilic † and c oruc † *ankara university, ankara, † ministry of health, ankara, turkey background: turkish ministry of health runs majority of the hospital blood banks in turkey. most of the moh hospital blood banks give service under the medical supervision of general practitioners. because there is no specific blood banking and transfusion medicine training in either under or post graduate medical education those general practitioners have limited knowledge about this field. aim: to give basic knowledge and practice to those general practitioners moh has established a special training programme with the collaboration of medical schools for 5 years. methods: this is a 6 months programme which has 2 basic components; theoretical education, laboratory and blood banking practice. the trainees are evaluated by a final examination. each group has limited cadets and the training is provided in selected blood banks with the collaboration of universities and moh training hospitals. conclusion: almost 100 general practitioners are trained by this programme in the last 5 years and those doctors helped to increase the quality of blood banking and transfusion service at their hospitals. background: it has been accepted that during cardiac surgery the complement-system is activated which enhances ischemia-reperfusion injury, leading to postoperative complications as infections and organ failure. the lectin pathway is one of the mechanisms that can activate the complement-system, this pathway can be mediated by mannose-binding-lectin (mbl). the level of mbl in serum shows a wide variation. a low level of mbl can lead to more infections in some conditions and a high level to tissue damage after ischemiareperfusion injury. the effect of transfusions of erythrocyteconcentrates and plasma on the mbl levels and thereby on complications after cardiac surgery are not known. aim of the study: the role of pre-and postoperative mbl levels and the effect of transfusions on postoperative complications after cardiac surgery. in a randomized controlled trial 474 cardiac surgery patients were included and blood samples were taken pre-and postoperatively. mbl measurements were performed by elisa assays. the data were linked with postoperative complications as mortality, infections and multiple-organ-dysfunction-syndrome-mods and with peri-operative erythrocyte and plasma transfusions. results: the mean pre-operative mbl-level was 837 ± 796 and postoperative 456 ± 347 mg/ml. there were no differences in preand post-operative mbl levels between patients with infections or mods compared with non-infected and non-mods patients. the difference in pre-operative mbl between non-survived (672 ± 587) and survived patients (853 ± 812) was not significant (p = 0.20). postoperative mbl levels between survived and non-survived patients was smaller. conclusions: pre-and postoperative mbl levels in cardiac surgery are not related to postoperative infections, mods and hospitalmortality. whether large amount of blood transfusions are affecting the mbl-levels and thereby the patients outcome is not known. introduction: patients undergoing cardiac surgery are receiving high amount of blood transfusions and are at risk for the development of infections and multiple-organ-dysfunction-syndrome (mods), these complications are influencing the survival of the patients. during cardiac surgery pro-and anti-inflammatory cytokines are released by different mechanisms. we found in a randomized trial in cardiac surgery a significant reduction in infections and mortality due to mods by transfusion of leukocytedepleted erythrocyte concentrates (ld) compared to leukocyte-containing, buffy-coat depleted erythrocytes (pc). aim of the study: the effect of ld on the concentration of proinflammatory cytokine il-12 and anti-inflammatory cytokine il-10 in relation to clinical outcome and complications after cardiac surgery. methods: in 474 participating patients blood samples were taken before and after surgery. using elisa il-10 and il-12 were measured in these samples. the results were linked with the endpoints of the randomized trial: postoperative infections, mods and in-hospital mortality. results: all pre-operative concentrations of il-10 and il-12 were low. mean postoperative level for il-10 was 70 ± 121 and for il-12 57 ± 74. compared with patients without mods and without infections and survived patients only the il-10 was significant higher in patients who died and in patients with mods. there were no differences in mean levels related to ld and pc. the levels of il-10 were higher in patients receiving more then 10 units blood transfusions compared to 0 transfusions. conclusion: there is an association between mods and mortality and il-10, not with il-12. ld has no influence on mean levels of il-10 and il-12. there is a correlation between transfusion of more then 10 units and il-10, not with il-12. these cytokine-profiles are not associated with the beneficial effect of ld on the postoperative outcome in cardiac surgery patients. anemia and blood transfusion practice in critically ill patients e grouzi*, e tsigou*, p evagelopoulou † , g baltopoulos † and i spiliotopoulou* *kat general hospital, transfusion service, athens, † athens university school of nursing icu, athens, greece background: anemia is a common problem in critically ill patients admitted to intensive care unit (icu), but the consequences of anemia on mortality and morbidity in the critically ill is poorly defined. aim: to define the incidence of anemia and red blood cell (rbc) transfusion practice in critically ill patients in the icu) of our hospital, and to examine the relationship of anemia and rbc transfusion to clinical outcome. patients and methods: the period study was from july 2003 to december 2004. patients were enrolled within 48 h of icu admission. follow-up time was 30 days, hospital discharge or death, whichever occurred first. results: a total of 169 patients (116 male, 53 female, mean age 57.6 ± 21.1 years, range 15-96) were included in the study. the mean hemoglobin (hb) level at baseline was 11.2 ± 2.1, which level was descending during the study. overall 63.9% (108/169) of the patients received one or more rbc units while in the icu stay (mean 3.1 ± 3.8 units per patients). the mean pretransfusion hb was 8.7 ± 1.8 g/dl and the mean time to first icu transfusion was 2.6 ± 4.1 days. more rbc transfusions were given in the first week of the icu stay (331 units vs 110, 61, 36 units in the second, third and forth week respectively). the number of rbc units which a patient received during the study was positive associated with longer icu length of stay and an increase in mortality (r = 0.19, p < 0.05 and r = 0.26, p < 0.05 respectively). baseline hb level was significantly related to the number of rbc transfusion (r = 0.55, p < 0.001), but was not an independent predictor risk factor of length of stay or mortality (r = 0.16, p > 0.05 and r = 0.13, p > 0.05 respectively). the mean baseline apache ii and saps scores were 19.6 ± 7.4 and 49.8 ± 17.4 respectively. furthermore both baseline apache ii and saps scores were significantly higher for patients with a baseline hb level of <10 g/dl (23.2 ± 7.9 vs 18.3 ± 6.7 and 54.6 ± 18.8 vs 48.1 ± 16.5 respectively), while the apache ii values were positive associated with a significantly increased likelihood of rbc transfusion (pearson correlation p < 0.005). conclusions: anemia is common in the critically ill patients, it appears early in the icu course and persists throughout the duration of the icu stay. rbc transfusion seems to be associated with worse clinical outcome. despite the intensive research for the transfusion practice, which taken place worldwide during recent years, data from our country is limited. the results of our study suggest that approaches to reduce rbc transfusion would be desirable. however, further well designed prospective studies with large number of patients are required to efficiently explore the risk of anemia, optimal transfusion hb threshold and the risk and benefit of rbc transfusion in the critically ill. consumption of blood products in a large, general hospital he heier*, j pillgram-larsen † , m hestnes ‡ , b gran*, a krog* and no skaga ‡ *ullevaal university hospital, oslo, † ullevaal university hospital, dept. of thoracic surgery, oslo, ‡ ullevaal university hospital, dept. of anestesiology, oslo, norway uuh is the largest general hospital in norway, and trauma referral centre for half of the norwegian population (2.5 mill) the trauma team performed initial assessment and resuscitation of 323 patients, 75% males, during the first six months of 2002. the aim of our study was to analyze transfusion practice at uuh with specific focus on trauma. materials and methods: blood consumption during this period was recorded. clinical data of trauma patients were collected from our trauma registry and anonymized before analysis. results: 7012 units of erythrocytes (er), 914 of thrombocytes (thr) (buffy coat preparations from 4 donors) and 903 of s/d-treated whole plasma (op) (octaplasò) were transfused in uuh during this period. 56.6% of er were given to surgical, 21.3% to medical and 12.2% to gynaecological and obstetric patients. 62% of er were given to patients above 45 years. er units were given to 1475 patients (mean 4.75 units/patient; range 1-63). mean age of trauma patients was 33 ± 18, median 30, range 1-89 years. for transfusion of this group local guidelines state that haemodynamically unstable patients should receive er if hgb <9 g/dl, irrespective of age and sex. eighty-eight patients (27.8%) received er, 15 of them as massive transfusion ( 3 10 er units in <24 hours) (17.0%), 6 of these died (40%). altogether trauma patients received 851 units of er (12.1% of total uuh consumption), 51.5 thr (6.1% of total) and 150 units of op (16.5% of total). massive transfusion consumed 340 er (40%), 29.5 thr (57.3%) and 110 op (73.3%) units. fourteen adult trauma patients (15.9% of those transfused) received 1 or 2 er units only. lowest pre-transfusion hgb was 3.3-11.3 g/dl, median 7.6, mean 7.5 ± 1; highest post-transfusion hgb 7.8-14.3 g/dl, median 9.5, mean 9.8 ± 1. five patients received er transfusion at higher pretransfusion hgb level than 9 g/dl, but in 4 er were given because of a hyperacute clinical situation. fifty-five% of issued er units had been stored for >15 days, while only 10% were issued before 10 days of storage. discussion: life-saving effect of transfusion would seem evident in the massively transfused survivors, while in the other transfused patients documentation of clinical effect is inadequate. transfusion practice in trauma at uuh seems fairly well in accordance with internationally accepted guidelines. the trauma unit consumed a surprisingly small part of total uuh transfusion resources. trauma patients deviate from the general uuh patient population by sex and age; transfusion is otherwise mainly given to more elderly patients. further analysis is needed on transfusion indications and results to optimize the total use of blood products in uuh. special focus should be on transfusion to non-bleeding patients without haematological disease. it seems that only a small part of transfusion efforts results in the saving of life. in croatia national guidelines for the use of rhd gamma globulin were laid down in the year 2000. in accordance with the guidelines, rhd gamma globulin is administered intramuscularly in doses of 250-300mg and should be given to rhd negative women after delivery of rh positive children, after abortions, in week 28 of their first pregnancy, as well as in cases pregnancies with an increased risk of fetomaternal hemorrhage. as to the latter, detection and measurement of fetomaternal hemorrhage are recommended. three years after the issuance of the guidelines a survey was conducted regarding the use of rhd gamma globulin that involved 33 health institutions across the country. as many as 38 601 deliveries and 10 974 abortions were reported in 2003. the institutions reported the usage of 5103 doses of 250 mg rhd gamma globulin or 110 doses/1000 deliveries + abortions. we compared our data with those obtained from similar surveys conducted in 1999, i.e. before the issuance of the guidelines. in that year 4407 deliveries and 1400 abortions were reported, and 6125 doses of rhd gamma globulin or 100 doses/1000 deliveries + abortions were administered. institutions were then divided into four categories: clinical hospitals, general hospitals with the capacity of 400-600 beds, general hospitals with the capacity of 100-400 beds, and institutions of rather limited capacity with gynaecology department. the range of the consumption of rhd gamma globulin/1000 deliveries + abortions in the first category was 73-167 with the median being 119; in the second category there were 73-115 doses with the median being 92, in the third category 55-128 doses with the median being 88, while in the fourth category the range was 10-191 doses with the median of 79 doses. the number of pregnancies which should have been protected with rhd immunization in 2003 year was obtained by the following formula: (frequency of rhd negative subjects) ¥ (frequency of the r gene) = 0.17 ¥ 0.59 = 0.10. if a complete antenatal and postnatal preventive measures involving rhd immunization had been taken in 2003, 12 287 doses of rhd gamma globulin or 250 mg/1000 deliveries + abortions should have been given. our research has shown that, despite of the national guidelines adopted in 2000, the prevention of the rhd immunization in pregnant women is still not adequate in croatia. in fact, it has not improved since a year prior to the adoption of the guidelines. it has also been established that the category of the institution is a factor influencing the implementation of the protective measures. we consider that much more should be done on informing both women and gynaecologists about the importance of prophylaxis of the rhd immunization. consumption of plasma derivates in croatia g jaklin*, b golubic-cepulic † and m dondur* *general hospital, varazdin, † clinical hospital center zagreb, zagreb, croatia a increasing consumption of plasma derivates, their limited supply and insufficient national reserves are problems that croatia is faced with nowadays, as well as many other countries. in 2003 a study was carried out regarding the usage of plasma derivates. as many as 34 health institutions took part having the capacity of 15 487 acute beds out of a total of 16 279. the data regarding the use of plasma derivates were collected as follows: albumin, i.v. gamma globulin, and concentrate of coagulation factor viii in two periods of time. we divided institutions into three categories: clinical hospitals, general hospitals with the capacity of 400-600 beds and general hospitals with the capacity of 100-400 beds. institutional practice patterns regarding the use plasma derivates were compared among them. the parameters considered were the total consumption of plasma derivates, consumption per bed and consumption per inhabitant. data on the use of plasma derivates was obtained from hospital transfusion departments and pharmacies across the country. we compared our data with similar study carried out in 1997. the consumption of albumin was 441.44 kg or 98 kg/million inhabitants in 2003 and consumption per bed for the first category institutions was in range 14.27-72.27 kg with the median being 39.42, for the second category the range was 1.24-44.64 kg with the median being 4.36, while for the third category the range was 0.32-23.13 kg with the median being 6.63. in 1997 in croatia the consumption of albumin was 436.50 kg or 97 kg/million inhabitants. the consumption of i.v. gamma globulin was 59.92 kg or 13.32 kg/million inhabitants in 2003 and the consumption per bed for the first category institutions was in range 0.03 g-8.89 g. with the median being 2.78, for the second category was in range 0.03-3.95 g. with the median of 1.56 and for the third category was in range 0.00-12.75 with the median 1.56 g. in 1997 in croatia the consumption of i.v. gamma globulin was 22.20 kg or 4.93/million inhabitants. the consumption of the concentrate of factor viii was 8807.000 iu or 1.96 iu per inhabitant in 2003. 1539.500 iu, i.e. 17.48%, was a recombinant factor viii. in 1997 the consumption of factor viii was 4876.350 iu or 1.08 iu per inhabitant. discussion: the use of albumin showed stagnation. however, the use of i.v. gamma globulin increased 2.6 times and the use of the concentrate of factor viii increased 1.8 times if compared with the results in 1997. self-sufficiency has not been reached in plasma derivates in croatia we needed 20 000 l plasma for gamma globulin and 58 000 l plasma for concentrate of factor viii for the level of usage of these plasma derivates in 2003. significant differences in the level of usage among hospitals have been observed. these reflected a considerable difference in hospital policy regarding the use of these products in defined clinical settings. therefore, we would recommend that the national society sets out guidelines for the use of the products. background: blood bank good practice requires avoidance of rh alloimmunization. several studies report a probability of immunization of more than 80% following transfusion with rhd+ rbc's and as many as 19% in the case of platelets. aims: the purpose of this study was to investigate the development of anti-d and the causes of alloimmunization in a university hospital (1300 beds), reviewing our practice on the use of rhd+ blood components in rhd-recipients. method: a retrospective study was performed whereby 15.872 rhd-patients, from 1990 to 2004, were evaluated for the development of anti-d analysing the data on the blood bank information system. results: from the 15.872 rhd-patients we found out 274 identified anti-d, 77% (211) detected before any transfusion in our hospital. of the 63 left, nine were excluded because no information was available during some years. 5 had history of pregnancy related to the immunization. 36 patients were exposed to rhd+ blood components: 12 were transfused only with rhd+ platelets (including two women of childbearing age); 24 with rhd + rbc's. the remaining 13 had been exposed exclusively to rhd -rbc's suggesting that some units could be mistyped. this idea was corroborated in three patients where there was a common donor (he was called for investigation). conclusions: transfusion services avoid as far as possible administration of rhd+ blood components to rhd-patients, although there are situations in which such transfusions are necessary. the retrospective review of records revealed the need for specific recommendations regarding the use of rh immunoglobulin to prevent anti-d immunization. the possibility of mistyping blood components also appeared in this review, emphasizing the role of these studies in the evaluation of methodologies used at blood banks. the purpose of the work: to present the usage of whole blood (wb) and blood products (bp) in the treatment of patients who are hospitalized in the internal disease department in gevgelija. material and methods: retrospective analysis is done according the data which were analysed in five years period (2000) (2001) (2002) (2003) (2004) . statistical methods which were taken from the history of the hospitalized patients in the internal department were used for analysis and the data of transfused wb units and units of bp were taken from the department of transfusion medicine. results: from the total 4285 hospitalized patients who have been analyzed, 759 (17.71%) received wb and bp, and there have been transfused 1411 units wb and bp. every patient, on an average, has been transfused with 1.86 units wb and bp. at the same time 326 (23.1%) units have been transfused as a wb, 928 (65.77%) have been transfused as red blood cells (rbcs) and 157 (11.13%) units have been transfused as a fresh frozen plasma (ffp). the data for every year particularly will be presented in our paper to the congress. the collaboration between doctors of transfusion medicine and health workers from the other medical branches is the most important thing in the medical practice. our aim is to raise the awareness among the health workers for the importance of blood safety and their role though adequate clinical usage of wb and rbcs, minimizing the non-useful transfusions, evaluation of reflexive information from undesirable reactions in the usage of blood and blood products, use the alternatives etc. the necessity of direct involvement of the doctors in transfusion medicine in the process of healthy workers' education from the other branches for regularly transfusion therapy via meetings and lectures is an imperative for regular function of the department of transfusion medicine. femoral neck fracture repair is one of the commonest orthopedic procedures. it mainly concerns intracapsular or intratrochanteric fractures and it may be considerably hemorrhagic, requiring blood transfusion perioperatively. the aim of our study was to investigate blood transfusion requirement in patients with femoral neck fractures repair at katerini general hospital during 2002-2003 and to compare them with other studies. one hundred sixty five (165) unselected patients of whom 116 (70%) were women and 49 (30%) were men with a mean age of 76 years (range 26-94 years) were studied retrospectively. seventy six (46%) patients had intracapsular fracture and 89 (54%) intratrochanteric fracture. the mean hb concentration on admission was 12.25 g/dl (range 6.8-17 g/dl) and the mean hb concentration at discharge was 10.78 g/dl (range 7.2-14.3 g/dl). a total of 297 units of rbc were transfused (a mean of 1.8 units per patient). blood transfusion occurred in 125 patients (75.8%), (45-69% in other studies) with a mean of 2.38 units per patient (range 1-10 units), (1.47-2.6 in other studies). patients with preoperative hb values <10 g/dl were transfused more often than those with hb values >10 g/dl (100% versus 73%) p: 0.017. women were transfused more often men (80.2% versus 65.3%) p: 0.042. patients aged >70 years were transfused more often than those aged <70 years (81% versus 60%) p: 0.008. finally patients with intratrochanteric fractures were transfused more often than those with intracapsular fractures (88.65% versus 60%) p: 0.001. conclusions: in our study blood transfusion requirement for femoral neck fracture repair are similar with these reported in other studies. blood transfusion frequency is greater in patients with hb <10 g/dl, in patients older than 70 years, in women and in intratrochanteric fractures repair. fresh frozen plasma guidelines and practices as saltamavros*, g talampouka*, c koumoundourou*, s dimitrakopoulos † and p tseliou* *st. andrews hospital patras greece, patras, † pyrgos general hospital, pyrgos, greece introduction: fresh frozen plasma transfusions should be done according to specific standards and guidelines. aims: we have reviewed the ffp transfusion practices followed in our hospital in an attempt to provide a set of guidelines and principles that will assist physicians and health care workers to make the right decisions for appropriate use of ffp transfusions. methods: retrospective review of ffp transfusions practices during the entire year of 2004. we classified transfusion practices according to the diagnosis of the patient and also of the clinical depart-ment that demanded transfusion. then we analyzed our results by comparing the requests with the internationally approved guidelines for ffp transfusion and counted the percentage of ffp to rbc units. finally we discussed with our fellow physicians the proper use of ffp and informed them about the accepted guidelines. as we can see from the underneath table, diagnoses and departments with high consumption were: internal medicine 22%, plasmapheresis to patients suffering from guillain barre and ttp (thrombotic thrombopenic purpura) 21.04% and trauma/surgery 20.68%, cancer 13.60%. summary/conclusions: the use of ffp corresponds to 27.88% of the rbc units transfused in our hospital. plasma units should be used properly and according to internationally accepted standards. plasma use should be justified by the physician, in order to avoid unnecessary risks on behalf of the patient for effective treatment and care. to show gradual discontinuation of using whole blood and increased use of red blood cells (rbc) in the management of patients in the transfusion department of the clinical hospital 'zvezdara' . methods: data of wb and rbc use from 1982 to 2004 in our hospital. results: our data are presented in table 1 . conclusion: after 1982, wb usage rates systematically decreased from 51% to 1.3%. however, in the period of 1991-2004, the wb usage rates increased slightly, and we arrived at a generally very low rate of wb use, namely only in about 3% of the cases. education about transfusion medicine and rational use of blood in the medical curriculum was generally insufficient and very poor. therefore, we started an education program and we established a hospital transfusion committee (htc) with the task of medical control and monitoring the quality of clinical transfusion practice in our hospital. members of the htc are a specialist in transfusion medicine, an internal medicine specialist, an anesthesiologist, surgeon and gynecologist. we have been organizing courses for the medical staff, doctors and medical technicians since 1995. background: platelet transfusions are given mainly to thrombocytopenic patients with malignant haematological diseases. currently the decision to give a prophylactic platelet transfusion is based almost exclusively on the number of circulating platelets in the patient although it is known that various clinical factors might influence the bleeding tendency. by free oscillating rheometry (for), using a reorox® 4 instrument, it is possible to monitor the coagulation over time in whole blood and obtain information about clotting time and coagulum elasticity. aim of the study: the aim of this study was to find out if for using the reorox® 4 instrument could be used to evaluate the hemostatic status of thrombocytopenic patients. methods: the change in elasticity over time in non-anticoagulated whole blood from leukaemia/lymphoma patients with thrombocytopenia was measured in the reorox® 4 pre and post a platelet transfusion (n = 10) and was compared with healthy controls (n = 40). the effect of platelet concentration on coagulation was studied by diluting platelet rich plasma from healthy subjects with autologous plasma to various platelet counts whereafter the coagulation was monitored with for (n = 8). the change in elasticity per minute (g'max slope) and maximum elasticity (g'max) were evaluated from the elasticity curves. results: the g'max, g'max slope and platelet count increased after transfusion for all patients. the thrombocytopenic patients had significantly lower g'max and g'max slope values both pre and post transfusion compared with healthy control subjects (p < 0.01). the measurement in platelet rich plasma showed that g'max and g'max slope increased with increasing platelet concentration. however patients with similar platelet count developed different clot elasticity. the reorox ® 4 instrument responds to changes in haemostatic function in form of the clot elasticity. the fact that patients with similar platelet concentration developed different elasticity shows that clot elasticity is a function of not only platelet concentration but also of functional properties. the for method seems promising to evaluate platelet function. quality control of pre-storage leukodepleted red cell concentrates vu urlep salinovic, k perbil lazic and l lokar teaching hospital maribor, maribor, slovenia background: the system of quality assurance including quality control (qc) in transfusion medicine is most important for safe blood supply. the safety and quality of blood components are increased by pre-storage leukodepletion of whole blood (wb). aim: the qc of leukodepleted red cell concentrates (rcc), prepared from pre-storage filtration of wb (quadruple blood bag systems imuflex wb-rp terumo) is performed with the aim to be sure that the quality of leukodepleted rcc is in accordance with the guidelines of council of europe. in three years (2002) (2003) (2004) , 35 559 units of wb were collected, among them 7765 (21.6%) units were collected for pre-storage filtration and in 376 (4.8%) of these qc was done. within 4 hours after collection, wb was filtered at room temperature. filtered wb was centrifuged at 4000 g for 20 minutes and separated in rcc and plasma. the samples for qc were collected before and after filtration. for each unit, volume, hemoglobin, hematocrit, % of hemolysis and residual white blood cells (wbc) count were measured. the number of residual wbc after filtration was determined in the nageotte chambre. for all parameters the mean value and standard deviation were calculated. the results of qc for the following parameters were: volume 320 ± 25.1 ml (89% corresponds with the guidelines of council of europe), hematocrit 0.61 ± 0.06 (96%), hemoglobin 60.9 ± 8.0 g/unit (99%), number of residual leukocytes 0.028 ± 0.16 ¥ 10 6 (100%), hemolysis 0.30 ± 0.19% (100%), the test of sterility was 100% negative. during filtration of wb, wbc were removed in approximately 99.99%, the duration of filtration was 16 ± 6 minutes and the loss of hemoglobin was 8.2 ± 4.3%. the pre-storage filtration of wb is highly efficient, 99.99% of wbc were removed and the loss of hemoglobin was small. background: the patients of cardiosurgery use big amount of blood components. there is no uniform approach by treatment with blood components in these patients. the safest and the most rational use of blood is based on the individual treatment of a patient and the evaluation of the most clinical and laboratory factors. aim: the aim of our study is to analyse the use of blood components from 1998, when the cardiosurgery department was founded in our hospital, to 2004. the data about use of red cell concentrates (rcc), platelet concentrates (pc) and fresh frozen plasma (ffp) in the period from 1998 to 2004 were collected from information system datec. the average use of all three blood components per patient is presented. we were interested, if the average use per patient was diminished in the analysed period. results: the use of three blood components and average use of all blood components are presented in the table. the data from the table shows, that the number of patients increased more than three times, but the average use of blood components per patient was not essentially diminished. conclusion: during seven years period the use of blood components per patient in cardiosurgery was not diminished. for more rational use it is necessary to apply all methods of autologous blood transfusion because of the increasing number of cardiac operations and decreased number of voluntary blood donors. background: the presence of leucocytes in blood components is cause for appearance of various adverse posttransfusion reactions such as nhfptrs, urticary, anaphylactic shock, alloimunization and platelet refractorines, infection with bacteria and leucothropyc viruses (cmv, htlv). the removal of leucocytes from blood components through filtration is especially important in the treatment of patients with malignant diseases who need frequent transfusions of blood and blood components. this is because of their lowered immunologic status which is a result of the disease and received immunosuppressive chemotherapy and radiotherapy. aim: to show the prevention of posttransfusion reactions and the positive effect from transfusion of leucoreduced er. concentrates, produced with filtration in therapy of anaemia in patients with malignant diseases, treated in our daily transfusion hospital at medical center -stip. methods: 123 patients with malignant diseases have been treated in our daily transfusion hospital in the last four years. most of these patients suffer from ca pulmonum, ca collonis, ca uteri, ca mammae. also, because of the secondary anaemia, the same patients were transfunded with at least two doses of leucoreduced er. concentrates. the blood donored by voluntary repeated blood donors was filtrated the same day after the donation, separation and the control of the same one. the blood was collected in baxter and terumo bags, and it was filtrated with baxter -sepacell rs -2000 and pall -purecell rn filters. the analyses of leucoreduction were made for every filtrated and transfunded unit before and after filtration. the analyses samples were taken from the tubing system before and after the filter. haemathologic parameters were automatically made in the central clinic laboratory. results: er. concentrates poor with the leucocytes for about 98-99.9% were produced with the use od these filters, and platelets from 88-100% with which side effects from frequent transfusions at these patients were prevented. with the routine monitoring of all patients none posttransfusion reactions were registered. the therapeutic effect is also important because of the fact that the number of er and the level of hg and hct remain almost unchanged. the aim of the blood banks is to help and to increase the safety of the blood components. the leucoreduction through of er. concentrates poor with leucocytes is a regular procedure in the therapy of malignant patients with remarkable clinical picture of anaemic syndrome and prevention the cancer recurrence end infection. the time of filtration is also very important which has to be shorter after collection of blood, because the leucocytes should be removed before they become disintegrated and relapse potentially dangerous substances end metabolites in the blood components. we have been applying so called ' prestorage filtration' because it has been proved that it is more efficient that bed -side filtration. background: the effect of leukocyte reduction of rbcs by filtration has been the topic of several rcts. these rcts investigated the effect of leukocyte reduction on mortality; post-operative infections and hospital stay in different patient populations. some rcts came up with answers that conflicted with the results of other rcts. aim of the study: with including the individual patient datasets of several rcts in one database, combined analyses are possible that may explain the differences in the reported results. using this technique, we may come up with answers (or questions) that cannot be obtained by performing standard meta-analyses of these rcts. methods: we coordinated several rcts comparing the perioperative use of buffy-coat depleted rbcs with the use of filtered rbcs. cardiac surgery patients were included in three rcts (1¥ cabg and/or valve; 1¥ re-cabg and/or valve; 1¥ valve with or without cabg). oncologic surgery patients were also included in three rcts (1¥ colorectal cancer; 1¥ gi-oncology or vascular surgery; 1¥ gi-oncology, vascular surgery or orthopedic surgery). the electronic data files of these rcts were uniformly recoded and entered in a single database. as different primary endpoints were investigated in the rcts, we focused on common endpoints, recorded in 4 or more of the rcts. multivariate analyses were performed on: in-hospital mortality, 30-day mortality, hospital stay, stay on icu, postoperative infections, and mods. results: in the rcts, individual datasets from 3875 surgery patients were collected (1630 onco; 1272 cardiac; 569 vascular; 352 orthopedic, 52 other). in the multivariate analyses, patients in the buffycoat depleted trial arm showed a higher mortality rate both in-hospital (p = 0.01) and at 30 days post-surgery (p = 0.03). no association between randomization and stay in hospital (p = 0.10) or stay on icu (p = 0.56) was seen in the combined study population. also, no association of randomization with the incidence or duration of mods was seen. the analyses of post-operative infections in the total population showed the trial arm to be associated (p = 0.016), and 'hospital' to be far stronger associated (p < 0.001). however, when the oldest rct, that had not yet used our standard definition list for scoring post-operative infections, was excluded, the association with the hospital was lost (p = 0.34) and the trial arm became more strongly associated with infections (p = 0.002). in the analyses of 3875 surgery patients, the use of filtered rbcs, compared to the use of buffy-coat depleted rbcs, resulted in reduced mortality (both in-hospital and at 30 days postsurgery) and a reduction in post-operative infections. the association of the variable 'hospital' with post-operative infections, as is frequently reported in literature, was initially confirmed in our analyses. however, when a standard definition for post-operative infections was used (excluding the datasets from one of six studies), this association was lost. background/aims: uk neqas for blood transfusion laboratory practice (btlp) operates an eqa service for 472 uk laboratories (including eire) and participants throughout europe, including major groups in denmark (n = 52) and portugal (n = 32). in november 2004 a questionnaire was distributed to determine the criteria used for selecting phenotyped blood for different patient groups, including pre-menopausal women (pmfs). results were analysed by country to establish any variation in practice relating to the selection of k negative (k-) and rhc negative (c-) blood, since antibodies to these antigens are now the major cause of hdn. results: overall return rate was 84% (uk) and 64% (non-uk), although only centres treating pmfs were included in this analysis. k-blood was selected for pmfs by 81% in wales (n = 16) and 71% in england (n = 291), but only 6% in scotland (n = 32), 8% in northern ireland (n = 12) and 10% in eire (n = 31). in mainland europe, variation was also observed: 12% in denmark (n = 17), 75% in portugal (n = 20) and 85% in other countries (n = 27 from 10 countries). fewer laboratories selected c-blood for pmfs: 8% in england and northern ireland, 0% in scotland and eire, rising to 81% in wales. mainland europe showed similar variation: 12% in denmark, 75% in portugal (all ccee matched) and 51% in other countries. there are no guidelines requiring selection of k-and c-blood for pmfs, but in the uk d negative blood is required for d negative females aged <60 years. trend analysis of questionnaire data in the uk, using theoretical clinical scenarios, shows a decrease in the number of laboratories that would select k + blood for a 30 year old female (with pre-existing antibodies other than anti-k), from 58% (1996), 42% (1999) to 26% in 2004. in this survey, k + blood was selected for a female aged 30 (no antibodies) by 31%, whilst 81% selected a k + unit for a female aged 54 (no antibodies), despite this patient being treated as a pmf for provision of d negative blood. a similar distinction was made between the 30 and 54 year old females in portugal and mainland europe. conclusions: variation in practice may at least in part be due to the availability of blood routinely labelled for rh and k. in the uk all donations are labelled, except for those from new donors, as are most units in portugal. uk questionnaire data (1999) suggested that >50% laboratories would change to selecting k-blood for pmfs if all units were labelled. however, labelling alone does not account for the differences seen within the uk, and perhaps the trend towards providing k-(and to a lesser extent c-) blood for pmfs is influenced by advice from transfusion services. it would be of great interest to monitor and compare the incidence of hdn due to anti-k and anti-c in different countries to measure the outcome of differing practices for provision of blood to pmfs and to thereby inform future policy. there is no ultimate in ex-vivo assay described, which can predict the outcome of plt transfusion in vivo. current in ex-vivo assays and animal studies are rather very complicated to carry out, cost effective and time consuming. objective: we hypothesized that the quantitative measurement of gpib expression by facs can be used to predict the outcome of platelet survival post transfusion. in our previous studies we demonstrated that our phagocytosis assay can predict the plt survival sensitively (blood dec-2004) . we isolated human washed plts by centrifugation and labelled with mepacrine and then incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. we measured gpib expression before and after plt-macrophages interaction by facs flowcytometry. results: gpib expression at surface of c22 fresh showed 90 ± 5 and after 48 hours storage 50 ± 15% expression. the gpib expression at surface of plt decreased and showed three populations with different densities high (gpib-1), low (gpib-2) and in-between (gpib-3). the binding and phagocytosis of plt showed an increase of 40 ± 10% which implicates an indirect and negative relation to gpib-1, and direct relation to gpib-2 expression. anti-human pselectin (cd62p) delayed 45 ± 10% the binding and annexing v 60 ± 12% the phagocytosis of stored plts, after 48-hour storage. these results show that gpib expression is rather easy, reproducible test which can be standardised and be used as a very sensitive in vitroassay to predict platelet survival posttransfusion. transfusion and lung injury jd dodig*, d sovic † , m tomicic † and h sager* *university hospital 'sestre milosrdnice', zagreb, † institute for transfusion med, zagreb, croatia background: the respiratory tree has been viewed as an infrequent site of injury arising as serious complication of transfusion. in recent years, this view has changed as investigators have shown that two complications-circulatory overload and transfusion related acute lung injury-are relatively frequent events. case report: a 59-year-old men was admitted due to chronic macrohematuria. he had no history or current evidence of cardiac failure. the hb level was measured 36 g/l. he received 500 ml 0.9% nacl and 500 ml ringer solutions. after that, he was transfused with 2 units of rbcs. during transfusion second unit he developed following symptoms: tachycardia, dyspnea, hypertension. massive pulmonary edema was noted. he was treated with mechanical ventilation, oxygen, diuretics, aminophillin, antihistaminic and corticosteroides. the patient recovered after being on ventilation for 6 hours. two days after the patient was operated. after surgery he was transfused with 4 units of rbcs. all transfusions were regularly performed. results: chest x-ray confirmed bilaterally pulmonary edema. the samples (patient and donors of first two units rbcs) tested were negative for the presence of hla specific and granulocyte antibodies. granulocyte agglutination and lymphocytotoxicity test were negative. tnf-a in recipient serum was slightly increased. conclusion: our patient is the first reported case suspected of trali, but all of the investigations didn't give us the answer. against neutrophils using flowcytometric detection of cell surface cd11b expression and l-selectin shedding. methods: a hundred microl of volunteers' heparinized whole blood were incubated for 15 minutes at 37°c with fmlp, lps or pma. monoclonal antibodies against hna1a and hla-i, or patient serum were incubated with whole blood for 30 min. surface cd11b and l-selectin were detected using facscalibur. results: neutrophil activation was detected after fmlp, lps or pma stimulation. p38 map kinase inhibitor reduced activation induced by fmlp and lps. anti-hna1a monoclonal antibodies induced neutrophil activation, which were also inhibited by p38 map kinase inhibitor. ten serum samples obtained from patients or donors who caused transfusion reactions were evaluated. five sera out of 8 samples having anti-neutriphil and/or hla antibodies exhibited neutrophil activation, while two samples without leukocyte antibodies had no effect on any activation. conclusion: these findings indicate that neutrophils activation is regulated through mak kinase and detection of neutrophil activation may be useful to predict transfusion-related reactions. results: 38 out of 50 transfusion reactions were febrile, 19 were anaphylactic, 1 were due to circulatory overload, 1 out of 50 transfusion reactions concerned the transfusion of incorrect blood component. 1 out of 50 transfusion reactions concerned acute haemolytic reaction. post transfusion purpura or suspected trali was not seen. fatal complication was not seen. the reactions to plasma were predominately anaphylactic. we detected no case of bacterial contamination among the cultures of transfusion bags. conclusions: the incidence of reactions among patients malignancy was high, while among surgical patients was lower. the incidence of transfusion reactions during the months had no statistically significant difference. we suggest that the improvement in prevention of transfusion reactions require a continual vigilance system for rapid recognition and information regarding these complications. measurements of ige immunoglobulin in thalassemic patients, before and after blood transfusion background: many studies have reported the implications of proinflammatory cytokines including interleukin (il)-1 beta, il-8 and tumor necrosis factor alpha (tnf-alpha) in febrile nonhemolytic transfusion reactions. il-8 has been shown to accumulate in packed rbcs even after the procedure of filtration, which is explained by the release of il-8 from rbc receptors into the packed rbc super-natant. stress induced elevation in tnf-alpha levels was demonstrated in healthy individuals. aim: to assess the level of proinflammatory cytokines in peripheral blood of blood donors. material and methods: immediately upon blood collection, plasma was separated from postdonation blood samples obtained from 75 blood donors and frozen at -80°c. upon thawing, the level of the il-1 beta, il-8 and tnf-alpha cytokines was determined in plasma samples by elisa method using commercial kits for cytokine determination (roche molecular biochemicals). the level of il-1 beta was at the test detection limit in all donor plasma samples. in 1 (1.3%) bd, the level of il-8 was 36.8 pg/ml, exceeding the test sensitivity limit of 6.2 pg/ml. in 12 (16.6%) bd, the level of tnf-alpha was within the range of 14-60 pg/ml, with a test sensitivity limit of 12 pg/ml. tnf-alpha levels >800 pg/ml were measured in plasma samples of 3 (4.1%) blood donors. the increased activity of blood donor's immune cells, indicated by elevated levels of the proinflammatory cytokines il-8 and tnf-alpha in peripheral circulation some blood donors, may lead to the occurrence of febrile nonhemolytic transfusion reactions in the recipients of the blood products manufactured from the blood of these blood donors. this hypothesis will be thoroughly investigated in our future studies. background: transfusion-related acute lung injury (trali) is a life-threatening complication of transfusion, under-recognized and underreported possibly lacking a consensus definition. aim: we report here a 'probable' case of trali syndrome in an elderly female patient. case presentation: an eighty-two year old female patient was transferred to the intensive care unit on the third postoperative day (pod) following the abrupt onset of acute pulmonary insufficiency (pao 2 = 49 mmhg, o 2 saturation = 79%), hypotension (bp = 90/50 mmhg) and fever (38°c). this occurred ten minutes after initiation of an infusion of a unit of packed red blood cells (prc). the patient had no history of any cardiac or pulmonary disease. she was intubated and placed on mechanical respiration and supported hemodynamically. the cvp (12 cm h 2 o) ruled out fluid overload. the chest x-ray revealed bilateral pulmonary oedema, the echo cardiogram and blood cultures ruled out cardiac and infectious aetiology of the episode. after treatment for 48 hours the patient improved significantly, was extubated and returned to the surgical unit on the 6th pod. reviewing the transfusion history we discovered that the patient did not receive any blood or blood component prior or during the correction of her ileum, but she was transfused a unit of ffp (fresh frozen plasma) five hours prior to the incident. the donor review revealed that the unit of ffp was from a 29-year old female with a history of multiple abortions whereas the prc unit was from a 52-year old male, a volunteer of several years who had no history of transfusions. there are some prerequisites for the implementation of a haemovigilance network and some of them have been met, so we can present the first results. methods: traceability of blood components is possible because there is unique information system in place in the whole country, identifying the donor, donation, each single blood component and identification of the recipient, but feed back information of the performed transfusion is still missing. cooperation between blood transfusion service and hospitals was established by introduction of hospital transfusion committees; the intensity and quality of their work is very different. homogeneity of reporting is achieved by the introduction of unique reporting form. a reporting route was defined: patients physician sends notification of atr to the local blood transfusion service. atr reporting form is prepared there and sent to the national blood transfusion service, where the data is collected for the centre for haemovigilance, which is going to be established very soon. data analysis will be responsibility of the centre for haemovigilance at the governmental level. type of adverse transfusion reactions and events is defined. education and information was passed to the health personnel by inclusion of haemovigilance in under and postgraduate programmes as well as seminars, scientific meetings and some publications. results: in the years 2002-2004 there were 410.000 blood components issued in slovenia and 333 atrs were reported (1 in 1232 blood components issued), in 25 of them the severity grade was 2 (1 in 16.412 blood components issued). in the year 2004 there were considerable differences between the number of atr reports compared to the number of blood components issued among slovenian hospitals, ranging from 1 in 3375 to 1 atr in 490 blood components issued. 9.6% of all atr were not classified. conclusions: although the number of reported atrs was increased by 24% and 31% a year in 2003 and 2004 respectively, it can be assumed that the collected data is not complete. feed back reports, much more information and cooperation is needed, especially in some hospitals, which should contribute to a better registration of atrs and events in the future. the slovenian haemovigilance system still needs upgrading, but despite this, some important work has been done in building a national haemovigilance system. . considering the multiplicity groups in cattle and since there was no research on repeated blood transfusions reactions in iran's native cattle, we decided to consider crossmatching in different processes of repeated blood transfusion from a head of blood donor to five recipients and observe the clinical and hematological alterations. six healthy iran's native cow, 2.5 years old, with average weight of 210 kg were used. the animals were dewormed by albendazole (15 my/kg bw) and were kept for two weeks under uniform managemental condition. three days prior to blood transfusion, vital signs registration (temperature, heart rate and respiratory rate) blood collection via jugular vein was done to indicate the baseline of research parameters. after that, blood transfusions were performed from one donor cow to five recipients three times at one-week intervals. cross matching was done at each transfusion. after each transfusion the research parameters do determined. results indicated that one of the recipients cows experienced anaphylactic shock, in the first step of blood transfusion and another cows in the second step and finally in the third steps two other cows showed the serious shock. this is in the contrary of this opinion that the first transfusion can be given safely without crossmatching in cattle practice ( van der valt, et al. (1969) background and objectives: blood transfusion may lead to the manifestation of anti-hla and platelet-specific antibodies that may in turn bring about different problems like platelet refractoriness. it appears that the study of antibodies against hla-class i and platelet-specific antigens are useful for the selection and success of the appropriate treatment protocol. the aim of this study was to detect anti-hla and anti-platelet-specific antibodies by flowcytometry in patients with hematologic disorders (including acute leukemia, aplastic anemia) and patients with itp. in this descriptive study, anti-hla and platelet-specific antibodies were detected by flowcytometric technique, using 62 sera drawn from patients with different haematological disorders who showed a poor response to platelet transfusion and 20 from patients with itp. the results of anti-hla antibodies were then compared by panel reactive antibodies (pra). results: our results showed 44 (53.7%) out of 82 (53.7%) patients had anti-hla class-i antibodies in their sera. the frequency of each antibody isotype was found to be as follows: igm (51.2%), igg (32.9%) and iga (1.2%). 36 (43.9%) out of 82 patients had platelet specific antibodies and the frequency of each antibody isotype was found to be as follows: igm (40.2%), igg (30.5%) and iga (12.2%). 27 (31.7%) out of 82 patients had both antibodies. no difference was found between the two groups in platelet specific antibodies. despite significant correlation between flowcytometry and pra methods, pra can only detect antibodies which react with complement. conclusions: with increase in the number of platelet transfusion, immunization to hla antigens occurs; moreover, immunization against platelet specific antigens may also occur during autoimmunity. the presence of these antibodies may be one of the reasons of poor response to platelet transfusion and platelet refractoriness in patients under study. conducting similar studies with higher number of samples, platelet cross-match, and the use of hlamatched platelets for these patients are recommended. post transfusion purpura (ptp) is a rare, severe thrombocytopenia that results from alloimunization to platelet specific alloantigens, following blood transfusion. in this condition, the patient's own platelets are destroyed by the alloantibody even though they are not supposed to carry the 'guilty ' antigen. the disease is rare occurring mainly in multiparous women. the majority of reported cases involved antibodies against the platelet specific alloantigen hpa-1a in a homozygous hpa-1b patient. in a minority of cases the offending antibodies were directed against hpa-1b, -3a, -3b, 4a, -5a, -5b. although self-limiting, the syndrome is characterized by severe bleeding with high morbidity and mortality; therefore prompt diagnosis and appropriate therapy is crucially important. ptp is a challenging diagnosis, because the patients are often critically ill or post-surgery, and have alternative explanations for thrombocytopenia such as infections or drugs. we present three patients with severe thrombocytopenia initially misdiagnosed. the first patient, a 54-year old women, had a past history of systemic lupus erythematosus and coombs positive autoimmune hemolytic anemia. at the present hospitalization, after antibiotic therapy for endocarditis, a severe hemolytic episode occurred and she need blood transfusion. when severe thrombocytopenia appeared, she was wrongly diagnosed as evans syndrome. the second patient, a 73-year old man, suffered from sepsis after vascular surgery and revealed clinical and laboratory picture of dic. the third patient, a 58-year old women, had end-stage renal failure and received heparin during hemodialysis, thus heparin-induced thrombocytopenia was first suspected. history of recent blood transfusion rose the suspicion of ptp in all this cases, and appropriate therapy with high dose iv immunoglobulin was started. adequate laboratory work-up confirmed the diagnosis. three different anti hpa-antibodies were identified: anti hpa-3a, anti hpa-1b and anti hpa-3b, respectively. the platelets genotype of the first patient was hpa-3b/3b, of the second hpa-1a/1a and of the third patient, hpa-3a/3a. the reported cases emphasized the importance of keeping in mind the possibility of ptp. incorrect diagnosis may lead to wrong treatment and fatal outcome. health sciences authority, singapore, singapore introduction: the haemovigilance programme in singapore was started by the centre for transfusion medicine (ctm) in 2002. the system covers registration of collected, produced and transfused blood components, and monitors adverse transfusion reactions (atr). the programme runs on a voluntary, non-punitive and confidential basis. aims of the study: (1) to gather and analyse reports of all adverse and untoward events occurring during transfusion of blood and components. (2) to use the information acquired to determine the morbidity of transfusion. (3) to provide guidance on corrective measures to prevent the recurrence of some accidents, and to improve transfusion safety. (4) to improve public confidence by demonstrating to public, patients and professionals the safety of the existing transfusion system. methods: (1) a common report form is used and made available to all participating hospitals. within the reporting system, the identification of the patient and staff involved are not required, to ensure confidentiality and protection of information belonging to the hospital. (2) reportable events include immediate reactions during transfusion (haemolysis, non-haemolytic febrile transfusion reaction, urticaria, anaphylactic shock, bacterial contamination, trali), delayed untoward effects after transfusion (haemolysis, post-transfusion purpura, acute gvhd), transfusion-transmitted infections, incorrect components transfused, and near misses. (3) within the hospitals, a responsible person ensures that all adverse events and untoward effects of transfusion are reported on the haemovigilance forms and provided to ctm for collation. within the ctm, the haemovigilance coordinator is designated to assist hospitals in investigating serious adverse events and advise on the reporting formats. results: (1) the total number of reported cases has steadily increased since the introduction of the programme. (2) the number of participating healthcare institutions has also increased to 100% (n = 12). please refer to table entitled 'summary of the haemovigilance report 2002-2004' . (1) the implementation of the haemovigilance programme in singapore is feasible with respect to the asian setting, and can significantly contribute to blood safety. (2) there has been very good participation from the 12 participating healthcare institutions, signifying greater awareness and willingness to partake in the programme. (3) the results obtained from the programme have given rise to initiatives and recommendations aimed at reducing (71%) were rated for seriousness. of these, 50 (6.5%) were rated as grade 2 (moderate to serious morbidity) or worse. 793 (72.6%) were rated for imputability to the blood transfusion. of these, a relationship to the transfusion was graded as 'certain' or 'probable' in 470 (59.3%) and as 'possible' in 245 (30.9%). overall relatively few errors were reported in comparison to other systems. a small number of reports concern (possibly) infected blood components, and imputability was deemed probable or certain only in a minority of these reports. autologous blood components gave rise to five reports (errors as well as mild transfusion reactions) which shows a relatively high risk associated with their use (9.98 per 1000 the rate of post transfusion hepatitis (pth) in israel in unknown. this information is important in order to learn about the residual infection risk in blood recipients. aim: to summarize the data on reported cases of pth. methods: suspected cases of pth are reported to mda blood services. the investigation procedure includes follow up testing of implicated donors and retesting of an archive sample of the transfused unit, if available. donors involved in suspected pth-b are tested for hbsag and anti-hbc. anti-hbc+ donors are tested for anti-hbs. hbv-dna testing is done if anti-hbs is less than 10 miu/ml. donors involved in suspected pth-c, are tested for anti-hcv and alt. since 2000 hcv-ag or hcv-rna are performed, when appropriate. investigation is considered complete if all the involved donors are retested >6 months following the implicated unit. results: between 1993 between -2004 suspected pth cases were reported: 74 (57%) were pth-b, 54 were pth-c (42%) and in 1 patient both hbv and hcv infections were reported. investigation was completed in 53/129 (41%) cases, with 70% of the involved donors (1222/1749) being retested >6 months after the implicated donation. hbsag was not detected in any of the 773 retested donors. anti-hbc was detected in 26 donors involved in 7 pth-b cases of which 21 were also positive for anti-hbs. pcr for the detection of hbv-dna was performed on the 5 'anti-hbc+ only' donors, and none was found positive. only in 1/449 donors suspected to be involved in pt-hcv, anti-hcv antibodies were subsequently detected. this donation was collected and transfused before the introduction of anti-hcv testing in israel, which was implemented in 1991. in another pth-c case, the implicated donor is still negative for anti-hcv, in follow up samples of up to 28 months, but was found positive for hcv-ag and hcv-rna. pth investigation of all the donors involved was completed in 49% (52/105) of the cases where the patient received up to 10 blood components. conclusion: in the past 12 years an average of 11 cases/year of suspected pth were reported to the israeli national blood services. investigation was completed in 41% of the reported cases. so far, there was no clear evidence of hbv transmission. in 2 cases (out of 2.99 million blood units collected nationwide during 1993-2004) hcv seemed to be associated with blood transfusion: one caseprior to the implementation of anti-hcv testing and the otherprior to the implementation of hcv-ag testing in a donor that did not develop anti-hcv antibodies. these findings suggest that other modes of hbv and hcv transmission should be sought in blood recipients in israel. 3-2.9, p < 0.05). the seroprevalence for a-hiv in the bd population was 0.096% (se: 0.028; ci: 0.034-0.153; p < 0.01) whereas in the a-hbc positive bd was 1.08% (p < 0.01) and in the a-hbc negative bd was 0.07%. from 113 a-hiv positive donations, 33 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-hiv positive was 29.2%. the relative prevalence (percentaje of positive donations for a-hiv in the a-hbc positive population divided the percentage of this marker in the donations a-hbc negative: rp) was 15.43, which indicates that the number of bd a-hiv positive is 15.43 times higher in the a-hbc positive that in the a-hbc negative population of bd. as regards a-htlv, the seroprevalence in the bd population was 0.042% (se: 0.019; ci: 0.01-0.084, p < 0.01), in the a-hbc positive bd was 0.42% (p < 0.01) and in the a-hbc negative bd was 0.031%. from 49 a-htlv positive donations, 13 were also positive for a-hbc, that means that the sensitivity of the a-hbc in the detection of a donation a-htlv positive was 26.5%. the rp for a-hbc as regards a-htlv was 13.54, which indicates that the number of bd a-htlv positive is 13.54 times higher in the a-hbc positive that in the a-hbc negative population of bd. our results suggest that, in our bd population the screening with a-hbc would be useful to prevent other infections transmissible by blood transfusion, like retroviruses, because of the high sensitivity and the relative prevalence. 1. despite the high specificity of currently available elisas, the positive predictive value is lower in blood donors. therefore, immunoblot tests and polymerase chain reaction (pcr) have been adopted for routine testing in elisa +ve blood donors, by our service. 2. our data show that the real anti-hcv prevalence of our donor population is very low (0.04%). 3. the selection and evaluation of appropriate assays of all donated blood for hcv infection ensure good laboratory practice and accurate post-notification counselling of infected donors. 4. given that donors who are elisa positive but persistently negative or indeterminate probably do not represent a risk for transmission, their deferral from donation increases the problem of availability of blood supply. 5. donor re-entry in the pool of donors is an issue for further discussion. 6. the introduction of nat technology may elicit more accurate responses and improve the screening process. background: the introduction of hcv antibody screening of all donor blood in 1992 represented a major step in the prevention of transfusion-associated hcv hepatitis and in identification of infected donors. the study of infected individuals provides a unique opportunity to define behavioral factors associated with infection. evaluating risk factors in hcv infected blood donors is essential for monitoring blood supply safety, donor screening effectiveness and developing appropriate prevention programs objectives: 1. to recognize the epidemiology of hepatitis c and how it differs geographically; 2. to investigate the risk factors for presence of anti-hcv antibody in blood donors; 3. to evaluate the effectiveness of our donor selection program in local level. methods: serological testing for hcv was performed according to standard procedures. initial screening was performed using secondgeneration eia and, after january 1996, using third-generation eia. our study included a confirmation test (riba) a questionnaire was used to collect data concerning demographic, social and sexual behaviors, and number and type of donations of blood donors. the study also included testing of sexual partners and family members. changes in rates of hcv infections were evaluated by comparing yearly prevalence estimates. the overall prevalence of anti-hcv (eia) was 0.11% out of 168 374 blood donations. the average prevalence of hcv infection by riba was 0.04%, which reaffirms the very low risk of transfusion-transmitted disease. the cumulative number of hcv infected donors was 68, with 54 cases in males and 14 cases in females. most infections were found among older persons (32% were aged 22-39, and 68% aged 40-59). the seropositivity was higher in family/replacement donors (63%) than in volunteers (37%). the annual prevalence decreased throughout years. the relative importance of risk factors for hepatitis c was: transfusion 10%, hospitalization 8%, immigrants 8%, occupational 5%, sexual transmission 6%, injection drug use 3%, household contacts 5%, other 3%, tattooing 2%, unknown 50%. according to the criteria for blood donation, certain donors should have been excluded in the predonation interview but these donors had denied risky behaviour when questioned. the importance of sexual activity in the transmission of hcv has not been well-established as we tested 30 sexual partners and 14 family members and none of them was found positive. conclusions: our results suggest that major improvement in the safety and quality of our blood supply has been made in our area. introduction: apart from immuno-haematological complications, blood transfusion recipients are exposed to the risk of viral and bacterial contamination of donor blood. the latter infectious risks are generally associated with the number of donations that are needed in the production of blood products: the pooling effect. one measure recently being discussed is the generalisation of the use of trombocytapheresis for the production of trombocyte concentrates. normally, the trombocyte product is a concentration of trombocyte extractions from a pool of 5 buffy coats: pooled platelet concentrates (ppc). in case of trombocytapheresis sufficient trombocytes for one transfusion can be collected from one single donor. aim of the study: in this presentation the effect of using 100% trombocytapheresis for the production of single donor platelets (sdp) instead of pooled platelet concentrates (ppc) on contamination risks will be assessed. these risks can be divided in 1) the risk of bacterial contamination, 2) the risk of viral contamination (e.g. hcv, hbv, hiv) resulting from window period donations, and 3) the risk of contamination with tse or emerging infections for which no screening test exists. the contamination probability of sdp versus ppc is assessed on the basis of the production characteristics of both products, e.g. the presumption that the contamination risk per trombocyte product will be reduced by a factor equal to the number of pooled donations (five in our case). reduction of the bacterial contamination risk was estimated using the results of the bacterial testing of pooled and apheresis platelet products. as patients are likely to obtain multiple blood products during treatment, the contamination risk reduction through sdp is not only dependent on the reduction of risk in trombocyte products, but also will also dependent on the total number of blood products transfused and their associated contamination risks. the platelet recipient risk reduction was calculated on basis of the distribution of blood products received by the patient population of the university medical center utrecht (umcu) in the year 2001. results: in the attached table the estimated risk reduction through 100% trombocytapheresis is shown for the general blood recipient patient and for the trombocyte recipient patients only. our analysis indicate that in platelet recipients, general application of sdp instead of ppc will reduce the risk for transfusion acquired tse infections by 49%, the risk of known viral infections by 56%, and transfusion acquired bacterial infections by 59%. the confidence intervals surrounding the results were obtained by bootstrapping. the large confidence intervals surrounding the reduction of bacterial infection risk is caused by the fact that only a limited set of 4300 apheresis trombocyte products were tested. conclusion: our analysis indicates that general application of sdp instead of ppc will not reduce the risk of transmitting infections to platelet recipients as linearly (1 : 5) as expected. whether it is a costeffective precautionary measure will have to be evaluated by a costbenefit analyses consideration clinical benefits and additional costs and risks of apheresis donations. introduction: hepatitis b is serious health problem world wide. its prevention, particularly in the population of blood donors is essential for providing good health care and protection. aim: the aim of this study is to present the distribution of hbsag(+) and hbsag(-) blood donors according to their profession. methods: specially designed questionnaires are used for interviewing the blood donors who has previously given signed consent for participation in this study. results: table 1 shows the distribution of the blood donors in different professions. administrative clerks with 43 (34.12%) and the workers with 40 (31.73%) registered in the group of hbsag(+) blood donors, as well as workers with 50 (50%) and administrative clerks with 30 (30%) from the hbsag(-) group of blood donors are dominantly more frequent than the other categories of professions. health care professionals, housewives and farmers in both groups of blood donors are least frequent. taking in consideration the distribution of blood donors by the given professions in both groups, for u = 21 and p > 0.05 there is no significant difference found. the analysis of the differences among the different frequencies, in distribution of blood donors according the profession, for d = 0.436 and p < 0.05 shows a significant difference, where the workers with 90 (39.82%) are the most dominantly represented. in relation to the issue of whether the type of profession of the blood donors is production or non production the results are presented on table 2 . in the group of hbsag(+) blood donors the number of those who work in production profession-92 (73.01%), is dominant over the number of those that has non-production profession-34 (26.98%). in the group of hbsag(-) blood donors there is no significant difference between the types of professions. conclusion: having in consideration the professions of blood donors in both groups, for c 2 = 14.8 and p < 0.01 there is a significant difference in the presented distribution. according to our study, which shows the horizontal transmission of hbv infection in the family in which there is an index case, the biggest number of participants in the study is workers, and the least number of participants are the farmers. introduction: blood donors, as part of the healthy population are tested for hbsag with each blood unit they give. therefore, they can be an epidemiological model for exploring the appearance of hbv infection in general population. aim: this study aims to show the distribution of hbv infection in blood donors in relation with their living conditions, space and facilities. the material needed for the study consists of the data obtained from 126 confirmed hbsag(+) blood donors and 100 confirmed hbsag(-) blood donors as control group. results: the table 1 shows almost equal number of hbsag(+) blood donors that live in houses or flats. in the hbsag(-) group 64 (64%) donors that live in a flat dominate compared to 36 (36%) of those that live in a house. having in consideration the presented distribution (table 10) for c 2 = 3.96 and p < 0.05 there is a significant difference, that comes from the bigger number of blood donors (128) that live in flat. as for the distribution of blood donors according the living space they use by member of the family (table 2) , we can show that 24 (19.04%) of the hbsag(+) donors have less than 10 m 2 living space, in compared to 5 (5%) from the hbsag(-) group. the bigger number of hbsag(+) blood donors with small living space gives bigger possibility for transmission of hbv infection in the family. the differences in the two groups for donors that have between 10 and 30 m 2 , and between 31 and 40 m 2 of living space per person, obviously are not very big. for u = 19 and p > 0.05 there is no significant difference in the number of the donors in the two groups, when the available living space in m 2 is discussed. introduction: when one of the sexual partners has hbv infection the other is also infected in 1 from 3 cases. aim: the aim of this study is to outline that the risk from transmission of hbv infection between sexual partners is smaller if they use condom as protection. methods: two groups of blood donors-hbsag(+) and hbsag(-) have been interviewed whether they are using condoms as protection, or not. results: table1 shows the distribution of blood donors concerning the use of condoms. table1: in the group of hbsag(+) blood donors those who have not used condoms dominate with number of 81 (64.28%), in compared to those 45 (35.71%) who used condoms. these data are in favor of eventually possible sexual transmission of hbv infection in hbsag(+) blood donors. in the group of hbsag(-) blood donors dominate those who have used condom-57 (57%), compared to 43 (43%) who have not. the given distribution of blood donors concerning the use of condoms for c 2 = 10.2 and p < 0.01, shows significant difference, which is due to the prevailing of the number of blood donors (124), who have not used condom. of er -concentrates are leukodepleted. the choice of bacteriological control of empty bags for blood, bags with er -concentrates in additive solution, universal and iso group plasma, as well as the systems for taking of blood are taken on free choice. the control of the erytrocyte concentrates is performed on the first day after the preservation and dekanting, and again between the 15th-21st day and 30th-35th day after the preservation. the pulled plasma is controlled on the day of pouring (spreading), and the control of the iso group plasma on the day of deplasming. three months later the iso group and the universal plasma kept on the temperature of -30°c is bacteriologically controlled again. bacteriological control is performed with standard procedures in the institute for health protection in stip. the transfusion transmitted infections are potentially dangerous complications of transfusion therapy in immunocompromised patients. the aim of this study was to determine the prevalence of transmissible infections in blood donor population in kashan, iran. a total of 600 consecutive sera were tested for cmv-igm antibody, hbsag, hepatitis b core (hbc) antibody, hepatitis c (hcv) antibody, and hiv antibody with standard methods. of the sera tested, 14 specimens (2.3%) were cmv-igm positive. the frequency of seropositive revealed no significant differences between male and female donors. the frequency rates of cmv-igm seropositive tests tend to decline with increasing the age. there was no relation between the frequency rates of cmv-igm seropositive with the educational level, socioeconomic status, marital status, urban dweller and rural resident patients. the prevalence of hbv, hcv, and hiv antibody were 0.5%, 0.5%, and 0%, respectively. these findings implied important clinical applications because detection of cmv positive sera may reduce the risk for transmission of cmv in blood transfusion and thereby decrease the risk on cmv-induced complications. introduction: the worlds problem, aids, steel can't be said that is a problem in these three centers in r. macedonia, in which blood is collected, controlled, and distributed. found negative and 5 of them were found positive for one of the three viruses (hiv-1, hcv, hbv). with the elisa/axsym assay 29 of the 992 blood units which were negative by the procleix ultrio assay were positive for anti-hbcag and negative for anti-hbsag and hbsag. from those 29 blood units 23 units were given for transfusion following our blood centre protocol and the remaining 6 units were discarded. the protocol consists of a good medical history, liver enzymes (ast, alt, ggt). we must take into consideration that from those 5 that were found positive by the procleix ultrio assay 1 was positive for anti-hcv and 4 were positive for anti-hbsag. summary/conclusions: despite the fact of the short period of time we perform this method, the ability of the nat technique for rapid use, reliability and sensitivity in detecting three viruses simultaneously, indicates the need for immediate use in blood donation as a screening method. in spite of the high cost of the method, it is clear that this assay is a valuable tool in our blood centre to provide fast and safer blood. bacterial contamination of blood products is a persistent, but often overlooked, problem in transfusion medicine. in greece it is recommended that platelets (plt) must be used or discarded within five days post-collection. recent reports from europe have advocated the use of bacterial culturing of platelets on day 2 or 3 and, in case of negative result, prolongation of their storage time to 7 days. aim of the study: to assess the prevalence of bacterial contamination of standard platelet units from whole blood, and to provide evidence that with the use of bacterial culturing it is feasible to extend the self life of platelets to 7 days. materials and methods: eligible blood donors were bled according to standard operating procedures used in greece. plt were prepared from whole blood, solely for the purpose of the present study, by the platelet-rich plasma method. plts were stored for up to 7 days at 20 to 24°c with end-over-end agitation. other 111 plts prepared from blood collected in triple-pack container system also provided with a predonation sampling device were also tested. plts were sampled in the bacteriology laboratory. plts were sampled on day 2, 5 and 7. both aerobic and anaerobic culture bottles were inoculated with a 7-ml platelet sample. culture bottles were incubated at 35°c in an automated microbe -detection system (bact/alert system) until a positive reaction was detected or for 10 days. all samples that were reactive were confirmed by routine culture. each reactive sample with bacteria growth on the routine culture was sub cultured for identification of the bacteria. results: a total of 1889 plt concentrates were cultured and bacterial contamination was assessed in each unit at day 2, 5 and 7 after collection. on of storage day 2 two out of 1889 (0.1%) plt units were found to be positive for bacterial growth. 7 cases of unconfirmed positive results were noted at the beginning of the study. out of the other 1887 units which were negative on day 2 and continued to be cultured for the next days, the assessment at day 5 found no other positive. after further storage, at day 7, defined as the end of the prolonged incubation period, 5 out of the 1887 plt concentrates (0.26%) grew bacteria although testing of the same units on day 2 and 5 gave no signal. from the 111 platelets units that were prepared from blood collected with a predonation sampling device, none of the plt concentrates gave a positive signal although 3 pouches were found to be positive, and subculture showed bacterial growth of coagulase -negative staphylococcus. despite the relative small number of tested platelet concentrate units, our findings discourage specialists in attempting platelet storage time prolongation to 7 days. bacterial contamination testing on day 2 and a storage time of maximum 5 days seems to be still the safest practice. bacterial screening of platelet concentrates using bact background: bacterial screening of blood components is a routine measure in the evaluation of blood product quality. at our institute bacterial screening is performed using bact/alert system. sampling and culturing of blood products is performed according to paul-erlich institute recommendations. methods: quality control data on the bacterial screening of platelet concentrates performed from 1999-2004 were retrospectively analysed. an initially positive (ip) and true positive (cp) rate, organisms isolated and time of detection are presented. results: a total of 5359 platelet products were tested during the 6year period. thirty (0.56%) were found initially positive by bact/alert. the cultures screening positive were subjected to bac-terial identification to distinguish false positive from real positive signals. bacterial contamination was confirmed in 14 (0.26%) plt concentrates. positive cultures were confirmed and identified in an independent laboratory ( hbsag, anti-core, anti-hbs, anti-hcv, anti-hiv i/ii, anti-htlv i/ii, rpr. these patients were transfused with 5-131 units of concentrated red cells, depending on their problem. a total 530 units were delivered from the beginning of their problem until december 2003. the control were done using last generation enzyme-linked immunoassay (dade behring, ortho, biomeurieux), as also using automated enzyme-linked immunoassay (axgym). the same patients were checked by their physicians before the initiation of transfusions for the same diseases. results: we found: 12 patients anti-hbc (+) and anti-hbs (+). 5 patients anti-hbc (-) and ánti-hbs (-) 3 patients anti-hbc (-) and anti-hbs (+) 1 patient anti-hbc (+) and anti-hbs (-) 1 patient hbsag (+), anti-hbc (+) and anti-hbs (-). all patients were negative for hcv, hiv, htlv, and rpr. the same results were found also from patients' physicians. conclusions: we conclude that the blood supply for blood transfusion-transmitted diseases is 100% safe in our centre. these results are in accordance with current international literature. this is due to careful selection of blood donors, to high quality of corporation between departments as also to internal and external quality control. all these factors contribute to safety of transfusions, the quality of life of the patients and the protection of patient's environment. neither in the pipetor nor in the extractor runs was found contamination. no false positive were detected and all the positive samples confirmed. (see tables 1 and 2) . for hiv and hcv the specificity was 99.99%. the validation criterion were met, so the system was implemented routinely in our laboratory. the purpose of our study was to analyse the applicability of the pall enhanced bacterial detection system (ebds) in the routine of our transfusion unit which is totally focused on apheresis platelet collection. methods: apheresis pcs, obtained by trima (cobe) and amicus (baxter) separators and re-suspended in 30% plasma and 70% ssp solution (macopharma), were submitted to microbiologic control using pall ebds system which uses oxygen percentage decrease as a surrogate marker of bacterial growth. the working steps were the following: 16-24 hour after donation, about 3 ml of pc were sampled into the ebds collection pouch and then incubated at 35°c under continuous agitation (incubator helmer 900) for 24 hours. after this period, oxygen percentage was measured using an oxygen analyser (pall bdso2). the test is based on the 'pass/fail' principle. in case of 'fail' result the microbiology department has drawn up the procedure to follow in order to confirm the data and to allow the micro-organism to be identified. the incidence of post transfusion hepatitis has been reduced by blood donor screening for hbsag, but the hbv infection is still responsible for a certain cases of post-transfusion hepatitis in world-wide. in this study the hbsag negative blood units were evaluated for anti-hbc and hbv dna by pcr method. an extra sample was collected from 2000 hbsag, anti-hcv, anti-hiv and rpr-negative blood donors. all of samples were examined by approved anti-hbc assay. all of anti-hbc positive samples were tested by hbsab assay and evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency and run control panels. 212 (10.6%) out of 2000 samples were positive for anti-hbc.166 (78.3%) out of 212 anti-hbc positive samples were hbsab positive, and 46 (21.7%) were hbsab negative. all of 212 samples were assayed for hbv dna (pcr) single and all of them were negative for hbv dna (pcr). further study for evaluation of anti-hbc test as a screening assay for blood unites in high hbv infection prevalence area strongly recommended. early detection of hepatitis b surface antigen: a comparison of ten assays hbsag detection is the corner stone of detection of hepatitis b virus infection in blood donors and patients with hbv infection. one of the most challenges is sensitivity of the technique and kit. in this study ten different assays evaluated by seroconversion and performance panels. some of them can not be used as screening assay due to low sensitivity. the sensitivity of ten hbsag assays from biorad, dade behring, biomeriux, diasorn, radim, diesse, thermo. biokit, gb and shanghai companies were evaluated by two or three seroconversion and two performance panels from boston biomedica ink. seroconversion panel is a series of samples that collected over a period of time from individual developing antibodies due to a primary infection. for evaluation of the assay sensitivity who and other notified body in the world-wide recommended the seroconversion and performance panels. the hbsag assays are two groups. group one with high sensitivity included six assays. they can detect ad and ay subtypes from 0.1 ng/ml bbi to 0.4-0.5 ng/ml bbi respectively. low sensitivity group included four assays and they can detect ad and ay subtypes more than 0.7 and 0.5 ng/ml bbi respectively. for blood safety, the high sensitivity hbsag assays recommended for blood screening and all assays should be evaluated by seroconversion and performance panels. diagnosis of chronic hdv infection is usually by antibody testing and hbv dna detected by pcr method. it is rare to find patients with two replicating hepatotropic viruses and if the accompanying hbv is replicating, prognosis will be very poor. to clarify the correlation between hepatitis delta virus infection and hepatitis b virus dna positivity, sensitive hbv dna (pcr) assay was used. the presence of hbv dna was investigated in 274 patients referred during the 21 aug. to 28 dec. 2003. all of them were hbsag positive. all samples were evaluated for hbv dna (pcr). the sensitivity of the hbv dna (pcr) assay was estimated 50 geq/ml according to vqc proficiency panel and run control. anti-hdv was tested by commercial available enzyme immunosorbent assay. 179 (65.3%) were hbv dna positive and 95 (34.6%) were negative. 8 (4.4%) out of 171 patients had evidence of delta infection and 13 (13.6%) samples of hbv dna (pcr) negative patients were positive for delta agent. the serum alanine aminotransferase (alt) levels in 5 out of 8 hbv dna (pcr) and anti-hdv positive patients were higher than reference interval, but only in 2 out of 13 hbv dna (pcr) negative and anti-hdv positive samples were higher than reference interval. the present data indicate that 7.7% of patients with chronic hepatitis b have hepatitisdelta infection. patients with hbv dna (pcr) negativity had a significantly higher prevalence of delta marker (13.6%) than those with hbv dna (pcr) positivity (4.4%). delta rna testing in positive hbv dna and anti-hdv patients is recommended. introduction: reduction of the window period of hepatitis c virus (hcv) infection represents an important goal in the transfusional and diagnostic setting and nucleic acid technology-based tests have been introduced in some developed countries to reduce the potential risk of transfusion-associated infection. a prototype assay designed to simultaneously detect circulating hcv antigen and anti-hcv has been developed by biorad (biorad laboratories limited, marnes la coquette, france). aim of the present study: to define the cut-off (co) value of the assay and to evaluate the specificity and sensitivity of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays. methods: in order to establish the co value and to evaluate the specificity of the assay, we tested 400 sera samples from the general population and 100 'difficult' sera from haemodialysis patients (n conclusion: the new assay shows high sensitivity and specificity and could be a useful tool not only in the diagnostic setting, where procedures to reduce the window period, such as antigen or hcv-rna detection, are not currently recommended, but also in the screening of blood donations, when nucleic acid technologies is not feasible due to costs, organization, emergency and/or logistic difficulties. introduction: in recent years the concern with the blood safety regarding the transmission of blood-borne viruses has been improved. this safety has been achieved with the combining of different strategies, such as a careful selection of donors, the screening for relevant virological markers and the viral inactivation/ removal methods. more recently, the implementation of the nucleic acid amplification technologies for the detection of hiv-1, hcv and hbv, has increase this aim by reducing the 'window period' of the infections. other viruses, such as parvovirus b19 (pb19) and hepatitis a virus (hav), can raise problems to the blood safety. these infections could provoke serious complications in some risk groups, like pregnant women, patients with haematological problems, children and patients with immunodeficiency. material and methods: an observational study was performed to determine the prevalence of pb19 and hav in portuguese blood donors. we gather, during four months, 5025 plasma donations and joined them into 505 pools, with no more than 10 donations each. [1999] [2000] [2001] [2002] [2003] in voluntary donors the anti-hcv prevalence ranged from 0.3% to 0.7%, in family replacement donors from 0.4% to 1.2%, autologous donors from 1% to 2.02%. we observed that the anti-hcv prevalence has a decline tendency during years in blood donors. according to sex the anti-hcv prevalence in men is 0.7% and women 0.6% (p = 0.004). over the 5 year periods the prevalence in men has a decline tendency (1.2% to 0.4%; p = 0.033) and increasing tendency in women (0.4% to 0.9%; p = 0.007). according to age group the anti-hcv is 0.6% in 17-29 age group, 0.65% in 30-39 age group, 0.8% in 40-49 age group, 0.86% in 50-60 age group (p = 0.001). the prevalence of anti-hcv is higher in fds than vds, but not statistical significant. [1999] [2000] [2001] [2002] [2003] . a total 14627 ftd and 684 ad have been tested for hbsag. a sample was considered as hbsag positive when found repeatedly reactive by 3rd generation. immunoassay method (elisa). the chi-square test was used for statistical analysis. results: the 5-year overall hbsag prevalence among first time blood donors was 8.9%. and ad 6.7%. among autologous blood donors was observed a decreasing hbv prevalence from 6.3% to 3.7% in 2003. according to age the prevalence was higher in 17-29 year group 7.7%, while according to sex was higher in man (8.8%) than female 6.2% (p < 00.5). among ad, a decreased hbsag prevalence according to age was observed in men and women. the same trends by sex and age were observed in ftd. the prevalence of hbsag in ad was lower than in ftd. however, from 1999-2003 hbsag prevalence has decreased in the same proportion in both population. this decreased can explain by to main factor: the improvement hbsag screening method in blood donors and decreased the hbsag prevalence in general population. (1 : 30 250) . the hbv dna-emia in hbsag negative samples was 1.14 ¥ 10 2 -9.55 ¥ 10 4 copies/ml. in two donors anti-hbc total was positive and in one anti-hbe was also detected. in one donor the glycin 145 alanin mutation in the s region was identified. the frequency of hbv dna pos/hbsag neg donors in poland is high (0.002%) therefore the decision to introduce routine hbv nat screening is justified. (1/1001) with stored apheresis and whole blood derived platelet concentrates. of these 118 failed results there were 23 confirmed positives (presence of bacteria in both the ebds pouch and the platelet mother bag by culture) representing 1/5133. the bacteria detected were staphylococcus or streptococcus sp. 76 of the 118 fail results were false positives (no presence of bacteria in the ebds pouch and the platelet mother bag by culture) representing 0.064% or 1/1554, and 18 were not confirmed initial positives (no bacteria in the mother bag by culture, ebds pouch not tested) representing 1/6559. there was one reported case of a missed detection with confirmed presence of bacteria (staphylococcus epidermidis) in the mother bag by culture. subsequently, the bacteria strain was isolated and inoculated into platelet units in our laboratory at levels as low as 5 cfu/ml. in all cases the pall ebds was able to detect. this supports the hypothesis that this missed detection was the result of a statistical sampling error rather than a system failure. the results from blood centers routinely using pall ebds demonstrated effective detection of bacteria in platelet products stored under routine conditions with a true positive rate of 1/5133, and with a low false positive rate (<0.1%). this is comparable to a recent survey result with other culture based systems. summary/conclusions: the minority group of pregnant women who come to labor without prenatal testing of hepatitis b and c revealed essentially similar prevalence of anti-hcv with healthy bd even if definitive confirmation is probably increased in this minority group. there is however markedly higher prevalence of hbv infection in the pw so that screening for hbv is essential for the prevention of vertical transmission. the systematic screening of bd with anti-hbc serves as further assurance for the prevention posttransfusion hepatitis eliminating only 1.21% of the possibly infectious, a percentage which can be restored to the blood pool after proving their immunity. methods: blood samples were screened for the presence of hbsag, hcv and hiv antibodies using enzyme immune assay and for syphilis using the tpha test. the results were analysed retrospectively. all samples with results at or above the minimum positive value were considered reactive. the tests for hbsag, anti-hiv and anti-hcv were repeated in duplicate in all reactive donations. blood units that were reactive in the primary or secondary assays were discarded. hiv positivity was confirmed by western blot analysis using hiv blot 2.2 (genelab diagnostics) results: results from a total of 77 903 screened donors were analysed. hepatitis b surface antigen rates was 1.99%; anti-hcv seropositivity was 0.54%; anti-hiv seropositivity was 0.01% and tpha seropositivity was 0.06%. one study calculated this risk to be one in 63 000 for hbv, one in 493 000 for hiv and one in 103 000 for hcv. it is therefore important to take a careful history from blood donors to eliminate those at high risk of infection. in view of the high infectivity of hiv positive blood, it is important not only to screen donated blood but also to exclude donations from high-risk individuals, such as males who have engaged in homosexual activity and intravenous drug users. a careful history should identify those who should not give blood. in turkey, among blood donors the average hbsag prevalence in 1985-1998 was 5.1%. but it had decreased to approximately 2.1% in 2003. anti-hcv positivity has been reported to be 0.6% between 1992 and 1999. but it was approximately 0.5% in 2003. rpr positivity in blood donors in turkey was reported to be <0.1% in 1973 and 0.0026% in 1990. in 2003, the rpr rates was 0.09%. in our study these rates are 1.99%, 0.54%, 0.09% and 0.06% respectively. anti-hiv seropositivity was found around 0. introduction: the serological detection of specific antibodies to treponema pallidum (tp) is an effective means of diagnosing syphilis, and an automated chemiluminescent assay is ideally suited to testing large numbers of specimens for the laboratory diagnosis of the disease. aims of the study: to develop a qualitative syphilis assay for the detection of tp immunoglobulin m (igm) and g (igg) antibodies. the assay will be used for the serological diagnosis of syphilis using the architect platform, which has the capacity to test 200 specimens/hour. the two-step assay is based on paramagnetic microparticle chemiluminescent technology, utilising microparticles coated with three recombinant tp antigens (tpn15, tpn17 and tpn47) and acridinium labelled anti-human igg and igm monoclonal antibodies as conjugates. in the first step, specimens, microparticles and diluent are incubated together, prior to a wash step; in the second step, acridinium labelled antibodies are added and after washing, pre-trigger and trigger are added to produce chemiluminescence, which is measured as relative light units (rlu). specimens yielding rlus less than the cut-off are considered negative, while those yielding rlus greater than the cut-off are considered positive. the sensitivity of architect syphilis tp was determined to be 100%, after testing 426 specimens that were previously screened as syphilis positive in fujirebio tppa; no prozoning was observed with high positive specimens (over 2 16 titer by tppa). the specificity generated from testing 564 hospitalised patients previously screened as tppa negative, was 99.6%. testing a mixture of sera and plasma from 5000 random donor specimens, generated donor specificity figures of 99.8%. the precision (cv%) with a positive control was 5.8% (95% confidence interval: 4.9-7.0%) by the standard 20-day nccls analysis (ep5a2). in a study conducted at asahikawa medical college hospital, in which, 81 positive and 82 negative specimens were tested, concordance with fujirebio tppa was determined to be 100%. no significant interference to the assay was observed from bilirubin (conjugated type and free type), haemoglobin or lipid. the architect syphilis tp assay is an automated, specific and sensitive test for the detection of antibodies to t. pallidum. background: hcv exposure of blood donors is serologically determined by the detection of anti-hcv antibodies in serum or plasma. however a 'window' period of 30-70 days after exposure exists during which specific antibodies to hcv antigens cannot be detected. hcv rna detection and/or hcv core protein testing result in dramatic reductions in the preseroconversion window period. the new bio-rad test, based on the simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, improves the detection of hcv infection in the early phase. aims: the aim of this study is to assess the performance characteristics of this new screening microplate immunoassay, monolisa hcv ag-ab ultra, by using the bio-rad evolis automated microplate processor system. methods: this two-step elisa assay is based on the combination of an indirect test for the detection of antibodies (core, ns3, ns4) and a sandwich test for core antigen detection. results are available within 2.5 hours, with sample addition monitoring and color coded reagents. no specimen pretreatment is required. evolis is a self-contained microplate processor designed for full automation of microplate-based eia techniques. the walkaway system can process four microplates at a time with continuous loading of samples and reagents. positive identification of samples, reagents and microplates, usage of disposable tips with clot detection, integrated quality control and complete traceability provide a high level of safety management. the monolisa hcv ag-ab ultra/evolis system performance is evaluated for clinical sensitivity on 30 commercially available and well-documented seroconversion panels. the results are compared to viral rna detection and conventional hcv ab screening assays. specificity is evaluated by using random blood donor samples. results: among the seroconversion panels that begining with samples negative for hcv rna and anti-hcv antibodies, the monolisa hcv ag-ab ultra assay detects exposure to hcv an average of 25 days earlier than the monolisa hcv plus v2 test. the mean delay of the monolisa hcv ag-ab assay in detecting hcv infection compared to hcv rna testing is around 2.5 days. the monolisa hcv ag-ab ultra/evolis system allows simultaneous detection of hcv core antigen and anti-hcv (core, ns3, ns4) antibodies, thus significantly reducing the time gap between the initial detection of hcv rna and the first appearance of detectable anti-hcv antibodies. the fully automated system combines high degree of assay performance with optimization of laboratory workflow and safety management. operational evaluation of pall ebds bacterial detection system l larrea gonzalez, ma soler and rj roig centro de transfusion, valencia, spain introduction: regulatory bodies are increasingly mandating the use of bacterial detection systems for platelet products (22 ed standards for blood banks and transfusion services). one system currently available is the pall ebds bacterial detection system which utilises percentage oxygen as a surrogate marker for bacterial growth. aims: to evaluate the pall ebds in routine use in our blood centre. in particular, to assess feasibility and adaptability to daily labour routines. the orbisac (gambro bct) system was used to produce leucocyte depleted buffy coat (bc) platelet pools (4 bc/pool) stored in platelet additive solution (ssp macopharma). mean platelet count was 3.2 ¥ 10e 11 /pool with mean leucocyte count 0.09 ¥ 10e 6 /pool. ebds installation and training occurred over a 2 day period. 500 platelet pools were tested for bacterial contamination over the subsequent 5 weeks. ebds pouches were sterile connected onto platelet pools 22 hours after blood donation. platelet samples were taken into the pouches and then the pouches were incubated for 24 hours on a shaking agitator at 35°c. after this time, percentage oxygen was measured. no positive results were found in this study. this was as expected due to the relatively low number of platelet pools tested and it also highlights the absence of false positive results. minimal training was required to use the ebds. the system was easy to use and did not require the use of a laminar flow cabinet to take samples. it was quick and simple to take samples and perform oxygen measurement. after pouch incubation, 1 technician was able to make 20 oxygen measurements in less than 13 minutes. the data management system allowed full traceability of product and work flow. results were very easy to interpret. conclusion: the pall ebds was found to adapt perfectly to a routine blood centre environment. (2004) was 8470. the percentage collected from volunteer blood donors was 56% (n = 4783) and the rest 44% (n = 3687) was given from patient-related donors. the age of donors ranged from 18 to 65 years old. the assay used for the detection of hbsag, hbeag, anti-hbc igg/total, anti-hbc igm, anti-hbe and anti-hbs was the automated microparticle enzyme immunoassay (axsym) of the abbot company. all the units were tested for hbsag, and anti-hbc igg. if the anti-hbc igg was detected, the specimens were automatically tested for anti-hbs. the units were wasted if the anti-hbs was negative, and the specimens were manually programmed for the testing of the anti-hbc igm, hbeag, and anti-hbe. from the total of 8470 tested units, 1195 of them were found to be positive to at least one marker of hbv infection, that means the 14.11% of the health adult population was infected in the past by the hbv. the 13.69% (n = 1160) was previously infected and now immunized with hbsag(-) and anti-core igg(+) and 0.41% (n = 35) were chronic carriers of the hbv with hbsag(+). the 68.57% (n = 24) of the positive donors were patient related donors and 31.42% (n = 11) were volunteer donors. in other words, 24 of the 3687 not volunteers (0.65%) and 11 of the 4783 volunteers (0.23%) were detected to be infectious. the combinations of the serologic markers for hbv are illustrated in the table attached. these results indicate that the incidence of hbv infection in the northeastern department of greece is equivalent to the incidence of hbv in other greek regions (0.42%) as it is referred to the national haemovigilance data and moreover, the percentage of infectious donors is bigger among replacement donors, 0.65% compared with the 0.23% of voluntary donors. as a consequence, the best source for safe blood collection is the population of volunteers. earlier detection of human immunodeficiency type 1, hepatitis c and hepatitis b viruses using the procleix® ultrio tm assay on the procleix® system and the study objective was to assess the ability of the ultrio assay and associated discriminatory assays to reduce the detection windows for hiv-1, hcv, and hbv. commercially available seroconversion panels were used for testing. methods: hiv-1 (n = 13), hcv (n = 12), and hbv (n = 16) seroconversion panels were tested neat and diluted (1 : 8 and 1 : 16) in the ultrio assay. panels were tested neat in the appropriate discriminatory assay. times to detection of hiv-1, hcv, and hbv nucleic acids in seroconversion panels were compared to the vendor's historical data on time to detection of antibody and/or antigen using licensed or validated serologic tests. p-210 effectiveness and limitations of methods for platelet bacteria screening -how to apply which screening method? the successful concept of virus safety in transfusion medicine is not suitable in bacterial contamination. bacteria can grow up in blood components to enormous amounts, whereas the initial number of contaminating bacteria is typically very low. therefore, sample drawing for bacteria screening must not be done immediately after blood donation. the established concept of relevance of clinical microbiology (pathogenic, non-pathogenic, facultative pathogenic species) is not valid for bacteria contaminating blood. here, the currently discussed criterion of clinical relevance is the ability of bacteria strains (not species!) to grow up in blood components. the paul ehrlich institute (pei) developed pei bacteria standards, which are characterized concerning their behavior in blood components. they contain a defined number of living bacteria, they are deep frozen, ready to use and shippable. there are two strategies to improve bacteria safety of blood: screening and pathogen reduction. neither of them is perfect, but screening methods are successfully established since several years in routine (belgium, the netherlands), and represent the current state of the art. further development and collecting of experience will produce the basis for assessments in the future. it is of high importance to apply the screening methods in dependence on their properties. methods implying an incubation/cultivation step ('early methods') have to be distinguished carefully from 'rapid methods' . for example, it is unreasonable to compare (or to advertise with) different sensitivities of methods not considering their detection principle or their informative value. both principles, cultivation methods as well as rapid methods, show advantages and disadvantages. selection of the method has to consider the respective conditions of the given blood service (including logistics up to time frame between issue and transfusion). results from the procleix hiv-1/hcv and hiv-1/hcv/hbv (procleix ultrio) assays for the detection of hiv-1 rna, hcv rna and hbv dna in blood donors of two blood transfusion centers of sw greece in discriminatory assay testing, 3 out of 5 (60% of the positive, 0.020% of total) were reactive for hcv rna only and 2 out of 5 (40% of the positive, 0.013% of total) were reactive for hiv-1 rna only. none were positive for both hiv-1 and hcv. the standard serological assays gave the same results for the above positive samples. two samples that tested positive by the standard serological assays tested negative in the procleix hiv-1/hcv assay. of the 4151 samples tested by the ultrio assay, 21 (0.5%) tested reactive for hiv-1/hcv/hbv. in discriminatory assay testing, 1 out of 21 (4.76% of the positive, 0.024% of total) was reactive for hiv-1 rna, 4 out of 21 (19% of the positive, 0.096% of total) were reactive for hcv rna, and 16 out of 21 (76.2% of the positive, 0.38% of the total) were reactive for hbv dna. all were single positive i.e. none tested positive for more than 1 virus. three out of 16 positive samples for hbv dna tested negative by the standard serological tests. the opposite was not observed. the procleix ultrio assay is a definite improvement over the procleix assay in a region with a high incidence of hbv carriers. up until its use, it is obvious that hbv positive blood with very low antibody titers was transfused into patients. more results will show whether procleix ultrio can eventually replace the standard serological tests. the introduction: patients with hemophilia represent a high-risk group for post-transfusion hepatitis whose frequency is closely linked with the number and quantity of blood products used. in albania, the frequency of hepatitis is also linked with hbsag testing with elisa (introduced in1993), and hcv testing (introduced in 1997). aim of the study: evaluation of the prevalence of the markers of hepatitis b, c, and d in patients with hemophilia. methods: our study included 145 patients with hemophilia treated with cryoprecipitate and commercial clotting factors. blood testing for anti-hcv, anti-hdv, and hbsag was performed with elisa -gen. iii. results: of 145 patients tested, 26 cases (17%) were hbsag positive, 87 cases (60%) were anti-hcv positive, and 7 cases (5%) were anti-hdv positive. co-infection of hbsag and hcv was found in 16 cases (11%), whereas co-infection of hcv, hdv, and hbv was found in 4 persons (3%). the highest rates of infections and coinfections were found in patients above 30 years of age. conclusion: mandatory blood testing has decreased the levels of post-transfusion hepatitis. in albania, hemophilia is also still treated with cryo-precipitation, thus patients are at a particularly high risk during the 'window period' . results: 2/16 760 (0.012%) samples from rbd were anti hiv 1 + 2 nonreactive and rr for p24 ag both being nonreactive in the neutralization test, they were interpreted as false positives. 1/134 617 (0.0007%) sample from fbd was rr for p24ag/anti hiv 1 + 2 nonreactive and it was confirmed positive by neutralization. this bd had been autoexcluded himself after blood donation. he showed seroconvertion 21 days later: p24ag nonreactive, anti hiv 1 + 2 reactive and western blot positive. the only bd p24ag positive/anti hiv 1 + 2 nonreactive during the analized period, was an first time donor and the post donation autoexclusion was effective en this case. although a larger populations of bd is necessary to be studied and in spite of the low prevalence we have found, we consider p24ag screening is an alternative up to implementation of nucleic acid testing and simultaneously we should increase the quantity of altruist repeat blood donors, undoubtedly, the best population to give blood. owing to the rather short interval between successive donations (~70 days), this suggests that some 260-310 infectious units escape the screening annually. to these, one has to add the (now unknown) proportion of potentially hbsag negative + hbv dna positive ftbds. hcv: since the introduction of the screening in 1995, the general incidence in rbd has dropped from 13.1‰ to 0.001‰, suggestive of a 1 : 10 000 escape rate. the prevalence in ftbd has stabilized at 12 ± 2‰. based on reasons similar to these employed for hbv, the residual incidence in rbd suggests that 1 potentially infectious donation in 10 000 rbd escapes the screening (= to a total of aprox. 40, annually). a limited investigation using hcv-antigen eia evidenced a 1‰ escape rate in ftbds (= to a total of aprox. 50, annually table 1 and 2 are concerned to the fist two months of the implementation, where we had to adjust the volume of the eluate. conclusion: these system adjusts to the laboratory daily routine in the blood bank, with the pools released after first analysis in less than 18 hours. background: the hbsag, anti-hcv, anti-hiv1/2, p24 antigen, alt and syphilis tests are performed for blood donations in czech republic. no nat tests are mandatory in czech republic. the aim of this pilot study was: 1. hcv rna pcr testing in anti-hcv negative blood donations; 2. correlation between hcv nat and anti-hcv testing results. methods: blood samples (anti-hcv serologically negative, alt not elevated) were pooled using the guardian plus spii into pools of 24 samples. pools of 1 ml were tested using the cobas ampliscreen hcv test v. 2.0 (roche). results: 891 pools of 24 samples from 21 384 a-hcv serologically negative donations were tested from october 2002 to july 2004. no one pool was initially reactive. invalid tests: 14 (1.6%) run failures were observed, due to: invalid internal controls 10 (1.1%) and invalid positive controls 4 (0.5%). invalid tests were repeated. in none of 891 pools a positive hcv nat result was observed. conclusions: no discrepancy between hcv nat and a-hcv results was observed in our study. all of the nat tested donors in our study were regular voluntary whole blood or plasma donors who were repeatedly a-hcv serologically negative. the hcv incidence in the czech republic blood donor population is low but it is slightly growing up in general population. hcv nat testing could improve the safety of blood supply by reducing the window period for hcv. introduction: parvovirus b19 is the only parvovirus known to be a human pathogen. most commonly, it causes a mild childhood rash, erythema infectiosum, but in some cases more serious symptoms can be linked to b19, such as acute or persistent arthropathies, critical failures of red cell production, hydrops fetalis, fetal loss, myocarditis or hepatitis. inactivation of the non-enveloped virus has proven difficult. as a consequence, manufacturers of blood products have implemented screening measures to reduce the load of parvovirus b19 in manufacturing plasma pools by the use of nucleic acid amplification techniques (nat). in our institute all blood donations were screened for human parvovirus b19 by nat since april 2000. methods: over the last 5 years 5.5 million donations were screened for b19 by nat. samples with a virus load over 105 iu/ml were defined as positive, whereas samples with a virus load between the detection limit (103 iu/ml) and 105 iu/ml were defined as weak positive. weak positive products were released, whereas positive products were discarded. in addition infection markers of 50 b19 positive donors (case group) were determined over a time period of one year. virus load and b19 antibody status was compared with 100 b19 negative donors (randomised control group). b19 antibodies (igg vp2, igm vp2, ns1) were analysed by two commercial antibody tests. results: overall 496 b19 nat-positive donors were identified with a virus load over 105 iu/ml out of 5.5 million tested. there was a seasonal accumulation during spring and summer, whereas a large epidemic occurred throughout the last year. vp2 igg was detected in 90.9% and 74% of the case and control group, respectively (p = 0.01). these data demonstrated statistically significance (p = 0.01). all donor samples which were b19 nat positive for more than three months developed neutralizing vp2 antibodies. in contrast, ns1 antibodies were observed in 83% of the case group and in 15% of the control group (p < 0.01). ns1 antibodies were detected more frequently in samples, which were b19 nat positive for more than six months. conclusion: b19 nat could be implemented in blood donor screening as release criterion without causing a shortage in blood supply. all b19 positive donors of the case group developed neutralizing antibodies within three months and virus load was dropped rapidly below 105 iu/ml. these data support our testing algorithm all components of high positive donations (virus load over 105 iu/ml) were discarded. donors with ns1 antibodies showed more often signs of a chronic disease with detectable levels of parvovirus b19 longer than six months. background: on recent years, the syphilis screening of blood donors has become increasingly important not only because of the transmission risk of this infection but also due to the risk behavior that this implies. on account of the importance of this screening the tests used are becoming more and more sensitive. aims: to evaluate an elisa screening test (the tmpa test recombinant is based on the sandwich principle, an immunoenzymatic technology in solid phase, for the measure of anti-treponema pallidum in serum or plasma). methods: in this study 1696 samples from blood donors were tested by the rotine 'cardiolipidic reagent for syphilis screening on microplates' -diagast laboratories as well as with 'hdtmpa recombinant' -hoslab diagnostics. positive samples were then confirmed with fta abs/tpha. results: using the mentioned tests we obtained the following results: 1. 1666 (98.23%)cases turned out negative with both technologies; 2. 3 (0.17%) cases were positive in both methods; 3. 27 cases were positive only using tmpa recombinant [of which 23 (1.36%) were confirmed positive by tpha/fta abs. as seen we found 23 samples (1.36%) that were only positives by tmpa recombinant test and that were confirmed by tpha/fta abs. we concluded that tmpa recombinant seems to be a suitable test for a quick and automated syphilis screening of blood donors and provides maximum safety for the recipients. background: in recent years, there has been substantial evidence indicating that typing and subtyping for hcv is clinically important in understanding hcv disease and its therapeutycal options. 'naive' viral load also seems to influence disease severity and responsiveness to therapy. therefore, viremia and genotype identification have been done routinely in molecular biology laboratory units. aims: the university hospital of coimbra studies and tests his own patients and patients from 11 other hospitals in the central portugal. we also collect and test blood donor candidates from this region. we proposed to analyse the distribution of hcv genotypes in this region, among 1578 patients with cronic hcv infection. we have simultaneously analysed the viremia and correlated it with age and severity of liver disease. methods: nucleic acid extraction was done using the semiautomatic 'xstractor' from biomerieux laboratories (boom method). the genotyping used reverse hybridization and was performed using probes from the 5¢ non-coding region (innulipa introduction: bacterial contamination of blood products remains a persistent problem. various techniques for the detection of bacteria in blood products exist but none of them has been widely accepted. bacterial detection systems could be divided into culture systems and rapid technologies. hemosystem has developed a rapid and sensitive technology for bacteria detection named scansystem tm . bacterial contamination of platelet concentrates is a rare event with an incidence between 1 : 2000 to 1 : 3000 per donation. therefore hemosystem developed a positive control in order to validate the scansystem tm platelet kit before use. aim of the study: the current study was designed to evaluate the performance of the scansystem tm positive control. the scansystem tm positive control is a capsule containing lyophilised lactobacillus casei subsp rhamnosus. the bacteria concentration per capsule is at least 8 ¥ 10 8 cfu. the positive control has to be stored at room temperature and is stable for 2 years. after dilution in pbs, the preparation has to be used within 1 hour. two capsules were tested for ten consecutive days with scansystem tm platelet kit as well as with optimised scansystem tm platelet kit. in an independent experiment three capsules were diluted in platelets stored in additive solution and were tested each with scansystem tm platelet kit and optimised scansystem tm platelet kit. results: 25 microscopic fields were analysed for bacteria specific fluorescence for each sample. the ratio between bacteria specific fluorescence signals and analysed signals was 1.0 in all samples for both scansystem tm platelet kit and optimized scansystem tm platelet kit. therefore by definition all tested capsules were positive. the lyophilized positive control capsules enable the user to validate the scansystem tm platelet kit before use. because bacterial contamination of platelet products occurs rarely, the routine use of positive controls improves safety of the screening method. scansystem tm is currently the only method that provides this safety measure. introduction: whereas implementation of nat for blood donor screening reduced the risk for transfusion transmitted hiv and hcv infections currently below one per 23 million transfusions, the risk for bacterial infections is estimated to be 1 : 200 to 1 : 3000. especially platelet products, which are stored at room temperature, are prone to bacterial contamination. aim of the study: several methods are currently developed to prevent the transfusion of bacterial contaminated platelet concentrates. the study investigates a new rapid bacterial detection method. material/methods: pool platelet concentrates were spiked with seven transfusion relevant bacteria strains under sterile conditions at concentrations of 100 cfu/ml to 106 cfu/ml. bacterial concentration was verified on blood agar plates immediately after spiking. five millilitres of spiked platelet concentrates were centrifuged, stained with thiazole orange dye and analysed directly by facs within five minutes after staining. aliquots of pool platelets spiked with concentration of 102 cfu/ml and 101 cfu/ml of each bacteria strain were incubated for two to eight hours in special bouillon at 37°c and were analysed by facs immediately after incubation. results: sensitivity of facs analysis differed between 103 cfu/ml for e. coli and 105 cfu/ml for klebsiella pneumoniae without preincubation and was enhanced to 101 cfu/ml when a pre-incubation step of two to four hours was included. conclusion: bacteria detection by facs analysis combined with a short pre-incubation (2-4 h) at 37°c is a quick and simple method with sensitivity comparable to other commercially available detection systems. the advantage of this new method is the rapid analysis, easy handling, high sensitivity and less expensive price. introduction: detection of bacterial contamination of platelet concentrates represents a major challenge in transfusion medicine. for blood transfusion services the method must have a high sensitivity, an easy performance and a low price. aim of the study: in this spiking study we evaluated the new optimised scansystem tm platelet kit detection method for use in apheresis platelets. methods: apheresis platelet concentrates (apcs) were spiked with strains of ten different bacteria species. after different incubation periods, apcs spiked with 10 cfu/ml were analysed by the optimised scansystem tm platelet kit. the number of bacteria was monitored by plating on blood agar. results: all bacteria strains were detected with the optimised scansystem tm platelet kit when the sample was collected 24 h after spiking. identity of the spiked bacteria was confirmed by gram staining and dna fingerprints. conclusion: in summary, the optimised scansystem tm platelet kit was able to reliably detect ten transfusion relevant bacteria species in apheresis platelet concentrates within 70 minutes when the sample was taken 24 hours after spiking. background: since year 2000 our laboratory started routine screening of hcv-rna in plasma minipools for all plasma intended for fractionation. although nat testing is not yet mandatory all blood products are released depending upon nat results. aim: to test and compare two different methods of rna extraction in order to make all the necessary adjustments to the test procedures while preserving the availability of blood products. methods: plasma minipools of 24 donations are prepared either on a tecan genesis robot or on a hamilton at plus. hcv-rna is isolated from 2 ml plasma by using either the qiagen biorobot 9604 and qiamp virus biorobot 960 kit or the manual extraction with cobas ampliscreen hcv pcr kit v 2.0. results: between march 2000 and december 2004 a total of 238 000 seronegative donations (9916 pools) were tested for the presence of hcv-rna. four pools were found to be positive for hcv-rna. of the four nat-positive pools with no eia-positive donor, four were confirmed as true positive by donor follow-up testing and/or testing of an independent sample from the index donation. all the positive donations were detected independently of the extraction method used (manual or automated). our experience shows that although the automated extraction method is 'off label' and it has to be validated, the use of biorobot 9604 does not pose a detectable contamination risk and it is possible to achieve a detection level for hcv less than 50 iu/ml. the advantage of the manual method is that it has better recovery of nucleic acids than the qiagen extraction. concerning the time needed for the extraction process the automated method runs 96 samples in 2 hours where the manual method needs 3 hours for 24 samples, needing, prior to extraction, an extra centrifugation step for one hour. the automated extraction method results in an assay with a high sample throughput, fast time, sufficiently sensitive, that can be successfully introduced into routine use in laboratories which have more than 800 samples/day while preserving the availability of blood products. anti-hcv similarly was high till 2002 (0.5-0.8%), but in trend to decrease afterwards (0.6%). anti-hiv reflected the low endemicity of the disease in public setting and was 0% through the mentioned years. rpr test for syphilis was around 0.1%. directed donors were 95% of all and volunteer donors consisted nearly 5%. donors in our blood center are being informed about donation prior to giving their blood and donor questionnaire forms (dqf) are filled out by the donor candidates. using dqfs have been mandatory at all blood banks in turkey by law since 1996. from that time infectious disease marker rates were dramatically reduced at all centers. donor information about the risks of transfusion and the importance of safe blood supply were detailed by the donation staff and physicians, consequently self-exclusion by the donor candidates who have risky behaviors was encouraged at our center. the interviewing staff was trained specifically for this topic. this steps were particularly emphasized in the last three years and the infectious screening results were displayed the outcome of this efforts. conclusion: education of the prospective donors, and recruit the voluntary, non-remunerated and regular donors will be the utmost goal of all blood banks. rigorous donor selection will contribute this ultimate success. we should spend more efforts to maximize enrolling voluntary donors to lower the serological marker results, consequently achieve safe blood. background: human t cell lymphotropic virus type i is endemic in japan, the caribbean, southeastern united states and parts of south america and africa. in non-endemic areas such as europe, htlv-i is less common and most infections are identified in immigrants. the epidemiology of htlv-ii is different, being predominantly found among indigenous american-indian populations and among ivdus, but the routes of transmission are the same. aim: our study's aim was to ascertain the prevalence of htlv i/ii in blood donors in order to understand the epidemiology of htlv in greece and initiate discussions of an acceptable level of risk and appropriate level of screening for rare transfusion-transmitted diseases. overall, anti-htlv seroprevalence levels among blood donors, are low. although the number of annual donations in this study is relatively small, the data for htlv indicate that rates of this infection are low and that infected donors will be seen infrequently. as all blood donations are screened for htlv i/ii during the last six years, a national survey is necessary in order to define the epidemiology of htlv in greece. introduction: toxoplasma gondii is the causative organism of toxoplasmosis. the disease transmitted by ingestion of either oocysts (in the feces of cats) or bradyzoites (in raw or undercooked meat). the parasite can also be acquired transplacentally by organ transplantation or from blood transfusion. the purpose of this study was survey of toxoplasma antibodies in some iranian blood donors at tehran blood center. blood samples were randomly collected for detecting of igg and igm antibodies (by elisa technique).the total numbers of donors was 217 of 52 (%24) were female and 165 (%76) male in age ranged from 18 to 55 years. results: 217 sera tested, 128 (59%) were found to be positive for toxoplasma igg antibodies and 6 (2.8%) were igm antibodies positive and 2 of them (0.9%) were borderline for igm antibodies. among males the frequency of positivity was higher than woman but this different was not significant. the most frequency of seropositivity was found in age group 41 to 50 years. conclusions: diagnosis of toxoplasmosis can be aided by serologic or histocytologic examination. the acute infection in healthy individuals is generally asymptomatic and not associated with any morbidity but in an immunocompromised host, toxoplasmosis be a very serious disease, and this can occur if a person is infection with toxoplasmosis before or after his/her immunosystem is compromised. in spite of the progress in the development of diagnostic, therapeutic and prophylactic methods, virus hepatitis still presents a serious global health problem. the possibility of transmission of these infections through transfusion of blood and blood derivates implies obligatory control of the donated blood. post-transfusion hepatitis is an important health problem in everyday practice, especially in patients who have to receive transfusion of erythrocyte concentrates as the only possible treatment for many years. objective: to show the prevalence of hepatitis b (hbsag) and hepatitis c (anti hcv antibodies) in multitransfused thalassemic patients. in our region there are 5 patients suffering from thalassemia major who are aged between 9 and 17, and who have been receiving erythrocytic transfusion 1-2 times a month since the age of one or two. they receive washed red blood cells, and in certain periods filtered red blood cells, controlled for viral markers and they mostly receive blood from voluntary, periodic and regular donors. the patients are tested periodically for the presence of viral markers (hbsag, anti hcv antibodies), using tests for hbsag (abbott auxyme monoclonal eia) and for anti hcv (abbott hcv eia 3.0). the presence of markers for hepatitis b and hepatitis c has not been detected in any of these multitransfused thalassemic patients who receive at least 20 transfusions a year. the tests in all 5 patients were negative. the blood used for transfusion must be tested for viral markers, and for the patients who have to receive blood for their whole life, the blood should be from voluntary, regular and periodic donors who donate blood at least three times a year, because then the risk of transfusion transmissible infections is very small. introduction: we observe yearly the prevalence of transfusion transmitted diseases following instructions of skae (national coordination haemovigilance centre). aims: to investigate the prevalence of the most important blood borne infections in our blood transfusion centre in the state achaia during the last five years (2000) (2001) (2002) (2003) (2004) . materials and methods: the detection of hbsag, anti-hcv, anti-hiv 1/2 was made by automated microparticle enzyme immunoassay (axsym, abbott) and by enzyme-linked immunoassay methods (dade behring, ortho, biomerieux) syphilis tests were made by using rpr kits. confirmation for anti-hcv positive samples was made riba or inno-lia, while the confirmation of anti-hiv1/2 positive samples was made by 'st. andrews' general hospital of patras reference centre. results: all the seropositive donors were first time donors. conclusion: (1) we observe that there is a decrease in all four infections. (2) the absence of anti-hiv seropositive donors is due to the high percentage of volunteer blood donation which approaches 70% in our centre during the last four years. methods: a prospective, one-year study has been set up in order to enrol at least 40 000 out of the estimate of 200 000 first-time donors, involving 21 blood transfusion centres from 9 of the 21 italian regions. each centre was required to enrol all first-time donors born before december 31st, 1978, and thus not included in the hbv mass vaccination campaign. the selected donors were tested for hbsag (mandatory by law) and for anti-hbc by commercial assays. all hbsag and/or anti-hbc positive specimens were stored frozen and sent to a reference laboratory for additional serological testing (anti-hbs, anti-hbe, anti-hbc/igm and anti-hbc avidity index by an experimental procedure) and for the determination of hbv-dna (both qualitative and quantitative) by real-time pcr. results: in the first 9 months of the study the 21 sites saw almost 29 000 first-time donors, of whom 70.5% belonged to the required age groups. among eligible donors, 0.4% were both hbsag and anti-hbc positive, and 10.3% were hbsag negative/anti-hbc positive. hbv positivity rates were higher in southern than in northern regions, although a high variability in hbv prevalence was observed between neighbouring areas in the north. hbsag positives were mostly males, 93% were positive for anti-hbe, 6% had raised alt and 8% were concurrently positive for anti-hbs. among hbsag negative/anti-hbc positive donors, 18% were negative and 82% were positive for anti-hbs. among anti-hbs positives, 35% showed values <100 miu/ml and 65% > 100 miu/ml. the avidity index results suggested that approximately 30% of anti-hbc positive individuals were recently infected. conclusions: our preliminary data indicate that approximately 70% of the italian first-time donors are older than 28 years of age and thus not belonging to the age groups who underwent to the mandatory vaccination against hbv, and that 10.7% of them have serological markers of ongoing or past hbv infection. anti-hbc alone was detected in nearly 2% of the study population. hbv-dna testing is underway at the time of this writing. in our country mandatory tests for each blood donations are: hbsag, anti-hcv, anti-hiv1/2 and tp ab. to c + ns3 + ns4, 9 (18.4%) to c + ns3, 4 (8.2%) to c + ns3 + ns5, 2 (4%) to ns3 + ns4 + ns5, 2 (4%) to c + ns4 + ns5, 2 (4%) to ns3 + ns5. the use of the hcv core ag elisa test system may provide substantially earlier identification of hcv infection than it is possible with current serological assays. although all of six anti-hcv assays are very sensitive and specific screening assays, they didn't detect hcv infection in one patient. majority of anti-hcv positive patients (77.5%) had anti-hcv ab for 3 or more different epitopes of hcv. international comparison of performance of abbott prism assays used for blood donor screening background: the national serology reference laboratory, australia (nrl), coordinates a quality control (qc) programme for laboratories that screen for anti-hiv 1&2, anti-hcv, hbsag and anti-htlv i/ii using the abbott prism assays. nineteen laboratories from australia, belgium, canada, ireland, israel, the netherlands, new zealand, norway, singapore, south africa and thailand have submitted data for this programme. aims: to determine the accuracy and precision of results from laboratories, individual prism instruments and different reagent lots by analysing data accumulated between october 2001 and january 2005. the multi-marker qc sample 'pelispy s2058 type 7' (s2058), produced by viral quality control (now acrometrix-viral quality control), was provided to participants. laboratories tested s2058 in each calibration run, in addition to the manufacturer's controls, on each sub-channel of the instrument. pelispy was used as a 'go/nogo control' and results were required to be reactive (s/co > 1) for a test run to be deemed valid. data were collected and analysed using the nrl's internet-based application edcnet (https://www.nrlqa.net). after submission, laboratories were able to compare their results with those submitted by other laboratories and investigate differences in results from reagent lots and instruments. data for five different s2058 lots were exported from edcnet and analysed. results: nearly 95 000 results were submitted: all results were reactive (s/co > 1). fifty of these results (0.0005%) were excluded from analyses because they were reported from invalid test runs [due to pipetting, aspiration or sampling error (n = 48) or due to unacceptable results (n = 2)]. a further 16 results were excluded because data provided by laboratories were inconsistent or incorrect. a total of 94 189 results, reported using 332 different prism reagent lots (145 for anti-hiv, 79 for anti-hcv, 55 for anti-htlv and 53 for hbsag), were analysed. results from prism hbsag and anti-hiv showed the least variation with coefficient of variations (cv) of <10% for all s2058 lots. results from prism anti-hcv and anti-htlv produced cvs between 9.17% and 15.38% for all s2058 lots. data reported for s2058 lot ps030618 (n = 38 662, range 8080 for anti-htlv to 10 225 for hbsag) were analysed further to review performance of prism reagent lots. hbsag showed the least variability between prism reagent lots with <8% bias for the 14 prism hbsag reagent lots used (bias: the difference between the mean ratio for the reagent lot and the weighted mean ratio for all reagent lots, expressed as a percentage of the weighted mean ratio for all reagent lots). prism anti-htlv showed greater variability between reagent lots with a single reagent lot generating a +63% bias. prism anti-hcv showed the greatest variability within reagent lot with results from 12 of 21 reagent lots showing a cv between 10% and 13%. conclusion: in 94 255 results in a qc sample distributed to 19 laboratories the abbott prism performance was found to be consistent over four assays. edcnet was robust in supporting laboratories' abilities to follow precision and accuracy of the assays in real time. introduction: since hcv rna testing of all blood donors started in finland in 2000, the nat screening process has continuously been improved. investments in process automation have made the work more efficient and blood safety has further increased since hiv-1 rna screening of all blood donors started late 2004. aim of the study: to implement hiv-1 rna testing in the nat screening program cost effectively, without increasing the throughput time of the samples and delay in the result reporting. to study if the sensitivity for both hiv-1 rna and hcv-rna will be sufficient when a single extraction is used and when the pool size of 96 donations and the sample volume are kept unchanged. methods: the nucleic acids were isolated from plasma samples of 1 ml with the magna pure lc instrument using the magna pure lc total nucleic acid isolation large volume kit. the internal controls from the cobas ampliscreen multiprep specimen preparation and control kit and the cobas amplicor hcv specimen preparation kit were added to the lysis/binding buffer (5 ml/ml of each). from the final volume of the nucleic acid eluate (100 ml) 50 ml was used for the detection of hiv-1 rna (roche cobas ampliscreen) and 30 ml for the detection of hcv rna (roche cobas amplicor). a run control containing both hiv-1 rna (200 iu/ml) and hcv rna (50 iu/ml) was included in all extraction runs. sensitivity of the both assays was assessed by testing dilution series of the who standards for hiv-1 rna and hcv rna. specificity was evaluated by testing fractionation plasmapool samples (n = 35) and minipool samples (n = 68). results: detection limits of the hiv-1 and hcv assays (95% hit rate) were calculated to be 100.1 iu/ml and 18.7 iu/ml respectively. specificity for both assays was 100% and during the validation phase also the robustness was good. the sensitivity of both assays with a pool size of 96 was below the recommendations by the council of europe for blood donor screening (for hiv-1 rna 10 000 iu/ml and for hcv 5000 iu/ml per individual donation). specificity of the assays was excellent, false reactive results were not observed. implementation of the hiv-1 nat assay in the screening program did not increase the throughput time of the donor samples when the pool size of 96 donations, 1 ml sample volume and a single extraction for two assays were used. the the very substantial increase in the number of industry-sponsored clinical trials has created challenges for medical schools, academic hospitals, faculty members of these institutions, and the journals that publish the results of these trials. in many cases, authors of reports of industry-sponsored clinical trials are paid consultants to the sponsor, have been paid by the sponsor to lecture on behalf of its products, or have equity in the sponsoring company. these ties to industry create a tension that actually is or can be perceived as • work internationally; • send young volunteers to international youth forums; • employ young people in your organisation; why use volunteers in blood donor recruitment? • they have networks to scout-groups, sports-organizations, tradeunions, rotary, staff of large companies etc. • they bring in fellow volunteers -with different prospects of society; • often paid recruiters are underpaid (!) and tend not to remain • you can not recruit by telephone! • 2 out of 3 donors are recruited by personal contact! • so you need direct personal contact = need many people (e.g. young ambassadors); • a large number of volunteer recruiters is a gift from heaven! paid donation gives the act of blood donation low status. the act of blood donation should be respected, and praised by role models, queens and presidents. efficient work and close cooperation of blood bank staff and volunteer organisations is the key to success in blood donor recruitment and retention! with such a prospect, it was important to evaluate the practices of blood donor selection in the eu. material and methods: a questionnaire was designed and sent in 2003 to the relevant institutions of the 15 eu countries plus switzerland. the questionnaire included questions on the interviewing practices before homologous blood donations, regarding non-specific risks to donors and recipients, identified risks to donors, infectious, bacterial, viral, parasitic and prionic risks to recipients and non-infectious risks to recipients. the questionnaire also inquired about each country's exclusion period for each contraindication (ci) to donation. results: predonation interviews were prepared, in all countries, by circulating informative documents to blood donors. they were supported by written questionnaires in nearly all countries. in half of the countries, those interviews had to be led by physicians (nurses or technologists in the others). the 18-65 age limits for blood donation (18-60 for a first donation) were the rule in 11 countries. in other countries the age limit could be brought forward to 17 and extended to 70 years old. the time interval between donations was identical for men and women in 11 countries, and varied from 8 to 16 weeks according to country. the questions of the questionnaires were very similar as regards the identification of risks to donors and recipients, and very close to the requirements that appeared later in the 2004/33/ec directive. this particularly concerned how to meet the expectations of european donors, so that they come back to your blood center! know your donors: make regular donor surveys. age, gender, number of donations, number of first time donors, media consumption, education, job situation, income brackets. use local donor organisations let volunteers help! they work for free, but donor recruitment and -retention costs money. each blood center should have a local donor organisation, run by volunteers. the donor organisation should receive a payment for each bag collected. the reputation of the blood system tell the donors, what the blood is used for. that all blood is tested. and that blood provides safe medical treatment safety of the donor. insurance is a must good quality and efficiency in blood services decentralized blood collection no waste, and minimal outdating efficient service: • a friendly environment, • donor friendly opening hours, • pleasant rooms, beds and well equipped waiting rooms, • parking-spaces, transport, • beverages and food, • letters with correct data etc. donors expect to be serviced by trained, medical professionals and that the medical check-up is taken seriously. donors should be recognized continuously. use directed press-coverage to higher the self-esteem of the donors. donors should be well informed: • leaflets, posters and questionnaires should be 100% correct; • use e-mail and web-sites for quick up-date of donor information. be visible! • have an offensive and comprehensive media approach; • have a yearly national campaign 14 june up to world blood donor day; • have an attractive home-page, constantly updated; • streamline your lay-out; • mail a donor magazine to all regular donors: • send newsletters regularly to volunteers and the press. • use recruitment cards. • easy phone-and fax numbers, e-mail addresses. directed campaign towards young people: • young ambassadors group; • special training sessions for young volunteers; • advertisements in media catering to young people; • poster competitions; • book and leaflets on blood addressed directly to young people; minimum bodyweight, blood pressure, pulse limits, questions involving viral risks, either sexually transmitted or linked to drug abuse, questions investigating risks of malaria transmission, questions aimed at identifying risk of prionic disease transmission. analyzing ineligibility times, on the other hand, revealed wide differences. for example, ineligibility for current multiple sexual partners, sexual relations with risk individuals, tattooing or body piercing, endoscopy, general anesthesia or invasive surgery, could vary from 3 to 12 months. previous transfusion history could not be a ci or could be one varying from 6 months to indefinite ci (this point recently changed in several countries). the results of that survey have revealed some differences between countries in the questions asked and especially in the ineligibility times. however, the conditions under which donor selection interviews are conducted were similar in all countries. the enquiry tool used in this study proved to be well adapted to evaluate the donor selection practices throughout eu. a next step will be to use it to appreciate their evolutions and especially the impact of the 2004/ec/33 directive on these practices in the 25 eu countries and furthermore to evaluate the results of this selection (rates and motives of deferral) which is a major factor of patients' transfusional safety. background: in europe on average 43 whole-blood donations are performed per 1000 inhabitants and year. whole-blood donation comprises a puncture of a venous vessel and letting of blood (usually 450 ml), which may be repeated several times a year. like other invasive procedures, blood donation has a range of effects on the individual who is subjected to it. aim: the aim of this paper is to review some aspects of the present state of knowledge on effects and complications of whole-blood donations. results: most studies of the effects of whole-blood donations on the donor have focused on negative or unpleasant events and on time in rather close association to the donation. complications related to percutaneous needle insertion (bruise assessed by inspection 0.66% -bruise assessed using post-donation interview 22.7%, sore arm 0-10.0%, nerve irritation 0.02%, arterial puncture/pseudoaneurysm/arteriovenous fistula 0.003-0.009% etc) are most commonly reported, while negative systemic reactions (vasovagal reactions 2.5-7.8%, syncope 0.08-0.34%) occurring in connection to blood donation are less frequent (newman bh: blood donor complications after whole-blood donation. curr opin hematol 2004; 11: 339-345.) serious complications requiring hospitalization (myocardial infarction, stroke) are extremely rare (0.0005%). fatigue (7.8%-10.0%) and diminished physical working capacity (7.0%) are reported to occur during days after the donation. a recent study of a consecutive sample of 528 swedish blood donors (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128.) (with a selfadministered questionnaire with an open-ended question: how does blood donation affect you? physically-bodily/psychologicallyspiritually/ethically-morally/socially during or after blood donation?) revealed that perceived negative effects (fatigue, diminished physical working capacity, vertigo/dizziness, susceptibility to infections etc) were less common (25% of the donors) than positive effects (feeling of satisfaction, being more alert, feeling generally better, less migraine, higher physical working capacity, respect from environment, feeling of relaxation etc; 35% of the donors). the duration of positive effects was regularly reported to be weeks, while negative effects lasted only days. investigations on the long-term effects of blood donation are scarce. yet, they may indicate that the donation of blood is associated with e.g. lower blood pressure and a reduced risk of myocardial infarction (nilsson sojka b, sojka p: the blood-donation experience: perceived physical, psychological and social impact of blood donation on the donor. vox sang 2003; 84: 120-128). conclusion: both the panorama and the frequency of occurrence of the different effects and complications of whole-blood donations vary as a function of how the information was gathered (openended questions, observations, interviews etc). serious reactions to whole-blood donations are extremely rare. more studies are needed in particular with respect to the long-term effects of regular wholeblood donations. t-pa-056 establishing a national adverse event reporting system for blood donors -a prospective study of 1. there was no national system and significant regional variation showed that the data was scientifically unsound. a coincidental initiative to remove the mandatory post donation rest period for regular donors further emphasised the lack of reliable, retrospective data to monitor and compare the impact of this new policy. aims: 1. to develop and implement a high quality, reliable national donor adverse events reporting (daer) system; 2. to define and categorise adverse events; 3. to record data systematically and prospectively using the existing computerised donor database. methods: from summer 2002, a small project team of senior clinical and operational staff took 9 months to agree a detailed policy for capturing and recording all donor adverse events, including precise definitions for grades of vasovagal reactions and bruising. detailed training material was written in may 2003 and the new protocol was validated in one region. from july to december a formal one day training programme was delivered to over 3000 staff working on 98 mobile collection teams, 19 static sites and 11 blood centres. daer was fully implemented by january 2004. adverse events are assessed by health care professionals on session and the relevant code entered onto the donor's computer record by clerical staff. information received after the session is entered by centre based doctors using the same system. the database is interrogated monthly for statistics. results: in the first 9 months 1.86 million donors attended sessions throughout england and north wales. results were very consistent month on month. 25 175 donors (1 : 75) had vasovagal symptoms but only 14% of these suffered syncope. 71% of all vasovagal reactions occurred in women. 21% occurred in donors aged 17-20 and a further 30% in donors aged 21-30. (donors aged 17-30 represent only 25% of the total donor base.) 831 donors (1 : 2250) reported a delayed reaction, 52% of whom did lose consciousness. nerve injury, unrelated to haematoma, occurred in 109 donors (1 : 17 000) and, more rarely, arterial puncture was diagnosed in 46 donors (1 : 40 000). bruising was reported after the session by 1 : 1300 donors. summary: a robust system has been developed and successfully implemented across a large, national blood service. based on the data already accumulated our next phase is to develop strategies to minimise adverse events, confident that any intervention will be effectively monitored. the community role in enhancement of voluntary, non-remunerated blood donation in the new millennium introduction: in the developing countries, about 80% of the blood supply comes from paid or replacement donors, where a high number of infected persons are in the donors population. only 16% of the global blood supply is donated as voluntary nonremunerated blood donors in countries with low and medium human development indices. and around 80% of the global blood population has access to only 20% of a safe blood supply. conclusion: blood is a national resource, it is the responsibility of governments through it's communities to ensure that the blood supply is safe and adequate to meet the needs of patients population and available to all who needs it. background: in response to a documented increase in the average age of donors, a survey was conducted to explore if young people had more unfavourable attitudes towards becoming blood donors. aim: to identify if the increasing difficulty in recruitment and retention of young people as donors, is linked to a low level of motivation for donating blood in this age group. methods: a national telephone-survey was conducted among a cross-sectional sample of the adult norwegian population (1000 participants). the survey was performed in november 2004. results: five percent reported being active donors (had donated during the last 12 months), 17% were passive donors (had not donated during the last 12 months), 22% were non-donors with a positive attitude towards becoming donors, and 55% non-donors with no intentions ever to donate blood. in the youngest age group (age 18-29), 1% reported being active donors and 8% were passive donors. however, 36% of the young non-donors reported having intentions of becoming a blood donor. fifty-five percent of young non-donors had a negative attitude towards ever donating blood. all non-donors were asked why they did not donate. thirty-six percent of all non-donors reported health related reasons for not donating blood. thirty-one percent of all non-donors claimed that they did not donate because no one had requested them to do so personally, and 4% reported they did not care about blood donation. in comparison only 28% of young non-donors reported medical reasons for not donating. thirty-eight percent claimed lack of personal request, and 7% reported of lack of interest as the main reason for not donating. summary/conclusion: although the youngest age group was under-represented among active donors, we found that a great proportion (36%) of young non-donors had a positive attitude towards becoming blood donors. the most important reason why young people do not donate was the lack of a personal request. indifference regarding donation was not very widespread. a relatively high proportion of young people considered themselves as not medically disqualified to donate. in light of these findings, efforts to recruit young people as blood donors are strongly recommended. background: the ever-increasing demand for blood, coupled with emerging new threats to blood safety, motivate the strengthening of the blood banking infrastructure. aim: employing new technology as an instrument for building relationships of trust between blood bank and blood donors. material: 1. a new software module supporting the management of magnetic cards was added to e-aima blood bank management application. the magnetic card supports the following information: • front-side: donor's photograph, surname, name and blood group (including rhesus phenotype & kell) and sign of blood collection centre. • magnetic stripe where data concerning donor's serial number, medical history (risk factors), test results for infections and the number of donations is stored. 2. specific hardware allowing reading from and writing to magnetic cards is integrated to the software module: • magnetic card scanners were added to pcs serving to donation collection, including laptop for mobile team collection. • web cameras to capture the photograph of the donor to be printed on the card. • card printer was deemed necessary to produce magnetic cards for donors on a need basis. 3. consumables: blank plastic magnetic cards, ink cartridges for printer. methods: in order to evaluate the performance of magnetic cards compared to the paper-based system, a questionnaire was distributed to 300 first-time and 200 repeat blood donors, in order to be used as an indicant of donor's satisfaction. 1. first-time donors increased 7.8% in the 6 months of application. among them, 4.3% were donors 'for relatives or friends' turned into volunteer donors and 3.5% were first-time volunteer donors. the questionnaire analysis further revealed: • 96% were motivated by the use of magnetic card. • 92% appreciated the presence of their photo on the card and they confessed that they had used it as a spill for recruiting their friends as donors. • 100% were persuaded that employing new technology would result in safer and more trustworthy procedures combined with reduced waiting time. • 100% considered magnetic cards more practical compared to paper cards because of their compact size and improved durability. 2. the turnover of repeat donors also increased 1.3% after replacing their plain old paper cards with new ones. further analysis revealed that: • 100% appreciated the quick cross-checking of donor's identity. • 100% were satisfied with the effectiveness and efficiency of magnetic cards in managing donor's data. conclusions: in 2004, greek health policy provided the legal basis for establishing the electronic national health card. the introduction of the national donor's magnetic card is another step towards this direction, being aligned with the modern national health strategy. apart from the positive impact on the number of both firsttime and repeat blood donors, it should be also pointed out that the use of a unique donor serial number on country level results in less error-prone procedures due to the reduction of administrative process overhead and facilitates interoperability between national blood banks using compatible technological infrastructure. t-pa-060 emerging technologies in transfusion. dna based assays until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. recent emergence of nucleic acid technologies (nat) has revolutionized viral diagnosis by not only increasing the sensitivity level but also facilitating the detection of several viruses in parallel, by multiplexing specific primers. however, in more complex biological situations when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. high throughput systems such as dna arrays permit a conceptually new approach. these miniaturized microsystems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, in additional confirmation tests and simplify data interpretation. however, the microsystems actually available require additional instrumentation and reagents for sample preparation and target-amplification prior to detection on the dna array. future technologies such as 'lab-on-a-chip' include channels, fluidics and thermal zones allowing extraction, amplification and detection. another major challenge in the area of dna detection is the development of methods that do not rely on target-amplification systems. almost all blood group antigens are bi-allelic and encoded by single nucleotide polymorphisms (snps). to facilitate the direct availability of typed red cells and platelets, we develop a high-throughput technique to genotype by dna microarray the whole donor cohort for all clinically relevant red cell and platelet antigens. methods: a multiplex pcr was developed to both amplify and fluorescently label 28 gene fragments of red cell and platelet antigens in one reaction. each array contains spots of short (17-27 nt) allelespecific oligonucleotides to discriminate between the two alleles of an antigen system. results: two blinded panels encompassing 94 donors were genotyped for hpa-1 through -5 and 15; no discrepancies were found. currently, arrays are prepared for the red cell systems. the fya/fyb, fy-gata1 mutation, jka/jkb, k/k, kpa/kpb, m/n, rhc/c, rhe/e, rhdpseudogen, rhdvi negative, rs, doa/dob, genotypes can be determined. the set up of genotyping assays for rare genotypes is difficult because of lack or insufficient amount of dna. the latter can be overcome by phi29 dna polymerase-mediated isothermal genomic dna amplification, from minute amounts of dna present in stored red cell fractions or antiserum. the results show that the blood group typing dna microarray will provide a reliable and fast genotyping procedure. the method can be further improved to obtain the necessary automated throughput for typing of large donor cohorts. and 15, all other weak d types should be regarded as potential anti-d immunizers. for correct determination of weak d both serological typing (polyclonal and monoclonal), as rhd dna typing are mandatory. when serology indicates weak d, more anti-d antibodies are tested (9 epitope model) to distinguish partial d from weak d. in addition, an rhd mpx pcr is performed to detect the presence of rhd exons 3, 4, 5, 6, 7 and 9. in all known weak d types, all six rhd specific exons are amplified (except for weak d type 4 which lacks rhd exons 4 and 5), whereas partial d phenotypes usually show aberrant patterns. aim: the aim of this study was to evaluate the diagnostic scheme for weak d typing. methods: between 2002 and 2004, 24 samples were investigated for weak d characteristics. four pcr-ssp assays were developed for identification of weak d types 1 (809t > g), 2 (1154g > c), 3 (8c > g) and 5 (446c > a). weak d type 4 was identified by the combination of serology and absence of exons 4 and 5 by rhd mpx pcr. rhd-specific exon sequencing was performed when serology and molecular typing were inconsistent. results: all 24 samples were subjected to the rhd mpx pcr and 1 sample showed absence of rhd exons 4 and 5, indicative of a weak d type 4 when combined with serology. the remaining 23 samples were analyzed by the weak d pcr-ssps, resulting in 12 weak d type 1 samples, 4 weak d type 2 samples, 4 weak d type 3 samples and 1 weak d type 5 sample. two samples remained undetermined and were sequenced for all 10 rhd exons and the rhd promotor region. one sample showed the mutations corresponding to the dau 2 partial d phenotype (209g>a, 998g>a and 1136c>t). the other sample had only one, not previously known mutation (1193a>t), which is located intracellularly at the coohtail. extensive serology using the 37 epitope model showed a pattern matching weak d. this new weak d variant was registered as weak d type 41. conclusions: based on these results it may be concluded that weak d phenotypes should be confirmed on molecular level to avoid misinterpretation of partial d that cannot be detected by rhd mpx pcr analyses. patients with weak d phenotypes, except for types 1, 2 and 3 should be regarded as being at risk for anti-d immunization after transfusion of rhd-positive blood products and should therefore be treated with rhd-negative bloodproducts. in this evaluation, 4 out of 24 patients carried such alleles. introduction: although kell antigens are expressed very early during erythropoiesis and a 0.1% incidence of anti-kel1 is found in obstetric patients, this is a relatively rare cause of hdn. anemia is produced by immune destruction of fetal rbcs and suppression of erythropoiesis. maternal antibody titers or amniotic/cord blood bilirubin levels are not relevant indicators of the severity of the disease, and the measurement of the fetal haemoglobin by cordocentesis is a procedure with risks of miscarriage and sensitization. pcr techniques for the determination of blood groups using fetal dna isolated from maternal plasma, allows the application of noninvasive methods. clinical cases: we describe two cases of pregnancies in women with anti-kel1 acquired by transfusion/previous pregnancies: 1st case: in july 2000, a 30-year-old woman (gravida 2, para 0), rhdnegative, kel1-negative was referred at 20 weeks gestation. the father's phenotype was rhd-positive, kel1-positive. a maternal antibody screen revealed d and kel1 alloantibodies. dna was extracted from amniotic liquid. the kel genotype was determined by pcr-rflp using the bsm i. pcr-ssp was used to studied intron 4 and exon 7 of the rhd gene. the results showed that the fetus was positive for rhd sequences and showed kel2 homozygosity; 2nd case: in august 2004, a 36-year-old woman (gravida 2, para 1) was referred at 28 weeks gestation. she had a history of transfusion with 2 rbcs units in 1996 -one of the donors was kel1positive. the woman typed rhd-negative and her husband typed rhd-positive. rbcs from both were kel1-negative. the maternal antibody screen revealed anti-kel1. doubts existed about the putative father of this child. dna was extracted from maternal plasma using the magna pure lc (roche). real-time pcr was applied to analyse: sequences of intron 4, exons 4, 6, 7 and pseudogene of the rhd gene and the sry gene by sybr green; and the alleles kel1/kel2 by hybridization probes. all rhd sequences were detected (with the exception of the pseudogene) and the kel genotype gave a kel2/kel2 result. in both cases the doctors choose not to use any invasive method to monitoring the fetuses regarding a hdn due to anti-kell antibodies, and the results of the molecular analysis were confirmed by testing the cord rbcs after birth. discussion: these cases illustrate the reliability of the molecular biology results, based on the collection of simple peripheral blood samples. a determination that the fetus lacks the relevant antigen obviates the need for expensive and invasive monitoring throughout the pregnancy. evidence-based medicine (ebm) is defined as: 'the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. the practice of ebm requires the integration of individual clinical expertise with the best available external clinical evidence from systematic research and our patient's unique values and circumstances. ' otherwise healthy individuals without cardiopulmonary dysfunction (cdf) tolerate acute reduction of haemoglobin concentration to about 5 g/dl, provided that blood volume is kept normal by a volume expander. however, individuals experience physical fatigue, and there is faint reduction of perception as measured by neurophysiological tests. symptoms are reversed upon retransfusion of fresh, autologous erythrocytes. acute, normovolemic anemia seems to be progressively less tolerated with increasing age and cdf. controversy has existed on whether or not to correct hypoalbuminemia in asb or icp by infusion of albumin. recently a large trial showed no outcome differences between icp patients treated with albumin or saline. thus in general there is no indication for albumin in asb or icp. however, albumin may yet be advantageous in e.g. patients with head injuries. furthermore, fractionated albumin is not equivalent to native albumin, since fractionation stabilizers remain bound to the albumin molecule. thus more refined albumin preparations may carry advantages still to be investigated. erythrocytes are given to increase the total oxygen transportation capacity of the organism. the effect of blood bank stored erythrocytes may differ from that of fresh, autologous erythrocytes, since changes of presumably important erythrocyte properties occur during storage. in the only large trial available, a transfusion trigger of 7.0 g/dl was found to be favourable to one of 10 g/dl in icp, except possibly in icp with unstable angina pectoris or heart infarction. however, the erythrocyte concentrates given were not leukocyte filtered, and side effects of infused leukocytes may have hampered the conclusion. on the other hand, a metanalysis showed transfusion as an independent indicator of unfavourable outcome in coronary bypass patients, but again, leukocyte filtered erythrocyte preparations were not applied. the effect on morbidity and mortality of 'top up' transfusions given e.g. to mobilize patients postoperatively has not been studied by trials, although this effect seems evident to many clinicians. grave anemia may reduce the haemostatic effect of thrombocytes because changes of blood rheology reduces the pressure forcing thrombocytes against the walls of small vessels. the transfusion trigger for thrombocytes in asb or icp remains to be established by clinical trials, however. the same applies for fresh frozen plasma, which is infused as a source of coagulation factors. on the other hand, the haemostatic effect of various fibrinolysis inhibitors is well established in asb and icp, but many clinicians appear hesitant to use them. another interesting haemostatic agent is recombinant fviia, the use of which to control asb in blunt trauma is supported by one well controlled clinical trial. evidence by systematic research is insufficient to decide what is optimal transfusion practice. the procurement of such evidence is one of the greatest current challenges to transfusion medicine research. . concerns about the transfusion-related complications, such as infections, tumour behaviour and immuno-modulatory effects, and the costs, necessitated a re-evaluation of the transfusion practice. aims: the goal of this study is to evaluate if a restrictive transfusion policy (hb transfusion trigger <4.5 mmol/l) reduces the amount of red cell transfusion compared to a liberal transfusion trigger (hb < 6.0 mmol/l) without a decrease in hrqol. because of concerns about the feasibility of this study early results were analysed and are presented in this abstract. material and methods: after a run in period of 3 months (hb transfusion trigger of hb < 6.0 mmol/l) patients are randomised for the restrictive or the liberal transfusion policy. patients are followed then 12 months. hrqol is measured after inclusion, after randomisation, 6 weeks, 3, 6, 9, and 12 months after randomisation. also anaemia related complications and red cell antibodies are scored. hb values were blinded for the patients during the study period. results: from july 2002 till june 2004 15 patients were included (4 ra, 5 rars, 4 rcmd, 1 raeb, 1 cmmol) in 2 general hospitals and 1 university hospital. two patients died in the run in period. eight patients were randomised for the restrictive transfusion policy and 5 patients for the liberal transfusion policy. the mean follow up period in the liberal group was 6.2 months (inclusive run in period) and 7.4 months for the restrictive group. two patients in the liberal group died after randomisation. one patient received growth factors. in the restrictive group 2 patients finished the study, 1 received growth factors and 1 patient withdrew informed consent. the mean hb level was lower in the restrictive group and after randomisation about 55% reduction in amount of transfused red cells was found (1.8 units per pt per month in the liberal group vs 0.8 in the restrictive group). no anaemia related complications were found, e.g. cardiac failure and cerebro vascular ischemia nor a decrease in activity performance. conclusion: there were some concerns after introduction of the restrictive transfusion policy. this preliminary results show, however, that a restrictive transfusion policy leads to a diminished use of red cell transfusion without an increase of cardiac complications or a decrease in activity performance. this study will be continued to compare hrqol scores in both groups. introduction: strong evidence supports the efficacy of blood conservation strategies such as autologous blood donation (abd) and erythropoietin (epo) for reducing exposure to allogeneic blood. however, use of these interventions is highly variable among institutions and frequently sub-optimal. the program to reduce orthopedic blood exposure (probe) evaluated a blood conservation program in patients undergoing total hip joint arthroplasty (thja) at ontario hospitals. aim of the study: the objective of probe was to determine whether a comprehensive blood conservation algorithm (bca) was more effective than usual care (uc) for reducing exposure to allogeneic blood in patients undergoing thja. methods: we randomized 30 hospitals that perform high volume elective primary thja to implement either a bca or to continue with uc. the bca consisted of three components: physician and patient education, blood conservation interventions (use of abd or epo), and transfusion guidelines. t-pa-071 table) . mortality for non-transfused patients was significantly lower than for patients receiving either lr-or s-prbc at all time points (p < 0.0001). t-pa-074 thalassaemia: the impact on blood transfusion services thalassaemia major is a genetically determined disease that causes severe chronic anaemia and further complications. it can be managed successfully in the vast majority of cases, so long as public health and other scientific and organizational infrastructures are adequate. although the progress achieved in the field of bone marrow transplantation and other disciplines promises cure of the genetic defect, regular blood transfusion from early childhood remains the cornerstone of treatment of patients with thalassaemia major. this presents national health authorities with the formidable task of assuring an adequate blood supply of high quality and safety for these patients and ensuring that it is transfused in the appropriate way. the basic principle in the modern management of thalassaemia patients is that of a global approach to care. within this approach, a standardized protocol for regular blood transfusions is a prerequisite for the patient's long survival and quality of life. if thalassaemia patients are not transfused effectively, the severe anaemia and over-expansion of bone marrow due to ineffective erythropoesis can lead to poor growth, bone deformities, organomegaly and impairment of normal physical activities. in countries or regions with large numbers of thalassaemic patients, the organizational and technical aspects of meeting their blood requirements represents a heavy additional workload for the blood transfusion services responsible for providing blood for this group of multi-transfused patients. the acquisition and preparation of blood, genotyping the patients' blood group (including at least rh, kell, kidd and duffy systems) preventing the transmission of infectious diseases and other transfusion associated complications, and assessing the patients' blood transfusion indices all have a tremendous impact on blood transfusion and treatment units. blood transfusion services are thus confronted with major challenges that can only be met if appropriate national transfusion policies are in place, both in the laboratory and the clinical setting of blood transfusion. the availability of safe blood is related to the effectiveness of donation programmes aimed at recruiting and retaining voluntary unpaid blood donors who are at low risk for the transmission of infectious diseases. sufficiency is further related to resources, organization and management of the blood transfusion service and continuous education of its staff. high technical standards for the transfused product and quality management systems are required to ensure that the product meets these requirements, as well as pre-transfusion, transfusion and haemovigilance systems and other more stringent quality measures in the whole chain of blood donation and transfusion. additional measures and continuous care are specifically required for the optimal transfusion therapy of the thalassaemic patient. the patients should be transfused with red cell concentrates, (rccs) preferably not more than one week's old and leucodepleted. other processes i.e washing of rccs, use of nutrient additive solutions, irradiation etc may be used to improve the quality and the safety of the transfused product, while other advances in red cell transfusion are expected to improve blood safety by preventing adverse reactions and reducing exposure of the patient to donor blood. should patients with thalassemia intermedia be regularly transfused? thalassemia major (tm) and thalassemia intermedia (ti) share mostly a common basic molecular mechanism, that is the reduced synthesis of the * globin chains. 1 the consequences of the resulting chronic hemolytic anemia are also common and include growth retardation, bone marrow expansion, extramedular hematopoiesis, splenomegaly, increased intestinal iron absorption, susceptibility to infections and hypercoagulability. what differentiates the two forms of the disease is the severity of the clinical phenotype, which in turn depends on a particularly heterogeneous molecular background and imposes diverse therapeutic strategies. the consequences of the genetic defect as well as the effect of the applied therapy seem to be mainly responsible for the clinical course of the disease. in untreated tm cases, the aforementioned consequences occur fast and patients die early in life mainly due to high output heart failure. over the past decades, the gradual adoption of the current transfusion and iron chelation strategies and the patients' compliance with this therapy have resulted in a significant improvement of survival, that according to recent statistics reaches 68% at age 35. this rate is even better in well-treated patients, almost 80% of whom survive at age 40. regular therapy extends survival mainly by preventing early development of cardiac complications. in addition, a multi-organ improvement is accomplished while patients' physical appearance is almost indistinguishable from that of the general population, hence permitting a normal social behavior with a high overall quality of life. bone marrow expansion and extramedular hematopoiesis are prevented; hepatosplenomegaly is substantially restricted and usually there is no need for splenectomy, while thromboembolic complications are rare and pulmonary hypertension is practically absent. patients with ti remain as a rule without regular therapy until a number of severe complications arise. the consequences of chronic anemia develop slowly compared to untreated tm cases and dominate patients' clinical picture usually by the third decade of life. at this time, all patients have developed hepatosplenomegaly and most of them have been splenectomized. bone marrow expansion results to bone deformities and fractures often occur. extramedular hemaotpoietic masses and bone deformities may lead to various complications depending on their bulk and location, such as neurological symptoms from masses arising in the paraspinal area or dyspnea from lung restriction. hypercoagulability, resulting from defects of native erythrocyte membrane phospholipids, together with the coexistent thrombocytosis in splenectomized patients lead to a wide spectrum of thromboembolic events. pulmonary involvement with respiratory dysfunction and hypoxemia as well as pulmonary hypertension leading to congestive heart failure are well documented in ti patients. nowadays, the beneficial effects of regular transfusion and chelation therapy in tm are beyond any doubt. the occasional application of transfusions in ti has a transient effect and does not seem to inhibit the consequences of chronic hypoxia. intensive and regular transfusion and chelation therapy in ti has proved effective in ameliorating the established complications such as spinal cord compression, hypercoagulability and pulmonary hypertension, without however reversing them. given the 30-year experience on intensive therapy in tm and the first encouraging data in ti, the earlier application of such treatment seems to be crucial in ti. the timing however of therapeutic intervention in ti in order to prevent anemia-related complications still remains an open issue that needs to be properly addressed. the impact of prestorage leucodepletion on the immediate transfusion adverse events of patients with thalassaemia major backround: regular blood transfusion therapy in patients with bthalassaemia major decreases the complications of anemia but it is associated to many immediate and delayed side effects. febrile non haemolytic transfusion reactions (fnhtr) are common complications due to alloimmunization of recipients against hla and/or specific antigens on donor's wbcs or to the accumulation during storage of biologic response modifiers (bmrs) that are directly pyrogenic or indirectly by stimulating recipients' white cells to produce pyrogenic mediators. post storage leucoreduction (lr) has reduced the fnhtr in these patients from 13% to 0.5% per unit. it is unknown whether introduction of prestorage leucodepletion (ld) has reduced the incidence of nhftr further. we analyzed the immediate transfusion reactions of 175 adult patients with b-thalassaemia major transfused with a total of 32.990 rbc units from january 1998 to december 2003. all units were fresh, stored less than 7 days. 11.950 units were lr-rbc, 8.370 units were ld-rbc and 12.670 were washed lr-rbc. results: the incidence of fnhtr and allergic reactions in patients receiving lr-rbc was 0.16% and 0.35%/per unit respectively, in those receiving ld-rbc was 0.10% and 0.36%/per unit respectively, while in those receiving washed lr-rbcs was 0.10% and 0.25%/per unit respectively. the relative risk (rr) of fnhrt and allergic reactions following transfusion of ld-rbc and washed lr-rbc compared to lr-rbc is shown in table. conclusion: prestorage leucodepletion and washing of rbcs reduced the risk of fnhrt in regularly transfused b-thalassaemia patients 1.6 times compared to poststorage filtration. these findings show that fnhtr after rbc transfusions are due not only to alloimmunization but also to accumulation of bmrs even in patients transfused with fresh rbcs. washing is as effective as prestorage leucodepletion in reducing fnhtr. prestorage leucodepletion has no effect on allergic reactions or other immediate adverse events in these patients. t-pa-078 viral inactivation/elimination of plasma derived medicinal products the safety of medicinal plasma products (mpps) relies on a whole range of measures from the quality of the source material to the release of the products after manufacturing under cgmp conditions. viral safety relies on careful donor selection, viral testing of the source material and viral inactivation and/or elimination during the manufacturing process. mpp manufacturing processes must include viral safety steps capable of inactivating, and/or eliminating, a large range of viruses covering the known blood borne viruses as well as anticipating possible future pathogens. it is recognized that one single step is often not sufficient to satisfy this requirement and manufacturing processes very often include two or even three complementary viral safety steps. very efficient methods have been implemented by manufacturers, for two decades, for the inactivation of major blood borne enveloped viruses (hiv, hcv and hbv). additional safety steps have also been introduced to provide a second step for enveloped viruses and to extend the efficacy to nonenveloped viruses (hav and parvovirus b19). pasteurisation (liquid heat treatment at 60°c) which has been historically used for viral inactivation of albumin solutions has been applied to some other plasma products. solvent-detergent (sd) treatment which is specific to enveloped viruses is used primarily for coagulation factors. since the introduction of sd-treated products, no hiv, hcv or hbv transmission has been reported. viral inactivation of coagulation factors can also be achieved using various conditions of dry-heating. acidic treatment is also an efficient means of inactivating viruses in igg products. nanofiltration using filters of less than 50 nm pore size was introduced in the early 1990s. this technique for viral elimination is based on the size of the agent and is independent of their resistance to other treatments. this property could be helpful in cases of new emerging pathogenic agents. new inactivation tech-niques are currently under development such as uv treatment or gamma irradiation with efficacy reported on enveloped as well as non-enveloped viruses. these new techniques can complement existing methods after careful validation that they do not have harmful effects on proteins in the product. the efficacy of existing techniques is well documented in controlled clinical studies and pharmacovigilance records. their application to each product is extensively validated at laboratory scale, according to international regulations and then carefully evaluated by health authorities. in this context, a recent european guideline established a viral risk assessment model to quantitatively estimate the theoretical safety margins of mpps, by taking into account the different safety measures, such as viral testing of plasma and the efficacy of viral inactivation/elimination steps. whilst technical limitations and some lack of scientific data lead to very conservative estimates, this model gives an overall assessment of the efficacy of the measures in the manufacture of a given product. developments in viral inactivation/elimination methods, in plasma testing as well as in evaluation procedures have together given mpps an excellent level of safety never previously achieved. to date the important safety measures needed to ensure a high safety margin to pooled plasma products are well understood by the plasma fractionation industry. safety nets rely on carefully done: donor selection to exclude high-risk donors, serological and nat viral testing of single donations and, pooled plasma testing using sensitive validated methods, and most particularly, efficient viral reduction treatments that must be validated and implemented at a large-scale following good manufacturing practices. over the last 25 years, successive key breakthrough in plasma product viral safety have included the use of solvent-detergent treatment to inactivate lipid-enveloped viruses, and nat testing of starting plasma pools and viral nanofiltration of products to reduce the risks associated to small non-enveloped viruses. the excellence of the system currently in place is illustrated by the demonstration that these safety barriers have virtually stopped the transmission of known viruses and avoided that of 'emerging' agents, such as west nile virus (wnv). however, multiple viral reduction treatments have generally decreased product recovery. in addition, although the implementation of viral reduction treatments have forced fractionators to introduce significant changes to product manufacturing methods, this period has been understandably followed by a period of relative conservatism of the plasma fractionation industry against further process changes. as time evolves and market dynamics changes struggle for improved economic balance of the plasma product industry is putting product recovery and diversified product portfolio at the forefront of r&d objectives. these developments in the plasma fractionation scene of the western world have been taking place in a context where many patients in the developing world are still treated with sub-standard, non-virally inactivated crude plasma fractions. in this specific area, one can expect that the developing world will bring innovative thoughts and take actions to find ways to improve the quality and safety of their own local plasma product supply. safety strategies adapted to the infrastructure and economy of less solvable countries may have to be considered. the intercept blood system for plasma uses a synthetic psoralen, amotosalen hcl, and long-wavelength ultraviolet light to photochemically inactivate a broad spectrum of bloodborne pathogens in plasma intended for transfusion (intercept plasma, i-ffp). phase 3 clinical trials have shown that i-ffp retains proteins necessary for hemostasis in the treatment of acquired and inherited coagulopathies, and in support of therapeutic plasma exchange for ttp. a prototype plasma processing set was used for the clinical trials. for commercialization, a new processing set has been developed to improve productivity. the prototype set accommodated approximately 250 ml of plasma, whereas the improved set accommodates up to 635 ml of plasma, resulting in up to three i-ffp doses per treatment. aims: this study was designed to characterize pro-and anti-thrombotic proteins in i-ffp prepared using the improved processing set. proteins of interest included components of the intrinsic and extrinsic coagulation cascade, the fibrinolytic pathway, the contact factor pathway, and the complement system, the vonwillebrand complex, endogenous inhibitors, and markers of thrombin generation. methods: six fresh jumbo (600 ml) apheresis plasma units, collected using the haemonetics pcs device (gambro), were photochemically treated. sodium citrate was used as the anticoagulant. plasma samples for analysis were collected before and after photochemical treatment, and were frozen below -60°c until batch analysis. standardized clinical assays were used for all analyses. results: (results in the table below are expressed as the percent activity in i-ffp in proportion to the activity in plasma before treatment [mean ± sd]). retention of procoagulant factors in i-ffp plasma ranged from 77% to 92%. factor viii and vonwillebrand factor activity, antigen, cleaving protease activity (vwf : cp, adamts-13), and multimeric composition remained within normal ranges after treatment. endogenous inhibitors of coagulation were retained 93% to 100%. plasminogen and alpha 2-antiplasmin were retained 94% and 78%, respectively. retention of contact factors was variable; some factors were below the reference range prior to pct. with the exception of tat, all markers of coagulation activation were well within normal ranges. the tat level in one i-ffp unit was slightly above the normal range; all other units had tat levels that were well within the normal range. the significance of this is unclear. cept plasma is similar to conventional plasma. the improved processing set, intended for commercialization, allows up to 3 doses of i-ffp to be produced from a single photochemical treatment. background: guidelines of the european directive 2004/33/ec require that fresh plasma prior to freezing contains <6109 residual rbcs per litre. this rbcs content is below the sensitivity limit of the automated cell counters used in routine laboratories. aim of the study: it was therefore essential to make available an alternative method to detect and quantify rbcs in plasma. we implemented a method by flow cytometry using a pe conjugated anti-glycophorin a (gpa) monoclonal antibody that recognises rbcs and erythroid precursors. to quantify residual rbcs in fresh plasma, the method uses the same trucount test tubes (becton dickinson) as those used to quantify wbcs and that contain a known number of fluorescent beads. after addition of plasma and pe-gpa antibody, cell counting is performed on flow cytometer (bd facscalibur). validation of the method: assessment of accuracy, linearity, and reproducibility with 2 different pe-gpa antibodies (immunotech and pharmingen) application of the method: quantification of rbcs in 105 fresh plasmas divided into 3 groups: group 1: 45 plasmas from leucoreduced whole blood, group 2: 30 plasmas from packed cells after removal of buffy coat and specific filtration, group 3: 30: apheresis plasmas. results: validation of the method: for both anti gpa antibodies, detection threshold is 0.05 ¥ 10 9 residual rbcs/l; linearity study with concentrations of 0.1, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0 ¥ 10 9 rbcs/l showed excellent correlation between observed and expected values (r > 0.99); reproducibility study showed c.v of respectively 5.7% and 6%. for values >2 ¥ 10 9 rbcs/l, it appeared necessary to introduce a correction factor of 1.3 for the anti gpa pharmingen. quantification of rbcs in plasma: group 1 (plasma from leucoreduced whole blood): 0.39 10 9 ± 0.23 rbcs/l; group 2 (plasma from packed cells after removal of buffy coat and filtration): <0.05°¥ 10 9 rbcs/l in all the cases; group 3 (apheresis plasma): <0.05°¥ 10 9 rbcs/l in all the cases. conclusion: quantification of rbcs in plasma by flow cytometry is a precise, quick and reproducible test. in addition this study shows that even if there are differences in residual rbcs counts according to the origin of plasma, the obtained values are much lower than regulatory requirements. 1. improving basic transfusion knowledge amongst health workers; 2. improving pre-operative preparation for surgery 3. strategies such as cell salvage autotransfusion combined with a conservative transfusion strategy for the use of allogeneic blood. my presentation will outline the approach adopted to ensure that the use of cell salvage autotransfusion both improves the use of allogeneic blood and preserves allogeneic stores. well-organised training can both minimise the risk of using such advanced techniques and decrease the overall risk involved in undergoing surgery where blood loss may be a significant factor in increasing morbidity and mortality. the various training methods employed to improve knowledge in this area will be described. the increasing current perception that the safety of allogeneic blood transfusion has dramatically been improved during the last decade is challenging autologous haemotherapy methods. in addition, growing concern about the unfavourable cost-effectiveness of most autologous haemotherapy methods requires a refinement of the application of these measures to well defined circumstances. in contrast, newly emerging transfusion-transmissible infections or periods of blood shortage might revive interest in these blood sparing techniques. the first two cases of transfusion transmitted vcjd provide a paradigm for this scenario; not so much with respect to a public fear of infection but rather a waning donor population due to more rigorous recruitment criteria. preoperative autologous blood donation (pabd) still plays a significant role in settings with high individual benefit for the patient, high transfusion probabilities and when all opportunities of cost minimization can be applied. adjustment to the individual situation of the patient is the main aim of a medically reasonable and economic use of autologous haemotherapy. this implies consideration of the patient's haematocrit, blood volume, tolerable blood loss, expected blood loss, etc. in order to choose the optimal method in the individual case. in this respect, double red cell apheresis may play a significant role. with this approach, donation schedules assumed to enhance erythropoiesis can be adopted. moreover, inconveniencies caused by long distances between patient home and donation service can be facilitated by withdrawing two red cell units during one session in selected patients. in conclusion, red cell apheresis can be used to promote the proposed approach towards individualized autologous haemotherapy preoperative plasmapheresis is considered to be a sensible adjunct if intraoperative retransfusion of salvaged and washed red cells is planned. acute normovolaemic haemodilution is valuable when the patient's tolerability of the haemodilution and the expected blood loss are carefully examined beforehand. intraor postoperative salvage of wound blood can also be regarded as useful measures to prevent allogeneic transfusions as long as the specific advantages and disadvantages of the different methods are taken into account. finally, alternative and supplemental measures such as iron or erythropoietin administration should always be considered in order to optimize the efficacy and effectiveness of autologous haemotherapy methods. the goal of a 'bloodless medicine' might not be reached but is supposed to be approached closely with an integrated concept exploiting all measures available. however, in times of restricted health care resources, regular sound costeffectiveness analyses, taking the availability and the cur-rent safety profile of allogeneic blood products into account, are always warranted and needed. compensatory fluid replacement of surgical blood losses: the transfusion of allogeneic blood is expensive and -although safer than ever before -still associated with potential complications. to reduce both, costs and immanent risks, allogeneic transfusion should either be completely avoided or at least minimized during surgical procedures. as a consequence an intraoperative blood loss is initially not replaced by red blood cells, but by erythrocyte-free, i.e. cristalloidal or colloidal solutions. when normovolemia is maintained the resulting dilutional anemia is compensated by an increase of cardiac output and enhanced arterial o2 extraction. however, once the hb has dropped to values recommended as the lower intraoperative limit, or once compensatory mechanisms of acute anemia become exhausted, as a rule transfusion of red blood cells (rbc) is initiated to increase arterial oxygen content (cao2) and to preserve a margin of safety for tissue oxygenation and organ function. as an alternative to immediate rbc transfusion, ventilation with pure o2 (hyperoxic ventilation) can be employed to rapidly raise cao2 by increasing the amount of physically dissolved o2 in plasma (hyperoxia). however, molecular o2 causes vasoconstriction, mediated by products of the arachidonic acid metabolic pathway. as a consequence hyperoxia has been shown to increase systemic vascular resistance and to decrease cardiac output and o2 consumption in subjects with normal hemoglobin concentration ( properly scheduled, three women who did not reach the necessary hct level after the first donation and consequently they got out of the protocol, and one woman who had unexpected intraoperative bleeding and received 5 homologous units in addition. the 3 patients undergoing tkr and the 11 patients undergoing removal of implants predeposited 9 and 21 units respectively, but finally 3 and 12 of them have been used. according to our patients data, the 55.5% of unused autologous blood units belongs to patients with tkr and implant removal. therefore a better schedule is needed for these type of surgery. all the autologous donors were supported by oral iron supplementation throughout the predonation and 1 month past surgery. fourteen of our cases were supported by erythropoietin s.c. in a dose of 300 iu/kg every other day. the majority of these patients was female, only one was male with multiple alloantibodies and was scheduled to predonate 5 autologous units. all of them underwent thr except one woman who also had a tkr 6 months later. the autologous blood donation was well tolerated by all patients and only one woman had a reaction during predonation. furthermore a group of 98 patients matched for age, sex and type of surgery, who did not predeposit blood, received a mean of 3.2 homologous units per patient, that is more than the patients on pabd program. our results show that autologous transfusion can be used in scheduled orthopedic surgical procedures and can reduce the need for homologous blood. however, every effort should be made to render the practice of pabd more efficient and to minimize its costs. colloid solutions and their establishment in clinical practice background: various situations like trauma, critical ill patients, sepsis, major surgical procedures and anaphylactic reactions are associated with disturbances in fluid homeostasis. this disturbance is related with reduced oxygen delivery, subsequent lactic acidosis and imbalance in oxidative status. the final result will most likely be an increased mortality and morbidity. aim: the important issue from clinical aspect is to define the optimal volume and type of fluid therapy. the debate for the ideal resuscitation solution lasts a couple of decades due to inconclusive and conflicting results. method: we searched the literature for clinical trials and major met analyses concerning patients undergoing scheduled surgical procedures, trauma patients and critical ill who received resuscitation fluids. results: dextrans reduce blood viscosity and von willebrand factor levels more, for the same degree of hemodilution, compared to other plasma expanders. in clinical setting they are effective in reducing the incidence of deep vein thrombosis and pulmonary embolism. after the initiation of dextran 1 for prophylaxis against anaphylactic reactions, they are considered the safest plasma substitutes, except maybe during pregnancy. dextran 40 10% is the most like to cause the 'hyperoncotic acute renal failure' syndrome. gelatins also cause a decrease in circulating levels of vwf : ag, vwf r : co, thrombin-antithrombin complexes and f1+2. in clinical aspect however, there are contradicted results about the effect of gelatins in bleeding diathesis, although they appear to exert a greater effect on rbcs protection from mechanical stress. in respect to anaphylactoid reactions, they have the greatest relative risk. hes has the same effectiveness in volume expansion with albumin and has the advantage of remaining intravascular even if there is an increased capillary permeability. in addition, it may improve splanchnic blood flow and tissue oxygenation. hes130/0.4 subsides the inflammatory response in patients undergoing major surgery, compared to a crystalloid-based volume therapy, but has conflicting results about it's effects on neutrophil respiration burst. clinical trials have so far failed to have a unanimous conclusion about the bleeding diathesis after hes administration, especially. caution must be held when administering hes during renal transplantation. albumin became the scapegoat of transfusion strategy during the past 20 years. resent met analysis have contradicted results about the safety of albumin infusion in variouw settings. a positive effect seems to have the early administration of hypertonic solutions in trauma patients, especially in combination with dextrans. conclusions: although some minor conclusions can be extracted, there is still a great lack of large scale multicentre randomized prospective clinical trials for extracting evidence based criteria. instead, we try to extract conclusions through met analysis. results show no evidence that resuscitation with colloids reduces the risk of death compared with crystalloids in patients with trauma, burns, major surgery or sepsis. also, there is lack of evidence that one colloid solution is safer -in clinical aspect -than any other. recombinant human erythropoietin therapy in critically ill patients -a dose response study* objective: the aim of our study was to assess the efficacy of two dosing schedules of recombinant human erythropoietin (rhuepo) in increasing hemoglobin (hb) level and reducing the exposure to red blood cells (rbc) transfusion in critically ill patients. design: a prospective, randomized, multicenter trial. patients: a total of 148 patients who met eligibility criteria were enrolled. intervention: patients were randomly assigned to receive intravenous (i.v.) iron saccharate alone (control group), i.v. iron saccharate and subcutaneous rhuepo 40 000 units once per week (group a) and i.v. iron saccharate and subcutaneous rhuepo 40 000 units three times per week (group b). rhuepo was given for a minimum of 2 weeks or until icu discharge or death. the maximum duration of therapy was 3 weeks. the requirement for rbc transfusions was significantly higher in control group than that in group a and b. no significant difference was observed between group a and b. the mean increase in hematocrit (dhct) and hb (dhb) from baseline to final measurement were significantly higher in group b than these in control group. dhct was significantly higher in group b than that in group a. dhct in group a was significantly higher than that in controls, whereas dhb did not differ significantly between control and group a. conclusion: administration of rhuepo in critically ill patients significantly reduced the need for rbc transfusions. the magnitude of the reduction did not differ between the low and high dose of rhuepo, whereas there was a dose response of hct and hb to rhuepo in these patients. in transfusion medicine, antibodies to antigens in the platelet membrane have traditionally been regarded as less significant compared with antibodies towards red cell antigens. there is an increasing awareness of antibodies towards platelet antigens. detection of autoantibodies to platelets can be a diagnostic challenge, but is seldom a problem in transfusion medicine because patients with such antibodies rarely are candidates for platelet transfusions. also, severe foetal thrombocytopenia is seldom present in pregnancies with autoantibodies to platelet antigens. the real challenge in transfusion medicine is related to patients with severe thrombocytopenia who are refractory to platelet transfusion due to alloantibodies towards platelet antigens. in our department, flow cytometry is used for compatibility testing and the choice of compatible blood donor is done without knowledge of antibody specificity. if crossmatch negative random donors cannot be identified, antibody specificity testing is performed and donors are chosen based on the specificity of the antibodies determined by a modified maipa procedure and with hla class i beads (flowpra from one lambda, usa) in flow cytometry. in some cases both hla class i and human platelet antigen (hpa) specific antibodies are detected and hla class i, hpa matched donors are chosen for crossmatch. if the crossmatch is negative, there is >90% chance of successful transfusions. in some cases drug induced anti-platelet antibodies are suspected and flow cytometry based antibody tests are performed in the presence and absence of the drug. in the case of suspected heparin induced antibodies, a beads assay is performed (diamed, switzerland). two percent of caucasian women have the platelet type hpa 1bb. ten percent of these women make anti-hpa 1a antibodies in their first hpa 1a incompatible pregnancy. 1 in 1000-2000 new-born has thrombocytopenia due to maternal alloantibodies which have crossed the placenta (neonatal alloimmune thrombocytopenia, naitp). results from a screening study covering the outcome of 100 000 pregnancies show that only 2 babies were born with intracranial haemorrhage (ich) and there was no still-born babies in the study. pregnant women with a-hpa 1a antibodies were diagnosed, received careful clinical follow-up and the delivery was performed by caesarean section in week 38 of the pregnancy with immediate transfusion of hpa compatible platelets if the new-born had platelet count <35 ¥ 10 e 9 /l. in previous studies, it is reported the ich appears in 10-30% of the pregnancies where antibodies are present and that 50% of the babies with ich, die. our results are different from what is reported from other studies and this may reflect the prospective approach and the clinical interventions. naitp represent a challenge in transfusion medicine both diagnostically, but also related to compatible blood products for the thrombocytopenic new-born and the mother who may have high level of antibodies towards platelet antigens. methods: apheresis platelets (4 ¥ 10e 11 mean) from donors with same blood group were pooled and divided equally into two bags, po-80 and control (pl2410, baxter), which have 2660 and 2024 ml/m 2 *day*atm of oxygen permeability, respectively. on days 0, 1, 3, 5, 7, and 9 of storage, swirling, mean platelet volume, po2, pco2, ph, glucose, lactate, aggregation, and p-selectin expression were evaluated. six experiments were performed. results: the swirling pattern was preserved better for up to 9 days in po-80 (6/6) than in control (3/6) bags. dropped ph less than 6.2 on day 9 was observed 1/6 in po-80 whereas 3/6 in the control. aggressive drop of glucose (0 mmol/l) with prominent lactate accumulation (327 mg/l) was also observed on day 9 in 1 of control bags. the po2 level in the control dropped more significantly by 45% (46.6 mmhg) on day 3 than in po-80 (65.8 mmhg) compared with the initial level (84.9 mmhg) (p < 0.001). these results suggest that aerobic metabolism of higher concentration platelets was maintained better in a container with higher oxygen permeability. and less lactate generation with slower glucose consumption is also suggested in po-80 bags than in control bags. the %hsr and aggregation decreased gradually in a similar manner in both bags until day 7, and became a detrimental defect in 2 of 6 control bags on day 9. p-selectin expression was higher in control bags than in po-80 on days 7 and 9 with no statistical difference. in two control bags p-selectin expression reached >84% and was accompanied by a loss of swirling. these functional and biochemical characteristics of platelets at a higher concentration were kept better for 7-9 days when stored in a container with higher oxygen permeability than in the best of marketed containers. t-pa-096 background: maintenance of a neutral ph in the range of 6.8-7.4 is essential for preservation of platelet function and viability during storage. furthermore, studies have also indicated that the presence of glucose in the platelet suspending medium is important for maintenance of platelet quality. however, a platelet additive solution (pas) containing glucose having a ph of 6.8-7.2 cannot be manufactured by steam sterilization due to caramelization of glucose. in order to have optimal ph, the currently available pas such as t-sol does not contain any glucose. this study describes a novel twostep approach to provide a glucose containing additive solution (pas-g) by using an acid, glucose containing electrolyte solution (ph 5.5) for resuspension and processing of the pooled buffy coats (bc), followed by transfer of the processed platelet concentrate (pc) into a storage bag containing bicarbonate for ph neutralization and maintenance during extended storage. aim: compare the platelet quality of pooled bc pc stored in pas-g with pooled bc pc stored in t-sol. methods: a paired study design was used, where a pool of 8 bcs obtained from standard day 1-old cpd-wb units, was divided into two equal parts: one part was resuspended and processed with a pall leukoreduction system (atsbc) using t-sol, the other part was processed in a similar manner with the acid part of pas-g. percentage plasma carryover ranged from 20-35%. both processed pc products were transferred and stored in elx tm bags with the pas-g pc elx bag containing a bicarbonate tablet. ten replicate studies were performed. results: the yields (3.2 ± 0.3 vs. 3.2 ± 0.4°¥ 10e 11 ) were similar (pags vs t-sol). statistically significant (p < 0.05 with paired ttest) improved platelet quality at days 5, 7, and 9 of storage was observed with platelets stored in pas-g as compared to t-sol. the table below shows results at 7 and 9 days of storage for ph, extent of shape change (esc) and hypotonic shock response (hsr). the results for t-sol stored platelets correlated highly with initial glucose (% plasma carry over) level (r = 0.91 for esc, and r = 0.68 for hsr at 7 days storage), while no significant correlations were found for pas-g stored platelets. conclusion: this study demonstrated the practicality of using a two step procedure to store bc pc in a glucose containing additive solution with neutral ph during storage, and confirmed the importance of glucose in the storage medium as nutrient for optimal platelet storage quality. the effect of irradiation on white cell reduced platelet concentrates, stored for 7 days background: the storage of white cell (wbc)-reduced platelet concentrates (pcs) can be extended from 5 to 7 days provided the quality has been validated and bacterial screening is performed. irradiation up to 50 gray (gy) does not affect platelet quality, but the effect of pre storage irradiation with subsequent storage up to 7 days is not known. method: two wbc reduced pcs, each made from 5 buffy coats and a unit of plasma, were pooled and divided into control group 'a' and study group 'b' . pcs in group 'b' were irradiated immediately after preparation with 25 gy. pcs in both groups 'a' and 'b' were then stored on a continuous flat bed shaker at 20-24°c. swirl, ph and cd62p expression were determined on day 1, 6 and 8. twelve experiments were performed and compared with a paired t-test, p < 0.05 was considered significant. results: see table (day 8 values; mean ± sd; n = 12). pooling and dividing of the pcs was successful with respect to volume and platelet number. on day 8, the ph in group 'b' was slightly lower than in group 'a', but the difference is not significant. in group 'a', ph on day 8 was <6.8 in 3/12 pcs, versus 5/12 in group 'b' (not significant). the cd62p expression in irradiated pcs is not significantly higher than in non-irradiated pcs. conclusion: irradiation had no significant effect on platelet quality when stored for up to 7 days after blood collection. -1b, -3a, -5b, pra 63%, 12 donors. in patient 3 -three transfu-sions were effective (two crossmatches neg by lct and pift, one pos lct, neg pift) but later on, when the patient was in severe clinical status (shortly before his death) two transfusions were ineffective in spite of neg crossmatches. it is very likely that for the same reason patient 4 and 5 were refractory to two and one hpa compatible platelet units respectively. in patient 6 and 7 compatible platelets were not transfused because they died before the whole procedure (diagnosis and finding a proper donor) was completed. conclusions: 1. the frequency of occurrence of anti-hpa antibodies in transfused patients was: -5b, -1b, -2b, -3a, -1a; in three patients they were monospecific, in four polyspecific. 2. in 2 patients who developed anti-hpa alone, transfusions of platelets without relevant hpa antigens were successful. 3.the effectiveness of compatible platelets in patients with both anti-hpa and -hla was more difficult to assess because of their severe clinical status, which might have been responsible for transfusion failure. in one of these patients, however, the transfusions of compatible platelets were successful when he was in relatively good clinical status, but shortly before death transfusions were ineffective. t-pa-099 the successful implementation of nucleic acid testing (nat) for hiv, hbv, hcv and further viruses as well as improved donor selection led to a dramatic risk reduction for viral transmission via blood transfusion over the last years. today, other risks get into the focus of haemovigilance. bacterial contamination of blood products can occur via the donor, suffering from a (clinically unapparent) bacterial infection, or via the donation process itself, storage and handling of the blood product. particularly platelet concentrates (pc) are vulnerable to bacterial growth due to their storage conditions. patients receiving such products have a potential risk of severe complications or even death. modern hygiene regimes, e.g. improved disinfection of the donors´ skin or preparation of blood products in fully closed systems as well as diversion, led to a significant reduction of bacterial contamination risk in the past. however, a small risk remains. therefore, two possible ways of further reducing the risk of bacterial contamination of blood products are feasible: (a) testing and/or (b) inactivation. testing for bacterial contamination is possible by different methods: direct detection methods for bacteria (microscopy, flow cytometry) have disadvantages regarding sample size and detection limit. bacteria might rapidly grow in a contaminated pc, so testing should be performed as close as possible to transfusion to the recipient. biochemical methods like oxygen consumption might not detect anaerobic germs. automated culture methods are still the most sensitive technique, but they have their downsides as well (e.g. time and size of aliquot drawn). novel molecular genetic test methods for detection of bacterial nucleic acid are in different states of development, but still have to proof their suitability for routine use. three different principles of pathogen inactivation can be distinguished: photodynamic reactions produce oxygen radicals which in turn inactivate bacterial structures by oxidation processes. examples for these chemicals are phenothiazines like methylene blue and thionin as well as vitamins like riboflavin (vitamin b2). photochemical reactants penetrate cell boundaries and irreversibly inhibit nucleic acid, thus blocking replication and proliferation of pathogens. chemicals of this group are psoralens like amotosalen as well as pen-110 or s-303. the third method, the solvent detergent (sd) method, is used for pooled plasma only and consists of the combination of both solvents and detergents, which interact with membranes and destroy bacteria. methods for inactivation of bacterial contaminants have to proof, that they effectively inhibit bacterial growth while maintaining full functionality of the blood product at the same time. these two qualities have to be fulfilled up to the end of the storage period. toxic or mutagenic compounds must not remain in the final product. the technology must be easily integrated into the existing work cycle of a blood bank. finally, costs per product must be acceptable. in summary, both testing and inactivation have their advantages and disadvantages, which have to be weighed up against costs and benefits of both procedures. pros and cons of introduction of inactivation methods in a blood donor service producing 1 600 000 blood components per year will be discussed. bacterial contamination of blood components, particularly platelets, is now recognized as a serious adverse reaction that is preventable. there are many studies that have documented that these reactions occur from platelets stored at room temperature, most commonly arising from a skin contaminant, but originating from donors with asymptomatic bacteremia in about 1/3 of cases. the problem is intensified for patients receiving pools of platelets compared to single donor platelets collected by apheresis. reactions are more commonly noted and more severe with platelets stored for greater lengths of time. previous studies at johns hopkins described a series of 23 reactions in 12 years with reactions more common in platelet pools (1 : 1600 transfusions) than with single donor platelets (1 : 15 000 transfusions). these reactions caused fatalities in 4 of 23 cases. although our data suggests that these reactions are more common than other studies using hemovigilance systems, our case definition requiring culture of all transfusion reactions to platelets led to a more reliable estimate of the incidence of sepsis from platelets, many potential solutions have been proposed to prevent these reactions. improved skin antisepsis should always be sought but will never eliminate the 1/3 of reactions due to asymptomatic bacteremia. the same limitation applies to methods that divert potential skin plugs from the collection bag. antibiotics in the bag would lead to manufacturing concerns or problems for patients with drug allergies. although cold storage of platelets is currently being revisited, it is not yet a practical solution. pathogen eradication systems have been developed but they remain unapproved in most of the world and have led to concerns about toxicity of additives, damage to treated cells, and cost. as a result of increasing recognition of the problem, there has been increased interest in bacterial screening to prevent sepsis from platelets. in march 2004, the aabb standards required testing of platelets for bacterial contamination. testing programs have been implemented in the us widely as a result of the aabb action. licensed systems based upon bacterial culture or growth characteristics are available for single donor platelets and have been commonly employed. although these systems have some difficulty with false positive reactions, the evolving national data suggest an incidence of 1 : 5000 true positive reactions. these data suggest that a number of serious reactions have been averted, although some cases have persisted due to incomplete adoption, problems with slow growing bacteria, or the use of inferior testing methods with inadequate sensitivity. whole blood derived platelets have become a more difficult issue, since the approved testing methods are limited. the use of ph monitoring, gram stain, glucose measurements, and inspection for swirling have all been attempted. these methods are not sufficiently sensitive or specific to interdict many contaminated units, so that screening for bacteria in pooled platelets is less effective. it is anticipated that new methods may become available to make screening of whole blood derived platelets easier to perform in a reliable manner. it is also hoped that bacterial screening of platelets may form the basis to permit seven day storage and prestorage pooling in the us. results: twenty four hcv rna (+)/anti-hcv(-) repeat donors were previously tested in routine hcv rna in mini-pools and were negative. twenty available look back samples were individually tested for hcv rna and in one the virus was detected. to make sure that the failure of hcv rna detection in routine nat was not due to the pooling procedure, the hcv rna was tested in undiluted look back sample and dilutions of this sample by hcv negative plasma: 2/2 repeats of 48x dilution and 2/2 repeats of 24x dilution were hcv rna negative, whereas 1/1 repeat of 8x dilution and 2/2 repeats of undiluted sample were positive. the results of cobas amplicor monitor (sensitivity 600 iu/ml) were negative, which means that viremia in hcv rna mini-pool negative donation was below 600 iu/ml. in the recipient of red blood cell concentrate from this donation hepatitis c was diagnosed. however, the possibility of pretransfusion hcv infection cannot be excluded as no hcv marker tests were performed before transfusion. the patient and the donor were infected with genotype 3a. the low hcv viremia (below 600 iu/ml) in the preseroconversion window period was responsible for no hcv rna detection in routine mini-pool hcv rna testing. introduction and aim of the study: bacterial contamination is a life threatening risk of blood transfusion, especially with platelet transfusions. bacterial culturing (bc) of platelets as well as pathogen reduction (pr) reduce the likelihood of such contamination. where the costs of bacterial contamination are far less than the costs of pathogen reduction, the latter will reduce not only the risk of bacterial contamination but also risks of other pathogens. therefore, the question arises whether this additional expenditure can be justified in the light of the additional effect achieved. this question we will answer by cost-effectiveness and sensitivity analyses. methods: the balance between costs and effects of preventing adverse events due to platelet transfusion is assessed using a mathematical model and assuming optimal effectiveness of pr. model parameters and valuations of health states were obtained from literature and information from dutch sanquin blood banks. . while the estimates in comparison to the situation without bc or pr are surrounded with large uncertainties, the conclusion that pr is not cost-effective in comparison to bc is very robust. the cost-effectiveness of bc and pr are very sensitive to the estimates concerning sepsis probability and associated complication rate, the cost-effectiveness of pr relative to bc is not. this conclusion is also insensitive to a wide range of assumptions regarding residual risks and costs associated with hiv, hcv and hbv. the estimates indicate that culturing in the netherlands is cost-effective, even with the deviation bag in place. the estimates however appear to be very sensitivity to the probability of sepsis. a decision to use pr will, after the introduction of bc and the use of a deviation bag, never meet cost-effectiveness criteria. even when assuming perfect protection, the conclusion that it is not cost-effective in comparison to bc is very robust and does not alter when varying underlying parameters within their margins of uncertainty. table) . of cb collection. two collections have been transplanted to date and this represents a 2.5% take-up rate (3.5% where a sibling is alive). this compares favourably with the numbers transplanted from unrelated cb banks. dcb collection is therefore at least as efficient a method as unrelated cb collection for transplantation albeit in the limited number of cases where a dcb collection is possible. dcb collection has the benefit of a possible immediate transplant combined with the availability of a sibling donor for future donation of both stem cells and lymphocytes. it is therefore a useful service to provide and complements the work of unrelated cord blood banks. increased yield of mature platelets in cultures of cd34-enriched cord blood cells maintained at 39°c introduction: the future use in transplantation of ex vivo expanded hematopoietic stem (hsc) and progenitors cells will facilitate the transplantation of adult patients and speed up hematologic recovery. also ex vivo cultures of hscs may eventually permit to produce donor-free blood components such as platelets for transfusion. culture of animal cells is routinely done at 37°c. however there is previous clinical evidence suggesting that hematopoiesis may be more active in hyperthermic patients. we have therefore compared the effect of hyperthermia on the ex vivo expansion and differentiation of cord bloodderived hsc in megakaryocytes (mk) and mature platelets. the cord blood-derived cd34 cells were cultured continuously at 37°c or 39°c for 14 days in cytokine conditions optimized for mk development and maturation. the cultures were regularly monitored for various parameters. results: compared to 37°c, the cultures maintained at 39°c produced significantly more total cells (4.9 fold) and total mks (7 fold), and showed accelerated and enhanced mk maturation with increased yield of proplatelets and mature platelets (16.6 fold). accordingly, the cells cultured at 39°c contained an increased frequency of cfc-mk (8 fold) at day 14. cultures done at 38°c and 40°c were also more efficient than at 37°c but less than at 39°c. platelets produced in 39°c cultures could be normally activated by thrombin. as expected, the cells cultured at 39°c contained an increased amount of the heat shock protein hsp70. control experiments showed that the culture of several cell lines was inhibited or unaffected by the 39°c temperature. the unexpected resistance of hematopoietic cells to the deleterious effects of heat and the stimulatory effect of >37°c temperatures on hsc proliferation and differentiation indicate that the routine culture of normal human cells at 37°c is a paradigm that needs to be revised. the responsible molecular mechanisms remain to be identified but the observation will facilitate the ex vivo expansion of the progenitors of the mk and possibly other lineages. the synchronous generation of a significant number of mature platelets in vitro will facilitate the study of the mechanisms of platelet formation and ageing and could eventually have important applications in transfusion medicine. it remains to be seen if the stimulatory effects of higher than 37°c temperatures represent a protective response against sustained body fever that is specific to the hematopoietic system. several countries have, in the past few years, included human tissue banking within a regulatory framework similar to that of blood. indeed, tissue safety has come to the forefront of the preoccupations of regulatory agencies after several well publicised morbidity and mortality cases have been reported in the press. tissue safety has many features similar if not identical to blood safety and a review of those common elements will be reported. as well, arguments in favor of integrating tissue banking within a blood system will be discussed, one of the more important aspect of which being the expertise of the blood centre staff with cgmps. the experience of a blood establishment (héma-québec) with the integration of tissue banking such as bone, skin, heart valves within its operations will be reported, emphasizing the medical as well as the management aspects of such an integration. finally, tissue banking is an activity which brings more expertise to a blood centre, expands its knowledge of its customers and gives more opportunities to its personnel. umbilical cord blood (cb) is an important source of stem cells for clinical transplantation and may cause less gvh disease than non-t-depleted bone marrow (bm). the relatively low numerical cell dose available from cb has usually restricted its use for transplantation in adults. only 25-30% of patients have an hla matched sibling and for others an unrelated bm or a stored unrelated cb donation may also not be available. for some children the collection of cb following the birth of a sibling may be the only opportunity for a transplant. directed cb (dcb) donations from matched siblings have been shown to give better long-term overall results than matched unrelated cb or bm. dcb collection is however not as easy to control as cb for banking where dedicated hospitals and trained staff are used. here we review dcb banking in oxford over a 2.5-year period. requests were received for 88 deliveries from 84 mothers and of these, 81 collections were successful including 4 pairs of twins. failed collection was most often due to a damaged cord at delivery. 61 collections were made for 57 siblings possibly requiring transplant (median age 4) with 24 for leukaemia, 16 for erythroid disorders, 13 for immune deficiency, 5 for enzyme deficiency and 3 others. the remaining 20 collections were mostly requested where there was a family history of an inherited disorder (majority scid). three collections tested positive for anti-hcv antibody but negative for hcv by pcr. collections were not excluded on the basis of volume or cell number. mean volume was 80 ml (range 22-174, 86% exceeded 40 mls) and mean tnc count was 1.0 ¥ 10 9 (range 0.2-2.7, 58% exceeded 0.8 ¥ 10^9). the mean cd34+ve count was 3.1 ¥ 10 6 (range 0.2-19.0). all collections were cryopreserved within 24 hours using dmso/dextran/saline without volume reduction. the mean tnc viability prior to freezing was 98% (range 86-100%) and the mean cd34+ve viability post freezing was 82% (range 71-96%). the reliance on the goodwill of midwives and the logistical difficulties that arise when organising collections from many different hospitals do not appear to reduce the success . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cyclesequencing using bigdye-terminators v.1.1 in an abi310 (applied biosystems). background: a number of adverse immune reactions associated with blood transfusion result from contamination of blood products by donor white blood cells. among these reactions, transfusionassociated graft-versus-host disease (ta-gvhd) has a mortality of greater than 90%. mirasol ® pathogen reduction technology (prt) has been developed for the reduction of viruses, bacteria, parasites and white blood cells loads in blood products. the technology is based on light and riboflavin photochemistry. this study was performed in order to evaluate the effectiveness of the mirasol ® prt process for inactivation of human pbmncs. methods: human pbmncs were collected from trima platelet apheresis disposable sets, purified by ficoll-hypaque discontinuous gradient centrifugation and divided into test and control samples. the test cells were treated with mirasol ® prt in autologous plasma on day 0. both test and control samples (n = 3) were tested on day 1 for cellular immunophenotype, t-cell activation using flow cytometry, proliferation in response to mitogen or allogeneic stimulator cells, ability to stimulate the proliferation of allogeneic responder cells and cytokine synthesis in response to lps stimulation was measured using a cba assay kit. results: although mirasol ® prt treatment did not significantly change the distribution of cd3+, cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ human lymphocyte subpopulations there were significant functional change. the expression of the activation marker, cd69, was observed in 65.4% (sd = 15.6%) of control t cells upon activation with pma, while only a 1.7% (sd = 1.3%) of the test t cells increased cd69 expression. proliferation assays showed that 3h-thymidine incorporation did not increase in the test cells in response to either pha or allogeneic stimulator pbmnc compared to the significant increase in thymidine incorporation levels observed with control cells. the test cells, when compared to the controls cells, demonstrated an inability to stimulate allogeneic responder pbmnc proliferation. the release of il-10, il-6, il-1b and il-8 cytokines after 24-h incubation in culture media increased significantly to 128 pg/ml (sd = 73), >5000 pg/ml, 404 pg/ml (sd = 301) and >5000 pg/ml for control cells, respectively. under the same conditions, these cytokines in test samples remained at background levels of 0.4 pg/ml (sd = 0.7) for il-10, 2.4 pg/ml (sd = 1.2) for il-6, 11 pg/ml (sd = 3) for il-1b and 1022 pg/ml (sd = 286) for il-8. addition of lps further stimulated the release of tnf-a, il-10, il-6, il-1b and il-8 in the control samples, but not in the test cell samples. in vitro studies demonstrate that mirasol ® prt treatment does not change lymphocyte immunophenotype, inhibits tcell activation by pma, abolishes pbmnc proliferative activity, eliminates pbmnc stimulatory activity for responder cell proliferation and suppresses the production of cytokines by pbmnc in both the absence or presence of lps. introduction: it has been discovered that vaccination of dendritic cells (dcs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. aim of the study: the purpose of the study was to investigate whether human monocyte-derived dendritic cells (dcs) were able to present p210bcr-abl protein and induce antigen-specific ctl responses in vitro after transfected with total rna of k562 cells (k562-rna). methods: dcs were derived from human pbmncs, which were incubated for 5 days in the presence of gm-csf and il-4, and then were transfected with k562-rna using electroporation or dotap lipofection. to verify the successful transfection of dcs with k562-rna, bcr-abl fusion genes expression of dcs was detected by rt-pcr and western blot. the immune phenotypes of the dcs were analyzed by flow cytometry. the cytotoxicity of ctl was assayed by propidium iodide (pi) staining and flow cytometry. results: it was shown that the bcr-abl fusion gene was detected in the dcs immediately after the transfection, but disappeared 24 hours later, while the cells were expressing p210bcr-abl protein and expressing increased cd80, cd83, cd86, hla-dr. moreover, the transfected dcs could significantly promote the t lymphocytes to kill the target k562 cells. conclusion: human dendritic cells transfected with total rna of k562 cells in vitro could induce effective p210bcr-abl proteinspecific immune responses and be used to induce tumor-specific immunity, which implies potential application of immunotherapy to tumors. appear to be relevant to the clinical response. ivig has a remarkably good safety record for long term administration, however the following side effects have been observed: mild, infusion-rate related reactions such as headaches, myalgia or fever; moderate but inconsequential events, such as aseptic meningitis and skin rash; and severe, but rare, complications such as thromboembolic events and renal tubular necrosis. judicial use of ivig based on results from controlled studies is recommended. t-pl3-09 donor-lymphocyte infusion: transfusion immunotherapy following allogeneic hematopoietic transplantation the notion that bone marrow containing immunocompetent cells is capable of mediating an antitumor effect was determined experimentally almost 50 years ago. subsequently, pooled leukocytes from patients with cml were found to effect responses in patients with advanced leukemia. response correlated with cell dose and with severity of gvhd. in the 1970's, the graft-versus-leukemia (gvl) effect was defined in the transplant setting using a lethallyirradiated mouse model and splenocyte infusions. such studies suggested that gvl could be enhanced without causing severe gvhd. the era of adoptive immunotherapy in the transplant setting began in the 1990's with reports of donor lymphocyte infusions (dli) for relapsed acute and chronic leukemias after bone marrow transplant. it is now clear that chronic myelocytic leukemia (cml) in chronic phase is highly susceptible to gvl effects mediated by dli which induce durable remission in 70-80% of relapsed patients. the success rate is 30% or less in patients with accelerated phase or blast crisis. since dli cell dose appears to be important in this setting, strategies of escalating dose infusions have been investigated to enhance gvl without exacerbating gvhd. unfortunately, the response to dli in relapsed acute leukemia and myeloma is less favorable (<30%) and less durable. dli have been used successfully to treat viral infections and virus-associated malignancies following transplant. both unfractionated dli and ex vivo-generated tcell clones have suppressed reactivated cytomegalovirus and eradicated epstein-barr virus-induced lymphoproliferative disease, a polyclonal proliferation of donor-origin b cells that occurs after transplant. where tumor-specific antigens have been defined, efforts to target dli have been undertaken and donor and patient immunization has been investigated. acute or chronic gvhd develops in approximately 60% of patients receiving dli for relapsed hematologic malignancies and for related, but not unrelated transplants, correlates with the donor t-cell dose. dli-induced pancytopenia occurs in approximately 20% to 50% of patients, is generally mild, and transient, but in <5% of patients, aplasia is severe and prolonged. complications of aplasia include infection, bleeding, increased transfusion requirements. efforts to limit the adverse effects of dli while retaining the therapeutic effects include insertion of 'suicide genes, ' selection of lymphocyte subpopulations, and targetting lineage-specific minor histocompatibility antigens. available clinical and experimental evidence suggests, that in addition to primary and secondary immune deficiencies, a wide spectrum of immune-mediated conditions could benefit from intravenous immunoglobulin (ivig), including acute and chronic/relapsing diseases, autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive t cells and inflammatory disorders e.g. an imbalance in cytokine networks. trimar-collected apheresis platelet concentrates (pcs) were exposed to 17.2 j/ml uv light in the presence of 50 um riboflavin, followed by storage under blood bank conditions with various concentrations of 2-deoxyglucose from 0 to 20 mm for 5 days. the control platelets were not stressed by uv light exposure and were stored under the same conditions without 2-dog presence. all test and control platelets were measured for in vitro cell quality including rates of glycolysis, morphology score and activation levels at days 0, 1, 3 and 5. results: lactate production and glucose consumption increased from 0.0378 mmol/1012 cells/h (sd = 0.0097) and 0.0268 mmol/1012 cells/h (sd = 0.0114) for control samples to 0.1371 (sd = 0.0281) and 0.0724 (sd = 0.0151) for uv-treated platelets, respectively. uv treatment also caused a decrease in ph from 7.50 (sd = 0.07) for controls to 6.55 (sd = 0.26) for treated platelets at day 5, hsr from 75% (sd = 12.4) to 33% (sd = 25.1), esc from 24.3% (sd = 3.9) to 3.8% (sd = 3.6), swirl from 2.8 (sd = 0.7) to 1.0 (sd = 1.0), and increased p-selectin expression from 21.2% (sd = 7.3) to 85.2% (sd = 9.4). addition of 2-dog up to 20 mm significantly reduced lactate production rate to 0.0515 mmol/1012 cells/h (sd = 0.0045) and glucose consumption rate to 0.0293 mmol/1012 cells/h (sd = 0.0060), and maintained ph above 7.35 (sd = 0.09) for 5 days of storage. the effect of 2-dog exhibited a dose-dependent response. however, the addition of 2-dog had no effects on hsr (28.1 + 11.4% at day 5), esc (2.86 + 2.75% at day 5), swirl (0.8 + 0.5 at day 5) and p-selectin expression (69.9 + 6.4% at day 5) during platelet storage. atp contents in both treated and control groups were maintained at a relatively constant level above 87% of the value seen in fresh platelets. furthermore, an exaggeration of uv-stressed platelet aggregation by addition of 2-dog was also observed. conclusions: increased glycolytic flux is not a direct cause for platelet morphology changes and spontaneous activation incurred during the development of the storage lesion. the results also suggest that a reduction in glucose utilization may foster an increase in platelet loss during storage. aim of the study: was to evaluate analytical sensitivity, sensitivity and inclusivity for subtypes and genotypes of hiv, hcv and hbv, the assay's effectiveness in closing the pre-seroconversion window period, clinical specificity as well as the effect of endogenous substances and microorganisms on the sensitivity and specificity of the assay. methods: secondary standard traceable to who international standard for hiv-1 (97/ 656), international standards for hcv (96/798) and hbv (97/746) were used to determine the analytical sensitivity. sensitivity and inclusivity for hiv-1 subtypes other than hiv-1b, for hiv-2 and for hepatitis b and c genotypes as well as specificity was evaluated with >1000 specimens. results: results from this study indicate that high analytical sensitivities (39 iu/ml hiv-1m, 114 cp/ml hiv-1o and 1.1 cp/ml hiv-2, 7 iu/ml hcv and 3 iu/ml hbv) and a specificity of >99.4% are accomplishable for the mpx test. the 95% detection rate for hiv-1m subtype isolates (a through h) was between 10 to 50 iu/ml, for hcv genotype isolates (1a through 6) between 2 to 10 iu/ml and for hbv genotype isolates (a through g and precore mutant) between 2 to 5 iu/ml. investigating seroconversion panels, hiv-1 rna was detected an average of 6 and 5 days earlier than hiv-1 antigen with abbott hivag-1 monoclonal and coulter p24 antigen tests, respectively, hcv rna an average of 31 or 21 days earlier than hcv antibody with the abbott hcv eia 2.0 or ortho eia 3.0 tests, hbv dna an average of 18 days earlier than hbsag with the abbott hbsag eia imx test. for all targets, no interference was detected with 17 microorganisms tested as well as elevated levels of triglycerides, albumin, hemoglobin, human dna or bilirubin. conclusion: automated pooling, sample preparation, and real time pcr using the blood screening system 200 taqscreen mpx test is an efficient and sensitive method to simultaneously screen for five important viruses in human plasma. the mpx test is another evolution step in the development of pcr automation by roche molecular diagnostics, and further represents roche's commitment to increasing the safety of the global blood supply. aim of the study: a prospective hemovigilance plan was set up in order to establish a registry for future reference, and to detect any unexpected side effect of ip that may occur with significant frequency in populations and indications that were not studied before and outside of a formal trial environment. methods: this plan is proposed to blood establishments and transfusion prescribers who have already decided to implement ip. this is an observational, non randomized, non controlled plan. no patient selection, inclusion or exclusion criteria are required. all ip transfusions are documented using an internet form, whether or not a reaction is observed. patient population data are collected anonymously, for epidemiological purposes. results: between october 2003 and september 2004, 2512 apheresis ip units have been transfused in 2 sites and registered in the database. ip platelets were considered leucocyte inactivated and were not irradiated, but were antigen matched as indicated (3.9%). the population of patients receiving at least one transfusion (n = 271) included 58.3% of males, 40.6% of females, the median age was 64 (range 0-89). the most frequent broad diagnostic categories were hematology-oncology (64.9%) and cardiovascular surgery (19.9%). the patients received their transfusions either in regular hospital wards (63.5%), intensive care units (27.3%) or as outpatients (9.2%). the number of transfusions by patient ranged from 1 to 106 (mean 9.3 ± 16.0, median 2). half of the patients (49.8%) had previous transfusion experience and 5.9% had previous history of transfusion reaction. transfusion reactions, defined as any deterioration of the patient's state of health observed following transfusion, were observed in 1.2% (n = 31) of the transfusions (95% ci 0.84-1.75), and 8.5% of patients. only 2 (0.1%) were considered serious. after further causality analysis including biological and clinical investigations by the transfusion physician, 0.9% (95% ci 0.58-1.37) of the transfusions were confirmed as having caused reactions in 6.3% of patients, none of them serious. the most often reported symptoms were chills (0.9%) and fever (0.6%). itching, skin rash or urticaria was observed in 0.5% of transfusions. of the 2 serious reactions, one was hypotensive shock in a patient with liver cirrhosis and haemorrhage, and one was septic shock, in which the platelet unit bacterial culture was negative. none of the reactions occurred in cardiovascular surgery patients. patients were more likely to experience reactions if they had previous transfusion history (odd ratio 2.17, p = 0.15). the active hemovigilance plan is a valid and feasible method to collect epidemiological data on transfusion safety. the risk profile of ip transfusions appears favorable. quality of theraflex mb-plasma during storage and treatment s reichenberg* and n müller † *maco pharma international gmbh, langen, † inst. for transfusion medicine, essen, germany background: although in the last decades thanks to the implementation of several methods like donor selection and testing procedures the risk of virus transmission from plasma has decreased, infection of patients still exists. additionally new viruses like west nile virus enter the transfusion chain. therefore, the treatment of therapeutic plasma with methylene blue (mb) is a technique used in several european countries for pathogen inactivation. macopharma has developed the proprietary theraflex mb-plasma bag system including a mb pill and a final mb filtration step. aims: aim of the study is to show the quality of the mb plasma during the preparation procedure and during storage using the theraflex system. methods: for the preparation process every single step was evaluated using 18 single donor plasma units. for the evaluation of the plasma factors 5 ml were drawn at different stages (before treatment, after plasma filtration with plas4, after dissolution of the mb pill, after illumination, after treatment). because the sample volume for single sample measurements would be too low the samples were pooled after drawing and measured for the specified factors. six samples of each stage were pooled at three days. a whole panel of plasma factors was measured for the resulting three pools. global tests: quick, inr, aptt, thrombin time coagulation factors: fibrinogen, factor ii, factor v, factor viii:c, factor ix, factor x, factor xi inhibitors: at iii, protein c, protein s fibrinolysis: plasmin inhibitor, alpha1-antitrypsin complement: ch50 activation: tat, factor xiia, d-dimer stability data were generated using three plasma pools. six plasmas were pooled and afterwards divided into six aliquots. each was treated as single unit and then each was divided into six storage samples. the same plasma factors as for the manufacturing process were evaluated. results: a moderate reduction for some coagulation factors during the preparation was found in the illumination step but not in the other preparation stages. this was mainly fibrinogen (17.5%), factor viii (22.2%), and factor x (13.4%). despite this reduction the values were within the ranges found in non-treated plasma. all investigated plasma factors remained stable during the investigated storage time. summary/conclusions: the investigation showed that plasma treated with the theraflex procedure showed slight reduction during treatment and no reduction during storage. all plasma factors remained within the threshold values. the treatment of therapeutic plasma with mb is a valid technique of pathogen inactivation. validation of intercept treatment of pooled platelets g santos, c silva, f pereira and g sousa lisbon regional blood centre, lisbon, portugal background: intercept blood system for platelets uses amotosalen hcl and uva light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet products. aims: the purpose of the study was to assess the feasibility of introducing this technology in the routine of lisbon regional blood center (crsl) and validate the procedure in our center. material and methods: whole blood units of 450 ml were collected from volunteer blood donors in quadruple top and bottom blood bags (optipure rc soft t& b baxter), kept in n-butanodiol plates; buffy coats with a volume of 55 ml were obtained in the opipress ii and kept overnight at room temperature before pooling. five buffy coats were pooled with 280 ml intersol using the octopus system intercept buffy coat pooling set with an integrated filter. the pools were treated using the intercept. samples were taken before treatment, after cad remotion, on days 2, 5 and 7. the following tests were performed: platelet count, mean platelet volume, ph, swirling. results: all pools met the intercept guardbands. platelet yield pre inactivation was 3.93 ¥ 10 11 (2.95-4.72 ¥ 10 11 ; sd-0.41). platelet pool volume was 362.3 ml (sd-10.7). plasma % was within 32.9% and 36.5%. all pools had leucocytes within council of europe specifications. after photoinactivation the platelet concentrates had 3.58 ¥ 10 11 (±0.37). the average platelet loss was 0.35 ¥ 10 11 . the ph was within specifications during all the storage period. conclusions: intercept treatment of pooled buffy coat platelets is feasible in the routine of crsl and in vitro parameters do not show significant changes, allowing us to proceed to clinical use. shown ip and conventional platelets (cp), stored for up to 5 days, exhibit comparable hemostatic efficacy and safety. extension of platelet storage duration to 7 days has the potential to improve platelet availability and reduce outdating and inventory shortages. clinical efficacy and safety of ip stored for 7 days were investigated. methods: a randomized, controlled, single-center, crossover, noninferiority design (pilot) study evaluated efficacy and safety of buffy coat ip vs buffy coat cp, each stored for 7 days. patients were randomized to receive one 7-day ip transfusion and one 7-day cp transfusion in random order. after each study transfusion, the 1hour platelet count, ci, and cci; time to next transfusion; bleeding response; transfusion reactions; and serious adverse events (saes) were assessed. the primary endpoint, 1-hour cci, was analyzed by a one-sided non-inferiority test for the per protocol population (patients with both transfusions and no major protocol deviations interfering with efficacy evaluation). the per protocol population included 20 patients, 9 randomized to the ip-cp sequence and 11 to the cp-ip sequence. more patients received allogeneic stem cell transplant in the cp-ip sequence than the ip-cp sequence (64% vs 33%; p = 0.37). mean platelet dose (¥10e11) was 2.8 for ip and 3.0 for cp (p = 0.23). there was a significant period by treatment interaction (p = 0.07) at the 0.10 significance level; therefore, the first period only was also analyzed for the primary endpoint. including both treatment periods, mean (±sd) 1-hour cci (¥10e3) was 6.6 ± 4.5 for ip vs 8.9 ± 5.5 for cp. the mean paired difference for both sequences was 2.4 ¥ 10e3 (p = 0.55 by non-inferiority test; upper bound of the 95% confidence interval = 4.0). for the first period only, mean 1-hour cci (¥10e3) was 8.7 ± 3.8 for ip vs 7.4 ± 5.4 for cp) the mean paired difference for the first period sequences was 2.4 ¥ 10e3 (p = 0.06 by non-inferiority test; upper bound of the 95% confidence interval = 2.4). the non-inferiority margin for the study was 2.2 ¥ 10e3 for mean treatment difference in cci (cp-ip). median time to next transfusion was 25 h for ip vs 24 h for cp following the first transfusion (p = 0.87 log-rank test; data censored at 7 days after transfusion) and 16 h ip vs 34 h cp after the second transfusion (p = 0.02). bleeding pre-or post-transfusion was uncommon, usually mucocutaneous, and grade 2 or lower, and responded similarly to ip and cp. no significant transfusion reactions or saes were reported. in this double-blinded, two-treatment crossover study the primary endpoint regarding 1-hour cci was not met. however, transfusion with 7-day-old platelets treated by the intercept blood system showed only a marginally and probably clinically insignificantly lower 1-hour cci compared to 7-day-old conventional platelets. methods: new zealand white rabbits were transfused with syngeneic blood (10 ml/kg), across a major antigen (hgd) mismatch. high anti-s-303 ab titers (≥1 : 1000) were induced after repeated immunization (days 1, 8, 15, 36, 64) with klh-(s-303) hapten (klhhapten) in complete freund's adjuvant. ab titers against srbc were determined by gel card agglutination, or by facscan with fitc-goat_anti-rabbit_igg. survival of infused rbc (4 ml/kg) treated with different methods was assessed by rbc biotinylation. blood samples were taken 1, 3, 7, 15, 21 and 28 days after transfusion, analyzed using streptavidin_pe and facscan to determine the proportion of circulating biotinylated rbc. results: groups (g) of rabbits were transfused with control rabbit rbc (crbc; n = 4, g1), or o-srbc (n = 6, g2). no ab against o-srbc developed after biweekly transfusions over 24 weeks in g2 rabbits. high ab titers to o-srbc could however be induced by klh-hapten immunization in a different group of animals (n = 2; g3). transfused rabbits (g1 & g2) exhibited no change in hematocrit or body weight and maintained good vital signs. high titer anti-s-303 abs were then induced by klh-hapten immunization in rabbits from g1 (n = 2) and g2 (n = 4), and in a group (n = 6, g4) of naïve rabbits. non-immunized rabbits (g1, n = 2), and (g2, n = 2) were maintained on the biweekly transfusion schedule of crbc and o-srbc, respectively. all rabbits treated with klh-hapten developed comparably high ab titers. klh-hapten immunization did not affect the viability of crbc in g1 rabbits. transfusion of o-srbc demonstrated reduced viability in hyper-immune g4 rabbits, but not any of the g2 rabbits. g2 rabbits exposed to 12 o-srbc transfusions prior to hyper-immunization with klh-hapten, had viability of o-srbc comparable to crbc, suggesting induction of immune tolerance by repeated exposure to o-srbc. after depletion of labeled o-srbc from circulation, g2 and g4 were transfused with m-srbc. viability of m-srbc in all g2 rabbits (hyper-immune or not) and the hyperimmune g4 rabbits was equivalent to crbc circulation in g1 rabbits. in pre-immunized rabbits with high titer anti-s-303 ab, o-srbc are cleared faster than control. in contrast, m-srbc survive normally in rabbits with high anti-s-303 ab titers. repeated transfusion of o-srbc does not result in alloimmunization of naive rabbits. the modified s-303 rbc process offers the potential for pathogen inactivation with elimination of immunoreactivity and retention of rbc viability. introduction: the bombay phenotype is extremely rare and characterized by complete absence of abh activity both on erythrocytes and in secretions. those individuals can produce anti-h, which is active over a wide thermal range. method: using liss indirect antiglobulin technique, the patient's serum showed +4 reaction by panel of eleven cells at room temperature phase as well as indirect phase while the auto reaction is negative. a cold adsorption using rabbit erythrocyte stroma was done to remove the cold antibodies from the serum; +2 reaction of an antibody was detected in the patient serum after five folds of rabbit erythrocyte stroma adsorption. introduction: immunohematology reference laboratory in kuwait central blood bank receives samples from all hospitals in kuwait both governmental and private sector. the laboratory performs the tests according to international standards and it is monitored by internal and external quality assessments on periodic basis. material and method: a tube and gel cards are two methods in the reference laboratory for antibody identification. the laboratory can identify the most commonly encountered clinically significant antibodies and investigates causes of positive direct antiglobulin test that occur mostly in autoimmune hemolytic anemia. there are facilities to phenotype most of rare red blood cell antigens. results: records of all patients investigated in the laboratory since the year 1985 are kept in computerized system that has 7528 patient's records. central blood bank has the potential to identify rare phenotypes such as kpb-, jsb-, lan-, bombay, rzr2, rzr1, r¢r¢, r¢r≤, r≤r≤, ge-2,3,4 and rare red blood cell antibodies such as high frequency antibodies anti-k, anti-ge2, anti-h, anti-lan, anti-kpb, anti-jsb, anti-wrb, anti-ena, anti-csa and low frequency antibodies anti-kpa-, anti-jsa-, anti-dia, anti-lua, anti-cob as well as hightiter-low-avidity antibodies such as anti-chido. samples of rare red blood cells and rare serums are kept frozen either by glycerol or liquid nitrogen technique to be used for pre-transfusion compatibility testing and continuing educational program. purpose of the work: to present the substitution of the blood groups o, a, b, ab in abo blood group system and rh (d) blood group in rh (d) blood group system in the population in the gevgelija-valandovo region. material and methods: a retrospective analysis was done on the data of following the blood groups o, a, b, ab and d in the blood group system abo and rh in the gevgelija-valandovo region. the asked population are voluntary blood donors, candidates for drivers, patients, pregnant women and newborn children. the period (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) was analysed. the examinations were done with two standard methods (on the plate and in tube), to define blood groups from the most represented blood group systems abo and rh in the population. tests were done with different series of commercial anti serum tests from domestic and foreign origin. results: totally 43.395 examinees were typified. with o blood group were 15.761 (36.31%); with blood group a were 17.675 (40.73%); b blood group 7.119 (16.4%) and ab blood group were 3.019 (7.52%) examinees. totally 37.365 (86.11%) were d positive and 6.029 (13.89%) were d negative. discussion: the given results from our examinations for the frequency of o, a, b, ab and rh (d) blood groups from abo and rh (d) blood group systems in the region gevgelija-valandovo show that the most present is the blood group a from abo blood group system 17.675 (40.73%) and d blood group in rh system with 37.365 (86.11) form examined population. the results are in correlation with data from the literature for other european nations. introduction: the policy of our center is to transfuse all heamatologic multitransfused patients with their own rhesus and kell phenotype. in addition, thalassemic and young leukemic patients are being transfused with compatible phenotype of the most clinical important duffy and kidd systems while all the other patients receive blood compatible only with abo and rhesus system. nevertheless, it is observed a significant positivity of the indirect antiglobulin test, due to alloimmunization. aim of the study: in this study we tried to evaluate the prevalence of alloimmunization in patients of our region. the most frequent detectable alloantibody remains the anti-d, with high prevalence 88.9% of anti-d in females versus 11.1% in males, due to alloimmunization during the pregnancy. as a consequence, the high incidence of anti-d is not transfusion related and anti-e is evidenced to be the most frequent transfusion related alloantibody, followed by anti-kell. care must be taken in order to transfuse as more patients as possible with their own phenotype regarding, at least the most immunogenic antigens, like anti-e and anti-kell. severe hemolytic reaction due to anti-j k3 background: red blood cell alloantibodies directed against antigens of the kidd system are notorious for causing delayed hemolytic transfusion reactions. the antibodies are formed because of pregnancy or transfusion. blood donors with the red blood cell (rbc) phenotype jk(a-b-) are extremely rare in the white population and exhibit a frequency of less than 0.1%. however, the rare phenotype jk(a-b-) is more common in polynesians (1.4%). individuals with jk(a-b-) phenotypes typically form anti-jk3 with inseparable anti-jka and anti-jkb activity. some jk(a-b-) patients' sera may show an additional distinct anti-jka or anti-jkb component when examined with adsorption studies. case report: a 63 years-old caucasian female with a negative antibody screen, no prior history of transfusion, presented with gastrorrhagia. it is reported four pregnancies with no history of haemolytic disease of the newborn (hdn). on admission, her haemoglobin was 5.5 g/dl. she was given 4 units of crossmatchcompatible rbc. on day 6 her haemoglobin was 12.1 g/dl, with a total bilirubin of 0.72 mg/dl and lactate dehydrogenase of 141 u/l. on 10th day an unexpected fall in hb (5.6 g/dl) occurred with an increase of bilirubin to 1.9 mg/dl and of lactate dehydrogenase to 1042 u/l. a new blood sample obtained for antibody screening and additional crossmatches showed a pan-agglutination and incompatible crossmatch. anti-jk3 antibody high titer was detected in the plasma by gel-test using liss/coombs cards (id-diamed). the dat was negative and the antibody reacted equally with jk(a-b+), and jk(a-b+) panel cells (jka:1/16 384 and jkb:1/16 384). other alloantibodies could not excluded, because jk(a-b-) cells are not available. she was started with erythropoietin-a (epo), folic acid, fe iv and high dose intravenous immunoglobulin (ivig). the epo was discontinued after four week of therapy when the haemoglobin was 12 g/dl. two months later her haemoglobin was 13.5 g/dl and anti-jk3 was present in the same titer. a year later her blood cell count was normal and the anti-jk3 was detected in a lessened titer (1/16). no additional distinct anti-jka or anti-jkb component was shown after two adsorptions at 37°c using carefully selected phenotyped red cell compatible with patient's rh, fy, mnss, lu, le system and jka(+) and jkb(-), but two additional alloantibodies anti-c and anti-e of low titer (1/4) were revealed. the rare anti-jk3 alloantibody found in this case displayed the erratic nature of many kidd system antibodies. although anti-jk3 may cause mild hemolytic disease of newborn, she did not have a history of hdn. our patient was sensitized to a kidd antigen during pregnancy, but showed no serologically detectable antibody until challenged with a massive transfusion following a gastrorrhagia. the use of epo and high dose intravenous immunoglobulin succeeded to avoid transfusion with incompatible rbc unit. background: differential warm adsorption is used in the investigation of patients with red cell autoantibodies for searching of underlying alloantibodies, but it is also useful in the detection of clinically significant alloantibodies in patients with alloantibodies to high frequency antigens such as k, kpb, lub and inb. this technique is especially useful in cases when patients when patient's phenotype cannot be identified due to recent transfusion. purpose: differential warm adsorption is performing on cases presented with an antibody reacting with all red cells of the panel and having a negative auto control test. in these cases, even rare cells panels, which allow the identification of a pan antibody, are available, other more common clinical significant antibodies cannot be excluded. methods and result: case 1. a 63 years-old caucasian female with preexisting myeloproliferative disorder (polycythemia) presented with pancytopenia. anti-k was detected in the plasma. it is reported two pregnancies and no history of transfusion. the dat was negative and the plasma did not react with one k-cell present on the red cell panel in use. the anti-k specificity was confirmed using additional k-cells. the patient's red cell were group a, d+, k+, k-, c-, e-, fy(a-), s-, le(a-), kp(a-), cw-. she was transfused with two units rbc k-. ten days after the first transfusion the dat became positive and the one k-cell present on the red cell panel reacted with her plasma. two adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). an anti-fya was identified in the presence of ant-k. . an antik (titer 1/16) was suspected. because k-red cell was not present in the panel in use, adsorptions were carried out at 37°c using carefully selected phenotyped red cell (compatible with patient's rh, fy, jk, mnss, lu, le system and k positive). after the anti-k antibody was totally removed, no additional alloantibodies were revealed. conclusion: differential adsorption in cases with alloantibodies to high frequency antigens represents a useful application of the technique and helps in the identification of clinical significant antibodies present, allowing a more accurate decision for transfusion. evaluation of validity of the expired enzymetreated 0.8% red cells in antibody identification gel tests using nacl cards p chalkia, s intzepeli, v avgoloupi, a tsoukala, e ntinopoulou and p didoudi ahepa hospital, thssaloniki, greece background: expired red blood cells of required phenotypic profile is often used to identify antibody specificities in patients with multiple anti-erythrocytes antibodies. accurate results depend on the integrity of the antigens. purpose: to validate the expired enzyme-treated 0.8% red cells for use in antibody identification gel tests using nacl cards. the serum of nineteen patients with common specificities antibodies in rhesus, kell, duffy, kidd, and mnss systems tested with commercially prepared 0.8% enzyme treated cells rbc panel (id-diamed). gel tests were performed according to the manufacturer's instructions on in-date rbc and simultaneously on rbc 1 month to 5 months past expiration. reactivity of the expired antigen positive and antigen cells was compared to in-date cells. results: twenty antibodies detected with enzyme treated red cells in neutral gel cards [d(4), c(3), e(2), k(7), cw(4)] and were tested with enzyme treated red cells in use and rbc 1 to 5 months post expiration. seventeen antibodies tested with enzyme treated cells gave acceptable results with antigen positive cells 1 to 5 months post-expiration, except 3 anti-k antibodies (negative with k positive cells 2-4 months post expiration). conclusion: most rbc antigens studied were detectable 5 months after rbcs expiration date. tests with 0.8% cells were valid in gel test (nacl/enzyme) for at least 5 months after manufacturer assigned expiration date and may be helpful for complex identification studies. studies for more antigens specificities are needed to testify the validity of the expired enzyme-treated 0.8% red cells. background: wr(a) is a low-incidence blood group antigen (1 : 1000) in the caucasian population. despite that anti-wr(a) is a common antibody type, it may cause severe transfusion reactions, haemolytic disease of the new-born. anti-wr(a) may occur as an autoantibody or arise without immune stimulus. we report a case of a naturally occurring anti-wr(a) antibody. case report, methods and results: 56-year-old non-transfused male patient with acute pancreatitis and severe anaemia had been transferred to surgery from a county hospital. the serological status identified by their blood bank was: b rhd positive, with anti-wr(a) antibody in the serum (cellbind card method). our results: the patient's cells were group b rhd positive (microplate method), dat negative (tube and gel test method). the antibody identification showed positive antibody reaction with all enzyme treated test cells but negative reactions in liss iat (tube test) and gel iat (scangel and diamed). discussion: our routine tests for antibody detection didn't detect any specific antibody in patient's serum. he was transfused several units of blood, that was wr(a) negative and showed negative crossmatch reactions. the patient had no transfusion reactions. 10 days after the transfusion anti-wr(a) specificity was confirmed in the serum with cellbind test. only few test cell panels contain wr (a) positive cells, which are usually not present in commercial screening cells. in our case the cross-match was only performed for this patient because of the detected nonspecific antibody reaction in enzyme. the risk of transfusion reactions caused by rare antigens are particularly high in the type and screen cases. background: according to requirements of the french committee for accreditation (comité français pour l'accréditation cofrac, iso 17 025 standards), it is essential to use validated and standardised methods in immunohematology. this imposes, among various requirements, the knowledge of metrological tolerances for all the techniques. aim: a multicentre study was carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and of tests cells for antibody screening using indirect antiglobulin test (iat) in filtration technique. the antibody screenings were performed manually in 3 blood centres using 3 different filtration systems: id diamed, biovue ortho and scangel biorad, the same tests cells, a standard 20 ng/ml anti rh1 (provided by cnrgs), a positive control anti kel1 and a negative control. all equipment used (oven, chronometer, pipettes) were calibrated according to cofrac standards. each antibody sample was tested under the following combined conditions (243 tests/sample): results: all the tests of antibody screenings from the multiples combinations of the above parameters gave the same results with a 2+ intensity agglutination for positive samples and the absence of agglutination for the negative control. conclusion: this study allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening in iat using commercial filtration systems maryvonne. the authors present a retrospective study involving 12 091 blood donors from the university hospital of coimbra during the year 2004. the incidence of weak d and rh (d) phenotype was determined in 1729 individuals who were rh (d) negative. the ab0 and rh(d) blood grouping was performed using a column gel agglutination card (diamed). the rh (d) typing was done using a anti-d polyclonal and a anti-d monoclonal antibodies. all donors that gave negative or poor agglutination results were tested for weak d with an indirect antiglobulin test, with anti-igg (gel matrix card) plus anti-d serum (diamed). within our target group of blood donors the rh (d) negative represented 14.3% of the total sample. we also describe ab0, rh(d) phenotype (c, c, d, e, e) group frequencies and establish reliable estimates frequency for weak d and rhesus haplotypes. the background: in 80s and 90s several authors tried to assess the relationship between the number od igg molecules per rbc and in vivo haemolysis, but determinations usually concerned small groups of tested patients. some of the investigators suggested that the number of igg autoantibody molecules per rbc was a major determinant of the severity of the haemolysis, whereas others found aiha patients with severe haemolysis and undetectable autoantibodies. aim: presentation of our experience with the quantitative elat performed on a large group of aiha patients during long-term observation. material and methods: six hundred fifty eight blood samples from 268 warm-type aiha patients were randomly tested for the number of igg molecules per rbc. eighty six of the patients were tested periodically from 3 to 15 times at one-month intervals. autoantibodies on rbcs were detected by the direct antiglobulin test (microcolumn technology) and measured by the enzyme-linked antiglobulin test (elat). results: in about 1/3 of tested samples the number of igg molecules per rbc was small (<190) and the laboratory signs of haemolysis were present in 65.7% of them as well as in 70.4% samples with moderately coated red cells (200-980 igg/rbc). the large number of igg molecules per rbc (>1000) was significantly associated with high frequency (87.9%) of severe haemolysis and it was also associated with presence of multiple igg subclasses on rbcs and c3d. in 79% of patients tested periodically, the number of igg molecules per rbc decreased and it significantly correlated with improvement of haemolysis parameters. in 21% of aiha patients the number of igg fluctuated and it was a poor prognostic factor. conclusion: in aiha patients the dynamics of the changing number of igg autoantibody molecules per rbc is a more helpful diagnostic and prognostic parameter than the number of igg molecules per rbc evaluated in one test. -b+) , -h-negative (using the anti-h lectin). antibody work-up showed a positive antibody screen (liss and peg-tube methods) reacting 4+ with o rbcs at all phases and 2+ with a1 rbcs. the direct antiglobulin test (dat) was positive with polyspecific ahg as well as anti-c3b,-c3d (table 1) . prewarming of test system did not change the reactivity (4+ at antiglobulin phase). a treatment with dithiothreitol (dtt) was performed and abolished all reactivity of the serum ( table 2 ). autoabsorption of the patient's plasma was performed. the absorbed plasma showed a decrease in reactivity from 4+ to 2+ when tested with o red cells, as well as a significant reduction in antibody titer from 1 : 256 to 1 : 2 tested at immediate spin (table 3 ). the patient remained crossmatch incompatible with o and a rbcs, but was compatible with oh rbcs. summary: we report an unusually strong igm anti-h antibody in this patient, who may require oh phenotype units. the patient is not a para-bombay since her red cells type strongly as group a. the cause for the auto-anti-h remains unknown at this time. if a thermal amplitude test shows that the antibody appears to be clinically significant the patient should receive h-units if transfusion is required. introduction: fetomaternal haemorrhage may determine an alloimmunization, in fact the transplacental passage of antibodies may cause the haemolytic disease of newborn. for this reason, in pregnant women, a screening for irregular antibodies research is routinely performed. however the indirect antiglobulin test (iat) may result falsely positive or negative for various causes, as operative mistakes or low specificity/sensitivity of the used techniques. aim of the study. in this study we have retrospectively evaluated the real incidence of alloimmunizations occurred in women screened by private laboratories. methods: we have studied 1.560 women, 19-43 years old, resulted iat positive at the first screening and successively assisted by our two hospitals. all women were re-tested, using gel-agglutination technique, for both direct antiglobulin test and iat. results: a positive iat was confirmed only in 64 cases; moreover a rbc autoimmunization was found in 7 women. anti-d (16 cases), e (10), c (8), k (8), c (6), s (5), d + jka (1), d + s + e (2), d + c + k (2), m (3), c + e (1), d + c + g (2) were the identified alloantibody specificities. anti-s, -e, -k, -c and -jka were the specificities in autoimmunized women. conclusion: in conclusion, a real alloimmunization is occurred only in 4.1% of screened women, while in the remaining cases iat resulted falsely positive: this observation forces us to affirm that, in order to minimize errors and alarmisms, the screening for antibody research in pregnant women must be performed only by immunohematology qualified center. background: one of the problems of the rbc transfusion is the alloimmunisation and the delayed haemolytic reactions (dhtr). besides rhesus and kell systems the antibodies against kidd antigens cause both dhtr and difficulties in their detection. aim and methods: the exact recording of all blood units according to abo rhesus kell and kidd systems. the abo-rh-kell systems are identified through automated microcolumn method (autovue, ortho), while kidd antigens are identified manually using microcolumn gel (diamed). results: the percentage of jka+ and jkb+ found in our department (75.8%) and (70%) respectively is similar to that of the caucasian population. conclusions: given the fact that there is lack of available freezing rbc system in greece, detailed recording of all units to the above antigenic systems can be proved extremely useful under circumstances of incompatibility. in the latter case suitable donors can be called and cover the shortage. the identification of all antigentic systems of the donated rbc units is underway. background: in many countries transfusion recipients are currently typed and transfused d-positive, if their red cells are agglutinated by 2 igm monoclonal anti-d that do not react with dvi. the transfusion strategy in weak d patients is not clear defined and it depends on the chosen monoclonal reagents and methods. patients who are carrying dw types 1, 2 and 15 were prone to develop anti-d. aim: the aim of this pilot study was to estimate capability of commercially available monoclonal anti-d reagents to recognize this weak d types as rhd positive. material and methods: edta anticoagulant blood samples were collected from 50 blood donors, previously typed as weak d positive by indirect antiglobulin test. molecular genotyping of rhd gene and weak d alleles by cde-ssp and d weak-ssp kits (inno-train, germany) were performed. direct agglutination was tested in a tubes and microplates using the following antibodies: rum-1, th-28, ms-201 (bioscot/serologicals) and d415 1e401/175-2 (immucor). results: out of 50 samples molecular typing results were as follows: in 5 samples dw were not determined, in 45 samples, 20 weak d type 1; 12 weak d type 2; 8 weak d type 3, 1 weak d type 11; 3 weak d type 14 and 1 weak d type 15 were determined. by all monoclonal reagents 95% weak d type 1, 100% weak d type 2, 10% weak d type 3 and 100% weak d type 15 negative results were given. by all monoclonal reagents weak d type 11 and weak d type 14 positive results were given. conclusion: according to weak d types, which were known to be at risk for anti-d immunization further advances may be brought by improved patient's monoclonal typing reagents with a low and donor's monoclonal typing reagents with high affinity for weak d type 1, type 2 and type 15. such improved typing strategies with novel reagents would enhance the transfusion safety. background: vel is a high-incidence antigen found in >99% of the population. anti-vel can be igm or igg and reacts optimally at iat, although it can also react at immediate spin and 37c. anti-vel may or may not cause severe hemolytic transfusion reactions and mild to severe hdn. autoanti-vel has also been reported. the aabb technical manual 14th edition states that the vel antigen is unaffected by protease and sulfhydryl treatment. we have reason to believe that sulfhydryl treatment may have an effect on the vel antigen as evidenced by a recently referred case. case report: a 67 year-old caucasian female presented with symptoms of anemia and renal vascular hypotension. transfusion history indicated multiple red cell transfusions in 1959. according to the patient, previous attempts to locate compatible units were unsuccessful. the case was referred to our laboratory. the patient's red cells (rbcs) typed as group o, d+ with a negative dat. the serological picture revealed an antibody reacting 2 + -3 + s at 37 c/liss, as well as at the antiglobulin phase. the antibody reacted with all rbcs tested and the autocontrol was negative. further characterization of the antibody showed similar reactivity using enzyme-treated rbcs (0.1% ficin) and no reactivity using 0.2 m dithiothreitol-(dtt)treated rbcs. a high incidence negative red cell panel (untreated) was selected that lacked antigens reported to be destroyed by dtt. the antibody reacted with all rbcs tested. additional rbcs were tested that lacked high-incidence antigens, including vel. the antibody did not react with four vel-rbcs tested using liss and peg methods. the patient's rbcs typed as vel-negative. all other clinically significant antibodies were ruled out using vel-or dtt-treated rbcs. based on the unusual reactivity demonstrated by the anti-vel, we tested 12 different examples of anti-vel (frozen in our rare sera inventory) against two sets of known vel+ and vel-rbcs. one rbc set was dtt-treated; the other set was tested neat. liss enhancement was used to test both sets. one of the 12 antisera failed to react with the positive control and one reacted with the negative control. both were disqualified from the study. four of ten remaining antisera demonstrated a decrease in reactivity >1 grade, between the neat and the dtt-treated rbcs. the remaining six antisera showed no change in reactivity. conclusion: contrary to the statement in the aabb technical manual, we discovered that sulfhydryl treatment (0.2 m dtttreatment) can have an effect on the vel antigen. our experience has demonstrated that in some cases anti-vel may not react or may show reduced reactivity when tested with dtt-treated rbcs. therefore, the presence of anti-vel should not be ruled out if negative reactivity with dtt-treated rbcs is encountered. additionally, dtt treatment may be a useful tool obtaining rule-outs of other clinically significant antibodies in the presence of anti-vel. additional data is needed to confirm these findings. but with no identified specific antibodies were investigated by repeated screening/crossmatch, papainized panel identification, hla antibody screening by lymphocytotoxicity test (lct) and elisa in some cases. patients' age, sex, department, diagnosis, previous transfusions/pregnancies, techniques, reactions' strength, number of positive cells, urgency, subsequent antibody tests, identification and lct were noted. antibody tests were performed: at pretransfusion testing (pt) by liss-coombs (diamed) and at blood grouping (bg) by biovue polyspecific (ortho) microcolumns -manually in urgency and routinely by sampler iif (diamed) and mitis (ortho) systems. results: investigated reactivity was recorded in 241 samples from 186 patients; 29 (15.6%) patients had > 1 episode. these findings comprised 15.1% of 1598 unexpected results found at pt and bg. incidences were 0.18% at routine and 0.21% at urgent bg (37 339 and 16 003 bgs, respectively), and 0.35% both at routine and urgent pt (15 803 and 23 705 pts, respectively). 56.5% patients were female, 44.6% over 60, but 19.9% < 30 years, coming mostly from surgery (19.4%), internal medicine (15.1%), hematology (10.8%), ginecology (10.2%), cardiac diseases (9.1%) and cardiac surgery (8.6% patients). frequent diagnosis were solid tumors (16.1%), cardiac diseases (13.4%), hematologic malignancies (8.6%), uraemia (4.8%), orthopedic surgery (4.3%) and hepatic diseases (3.2% patients). 43.0% patients were previously transfused, with only 9.1% patients proved as not transfused or pregnant. subsequently positive antibody test during the study had 27.6% tested patients. at pt positive crossmatch was found in 38.8%, antibody screening in 43.9% and both tests in 17.3% cases. majority of reactions were '1+' (50% at pt and 38.6% at bg); reactions '3+' or '4+' were found in only 5.1% cases at pt, compared to 17% at bg. one crossmatch only was positive in 75.2% positive crossmatches, with 38/43 patients having > 1 crossmatched unit. ahg identification was positive in 27.6% tested patients; in 26% of them papainized panel was also positive. lymphocytotoxic antibodies were found in 45.5% tested patients; 72.6% (8%-100%) of lymphocytes were reactive. finally, the cause of reactivity in antibody tests was determined as 'laboratory mistake' in 42.5%, hla lymphocytotoxic antibodies in 9.7%, 'igg antibodies of unknown specificity' in 7.5% (hla noncytotoxic antibodies in 2/2 elisa tested samples!), contaminated sample in 5.9%, anti-bga in 4.8%, non-specific cold antibodies in 3.8%, subsequently recognized specific antibodies in 3.2% (2 lua, 2 m, 1 kpa, 1 yka), non-specific autoantibodies in 3.2%, carry-over of dat-positive cells and antibody to reagent in 2.2% cases each, while in 4.3% cases antibody screening and in 4.8% cases crossmatch was repeatedly positive without confirmation in panels. discussion: after introducing of sensitive microcolumns, positive antibody tests without detectable specific antibodies require significant laboratory activities, particularly in older patients with malignancies or surgery. such reactivity was frequently caused by laboratory mistake, but often hla and sometimes specific antibodies were later recognized, or reactivity continued without confirmation in panels. relationships that may be helpful are further discussed in abstract part ii. results: significant differences (p < 0.05) and relationships of interest were noted. sex. in female vs male patients frequent features were: crossmatch as only reactivity at pt (45.8% vs 28.2%), positive lct (59.4% vs 26.1%), lymphocytotoxic hla antibodies (lytxab) (13.3% vs 4.9%) and reactivity 'positive screening, negative panels' (5.7% vs 2.5%); in males non-specific cold antibodies (7.4% vs 1.0%) and antibodies to reagents (4.9% vs 0) were noted. age. in patients > 60 vs < 30 reactivity was often found at pt (65.1% vs 35.8%), caused by lytxab (21.7% vs 5.4%), anti-bga (8.4% vs 2.7%) or 'positive crossmatch, negative panels' (9.6% vs 0), but rarely by laboratory mistake (37.3% vs 59.5%) or later recognized antibody (1 of 6 patients). subsequent antibody tests: tests were subsequently positive more often if reactivity was found at pt (79.3% vs 20.7% at bg), as positive antibody screening (41.7% vs 29.2% if positive crossmatch), with positive panels (48.3% vs 12.2% if negative). subsequent tests were positive in only 43.8% patients with lytxab, 50% with anti-bga, 1 of 3 with antibodies to reagent and in no case with 'positive crossmatch, negative panels' . techniques. lct was positive in 60% and 45.5% tested samples found at urgent and routine pt (diamed), vs 0 at bg. all 18 cases due to lytxab, 8 of 9 anti-bga and 6 of 8 'positive antibody screening, negative panels' were found by diamed. at bg (ortho) 71.4% cold antibodies and 64.6% laboratory mistakes were found. positive antibody test: identification was negative in 78.6% screening-only cases; 69% of them were caused by laboratory mistake. lytxab were found in 67.9% crossmatch-only cases; 77.8% reactivities caused by lytxab were crossmatch-only. identification. ahg panel was positive in 60% cases with lytxab (in 2 with papainized panel) and often due to 'igg antibodies of unknown specificity' (29.7%), anti-bga (24.3%), contaminated sample (16.2%), but also to later recognized specific antibody (10.8% cases). strength of reaction: laboratory mistake was noted in 61.6% of 'w', 41.3% of '1+', 46.4% of '2+' and 58.8% of '3+ and 4+' antibody screenings (ns). diagnosis. in patients with solid and hematologic malignancy reactivity was often found at pt (80% and 81.3% of patients, respectively), due to positive crossmatch (40% and 50%; 0 and 4.3% patients with liver and cardiac diseases) and caused by lytxab (16.7% and 31.3%) or 'crossmatch/screening positive, panels negative' (16.7% patients with solid tumors). in patients with liver and cardiac diseases reactivity was often found at bg (83.3% and 68.0% of patients), due to laboratory mistake (50% and 60%) or cold antibodies (16.7% and 12%), respectively. discussion: features of non-specific reactivity depended on sex, age, positive antibody test, diagnosis and, moreover, used techniques, sometimes in very distinctive manner. this analysis might be of considerable help in planning of laboratory tests, but also in quick analysis of unexpected results and choice of further testing, particularly in urgent situations. background: worldwide screen and type is a very usual method for pre-transfusional testing. the ultimate objective is to prevent not only the clinically expressed delayed hemolytic transfusion reactions but also the serologically revealed ones. aim: the aim of this study was to determine the frequency of red blood cell (rbc) alloantibodies in patients undergoing cardiac surgery or cardiac procedure, during the pre-transfusion screening. materials and methods: blood samples of 16 291 patients (12 628 male and 3663 female) were evaluated. the mean age of the patients was 57 years. pre-transfusion samples were examined for clinically significant alloantibodies, using antibody screening with gel test (liss -enzyme). in case of a positive result, identification was performed (panel with autologous control). in addition, the serological testing included cold agglutinins´ detection (tube test), as well as titration (tube test) and identification (gel test) in case of a positive result. when the result was marginal (1/32) a new test was carried out after a seven days period. in the presence of a positive autologous control or an autoantibody, samples were examined with direct antiglobulin test (dat). results: alloantibodies were detected in 580 patients with the incidence of 3.56%. antibodies were registered more frequently in females (190/3663, 5.18%) than in males (390/12 628, 3.09%). 183 patients (31.49%) developed single antibody with anti-kell being the most frequent. the incidence and the specificity of the detected antibodies are summarized in the following table (table 1 ). in 23 patients (3.96%) multiple antibodies were detected, with most frequent the anti-d and anti-c combination. 35 patients (6.03%) were dat positive. autoantibodies were found in 10 patients (1.72%), all of which had specificity to rhesus system. cold agglutinins were positive in 53 patients (0.32%). no specificity could be assigned in 245 patients (42.24%), while in 110 patients (18.96%) non specific reactions in enzyme treated rbcs, were observed. one patient developed delayed haemolytic reaction 15 days post-transfusion, due to anti-jka. the antibody, however, was not detected in the pretransfusion sample re-testing. the frequency of the pre-transfusion detection of red blood cell alloantibodies in our center, was 3.56%. the most frequently identified were the anti-kell and anti-rh. the high rates of unidentifiable antibodies and non specific reactions in enzyme treated rbcs are probably attributed to the kind of medication that most of these patients receive, as well as to the degree of inflammatory process which usually accompanies such diseases. the high frequency of unidentifiable antibodies indicates that a larger and more complex erythrocyte panel would be useful for routine testing. the routine pre-transfusion screening for alloantibodies probably assures the prevention of dhtrs and provides sufficient time for blood selection for transfusion. introduction: in the united kingdom, about 1% of women form red cell allo-antibodies in pregnancy and 0.07% of all pregnant women produce anti-c. before the introduction of prophylactic anti-d, it was reported that 3% of the total haemolytic disease of the newborn (hdn) cases were due to anti-c. currently, cases of hdn due to anti-c are half as frequent as anti-d and 14% of the uk population are rhc negative. we present two unusual cases of pregnant women who are d and c negative and have anti-c and anti-d detected in their serum. case studies and results: case 1: a 38-year-old asian woman had three previous uneventful pregnancies. in her 4th pregnancy she presented with miscarriage at 19 weeks gestation. she was group b, d and c negative. her serum contained anti-c and anti-d. anti-d was detected by liss tube iat and anti-c was only detected by manual polybrene technique (0.9 iu/ml by quantification using r2r2 cells). in a 5th pregnancy, no antibodies were detected until 34 weeks gestation when this patient presented in early labour. anti-c was then detected by two-stage papain technique only as well as anti-d. case 2: a 33-year-old asian woman had a positive antibody screen post caesarean section in jan 2005. no antibody had been detected during the pregnancy. standard prophylactic anti-d was given at 28 and 34 weeks gestation as this patient was d neg. anti-c and anti-d were confirmed in her serum. the anti-c level was 0.7 iu/ml using rr cells and the anti-d level was <0.1 iu/ml using r1r1 cells (prophylactic anti-d ig). she was phenotyped as o r¢r¢. at delivery the baby was found to have a negative dat and was phenotyped as r¢r. discussion: cde/cde (r¢r¢) is an uncommon phenotype in the uk population with a frequency of 1/10 000. in routine antenatal testing, abo/d grouping is only performed for pregnant women at booking and 28 weeks gestation according to bcsh guidelines. full rh phenotyping is not carried out unless the pregnant women has a positive antibody screen. in routine testing of the above cases, this extremely rare phenotype is missed. prophylactic anti-d was given to both patients and immunisation due to anti-d was prevented. there is currently no prophylactic regime developed to prevent anti-c allo-immunisation by the fetus in pregnancy. antenatal management of patients with anti-c and anti-d during pregnancy can be problematic: (i), problem in antibody identification; (ii) monitoring of antibody level (i.e. quantitation by auto analyser for anti-c and anti-d) with two different cells and (3) provision of blood during pregnancy, at labour and post delivery for both mother and newborn. study on the frequency of red cell phenotypes (e.g. duffy, kidd and mns blood group system) in our local population l leou, yf wong, mbc koh and d teo health sciences authority, singapore, singapore background: the frequency of various red cell antigens in the caucasian population has been well studied. to date, the frequency of these antigens in the local population composed of a multi-racial mixture of chinese, malay, indian and others is still unclear, especially in the malays with paucity of data in the literature. aims: to investigate the frequency of clinically significant red cell antigens duffy, kidd and mns across the local ethnic groups. to investigate the occurrence of rare phenotypes. to be aware of these rare phenotypes so as to facilitate planning of blood inventories and supplies. typing for the duffy, kidd and ss antigen on blood donors was performed using monoclonal as well as polyclonal anti-sera by manual tube method. a total of 1427 blood donor samples were tested using specific anti-sera that will agglutinate red blood cells that have the corresponding antigen. agglutination is demonstrated by the indirect antiglobulin technique. result and discussion: table 1 -a higher frequency of the fy(a+b-) phenotype is seen in chinese, malay and others in contrast to the indian and caucasian population. the clinically significant allo-antibody anti-fya is rarer in our local population. it occurs predominantly in malays and indians and usually in combination with other antibodies. it means that provision of antigen negative blood may be difficult. table 2 -all groups show similarity of distribution of the kidd phenotype with the caucasians and distinct from the american blacks. the jk(a+b-) and jk(a+b+) phenotypes are relatively equal in frequency and there should be no problem looking for such a phenotype in the local population. table 3 -the s-s+ phenotype is most common amongst all races. the indians are more similar to the caucasians with a relatively high frequency of s+s+ phenotype. the chinese and malay distribution are unique with > 99% being s+. conclusion: there is a unique distribution of red cell antigen groups in the 4 races and the data on malays is especially useful. this data will allow the national blood service in its inventory planning and the potential difficulties of providing antigen negative products due to clinically significant allo-antibodies. miltenberger phenotypes among taiwanese table 1 . conjointly, in order to obtain the frequency of mi.v phenotype, we screened 543 samples among 1230 with anti-hil and four additional cases of mi.v were found. conclusion: in this study significant miltenberger polymorphism was seen among the taiwanese population. besides previously described mi.iii phenotype (1.9%), there were also mi.i/ii phenotype (0.4%), mi.v phenotype (0.7%), mi.vi phenotype (0.7%), mi.x phenotype (0.1%), and most interestingly two miltenberger related not yet classified variants (1%). related variant a (0.5%) was phenotypes as mia+, anek+ and hil+. related variant b (0.5%) was phenotyped as mia+, anek+. interestingly, both variants were mur-(negative). the total estimated frequency of miltenberger variants in taiwanese population (including related variants a and b) is therefore 4.8% (table1). the discovery of 2 unclassified variants (most likely not yet described in the literature) is of great interest in the field of immunohaematology and warrant further molecular genetic study. introduction: the use of column technologies for the detection of rbc antibodies improved significantly the screen test sensitivity. each column-based method has its advantages and disadvantages. aim: to compare antibody detection by two column agglutination tests; the fully automated ortho auto vue tm method and the manual diamedᮀ id-micro typing system. material and methods: during the study period 57 375 patient samples were screened, 414 of the positive results were evaluated. 377 blood samples with positive screen tests by the ortho auto vue tm method (av) performed with 3% cell suspension, and 37 patient samples with antibodies identified by the diamedᮀ system (dm), were reciprocally re-screened, respectively. positive samples were tested by diamed panels for antibody identification. sera samples were divided into 6 categories according to antibody specificity; 1. rh system antibodies (n = 150), 2. clinically significant non rh system antibodies (n = 85), 3. clinically non significant antibodies (n = 28), 4. auto antibodies (n = 13), 5. not identified (ni) antibodies (n = 48), 6. negative screening results by the diamed technique (n = 90). the 6 categories were divided, according to the intensity of agglutination in the screen test, into those exhibiting stronger reactions by the auto vue (av > dm), those exhibiting equal strength reactions (av = dm) and those with weaker reactions by the auto vue (av < dm). statistical analysis was carried out using the wilcoxon signed ranks matched-pairs test. results: a total of 414 samples were compared, 31.2% of them gave stronger reactions by av, 45.4% gave equal reaction strength and 23.4% of them gave weaker reactions by av. positive screen tests by av only were detected in 90 samples, no specific antibodies were identified. in contrast to that, positive screen tests by dm only were detected in 9 samples, 5 were rh system related, 3 kell system related and 1 not identified, results are summarized in the table. discussion and summary: the antibodies detected by dm only, are anti-d and antibodies directed to low frequency antigens. the failure of av to detect rh system antibodies is further sustained by the fact that statistically significant weaker reactions for this antibody system were obtained by av technique. recently ortho-clinical diagnostics modified the screening reagent red blood cells to 0.8% suspension, in order to improve the sensitivity of the method (unpublished data). the possible explanation for the failure to detect low frequency antibodies is that ortho screen cells do not consistently carry the low frequency antigens cw and kpa. positive screen tests detected by av only, can be explained either by the fact that antibody identification was carried out on dm panels, or these are false positive reactions. in order to clarify this question we recently repeated these av only positive samples by the manual ortho bio vue technique. preliminary results indicate that no specific antibodies were detected. it can be assumed that these results are false positive. further study of this issue is required. aim of the study: we have detected blood donor with rohar variant which was mistaken as d-and his donations used for d-recipients. we tested these patients in order to evaluate possible anti-d or anti-lfa immunization. methods: rohar variant was tested serologically (commercial and workshop moabs) and on dna level (pcr-ssp). involved recipients were tested by diamed column agglutination (gliat with normal and enzyme treated rbcs) with commercial rbcs and with rh33 and rh50 rbcs. results: rohar variant was confirmed on phenotype and genotype levels. in four d-and two d+ recipients of rohar positive units no anti-d not anti-rh33 or -rh50 antibodies were detected. in one case anti-le(a) antibody was found. conclusion: in our cases massive exposition of recipients (whole transfusion unit) by rohar red cells did not lead to production of detectable anti-d or anti-lfa. the immunogenic potential of this variant seems to be low. unusual ab0 grouping discrepancy -inhibition of anti-b reagent by isolated increase of plasmatic b substance in a patient with group ab and pancreatic cancer m pisacka*, k petrtylova † , m kralova* and h flidrova* *uhkt, prague 2, † blood bank, faculty hospital m, prague 5, czech republic introduction: in rare pathological conditions excess amount of blood-group specific substances can be observed and can cause neutralization of grouping reagents. changes in abh and related histo-blood group antigens in malignant tissues were described but there are few information about similar changes in secreted bloodgroup specific substances. aim of the study: we describe a case of isolated increase of b group substance in plasma of a group ab patient with pancreatic cancer. methods: ab0 grouping was performed with registered immucor reagents (immuclone: anti-a birma 1, anti-b lb2) by slide and tube tests. neutralizing effect was quantified by (i) inhibition od anti-b reaction by titrated patient's serum; and (ii) inhibition of titrated anti-a and anti-b reagents by patient's serum, compared to ab serum of a healthy donor. results: slide test: unwashed rbcs: group a, washed rbcs: ab. tube test (washed rbcs): ab. titrated patient's serum when added in aliquot to anti-b reagent inhibited agglutination up to titre 64. titration of reagents /+ aliquot of serum added/: anti-a: titre 4096 (both patient's serum and control); anti-b + patient's serum: titre 4, anti-b + control: titre 4096. conclusion: pancreas is known as rich source of blood group specific substances. malignant transformation is known to be associated with either loss or re-expression of cell-bound abh antigens. reported excessive increase of b substance in group ab patient could be either due to loss of a-transferase activity in malignant pancreas cells or isolated increase of b-transferase activity. further studies on larger groups of pancreatic cancer patients will help to understand this observation. weakened abh reactions of unwashed rbcs could be of diagnostic importance, because in other case this observation preceded several years the pancreatic cancer clinical manifestation. implementation of the autovue innova, an upgraded column agglutination technology (cat) for pre-transfusion testing in a large blood establishment c politis, k armyros, a antypas, v malamou and p katsea g. gennimatas general hospital, athens, greece objective: automated cat testing in a blood transfusion laboratory aims at standardization and savings in labour as well as reagents. a new technology is evaluated in comparison to standard methods. materials and methods: we used column agglutination technology with the innova autovue system, ortho, ratrian n.j. according to the manufacturers this system provides priority to the management of the stat samples while the random access feature of the system enhances the system throughput. it provides an extended test menu as well as automated antibody identification with the red cells panels. the autovue innova system supports the bi-directional communication with the lis interface for all the tests including the crossmatches and it provides a continuous traceability and notifying of the instrument's status concerning either the required and available resources or the proper function of the system's submodules. in this study we performed 1423 tests including forward and reverse abo group, rh type and phenotype and kell in randomly selected blood donors, as well 895 tests in haematological patients for antibody screening using an untreated three-cell panel and autocontrol in the indirect antiglobulin test (iat). the results and the time performance (specimen handing, operation of testing and recording of results) were compared with those obtained by the semiautomatic id-diamed gel agglutination microtyping system and by standard manual methods. results: test results showed 100% agreement between all three methods. 68 samples were tested per 60 min with the autovue innova, compared to 90 min required for the same number of tests performed with manual testing and 70 min with the semi-automated method. the technical execution was easy with the autovue innova procedure and it appears that the consumption of testing reagents is smaller with the automated system comparing with the other methods. computerization of the test results with the autovue innova provides an important advantage in record keeping in the blood establishment. conclusion: standardization of sample collection and tests performance in pre-transfusion testing, as well as computerized records and time saving are advantages offered by the autovue innova, a new automated column agglutination technology, comparing with a semi-automated and the classical manual methods. a heavy workload is expected to significantly decrease time performance. clearance of senescent erythrocytes in young and old individuals al racca, a ensinck, c cotorruelo, s garcía borrás, l racca and cs biondi universidad nacional de rosario, rosario, argentina introduction: after a lifespan of 120 days, human red blood cells (rbc) are captured and phagocytized by monocytes/macrophages. the accumulation of autologous igg on rbc membrane provides a direct mechanism for the removal of senescent (se) rbc. an alternative pathway, immunoglobulin-independent, with participation of sialic acid, has been proposed. the physiological elimination of serbc might be modified by individual's age. aim of the study: to investigate in young and old individuals, the interaction between monocytes and different erythrocytes suspensions: serbc, rbc stored with or without serum and desialinized rbc. methods: healthy individuals blood samples (20-40 years old, n = 50 and > 70 years old, n = 49) were studied. different suspensions from each sample were obtained: (i) se and young (y) rbc by differential centrifugation; (ii) rbc stored with its own serum (rbcs) and without serum (rbcws); and (iii) rbc desialinized with neuraminidase (ne) and tripsine (t). the suspensions were subjected to the erythrophagocytosis assay: peripheral blood monocytes were incubated with the different erythrocyte suspensions for 3 h at 37°c. two hundred cells were analyzed to determine the percentage of active monocytes (am) with phagocytosed and adherent red cells. non sensitized rbc (nrbc) and ex vivo sensitized rbc (srbc) were used as negative and positive controls respectively. results: the% of am obtained with old individuals were: serbc: 21.9 + 1.0, yrbc: 2.9 + 1.2, rbcs: 16.2 + 1.4, rbcws: 3.1 + 0.8, nerbc: 11.1 + 1.3, trbc: 3.2 + 1.1, nrbc: 3.0 + 1.2; srbc: 31.2 + 1.8. the values of am obtained with young individuals were: serbc: 17.1 + 1.5, yrbc: 3.1 + 0.9, rbcs: 11.1 + 1.1, rbcws: 2.8 + 1.2, nerbc: 10.8 + 1.4, trbc: 3.5 + 1.0. nrbc: 2.9 + 1.3; srbc: 30.5 + 1.7. conclusions: no differences in the% of am were found when compared to positive and negative controls, indicating that this assay would not detect variations in the phagocytic activity of monocytes from young and old donors. the values of am with serbc were higher (p < 0.001) than those obtained with yrbc in both populations analysed. the rate of erythrophagocytosis with serbc in old individuals was significantly higher (p < 0.001) than that obtained in young donors. the increase observed may be due to agedependent changes of rbc that occur with human ageing. the% of am with rbcs were higher (p < 0.001) in old individuals. no modifications were observed with rbcws. the significant increase in the rate of erythrophagocytosis with serbc and rbcs show the involvement of autologous igg in the selective removal of erythrocytes. these values were higher in old individuals indicating that this process would increase in aged donors. the tripsine activity was not enough to modify the% am. the values of am obtained with neuraminidase treated rbc were higher than those observed with yrbc (p < 0.001). however these values were similar between young and old individuals, suggesting that the desialylation would not participate in the increased removal of erythrocytes observed in old donors. cell-cell adhesion is a crucial phenomenon for the relationship between the cell and its environment. therefore, the development of experimental methods to obtain quantitative parameters of cellular adhesion is important. erythrocytes are widely available and their membrane properties are well known, so these cells are used as an ideal model for studying the cellular interaction mechanisms. the study of the formation and break-up of receptor-ligand bonds in sheared erythrocyte suspensions is a subject of considerable importance in the blood circulation, where formation and break-up of blood cell aggregates occur in a variety of physiological and pathological conditions. the main two objectives of this work were to study the cellular adhesion phenomenon using erythrocyte adhesion mediated by monoclonal anti-a antibody as a model and to achieve quantitative values of the parameters involved in the intercellular binding and its possible relationship with cellular deformability parameters. agglutinates of two a erythrocytes (doublets) induced by specific monoclonal antibodies anti-a were used. adhesion energy was indirectly quantified by the study of doublet dissociation under the effect of a given shear stress using a controlled flow chamber system. the chamber was installed on the stage of an optical inverted microscope (union optical, magnification 100¥) in such a way that the cover glass was the floor of the microchannel. a ccd (charge coupled device) camera was placed in the ocular tube of the microscope and connected to a digital image processor (ipplus system) to digitize, record and analyze microscopic images. the doublets were initially immobilized inside the chamber, and then put under different shear stress values by a controlled laminar flow during a definite time. in this way, a shear stress parallel to the contact surface between both cells was applied, observing that the upper cell detached progressively with time and also with a gradual rise of the shear stress. the sequential microscopical images were registered and digitally processed, measuring the geometrical dimensions that the cells acquire during the deformation process, and the dissociation of the antigen-antibody bond. digital image processing allows the analysis and the quantification of the red blood cells doublets dissociation phenomenon. these results show that, while the shear stress applied is raised, the contact area between both cells diminishes. the obtained parameters give important information that lead to the estimation of the value of adhesion energy between two red blood cells agglutinated by monoclonal antibodies. as consequence, they will allow the characterization of the antibodies used, since it would evaluate their association capacity with cellular antigens. glycophorins (gp) a, b and c are abundant transmembrane integral proteins in red blood cell (rbc). their highly glycosylated nature and high sialic acid content account for the net negative charge of mature rbc membrane, which is physiologically important because it impedes any tendency to stick together in the circulation. in this study, we have tested anti-gp specific mouse monoclonal antibodies (moab) as primary antibody to directly agglutinate rbc. fret technique has been developed to characterize the hemoagglutination based on the interaction of fluorophores (alexa488tm, dio and dii) located in the rbc membrane or combined to secondary antibody directed against the primary agglutining moab. combined intensity (spectral) and lifetime (flim) imaging was used to discriminate the fret signal of molecules on their different lifetimes whereas their emission spectra overlap (alexa488tm or dio/dii) as independent phenomena of the fluorophore concentration and photobleaching. furthermore, gp-cytoskeleton interactions were analyzed by 3d-fluorescence microscopy after lipid extraction with triton x-100 in red cell. all the antibody used was found to directly agglutinate the human rbc. in view of the fluorescence properties depicted in rbc for the pair of fluorophore alexa-488tm (donor) and dii (acceptor), it may be expected that upon agglutination of rbc, effective fret should be observed. flim in dynamic-state provides a discrimination of molecules in their fluorescence lifetime, which allows to evaluate the underlying mechanism of energy transfer process in the agglutinated erythrocytes. the results demonstrate that's upon excitation at 780 nm the flim-fret from alexa488tm to dii for the anti-gpb moabs only with a lifetime distribution in the picosecond range. similarly, effective fret was not observed for the anti-gpa moabs. the contrast in measured lifetime image is a reliable indicator for spatial variations in donor-acceptor association. 3d-fluorescence microscopy images showed interactions between gpc or gpb and cytoskeleton and did not show interactions between gpa and cytoskeleton. all together, these results only revealed by fret-flim and 3d-fluorescence microscopy strongly support the importance of the specific reactivity with glycophorin a or b of agglutining moab. introduction: the frequency of alloimmunization to red blood cell antigens in transfused sickle cell patients can range from 10 to 40%, but the development of autoantibodies is much less recognized and descried. aim of the study: in order to evaluate the autoantibody formation and observe the blood transfusion association, we analyzed the direct antiglobulin test (dat) as well as the antibody screening results in 612 transfused sickle cell patients. methods: all the patients included in the study had received at least 1 unit of red blood cell concentrate, on the majority of the cases matched for the c, c, e, e, k antigens. the transfusion range was 1-94 units. the dat performed was a polyspecific gel test. in cases of positive dat, was performed the monospecific gel test (igg, iga, igm, c3c, c3d) . in almost all of cases an acid glicin elution was performed and the eluate was tested against a red cell panel (liss/coombs and papain). an antibody screening by gel test (liss/coombs and papain) was performed in all the 612 patients. the dat was positive in 98 (16%) of the 612 patients. in 96 cases (98%), the dat was igg type, in 1 case (1%) igg + c3d and in 1 case (1%) igg + c3c + c3d. the acid elution was performed in 97 cases. the eluate was positive in 38 cases (39%). in 32 (84%), of the 38 cases, autoantibodies were pointed out whose specificity were 25 specificity public, 5 anti-e (rh5), 1 anti-c (rh2), 1 anti-ce (rh7). in 6 cases (16%) we found alloantibodies whose specificity were 2 anti-e(rh3), 2 anti-k(k1), 1 anti-c (rh2) and 1 anti-jka (jk1). in the 59 cases (61%) no antibody was identified on the eluate and the cause of positive dat is unclear. we could correlate the positive dat with a presence of autoantibodies in 32 (5.2%) of the 612 patients. of these, 13 (40%) had a blood transfusion association. the global frequency of red cell alloimmunization in this set of 612 patients was 15%. seventeen patients (53%) of the 32 patient who had autoantibodies had also alloantibodies associated. conclusion: the mechanism by which erythrocyte antibodies form in association with blood transfusion are not well understood, but even though the medical literature indicates strong association with blood transfusion as well erythrocyte alloantibody formation, we could not find support for this association. the loss of splenic to sickle cell patients could be important because experimental studies suggest that the spleen is involved in the regulation of autoantibody formation. forward and reverse blood grouping with lateral flow based assays p schwind*, i aebischer*, k loester † and p monod* *medion diagnostics gmbh, duedingen, switzerland, † prisma diagnostika gmbh, berlin, germany background: recently, a lateral flow assay for simultaneous typing of abod, rhesus subgroups and kell with stable end-point and without a centrifugation step was presented (loester k, fleischhauer s, schwind p: lateral flow assay for simultaneous typing of of abo, rhesus subgroups and kell. vox sang 2004, 87 (suppl. 3), 40). in many countries, the determination of isoagglutinins in addition to the red cell antigens is mandatory for abo grouping. aims: to develop a lateral flow test for reverse grouping, supplementing the lateral flow typing assay. a lateral flow device was constructed with a separation membrane equipped in a cassette housing having 4 distinct incubation wells, application zones and detection areas. 25 microliters of 5% suspensions of reagent red cells for reverse grouping (reverse-cyte a1, a2, b, 0, medion diagnostics, switzerland) are mixed in each incubation well with 100 microliters of plasma. the resulting suspensions are incubated for 2 min, followed by the transfer of 50 microliters each to the application zones, where migration starts immediately. results can be read after 3 min in the detection areas. a positive result is recognized as a distinct red dot, a negative result is monitored by the absence of a dot. results: the plasmas of 80 donors, previously determined for the respective blood groups and isoagglutinins by the tube technique, have been tested with this method. the results for both methods were in full agreement. conclusions: a simple, rapid and flexible lateral flow method for reverse grouping is presented, allowing now for the determination of forward and reverse typing in similar formats. both methods give results after 5 min with stable end-points without the need of a centrifugation step and are easily applicable to non-laboratory environments. the performance of the autovuetm system for red cell antibody screening of blood donors during background: in israel every donation is tested for the presence of red cell antibodies (rbc abs). screening is performed on the autovuetm system, using ortho 2 screening rbc since the year 2000. aim: (i) to summarize the performance of the autovuetm system for rbc abs, during 4 years (2000) (2001) (2002) (2003) (2004) . (ii) to evaluate if an initial low positive result, with two negative repeats, could be considered a negative result. methods and results: rbc abs screening was performed using igg cassettes. positive results were confirmed by diamed gel cards, with 3 screening rbc, followed by diamed panels for abs identification. results: summary of the results is presented in the table. (i) in 0.57% of 1358,769 blood donations that were screened, during 2000-2004, using the autovuetm system, a positive initial result for rbc abs was detected, which was confirmed in 40.2% (0.22% of the donations). an increased percentage of confirmed tests is noted, since 2003, probably due to an improvement in the manufacturing of the cassettes by ortho. (ii) during the validation, 83 samples with low positive results (agglutination degree of 0.5) were retested twice on the autovuetm. 57/83 (69%) gave negative results in both repeats. only 1/57 was confirmed positive and anti-m was identified by diamed gel test. the remaining 26 samples had at least one positive repeat. 5/26 (19.2%) of those samples were confirmed positive. summary: autovuetm can be used as a competent method for rbc abs screening, in blood services which require high thoughput automated systems. samples with a low positive agglutination, which were negative in two repeats, can be considered negative. retesting these samples on the autovuetm, can reduce the number of tests sent for confirmation and allow early release of blood components. rk tagi-zadeh*, aa karimov*, ar hasanov † , ly novruzova* and si donskov ‡ *hematology and transfusiology, baku, † blood transfusion centre, ganja, azerbaijan, † centre for hematology, moscow, russian federation background: the main method of treatment of severe homozygous thalassemia forms at the moment is still an anemia control with regular rbc transfusions. the use of adequate transfusion regime for these patients not only prolongs their lives but also promotes normal physical development of the children and improves the quality of their lives. however, necessity to do multiple transfusions increases a risk of post-transfusion reactions and complications due to alloimmunization to rbc antigens. in order to prevent posttransfusion reactions it is essential to know the frequency of blood group distribution in the region and then implement organizational procedures to enhance the transfusion services for these patients. objective: examine the distribution of rbc antigens among thalassemic patients and blood donors. methods: 108 blood samples of homozygous betta-thalassemia patients (who stayed at the daytime inpatient wards of scientific research institute of hematology and transfusiology baku, azerbaijan) and 1690 blood donors of azeri nationality were examined. 108 patients' blood samples were typed on rbc rh (c, c, d, e, e) and kell (k, k) antigens. gel test bio-rad (france) was used to detect rbc antigens. results: phenotyping of the patients' rbc rh antigens (d, c, c, e, e, cw) revealed that frequency of occurrence of the antigens in general was higher than in the donors. studying of rh phenotype distribution showed that the patients' ccdee (44.1%) •• ccdee (30.4%) phenotypes were twice as much higher than the occurrence of those in the donors (20% and 16.5% respectively). phenotype ccdee occurs more frequently in the donors (32%), whereas it is significantly rare in the patients (8.8%). the similar picture was observed in relation to ccdee and ccdee phenotypes, which occurred more frequently among donors (11.8% and 10.6%) than in thalassemic patients (6.86% and 2.94%). additionally, the results demonstrated that k antigen of the kell system was not detected in the thalassemic patients whereas k antigen was detected in all the patients. the same picture was observed with two other antigens of this system. thus among the patients typed on rbc antigens kpa antigen was detected in just one case whereas kpb was detected in the rest the patients. the results of the analysis showed that for thalassemic patients with phenotypes ccdee and ccdee it is easier to find compatible blood than for patients with phenotypes ccdee, ccdee and ccdee. furthermore, it is known, that some congenital diseases are genetically connected with the specific group systems, for example, the mcleod syndrome is genetically associated with the sharp suppression of the kell alloantigen expression (absence of gene kx). the fact of the discovered absence of antigen k in homozygous bthalassemic patients makes it possible to assume, that this group of patients suffer from scarcity along the kell system, like the scarcity in mcleod syndrome. all the mentioned above make it possible for us to conclude, that prior to blood transfusion to thalassemic patients phenotyping of rbc rh and kell antigens must be carried out. hyperhaemolysis in sickle cell disease due to complement activation. tessa thorp rci national blood service manchester uk tm thorp national blood service, manchester, uk introduction: sickle haemoglobin is caused by a genetic mutation in codon 6 of the beta globin gene, resulting in the conversion of glutamic acid to valine. when critical amounts of polymer accumulate within the sickled erythrocyte cellular injury results. clinically sickle cell disease is characterised by chronic haemolysis and intermittent vaso-occlusion. mold et al. demonstrated that deoxygenation and sickling of erythrocytes is related to membrane phospholipid changes and these changes result in the activation of the alternative complement pathway. aim: the objective of this study was to measure the amount of c3c and c3d bound in vivo to red cells of homozygous and heterozygous sickle cell patients. these levels were then compared to 'normal' sickle negative blood donors. haemoglobin levels and red cell morphology were also examined for signs of active haemolysis. the hypothesis was that an increase in red cell bound complement in vivo could provide an indication of hyperhaemolysis syndrome is sickle patients. method: flow cytometric analysis using rabbit anti-c3c or anti-c3d and fitc labelled goat anti-rabbit was developed using a beckman epics xlmcl flow cytometer. control samples were prepared using a fruitstone buffer technique of complement coating. results: a total of 16 heterozygous sickle patients and 11 homozygous patients were analysed. of the 11 homozygous patients analysed only one was undergoing a sickle crisis. a positive result was indicated if the mean trait or homozygous sample was coated with complement to a greater degree that the mean normal donor plus two standard deviations. the results obtained in this research project indicate an increase in c3c levels bound to red cells of homozygous sickle patients in vivo. statistical analysis of results obtained suggest that there was no increase in c3d levels in either sickle trait or homozygous sickle patients. conclusion: these findings support research carried out by mold et al. (1995) and are present in more than 90% of caucasian population. it has been reported that anti-chido and anti-rogers don't cause hemolitic reaction but may be responsible for life-threatening anaphylactic reaction during transfusion of plasma proteins. case report: positive iat and dat were detected in a 34 years old swedish woman, a rh negative, at 34th week of pregnancy. previous iat and dat determinations were negative. at time of detection dat wsa weakly positive and igg subclasses identified. antibody identification revealed the presence of high titre (1 : 64) anti-chido and anti-rogers antibodies. a slight decrease in platelet count (from 210.000/ul to 80.000/ul) was observed. this pattern showed no variation until the end of the pregnancy. at 39th week a healthy baby was delivered, dat negative. the mother dat and iat remained positive for four months after delivery. the platelets count raised again to 200.000/ul. the same laboratory findings were detected in a previuos pregnancy in sweden. the patient reported the presence of antibodies at 35th week that disappeared few months after delivery. a healthy baby, dat negative was delivered in this case too. conclusions: this is the first report of the presence of chido and rogers as autoantibodies during the last months of pregnancy. the association with decrease in platelets count and the lack of evident clinical symptoms need further investigation. p-289 rbc alloantibody frequency and their prevalence within chinese, malay and indian community in singapore e widjaja, mbc koh and d teo health science authority, singapore, singapore background and aim: there is a recognised existence of different alloantibodies in different ethnic groups. while this has been studied in the caucasian population, their frequencies remain less well documented in the asian population. the frequency of different alloantibodies and their distribution in terms of age, sex in singapore population is studied, as were their prevalence within the chinese, malay and indian races in singapore. design and method: we conducted a retrospective study for the frequency of 28 alloantibodies on 1856 blood samples over 2004. these blood samples were largely sent by hospitals where preliminary antibody screening had been done and positive result obtained. they were sent to the centre for transfusion medicine for antibody identification. small number of these samples came from hospitals where preliminary antibody testing was not done. the antibody distribution across different ethnic groups (chinese, malays, indians) age and sex were studied. results: the most frequent alloantibodies in the population is mia (40.7%), e (20%), le a (17.5%), le b (14.8%), p1 (6.1%), m (5.4%), d (4.5%), c (2.7%), jka (1.9%) and c (1.4%). within the chinese community, the most frequent alloantibodies were similar: mia (51.9%), e (24.1%), le a (11%), le b (8.8%) and p1 (6.1%). in the malays, the most frequent alloantibodies were le a (35%), le b (26.4%), mia (24.2%), e(13.4%) and p1 (8.2%); while in the indians, these were mia (28.9%), le b (28.9%), le a (27.8%), d (17.5%), and e (12.4%), with the anti-d reflecting the higher incidence of rh d negativity in the indians race. for the lower incidence antibodies, anti-c was more common in the malays and indians (4%) compared to chinese (1.9%). anti jka tended to occur mainly in the malay race and anti-c was rare in all (<2%) reflecting the high prevalence of c in the singapore population (r1r1 phenotype). the ratio of alloimmunised male to female (m : f) is 1 : 3. most alloantibodies demonstrated significant skewing towards to the female, although relatively less so for mia where m : f ratio is almost equal at 1 : 1.9. alloimmunisation increased with age for mia, e, k, p1, jka and fyb while the frequency of alloimmunisation to lea, leb, d, m and c decresed with age. the prevalence of patients with multiple alloantibodies (2 or more) within the alloimmunised subjects is 21.7%. conclusion: anti mia is very common within the asian population especially in the chinese. anti d is common in the indians. most antibodies show increased frequency with age except for anti lea + b, d and m. the majority of alloimmunised patients are females. study aim: to identify the present antibodies in newborns, after a positive direct antiglobulin test (dat). material and method: during a 7 years period, from 1/1/1998-12/31/2004, we studied 2652 newborns and their mothers. each newborn was examined for abo group, rhesus with phenotype, kell and dat. each mother was also tested for abo group, rhesus with phenotype, kell and indirect antiglobulin test (iat). after each positive dat, the study was continued with the elution test, in order to identify the present antibody. dat test was performed with dc screening i-diamed sa, switzerland. for the laboratorial analysis of newborns the sample used was cord blood and in certain cases venous blood. results: the dat test was positive in 132 (5%) of newborns and the antibodies found were igg type immunoglobulin. anti-a antibody was detected in 89 (67.4%) (newborns of group a with mothers of group o), anti-b antibody in 25 (19%) (newborns of group b with mothers of group o), anti-d antibody in 2 (1.5%) (newborns d+ from d-mothers), anti-e in one case and anti-jka in another one. the eluate test was found negative in 3 newborns and in the rest 11, no special antibody could be identified. the results are presented in the following table. conclusion: the majority of antibodies in newborns with a positive dat test, is due to abo incompatibility (the mother belongs to group o and the newborn to group a or b). anti-d (2), anti-e (6), anti-k (7), anti-fya (5), anti-s (2), anti-jkb (1), anti-n (1), anti-kpb (1). in two patients 2 antibodies were identified, while in 24/97 (24.7%) no antibody was identified (unspecific). it is remarkable that only in 10 out of 30 patients with both dat and iat positive, an irregular antibody was identified, while the rest 20 patients had unspecific antibodies. in 19 patients with only iat positive, 15 had an irregular antibody and 4 had unspecific antibodies. in 3 out of 31 patients with both dat and iat negative the cause of incompatibility was the positive dat in the corresponding sample of the blood donor, while in the rest 28 patients the reasons were technical problems that include the inappropriate blood sample of the patient, patients under medication and errors during the crossmatching procedure. the results show that the incidence of red cell incompatibility in our hospital is 2.96% and the most common antibodies are anti-k, anti-e, anti-fya, while anti-d is important for d negative patients. 24.7% of the detected antibodies were unspecific and this is still a problem that possibly was due to the lack of additional panels of reagent red cells or antibodies to low-incidence antigens. finally, in some cases the reasons for incompatibility are due to factors affecting the blood donor or to technical problems in the crossmatching procedure. multiple isoforms excluding normal rhd mrna detected in rh blood group del phenotype with rhd 1227a allele introduction: del phenotype is very common in rh-negative chinese. the rates in hans (more than 91% in china) were reported from 26% to 30%. moreover all del individuals in this population were found mainly carrying a same allele, rhd 1227a, through genomic dna analysis. those individuals always possess one or two of this allele with ccee or ccee phenotypes. aim and the study: we focused on the mrna investigations of del individuals carrying rhd 1227a alleles, in chinese, to expect it could be explained that why a silent mutation is associated with del phenotype. the full-length rhd mrna was analyzed in 2 rh-positive donors with cde/cde and cde/cde genotypes, respectively, and 4 del phenotype individuals carrying rhd 1227a allele with cde/cde, cde/cde, cde/cde and cde/cde genotypes, respectively, through reversed-trancriptase pcrs and cdna direct or cloning sequencing. results: five transcripts and 6 isoforms were detected in rh-positive and del, respectively. among them, 4 isoforms have identical sequences, which are transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7 to 9 spliced out. the normal rhd mrna was only observed in rh-positive, but not in del individuals. in stead, two additional transcripts were found in del individuals. its exon 9 or exons 8-9 were spliced out, but both possess a 170 bp segment of sequence from intron 7 of rhd. through 2 additional reversedtrancriptase pcrs, which amplified exon 8 to 3¢-region and exon 9 to 3¢-region, the results showed that exon 9 did not exist in del anyway. conclusion: (i) a normal rhd protein does not exist in a del individual with rhd 1227a allele since the exon 9 was always spliced out in all isoforms. all 6 transcripts in del maintain a normal open reading frame and encode 6 proteins with different numbers of amino acid residues and different c-terminals (genbank ay751491, ay751492, ay751493, ay751494, ay751495, ay751496). among them, the sequence of del9 (isoform with exon 9 spliced) transcript was the most similar as normal rhd mrna. this isoform was first described by chang et al. in taiwan in 1998 . it encodes 463 amino acid residues and has 46 amino acids more than normal rhd. it is different from rhd after codon 384. in normal rhd protein, the amino acids after 384 (33 residues) are mainly the trans-membrane and intracellular regions. therefore a further study on if a del red cell possesses all epitopes of normal d antigen may be significative. (ii) a normal rh-positive individual has also the transcript of del9 that was found in del. (iii) there is only one polymorphism in the region of 170 bp segment between rhd intron 7 and 2 of the del transcripts, which indicated that other polymorphisms may exist in intron 7 of rhd 1227a allele compared rhd to explain that this situation was not happened in normal rh-positive individuals. total wbc were enumerated by flow cytometry and cell counting. wbc subsets were analyzed by flow cytometry with three-color fluorescence. in this study, the third generation bags and filters are used. results: before filtration, the total number of wbc, was significantly higher in fresh units compared with stored units, whereas in postfiltration samples the number of white cells was significantly lower in the fresh compared with the stored units. although absolute numbers were significantly reduced, filtration also induced significant changes in the proportions of subsets in hoth fresh and storod units, the percentage of t cells was decreased, whereas the percentage of b cells and monocytes was increased after filtration. in conclusion, both pre and post storage wbc filtration affect the proportions of wbc in the final product but pre storage wbc filtration of platelet concentrates is superior than post storage wbc filtration. the effect of pre-and post-storage filtration on platelet rich plasma: derived platelet concentrations background and objective: the white blood cells (wbc) within transfusion products are a major stimulus for a number of detrirmental biological reactions, including febrile nonhemolytic transfusion reactions, alloimmunization against hla antigens and cytomegalovirus transmission. in this work, our objective was to study the effect of storage time on the filtration of platelet concentrates (pcs). the total number of white blood cells as well as the distribution of wbc subsets, in units filtered before and after storage were compared. materials and methods: platelet rich plasma -derived pcs were filtered either fresh (5 pooled we reported earlier that metabolic arrest followed by incubation at 4°c reduces the platelet storage lesion (badlou et al. transfusion 2005) . here we report that this treatment also reduces binding and phagocytosis by macrophages. metabolic suppressed platelets (msp) were prepared by incubation in glucose-free, antimycin a containing medium (40 min, 37°c) followed by storage (48 h, 4°c) and recovery with glucose (1 h, 37°c). controls were (i) platelets in glucose-rich medium stored for 48 h at 22°c and recovery with glucose (c22) and (ii) platelets stored for 48 h at 0°c (c0) with rewarming. platelets were labelled with mepacrine and incubated with pma-matured thp-1 cells (37°c). binding was measured by facs analysis of cd42b/cd14 positive particles, and phagocytosis by counting mepacrine/cd14 positive particles. binding of msp, c22, c0 was 30 ± 4, 39 ± 10, 50 ± 12% of total platelets. phagocytosis of msp, c22 and c0 was 32 ± 9, 40 ± 8, 50 ± 9% of total macrophages (means ± sem, n = 4). before recovery of msp, binding/phagocytosis was 30% higher than thereafter, revealing energy-dependent control of the mechanisms that trigger plateletmacrophage interaction. these data show that metabolic suppression prior to cold storage attenuates binding and phagocytosis by phagocytes and may help to develop means to improve platelet survival post-transfusion. platelet compatibility testing and alloimunization in multiply transfused hematologic patients purpose: multiply transfused patients with heamotological malignancy often become refractory to platelets due to alloimmunization. refractoriness is usually defined as an insufficient platelet increment after consecutive platelet transfusions. two major causes of a decreased platelet increment can be distinguished, immune and nonimmune factors. alloimmunization occurs most frequently against the hla, and rarely against the hpa system. nonimmune factors been identified are splenomegaly, fever, sepsis, and disseminated intravascular coagulation as well as the quality of the transfused platelet concentrates. we performed this study in order to investigate platelet crossmatching, compatibility, and antibody determination among thrombocytopenic patients multiply transfused. we performed 164 crossmatchingcompatibility tests of single donor leucodepleted, abo compatible, platelet concentrates been transfused in 23 patients with leukemia and lymphoma, 18 males and 5 females with mean age 50 ± 25 years old. we also obtained samples from the patients for platelet antibody detection. we evaluated the cci (corrected count increment) 18 h after the transfusion. the solid phase rbc adherence assay (modified capture p system/immucor) was used for platelet compatibility and antibody detection. a total of 164 compatibility tests were performed, of which 84 were compatible. twenty five compatible platelet concentrates out of 84 were clinically evaluated. twenty from 25 compatible crossmatches (80%) were resulted in successful transfusion while only 5 from 25 (20%) in unsuccessful. the 80 incompatible platelets been transfused was resulted in unsuccessful transfusion. we found statistically significant difference among patients successfully transfused with compatible and incompatible platelets (p~0.01). additionally 14 patients out of 23 (60.8%) had been alloimmunized against multiple hla antibodies. three patients transfused with compatible platelets, during the study, developed alloantibodies. we found a large number of incompatible platelet concentrates that result in unsuccessful transfusion and clinical response. the platelet compatibility testing as well as alloantibody determination of multiply transfused patients is necessary for the identification and selection of compatible, with the patients, donors in order to result in succesful transfusion and clinical outcome. furthermore compatible platelet concentrates provide optimal support for refractory patients and it is known that they are acceptable as an alternative component. naitp is a rare clinical syndrome characterized by marked thrombocytopenia shortly after birth. it is caused by maternal immunization against paternally inherited antigens present on foetal platelets. screening and identification of antibodies in the maternal blood sample is the main support in the diagnosis and management of naitp. we have evaluated the frequency of maternal alloimmunization, the role of the antibodies involved (hpa and/or hla systems) and the pertinent risk of naitp in neonates using a fully automated system with a solid phase red cell adherence methodology (sprc-capture p) and paternal or random donors (indirect test). screening started in june 2003 and is still in course: in january 2005, 2300 blood samples were examined. identification of antibodies in maternal serum was carried out using elisa methodologies: maipa and commercial kits (pak-plus and quick screen-gti). of the 2300 blood samples analysed, 43 were reactive and the specificity of the antibodies were: anti hpa1a: 8, anti hpa1b: 2, anti hpa1a + hla: 2, anti hpa5b. 6, anti hla: 24, auto hpa-5b: 1. specificity of hpa antibodies was confirmed by determination of parents' hpa genotype (hpa-1,2,3,4,5,6) using pcr-ssp or pcr-rflp. the infants with hpa 1 immunization suffered from severe (plt count 5-103/ml) and symptomatic naitp (bleeding and petechiae were present), therefore they were treated with platelet transfusion and administration of high doses of intravenous immunoglobulin. we confirm that naitp due to hpa-5 and hla immunization is clinically less severe: all neonates had mild and self limiting thrombocytopenia at birth; no therapy was administered. it would be advisable to carry out pre-natal screening, at reasonable cost, using maternal serum versus paternal platelets and to proceed to the identification of antibodies only in presence of positive results. background: fetal or neonatal alloimmune thrombocytopenia (fmait) results from a maternal alloimmunization against fetal platelet antigens. it is the commonest cause of severe thrombocytopenia in the neonatal period. the diagnosis of fmait is made initially on clinical grounds, depending on exclusion of other causes of neonatal thrombocytopenia. in caucasians, hpa-1a is the most frequently implicated antigen. other antigens such as hpa-3a, or hpa-4a are less often implicated. during the past few years fmait has been reported associated with rare or private antigens. the diagnosis is straightforward when a maternal alloantibody with a corresponding parental antigen incompatibility is present. however it could be equivocal in the absence of such an antibody or difficult when a private antigen is implicated. if the father is heterozygous for the considered antigen, the infant's platelet typing should be performed to confirm the diagnosis. due to the risk of hemorrhage, particularly intracranial hemorrhage (ich), during the course of severe thrombocytopenia, specific therapy is mandatory. because subsequent siblings may be more severely affected, accurate diagnosis will allow better management of subsequent pregnancies. study design and methods: since the first documented case of feto-maternal alloimmune thrombocytopenia (fmait) due to anti hpa-9bw (maxa+), no additional cases have been reported. we present here a retrospective analysis of the cases referred to our laboratories in recent years. since 1999 we have screened for rare or private antigens in suspected cases of fmait when there is no incompatibility for the most frequently implicated antigens. the diagnosis was performed by genotyping and identification of the maternal alloantibody by the maipa technique. results: parental genotyping showed hpa-9bw (maxa+) mismatch as the sole antigenic incompatibility in 7 out of 8 families. in the last one, incompatibility was found for hpa-3 without anti hpa-3b maternal alloantibody. as the father was found to be hpa-9bw (maxa+) heterozygous in all the cases, the infant or fetus was genotyped to ascertain the diagnosis. the maternal alloantibody was identified in the maipa technique. however, our data strongly suggest that recognition of the hpa-9bw (maxa+) epitope is not uniform. the neonatal thrombocytopenia was severe in most cases with bleeding. the outcome was good in all the cases but one. conclusion: this analysis confirms that anti hpa-9bw (maxa+) fmait is not uncommon and was found to be around 2% of our confirmed fmait cases with parental incompatibilities and presence of maternal alloantibodies. it is a clinically severe syndrome which requires prompt diagnosis, albeit difficult, and maternal platelet transfusion therapy. laboratory investigation of a suspected fmait case should be carried out in a specialist laboratory wellexperienced in optimal testing. therapy requires strict collaboration between clinicians and blood bank services. appropriate management and antenatal therapy should be considered for successive pregnancies to prevent fetal bleeding. introduction: the human platelet (plt) antigen (hpa) system is independent of the hla system. therefore, host-or donor derived alloimmune thrombocytopenia can develop after allogeneic haematopoietic stem cell transplantation (hsct) even in hlamatched donor-recipient pairs. we report the first case on a stem cell recipient developing thrombocytopenia due to host-derived hpa-1a antibodies after non-myeloablative allogeneic hsct. a 52year-old male patient was diagnosed with multiple myeloma in 6/2003. treatment consisted of 3 cycles vincristin, adriamycin and dexamethason followed by tandem autologous stem cell transplantation. because of progressive disease he received 4 cycles of bortezomib, and after complete remission a stem cell allograft (7.99 ¥ 106/kgbw cd34 + cells) from his histocompatible (hla a,b,c,dr identical) brother after reduced intensity conditioning regimen with fludarabine (3 ¥ 30 mg/m 2 ) and alkeran (100 mg/m 2 ). he had received only twice packed red blood cell concentrates and one plt concentrate before allogeneic hsct. stable bilinear engraftment occurred around d12 but was accompanied with a continuous decrease of plt counts. between d10 and d19 the patient received seven plt transfusions, containing a median of 2.96 ¥ 1011 plt/unit (range 2,7-3,21 ¥ 1011 plt/unit) from random donors. the corrected plt count increments at 12 to 24 h after these transfusions were <2500/ml. therefore, and because of even a further decline of platelet counts to 1000/ml on d19 we investigated the presence of plt antibodies. methods: the patient's serum was tested by antigen capture elisa assays (pakplus® and pak1®, gti) and a solid phase assay (capture-p®, immucor). the maipa assay was used to confirm the results obtained by the above mentioned assays. in addition, we tested the patient's serum by the maspat kit (clb) against plt from the donor and against homozygous hpa-1a plt obtained from our donor pool. stored recipient's dna from the time before hsct was used for genotyping. genotyping for hpa-1, -2, -3 and -5 of the donor and the recipient was performed by pcr-ssp (hpa, protrans). the patient's serum obtained on d19 after hsct reacted strongly with the donor's plt due to anti-hpa-1a antibodies and antibodies against hla class i antigens. the patient's genotype before transplantation was hpa-1bb, -2aa, -3ab, and -5aa; the donor was hpa-1ab, -2aa, -3aa, and -5aa. thus, the antibodies were host derived and directed against the donor's plt. serum samples obtained on d38, d45 and d65 after hsct contained antibodies against hla class i antigens but hpa-1a antibodies were not anymore detectable. no hla antibodies were detectable on d149 after hsct. the severe thrombocytopenia was caused by hostderived hpa-1a antibodies. fortunately, plt counts started to increase on d20 spontaneously and the patient could be discharged at d27 (plt 51.000/ml) with a complete donor chimerism. the decrease of the serum antibodies parallel to the increase of the plt count strongly suggests a progressive elimination of residual host cells. we conclude that the hpa mismatch between recipient and host affected thrombopoietic engraftment and the success of plt transfusions. severe neonatal alloimmune thrombocytopenia with anti-hpa-5b antibodies: case report p moncharmont, m vignal, y mérieux and d rigal efs rhone alpes site de lyon, lyon cedex 07, france usually, in case of feto-maternal incompatibility, the platelet (plt) specific anti-hpa-5b antibodies (ab) induce only sometimes a mild neonatal alloimmune thrombocytopenia (nait). contrary to this observation here is reported the case of a severe nait. a 34-year old mother, gestation 2/partum 2, gave birth to a male neonate by caesarean section at 34 weeks of gestation because of intra-uterine growth retardation (iugr) and anamniotic fetus. five years before, she had had a first pregnancy with iugr of the fetus but no nait. the second neonate weighted 1.350 g, was 41.0 cm tall and had a head circumference of 27.5 cm. the apgar score was 9,10,10,10 at 1, 3, 5 and 10 min respectively after birth. no bleeding, hepatomegaly, splenomegaly or infectious signs were noted. five hours after birth, a respiratory distress syndrome appeared and an oxygenotherapy was performed during 44 h. the plt count which was 82.0, 79.0 and 73.0 giga/l at day (d) 0, d1 and d2 respectively dropped dramatically at 9.0 giga/l at d5. simultaneously, an intracranial hemorrhage grade ii was diagnosed on ultrasound scan. because of the clinical signs and of the decreasing plt count the mother's serum was tested for plt-specific ab by immunocapture and the ab identified by the monoclonal ab-specific immobilization of plt antigen (maipa) assay. a plt genotyping was performed in the neonate and his parents by sequence-specific primers polymerase chain reaction. the mother was hpa-5a/a and anti-hpa-5b ab were detected in her serum. the baby was heterozygous, hpa-5a/b. plt were transfused to the baby and the plt count rose to 80.0, 115.0 and 315.0 giga/l at d6, 10 and 18 respectively. no further transfusion was needed and the development of the baby was satisfactory with a normal electroencephalogram. in conclusion, when a mild thrombocytopenia with iugr and hypoxia but without bleeding signs is present in a neonate immediately after birth, a maternal plt specific ab screening must be performed in case the thrombocytopenia became severe during the newborn monitoring. anti-hpa-5b ab can be detected. partial results of the incidence of heparin induced thrombocytopenia type ii osc oliveira*, ra rached*, c cavalheiro-filho*, jc nicolau*, shgl pasqualucci*, daf chamone † and sp bydlowski † *heart institute, university of são paulo, † university of são paulo, são paulo, brazil heparin induced thrombocytopenia type ii (hit) is a severe side effect of heparin, associated with heparin-platelet factor 4 antibodies. hit type ii occurs in up to 5% of patients who are exposed to unfractionated heparin (ufh). in our institution patients that are under heparin treatment are mostly cardiac patients. the purpose of this study is to determine the incidence of hit type ii in these patients. material and methods: 111 patients from the intensive care unit and cardiac care unit treated with ufh or low molecular weight heparin (lmwh) for 5 or more days were studied. known causes of thrombocytopenia were excluded. platelet count was monitored pre and post heparin therapy. all selected patients were tested for detection of anti heparin/pf4 antibody test (diamed id-card). results: from the studied patients, 63 (56.8%) developed thrombocytopenia (determined by a decrease in the platelet count below 30%, after the introduction of heparin therapy); 48 (43.2%) did not show decrease in the platelet count. six (5.4%) out of 63 thrombocytopenic patients were positive for anti-heparin/pf4 antibody. three (2.7%) out of 48 non thrombocytopenic patients were positive for anti-heparin/pf4 antibody. the results demonstrate that 9 (8.1%) patients were positive for anti-heparin/pf4 antibody and they were no different from those described in the literature regarding the frequency of heparin induced thrombocytopenia. moreover, a higher frequency of patients with heparin/pf4 antibody was noted without the presence of thrombocytopenia, indicating that other factors should be considered. introduction: neonatal alloimmune thrombocytopenia (natp) due to maternal immunization against fetal platelet antigens affects 0.8-1 in 1000 live births. although it is usually a self-limiting condition, a major complication in cases of severe thrombocytopenia is the occurrence of intracranial haemorrhage leading to death (in up to 10% of reported cases). the commonest antibodies are anti-hpa-1a. treatment consists of ivig, compatible donor platelet concentrates or washed maternal platelets. the administration of random donor platelet transfusions is controversial but has been used successfully in some urgent cases when compatible platelets were not available. case report: a baby born in week 40 of gestation to a healthy mother after first uneventful pregnancy; birth weight 3000 g, apgar score 8. immediately after birth, severe thrombocytopenia (7 ¥ 109/l) and signs of haemorrhagic diathesis (generalized petechiae and ich gr. ii-iii) were observed. coagulation tests were abnormal and k-vitamin, fresh frozen plasma and random donor platelet concentrate (retrospectively genotyped as hpa-1a/a) were given. twenty-four hours later platelet count rose to 26 ¥ 109/l and no new petechiae were observed. on third day of life the blood platelet count was 13 ¥ 109/l and the newborn received ivig 2 g/kg and corticosteroides. twenty-four hours later the platelet count rose to 107 ¥ 109/l and further clinical course was uneventful. natp due to hpa-1a was serologically confirmed. conclusion: optimal therapy for an infant with severe thrombocytopenia during the first 24 h of life is the transfusion of platelets that will not be destroyed by the maternal alloantibody in the infant circulation. random donor platelet concentrates are controversial in a setting where optimal treatment is not available, however, in this case they led to a significant platelet count improvement in spite of hpa-1a incompatibility. accordingly, random donor platelets may be considered appropriate in emergency situations. background: rhd is the most immunogenic blood group antigen, and its correct identification is essential in the blood bank and in the prevention of the haemolytic disease of the newborn. the weak d phenotype is the most common d variant, with a frequency of 0.2% to 1% in caucasian individuals. there are several weak d types, with different frequencies in european countries, which may pose serologic problems and have the potential for alloimmunization. the objective of the study was to determine the frequency of the principal weak d types in portugal. study design and methods: lisbon regional centre of the portuguese blood institute and oporto são joão university hospital selected samples from blood donors and patients. rhd was tested by two (oporto) or three (lisbon) distinct anti-sera, in direct agglutination tests, at room temperature. when discrepant results were observed, the samples were tested with panels of monoclonal anti-d by liss-iat. samples that reacted weakly with igm anti-d but positive with igg anti-d were sent to the molecular biology centre. pcr with sequence-specific primers was performed using two commercially available kits (inno-train and bagene). real-time pcr, carried out on a light cycler, was applied when the interpretation was dubious. results: 101 samples were referred after being characterized as weak d. in 99 cases we obtained a positive result, with a preponderance of weak d type 2 (63.6%) over type 1 (16.2%), 3 (14.1%) and 4 (6.1%). two samples were not categorized. the high incidence of weak d type 2 in our population is in marked contrast to studies performed in other european populations where weak d type 1 was the most frequent. this might be due to our sample selection criteria or ethnic variation in the causes of weak d. there are advantages in genotyping serologically depressed d samples: to avoid the waste of d-negative rbc units and the use of immunoglobulin in pregnant women, who have no risk of alloimmunization. analysis of rhd zygosity in different rh phenotypes cal except for a 4-bp t insertion. the deletion of the rhd gene, found in most rhd-negative caucasians, was theoretically due to recombination of the upstream and downstream rhesus boxes resulting in the formation of a hybrid rhesus box. thus, the detection of a hybrid rhesus box in an rhd-positive individual denotes an rhd heterozygous status. aim of the study: to determine the rhd zygosity in different rh phenotypes. methods: blood samples from 106 white trios (father, mother and child) were studied. the rh phenotype was performed by hemmaglutination and the rhd zygosity was inferred in each member of the family groups. the rhd deleted allele was determined by a pcr strategy using a forward primer complementary to 5¢ end of the identity region of the upstream and hybrid rhesus boxes and a reverse primer complementary to the 3¢ end of identity region of the downstream and hybrid rhesus boxes. these primers selectively amplify a 1980-bp segment of the hybrid rhesus box in rhdnegative and rhd-positive heterozygous samples. serological and pcr inconsistencies were studied by a pcr-rflp method to detect another polymorphic site of the hybrid rhesus box. frequencies were obtained analysing only unrelated individuals (fathers and mothers, n = 212). results: 179 (84.4%) rhd-positive and 33 (15.6%) rhd-negative samples were phenotyped. of the rhd-positive donors, 75 (41.9%) were rhd homozygous and 104 (58.1%) were rhd heterozygous according to pcr. pcr-rflp analysis confirmed the results of pcr in serological and molecular discrepancies. these results were coherent within each family group and did not differ from those published in the literature for caucasians based on the most probable genotype method. however, the homozygosity indexes were significantly higher in the dccee (20.0% vs 6.6%) and dccee (16.6% vs 3.3%) phenotypes due to an increase of the dce haplotype. in all samples with the dce haplotype the rhces allele, frequent in individuals of african descent, was investigated by pcr-ssp. this allele was found in 33.0% of the dce haplotypes. . rhd and rhce typing was performed by multiplex-pcr with 24 fluorescent primer pairs. positive results were obtained for rhd-exons 2-7, 9, 10 and polymorphisms associated with antigens c, c, cw, e and e. sequencing of rhce-sequences, including exons 1 to 10 and intron/exon borders, were done by direct taq cycle-sequencing using bigdye-terminators v.1.1 in an abi 310 (applied biosystems). results: see table 1 . the substitutions of rhce-specific nucleotides in exons 3, 5 and 6 with their rhd-specific counterparts lead to 3 different new rhc-antigens with weakened expression. since one amino acid change in allele 3 lie in extracellular loop of the antigen suggested that this antigen may be involved in allo-immunization. inheritance of a rhd cat vi type ii in a twin pregnancy: a case report introduction: determination of hdn-relevant fetal blood groups in amniocentic fluid with pcr is a routinely used method. misinterpretation of test results -e.g. overlooking rhd category -decreases depending on the number of examinated rhd-specific exons. case report: a 28-year old mother (w.o.g. 23) pregnant with twins was regularly tested for irregular antibodies and was shown to have an anti-d. after amniocentesis of both foetuses tests for the occurrence of rhd intron 4, exons 7 and 10 were performed in the hospital's lab. the sample of fetus i showed pcr-products for intron 4, exon 7 and exon 10 while sample of fetus ii lacked rhd-specific intron 4. therefore we investigated blood samples from the parents as well as amniocentic fluid of both foetuses. methods: total dna was amplified in a multiplex pcr with fluorogenic primers for rhd exons 2-7, 9 & 10; polymorphisms for dweak type 1-5, d-vii, d-hmi and rhce polymorphisms for c, c, cw, e, e. capillary electrophoresis for size fractionation and fluorigeneic analysis was done in an abi 310. rhd zygosity was determined by quantitative real-time pcr with co-amplification of rhdand rhce-specific exon 3 and subsequent calculation of d ct value (ct rhce minus ct rhd). results: while maternal dna sample has been genotyped as rhdnegative, amplification of paternal dna for rhd-specific exons 2, 3, 7, 9 and 10 were possible and failed only in exon 4-6. determination of rhd-zygosity revealed a homozygous constellation of the rhd-gen. investigation of amniocentic fluid from both foetuses resulted in a rhd-wildtyp for fetus i and a rhd cat. vi type ii for fetus ii which was the reason for missing amplification in rhd intron 4 pcr. results: weak d type 3 was not identified in our research population. weak d type 1 was identified in 2 cn, weak d type 2 was identified in 1 cn and weak d type 4 was found in 1 cn, 3 bsa, 4 be, 1 bc and 1 saa. conclusions: although weak d types 1 to 3 represent the majority of all weak d phenotypes in caucasian populations, none of these weak d alleles were found in black populations. since none of the frequent weak d types were identified in non-caucasians one might not expected to find type 4 in all populations. however, regarding rhd phylogeny, the weak d type 4 mutations (602c > g and 667t > g) form the basis of a cluster of aberrant alleles that are predominantly observed in blacks. therefore, it is not surprising that weak d type 4 was identified in non-caucasians. based on these results it may be concluded that weak d phenotypes have evolved relatively recently since they are present in caucasian and asian methods: dna was extracted from 9 a, 14 b, 11 ab subgroups, and 21 provisional cis-ab after serological abo typing. allelespecific pcr, rflp, direct sequencing of exon 6 and 7, and allele separation were performed on these samples. results: abo*a205 allele was observed in an aint subgroup. two new a alleles that showed 784g > a base change and 990c > t of intron 6 and a polymorphism of 532c > t in a(pro) intron 5 were discovered. o04 and o20 alleles were also observed. in b subgroups, a silent substitution 255c > t (leu85leu) was observed as a new b allele. another new b allele which showed 547g > a was also found in 4 b subgroups. conclusion: we discovered new abo alleles and polymorphisms in korean populations. many other polymorphisms and alleles already reported in japanese were also observed in koreans. evaluation of a multiplex snapshot-pcr method for red blood cell marker genotyping c jungbauer*, c hobel*, em dauber † , pk rabitsch*, dwm schwartz* and wr mayr* *austrian red cross, † medical university vienna, vienna, austria once conventional reagents for identification of rare red blood cell phenotypes are scarce, methods using current nucleic acid testing techniques to identify the patient's genotype and possibly to screen for donors would be desirable. the approach of multiplexgenotyping using pcr-ssp has apparently technical constraints so we are currently analyzing whether a modification using snapshot pcr-technique could provide a routine application for such purposes. compared to ssp-technique, the snapshot pcr only requires one single primer (labeling reaction) for the detection of two (or more) alleles instead of one pair of primers for every single allele. briefly, we perform snapshot pcr as follows: in a first step, dna segments covering the essential polymorphic regions for the abo, rhd, kel, jk and fy genes are amplified using a standard pcr step. after purification treatment of the pcr products (roche high pure pcr product purification kit or shrimp alkaline phosphatase (sap)/exo1 treatment according to the abi prism snapshot multiplex kit protocol) the labeling reaction is performed using the abi prism snapshot multiplex kit. after a second purification step the products are analyzed on a abi prism 310 genetic analyser in fragment analysis mode. our preliminary results show the feasibility of this approach as reliable typing results can be obtained for all tested single nucleotide polymorphisms corresponding to alleles. generally a better signal quality from controls and samples is obtained compared to the ssp-technique with consecutive gel electrophoresis. we also consider the possibility of the automated interpretation of the results as an important improvement, especially when aiming for an application for a large number of samples (donors) or markers (patients). in contrast, the method involves more manual steps and higher costs. we may conclude that the implementation of snapshot-pcr techniques for red cell marker genotyping is a promising alternative to pcr-ssp. the obvious quality improvements compared to pcr-ssp might be critical for a routine application in blood banks (donor screening) or complex questions in clinical laboratories. quantification and quality control of monoclonal antibody using an optical sensor based on the laser reflectometry technique the monoclonal antibodies (moab) are biological reagents of homogeneous activity. they are generally used in the recognition and quantification of very small amounts of biological substances (hormones, enzymes) present in a solution, ag identification, blood group determination, oncology, organs transplant, etc. moab can be characterized by its specificity and affinity. affinity may be expressed as the equilibrium association constant (k). several techniques are available to determine the equilibrium constant of the ag-ab interaction. in this work, is shown an optical sensor for monoclonal antibody quantification by reflectometry technique. the laser reflectometry technique (null ellipsometry technique) can give information about the kinetic of the interactions, stoichiometry of molecular binding and the concentration of molecules in a solution, and also offers detailed and accurate determinations of real-time adsorption kinetics of protein without labeling. a silicon wafer was chosen as reflectant surface. once fixed the principal angle of incidence, an amount of anti-ab is added to the sample. the reflected laser intensity is registered in real time as the protein is being adsorbed onto the wafer. the mathematical analysis of the results verifies that the antibodies adsorption follows langmuir's kinetics. from the curve analysis, the parameters related to the anti-ab concentration are extracted. from them, the calibration curve is constructed. this curve allows the desired commercial monoclonal antibody quantification. the developed technique shows to be sensible and precise. the obtained graphics are very well approximated (r > 0.94) verifying that the monoclonal anti-ab associa study aim: to prevent the sensitization to rh0(d), to a d-patient who was transfused with d+ blood. material and method: on september 2003, (9/28/2003), we admitted to our hospital through air-carriage, a female of 22 years old, badly injured after a car-accident. the patient was on an olighemic shock (ht: 16%) due to retroperitoneal, paracolic and procystic hematomas, had multiple fractures on the left feet, the main important of which was an acetabulum one. she had a blood group: o, d-and her phenotype was ccdee with du-. after her arrival she was urgently admitted to the surgery room and our blood bank was asked for condensed red cells. initially she was transfused with blood of the same group (o, d-) but when we were short out of dblood, we were asked for 7 more units. the necessity of blood was imperative, the patient was on a critical condition and the mechanism of blood transport, from another blood bank would take some time to be put in motion, and so it was decided that the patient would be provided with d+ blood. the indirect antiglobulin test (iat) and papaine were negative, so there would be no problem with the transfusion with d+ blood. the patient received finally 3 units (850 ml) of d+ condensed red cells, out of 7 that were initially asked. before the 48h from the surgery had passed, it was decided that human anti-d immunoglobulin (rhesogamma p) should be provided, in order to prevent the sensitization of the patient to rh0(d). the indicted dose was 500-1250 iu/10 ml of the transfused blood, provided piecemeal during a several days period. analyzed by real-time pcr amplification. based on a published report, we selected 12 primer pairs targeting insertion/deletion polymorphisms which are located on different chromosomes, unrelated to each other and not associated with immunocompatibility. we optimized the amplification conditions for all 12 primer pairs using our sybr green real-time quantitative pcr protocols, and investigated analytical sensitivity for each primer pair by performing spiking studies, in which a single copy of positive dna was added into 100 000 copies of negative dna followed by allele-specific pcr amplification. we also created a theoretical panel of donor-recipient pairs (n = 73) to evaluate the clinical sensitivity for detection of ta-mc using both hla-dr and indel panels. results: for the short-term samples, 10 additional mc cases was identified in non-lr group using indel panel; and one additional mc case was detected in lr group (table 1) . for the long-term follow-up samples, 4 additional mc cases were found. when evaluating analytical sensitivity, we were able to detect a single copy of positive dna mixed with 100 000 copies of negative dna in a single amplification tube for all 12 primer pairs. we were also able to calculated the clinical sensitivity that using 73 donor-recipient pairs. 99.5% of donor-recipient had at least one informative allele for detection of ta-mc if we consider both hla-dr and indel panels. conclusion: using our new indel panel, we were able to detect more instances of mc in this cohort of patients. we conclude that the dr assay underestimates the presence of mc. moreover, the tandem use of both panels provides a powerful tool for the detection of mc with 99.5% of recipients having at least one informative allele. background: we reported severe immunesuppression and longterm transfusion-associated microchimerism (ta-mc) in transfused trauma patients. we have also reported, in a murine transfusion model, that sensitivity to 1-chloro-2-4 dinitrobenzene could be transferred, albeit transiently, by transfusion of fresh blood from a sensitized donor to a naïve immunecompetent recipient. in order to mimic the immunecompromised status of trauma patients and further investigate the mechanism underlying ta-mc, we established an animal model using immunedeficient knock-out mice. aim: our objective was to test virus-specific immune functionality of the chimeric donor leukocytes in a murine ta-mc model. material and methods: female rag-2/common gamma double knock-out mice were transfused with fresh blood collected from male balb/c mice, which were either not infected (non-primed, np) or infected twice (primed, p) with 100 million viral particles of murine cmv (mcmv). at different post-transfusion time-points (1 h, 2 weeks, 4 weeks, 6 weeks, 8 weeks), different female recipients plus non-transfused female knock-out controls were challenged with 100 million viral particles of mcmv intra-peritoneally, and then monitored weekly for the concentrations of male donor cells as well as mcmv viral load in recipient's circulation. each female knock-out received only one challenge of mcmv. if the subject died, we quantitated mcmv viral load in the brain, spleen, lung and liver. we used real-time quantitative pcr targeting murine y-chromosome, h2k and mcmv to quantitate male donor cells, transfused recipient cell dna input, and mcmv vrial load, respectively. the number of recipient cell dna input served as a denominator to calculate the concentration of male donor cells and the mcmv viral load. results: results of overall mortality are summarized in the table. all female knock-out recipients transfused with primed donor blood, except for the post-transfusion 6 weeks, are able to survive mcmv infection. all non-transfused control and recipients transfused with non-primed donor blood died after mcmv infection; these two groups also had higher mcmv viral load in blood than the recipients transfused with primed donor blood. when the subject died, we were able to detect mcmv in all four organs we analyzed, with liver having the highest mcmv viral load. there was no significant difference for the concentration of donor cells in recipients' blood between recipients transfused with non-primed donor blood and recipients with primed donor blood. the preliminary data of our study showed that chimeric primed donor cells, but not non-primed donor cells, are able to protect immune compromised knock-out recipients from murine cmv infection. the time-point of 'post-transfusion 6 weeks' might represent a weak window for the functionality of chimeric donor cells, which requires further investigation and confirmation. aims: to compare the effectiveness and the cost of epoetin-a and darbepoetin in patients undergoing pabd. methods: seven adult patients scheduled for operations were administered aranesp (300 mg sc once or q3 weeks if needed) for pabd (aranesp group) and they were compared with a historical epoetin-a group of seven age-matched adults (eprex 300 iu/kg biweekly). the two groups were matched according to the ht, ferritin levels, number of the predonated units and type of the operation performed. cbc count and reticulocytes were measured weekly during the donation period, the day before, day 1 and day 6 after surgery while ferritin and biochemical indices were measured during the first visit. erythropoiesis-stimulating factor was administered when ht £ 36% during blood donations and blood donation was not performed if ht < 34%. results: there was no statistical significant difference in hematological parameters during the donation period, the pre-operation day and after surgery between the two groups. five of seven patients from both groups received one or two autologous blood units. both factors were well tolerated without any side effects. the cost per patient was 609.09€ in the aranesp group and 414.2€ in the epoetin group. conclusion: despite the small number of patients and the limitations of this preliminary retrospective trial we believe that subcutaneous darbepoetin-a is equally effective with epoetin-a in patients undergoing pabd. darbepoetin has the advantage of less frequent administration and it is possibly superior that epoetin-a in terms of patient compliance. however smaller doses should be examined in order to reduce the cost. larger prospective randomized trials are needed to estimate the cost-effectiveness of the use of darbepoetin-a in pabd. background: incompatibility with many blood units is a major problem in transfusion therapy. in selective operations, preoperative autologous blood donation could solve many problems, when of course the patient's condition and his haemoglobin levels are appropriate. we present here the experience of our blood transfusion centre from 4 operations in 3 patients with anti-erythroid antibodies. materials: three patients (1 male and 2 females), aged between 67-80 years old, had to undergo selective operations, 3 total hip replacement surgery and 1 aortic aneurysm. introduction: because of improvements in surgical techniques, preoperative autologous blood donation (pabd) in patients undergoing radical retropubic prostatectomy (rp) is contested. aim of the study: we wanted to develop and validate an algorithm to determine the patients who probably do not benefit from pabd. methods: we calculated the perioperative hb-loss of 153 consecutive patients (group 1) who donated two red cell units (rbc) of autologous blood 5 and 4 weeks before undergoing rp: hb-loss (g) = preop-hb ¥ bv + (n rbc ¥ 65) -(postop-hb ¥ bv) (bv = blood volume (l) = body weight (kg) ¥ 0.08; postop-hb = hb (12-18 h after rp); n rbc = number of transfused autologous and allogeneic rbc). hb of rbc was taken as 65 g. rbc requirement is probably if initial-hb -(hb-loss: bv) -trigger-hb (taken as 80 g/l) < 0 (initial-hb = hb at the first contact with the pabd-unit). this assumption was validated by the next 125 patients who were also assigned for pabd (group 2). pabd was refused if the probability of rbc requirement (prr) was <25%. between 25-50% one rbc was taken after considering the patient's individual risk of pabd. if prr exceeded 50% two donations were planned. results: both groups did not significantly differ in age or initial hb. preop-and postop-hb were significantly lower in group 1 (141 vs 151 and 98 vs 111 g/l). 79% of autologous blood of group 1 were discarded, 3/153 patients needed additional allogeneic rbc. hb-loss caused by rp was 269 ± 111 g. mean prr in group 2 was 8.5%. 5/125 patients donated one rbc, which was later discarded, and no patients donated two rbc. 7/125 of group 2 needed allogeneic rbc. mean prr of these patients was 12% (range 0.65-33.3). conclusion: postop-hb were lower in rp-patients with pabd because of the lower preop-hb and the restrictive indication for transfusion of autologous blood. the individual calculation of prr, for which only body-weight and initial-hb of the patient are necessary, shows that pabd in patients undergoing rp is indicated only in rare cases. the algorithm also may be used in other major operations, if hb-loss is known. use of darbepoetin-alpha in preoperative autologous blood donation: preliminary results background: preoperative autologous blood donation (pabd) is an alternative practice to eliminate complications of allogeneic blood transfusion although its cost-effectiveness has been questioned. darbepoetin-a (aranesp, genesis pharma sa) is a novel erythropoiesis-stimulating factor that it has been shown to be equivalent to epoetin-a (eprex, janssen-cilag) in patients with chronic renal failure and cancer. darbepoetin-a has a longer serum half-life and higher relative potency than epoetin-a. this property leads to less frequent administration and may reduce drug cost. so far, no clinical trials with darbepoetin have been published in patients with surgical anemia. other 44 (15.3%) patients who required autologous and homologous blood, had average predonation hb level of 13 509 (sd ± 9.01) g/l. we found a significant relationship between the need for postoperative transfusion and the predonation hb level (p = 0.003), predonation htc values (p = 0.006), weight (p = 0.003) and gender (p = 0.002): female patients and patients with lower predonation hb and htc, as well as patients with lower body weight more often needed additional homologous blood transfusion. no relationship was found between age of patients and the need for transfusion (p = 0.121). 104 (80.6%) patients with ptka were transfused with autologous blood only, and had average predonation hb level of 13 867 (sd ± 9.06) g/l. other 25 (19.4%) patients transfused with autologous and homologous blood had average predonation hb level of 13 588 (sd ± 8.93) g/l. the significant relationship was found between the need for postoperative transfusion and weight (p = 0.012): patients with lower body weight more often needed additional homologous blood transfusion. no relationships were found between predonation hb level (p = 0.099) predonation htc values (p = 0.243), gender (p = 0.241) and age (p = 0.276) of patients and the need for postoperative transfusion. conclusions: our results show that over 80% of patients needed only autologous blood. in our patients with ptha predonation hb was significant predictive factor for additional transfusion therapy, while in ptka it was not observed. in both groups of patients body weight was significant predictive factor, thus this feature seems important for planning of transfusion therapy in patients with ptha and ptka. aim: prevention results of loosen anastomoses on colon, with fibrin sealent (fs) application and influence on colagen production. materials and methods: investigations were done on rats, weight 350-500 g. in control group, after partial resection of left half of colons termino-terminal anastomosis was derivated. fs was applied in examined group. concentration of colagen was done indirectly, with quantitative l-hydroxyproline determination. place of anastomosis, 1 cm proximal and 1 cm distal of anastomosis, was analyzed iii, v, vii and xiii day postoperatively. results: analysis of hydroxyproline on the place of anastomosis showed higher hydroxyproline value in group with fs application. the highest approximate value of hydroxyproline was registered v day postoperatively. distal, 1 cm of anastomosis, the quantity of hydroxyproline is higher iii day postoperatively in control group but v postoperative day value is intensively growing in group with fs application. electronic microscopical was done v postoperative day in control group at the place of anastomosis detected a defect with detritus and absence of larger colagen fibres. in group with fs application on the place on anastomosis, in the shape of bundle, colagen fibres were grouped and completely fills the place of anastomosis. conclusion: fs application accomplish higher concentration of colagen in all segments of isolated colon, that enables better healing of anastomosis. the study of the use of the safest blood (autologous blood transfusion) through preoperative blood donation (pbd) in 100 surgery patients introduction: the use of the autologous blood is already under consideration in developed countries. thus, it is probable that autologous blood donation would be effective in one way or the other in reducing blood transfusion complications. in this study, pbd as the easiest method to use and the most cost-effective one was selected. aim of the study: it aims at improving blood safety and raising blood inventory. methods: in this study, 100 patients, including 75 males and 25 females, intended to undergo elective surgery were selected as subjects to donate their autologous blood. the subjects with hematocrite level of about 30-33 percent as ordered by their physician donated their blood by this method. blood collection procedure was followed at 3-7 day intervals. the blood volume taken from patients in every collection differed from 100-450 ml according to their weight. results: this study showed that in all patients undergoing plastic, gynaecological, jaw, and ent surgeries autologous blood transfusion was used with no need for allogenic transfusion. in other surgeries, including orthopaedics, the need for allogenic transfusion was estimated to be at about 50 percent of cases. to avoid the complications of allogenic blood transfusion, the safest way is the use of autologous blood which involves low cost and is easy to perform. the introduction: the purpose of this study is to describe a technique to perform labelling of autologous platelet-gel with 111in-oxine and to evaluate its usefulness, after in vivo graft implant, as a marker of bone osteoinduction by means of scintigraphy, in patients with jaws bone defects following the enucleation of cystic lesions and cystic lesion derived from extraction of deeply impacted lower third molar. methods: agp made. consent was obtained by patient to conduct hiv and hbv testing. briefly, 40 cc to 60 cc of blood is withdrawn from the patient. the blood was separated, by means of successive step of centrifugation, in to platelet-rich plasma (prp) , platelet-poor plasma (ppp), and red blood cells (rbc). the red blood cells were discarded. the prp [comprises of approximately 10% of the total blood volume withdrawn] had platelet counts of 500 000-3 000 000/mm 3 . the procedures of agp labelling were performed in laminar flow chamber. to 3 seconds the solution will assume a gel-like consistency forming platelet gel. imaging: the scintigraphy was performed 2 h after application of labelled agp (early scan) and at 24, 48, 72, 96 h (delayed scan) by means of a gamma camera equipped with medium energy collimator. a later scan was performed at 10 days after graft. the platelet uptake index (pui) was then calculated by dividing the cpm/pixel in the graft roi (recognized in and planar and trans-axial slice) for cpm/pixel in a mirror background roi. in vitro sampling: the radioactivity of the plasma samples collected at 2, 24 h and at lapse time = 24 h for 7 days, were used for the plasma clearance determinations and for in vitro studies of the platelet loss from the gel. results: all patients presented early high concentration of 111in oxine agp, at site of the graft, that was easy recognized at scintigraphy performed as in anteroposterior and lateral planar projection of the jaw as in spet slices reconstructions. all labelled agp was well confined within area of original implant and no activity was seen in the surrounding tissues or in the distant organ. conclusion: all patients studied well tolerate the implant of agp; no adverse reactions were observed and follow up -performed 3 months later -showed bone remodelling activity in the site of the graft. serial blood donations in a ko pregnant woman with the use of recombinant erythropoietin for intrauterine transfusions of severe hemolytic disease of the newborn due to anti-ku biweekly) were administered to the mother to ensure an adequate supply of compatible rbcs for intrauterine transfusions and possible perinatal haemorrhage as well. results: intrauterine transfusions were repeated every 1-3 weeks. by the 35th week of gestation the patient had donated four units of blood, her hematocrit was 40%, anti-ku titre was 1/2048 and four intrauterine transfusions had been performed. cesarian section was decided and the apgar of the newborn were 8 and 10 at 1 and 5 min. the newborn was treated with phototherapy but without exchange transfusions and two weeks later he was discharged. by the 15th day of life rh-epo was administrated to him due to anemia. the maternal red cells completely disappeared from the child's blood by the day 100. the experience of the use of erythropoietin in pregnancy is minimal. as illustrated by this case treatment with rh-epo and iv fe has effectively increased mother's capacity to donate rbcs' for autologous use and intrauterine transfusions as well, with no adverse effects to the mother or the child. however, further research is necessary to evaluate if rh-epo crosses the placenta. introduction: blood components should be transported by a system which has been validated. the containers used for transport should be well insulated, some form of temperature indicator should be used to monitor the in-transit temperature. whole blood should be stored at different temperatures above 2°c. aim of the study: bags with whole blood collected in a collection site are transported in containers without active cooling. we tested temperature of blood in containers put into the extreme weather conditions (+50°c, -20°c) during loading test for transport. methods: the container without active cooling was filled with the exact number of bags with blood and the exact number of passive cooling elements (frozen water cubes in plastic) placed in the exact positions without close contact with the blood bags. the bags with blood of the temperature +4°c, +20°c and +37°c were used. temperature indicators were situated in the bottom, centre and top of the container. filled container was placed into thermostat (+50°c) or freezer (-20°c). the temperature was observed in 10 min intervals for three hours, first measurement was 30 min after putting into freezer or termostat. results: (see table 1 ) nt, non tested; tmp, temperature. in the table there are shown minimum and maximum temperature parametres observed during tested time including increasing or decreasing trend. conclusion: loading test for transport of the bags with collected whole blood helps us to optimize transporting system, especially number of cooling elements in relation to the season and its place in the container. in the light of presented data we corrected transporting system to maintain the recommended temperature during transport. background: since 1999, we have produced pooled and filtered platelet concentrates out of four buffy coats in tsol platelet additive solution and have stored them in pall autostop clx bags made out of pvc/totm. the residual plasma content is between 30-40%, the mean volume about 260 ml and the mean platelet-content is 3.1 ¥ 10 11 per unit. for pathogen inactivation or bacterial screening it is necessary to extent the storage time from 5 to 7 days. new foliage like polyolefin is supposed to maintain a good quality environment for prolonged storage of platelets. aims: storage bags made out of polyolefin (pall autostop elx) were tested to prove their suitability for prolonged storage of platelets. methods: 18 twins made out of pools from 8 buffy coats were produced with the standard method, one twin was stored in the conventional bag (cb) the other in the new foliage bag (nfb). the platelet pools were stored on flatbed shakers at 22°c, and sampled at day 1, day 5 and day 7. ph, glucose, lactate, hypotonic shock response (hsr) and p-selectin expression were measured by standard in-house methods. results: mean ph on day 7 with cb was 7.21, with nfb 7.35; glucose with cb 4.4 mmol/l, with nfb 6.0 mmol/l; lactate with cb 17.4 mmol/l, and with nfb 14.8 mmol/l, hsr with cb 79%, with nfb 85%; p-selectin with cb 29% and with nfb 18%. the new platelet storage bag showed better results of in vitro quality markers, especially after day 5 of storage. prolonging storage time will make it easier to introduce bacterial screening or pathogen inactivation techniques into platelet transfusion. the possibility to filter rbc either at 4°c or rt simplifies the preparation process. filtration at +4°c enables to achieve a better leukoreduction performance. the nbs has successfully implemented this project which has the potential for improvement in patient safety and is predicated upon practical application and risk reduction rather than elimination. the impact of this work on the incidence of trali will require detailed, long term analysis of hemovigilance data using existing mechanisms. active communication, a team approach, perceived value of the initiative and the hard work of all staff involved were key success factors. quality assessment of buffy coat-derived platelets prepared from leucoreduced whole blood background: whole blood can be separated into; plasma, buffy coat and red-cell conc (rcc) by differential centrifugation and separation on a separation device. because of the high hematocrit of the rcc, 60% of the process time is needed for expression of the rcc. by increasing the internal diameter of the tubing at the bottom of a t&b system by 0.7 mm. a decrease of the process time is expected. methods: 220 units of whole blood were collected with the new t 3988 'wide boring' blood pack and separated on a routine base. quality control parameters were checked and the whole process time was monitored. free hemoglobine was measured up to 42 days. results: process time of a 'wide boring' bag is significant shorter compared to a standard blood bag. average decrease: 86 s. slightly increase in free hemoglobine is measured probably due to the increased express rate of the red cells. bloodproducts produced with the new t 3988 meet european guidelines. no significant increase of free hemoglobine due to the faster expression is measured. an significant decrease in process time is measured with the wide boring bloodpack. the new fresenius hemocare rcc in-line system: t 3988 can be used for routine production which will speed up the production process considerably. introduction: leukoreduction of blood components is required to prevent several transfusion-associated complications. aim: the aim of this study was the full process validation of the pall leukotrap wb system for the preparation of leukoreduced blood components. we collected 60 whole blood units from donors suitable for donation using a quadruple blood-bag, which includes an wbf3 in-line filter (pall) for the removal of leukocytes and platelets. mixer balances (baxter) were used and donation occurred within 12 min in all cases. after donation whole blood units were stored at room temperature for 2 h. subsequently, whole blood filtration was performed by gravity at a standard height of 150 cm using a blood leukoreduction cart (baby leuko cart, itl-corporation). filtered units were centrifuged at 5000 g ¥ 10 min by an heraeus cryofuge 6000i. an automatic extractor (bag press plus-bioelettronica) was used to prepare red cell concentrates in sag-m solution and fresh plasma units. air in the system was automatically expelled by the extractor. complete cell counts and hemoglobin concentration were evaluated in pre-filtration samples and at the end of the blood components preparation using an automated cell counter (pentra dx120-abx). we enumerated residual leukocytes in red cell units by flow cytometry (becton dickinson-leucocount kit). results: pre-filtration data of whole blood and end-of-process data of red cell and plasma filtered units, are summarized in table 1 . results are given as mean and standard deviation. whole blood filtration was completed within 10 min in all cases. red cell units were transfused after a mean of 6 days to 57 patients affected from transfusion dependent (45%), post-surgery (30%), and post chemotherapy anemias (25%). no cases of transfusion reaction were observed. the pall leukotrap wb system was easily introduced in our setting. all blood components prepared by the system fulfilled the council of europe requirements with regards to hemoglobin content in red cell units and post-filtration residual leukocytes. future studies are needed to evaluate its cost-effectiveness in the setting of routine blood component preparation. background: during an evaluation of the compodock (fresenius hemocare) sterile connection device (scd), we observed irregularities on the inside of the tubing at the site of the weld. it was our aim to investigate the effect of these observations on the quality of blood products. methods: three leukoreduced red cell concentrates (rccs) were pooled and divided over 3 bag systems: one without weld in the connecting tubing, one with a compodock-weld, and one with a weld made with the terumo scd. the rcc was transferred 10 times over this tubing to have maximum result if the weld had deleterious effects. the rccs were stored in pvc containers, and sampled on day 1, 28, 35 and 42, and free hemoglobin (hb) was measured. the same procedure was also performed using platelet concentrates (pcs), but these were stored in polyolefin containers, sampled on day 1, 6 and 8, and cd62 expression was measured. ten experiments were performed per blood component. according to who standards processing of blood into labile components are considered an expression of quality of transfusion service. in our practice, modern transfusion principles are successfully applied. they cover blood collection, serological processing of blood units, technological preparation of blood products (gmp, sop) and rational utilization of blood components and blood derivatives. in the past four years (2001, 2004.) aberrations from these principles have taken place (self-sufficiency). nbti collected x = 62 926 (249 778) blood units into blood bags. in serbia x = 230 537 (691 611) blood units. retrospective analysis: ldpc-bc was administered x = 97% with the satisfactory haemostatic effect. increase of the cyta and plasmapheresis-manual procedures was also noted (100%). increase of the use of leukocyte poor red blood cells was also registered (95% introduction: according to the relevant recommendations of the council of europe, whole blood is a source material used for the preparation of blood components and blood products. basic concept of the therapeutic use of blood components is the compensation of the lacking or deficient blood component. in that way, a possibility of the infusion of unrequired or deleterious components of whole blood is eliminated. objective of this presentation is to analyze the reasons of non-utilization of certain blood units, the actual quantity and ratio. aim of the study: the purpose of our in vitro study is to compare storage of platelet concentrates at 4°c with platelets stored at 22°c, and to determine the in vitro-effects of pre-incubation at 37°c for 1h prior to analysis on the basis of the maintenance of platelet metabolic and cellular integrity. methods: platelets concentrates (pcs) were prepared from pooled buffy coats (bc) for paired studies to be stored into different conditions. (i) at 20-24 degree on a flat bed agitator; (ii) at 20-24 degree on a flat bed agitator and pre-incubated for 1 h prior to analysis; (iii) at 4°c; and (iv) at 4°c and pre-incubated for 1 h prior to analysis. this paired in vitro study (n = 8) over 21 days include volume, platelet counts, mpv, volume, ph, po 2 , pco 2 , bicarbonate, glucose, lactate, swirling, leucocytecount, hsr, esc, atp, ldh and release of a-granule content (rantes, ß-thromboglobulin and pf4). results: platelet count (day 5 and 21; p < 0.05), mpv (day 5; p < 0.01), ph (day 14 and 21; p < 0.01), pco 2 (day 14 and 21; p < 0.01), bicarbonate (day 21; p < 0.01), glucose (day 5, 7, 10, 14 and 21; p < 0.01), atp (day 14 and 21; p < 0.01) was significantly higher in platelets stored at 4°c and platelets stored at 4°c with preincubation. ldh (day 21; p < 0.01), bicarbonate (day 5 and 7; p < 0.01), lactate (day 5, 7, 10, 14 and 21; p < 0.01), ph (day 5 and 7; p < 0.01), esc (day 5, 7, 10, and 14; p < 0.05), hsr (day 5, 7 and 14; p < 0.05) was significantly lower in platelets stored at 4°c and platelets stored at 4°c with pre-incubation. the concentration of rantes, ß-thromboglobulin and pf4 was significantly higher in platelets stored at 22°c than in platelets stored at 4°c (day 5, 7, 10, 14 and 21; p < 0.01). hsr (day 5 and 10; p < 0.05) and esc (day 5, 7, 10 and 14; p < 0.01) was significantly higher in preincubated platelets stored at 22°c compared with platelets stored at 22°c. conclusion: platelets stored at 4°c maintain metabolic and cellular characteristics to a great extent during 21 days of storage. we confirm the loss of platelet discoid shape and have shown that loss of discoid shape in platelets stored at 4°c is associated with decreased metabolic rate and decreased release of a-granule content. aim: as reference centre of the swiss blood transfusion service for new materials and blood products we evaluated that system for routine use and official registration in switzerland. method: 20 whole blood donations were collected in a whole blood filtration set with cpda-1 and stored at room temperature for 1 h before filtration at room temperature. the leucodepleted whole blood was stored for 42 days. following parameters were analysed on day 0, 35, 42: free haemoglobin in%, k. in addition leucocyte count was performed on day 0 and a blood culture on day 49 (see table) . blood cultures on day 49 remained negative and all counts of residual leucocytes were below 1 ¥ 10 (exponential) 6/unit. summary and conclusion: as expected there was a clear increase in k and free haemoglobin after day 35. however the results were within the required specifications from the european and swiss guidelines up to day 42. we conclude that autologous leucodepleted whole blood can be stored in cpda-1-for 42 days without loss of stability of the red cells. we will introduce the system to the offi-cial material list of the swiss blood transfusion service and then implement the procedure to our daily routine. results of ffp production from whole blood, and of ffp and pc produced by use of cell separator over a 5-month period before and after the introduction of measures for trali prevention are presented. the following measures were undertaken: (4) blood of female donors was not used for ffp production, and plasma was only used for fractionation (5) plasma of female donors was not used for kt-bc pools (6) platelets and plasma were produced on a cell separator only from the female donors without a history of pregnancy. female donors of whole blood: 16%*; 16%** ffp produced by plasmapheresis: 2%*; 2.5%** female donor units on cell separator: 9.4%*; 4.5%** ffp from total plasma units: 63%*; 58%** plasma units used for bc-pc pools: 4%*; 6%** *period before and **after the introduction of measures for trali prevention the exclusion of female donors had no major impact on the production of ffp and bc-pc pools from whole blood because of the very low rate of female subjects in the croatian blood donor population. the amount of plasma and pc collected from female donors by use of cell separator was significantly lower (~50%), however, without any major impact on total ffp store because of the small rate of plasma and platelets obtained by apheresis. background: platelet concentrates (pcs) are currently stored for a maximum of 5 days. extended storage to 7 days would increase the supply and reduce the waste of pcs. transfusion-associated graftversus-host-disease (ta-gvhd) is a severe transfusion reaction caused by t-lymphocytes in the transfusion product. the risk of developing ta-gvhd can be prevented by gamma irradiation of the pcs. various in vitro tests can be used to study the quality of pcs such as inspection of the swirling phenomenon, hypotonic shock response (hsr), detection of platelet surface markers (e.g. cd62p and cd42b), metabolic parameters and blood gases. free oscillation rheometry (for) using the instrument reorox® 4 can be used to monitor the coagulation over time in whole blood, pcs and plasma samples, and to obtain information about clotting time and coagulum elasticity. aim of the study: the purpose of this study was to evaluate the quality of pcs obtained by apheresis technique during storage for 7 days and to study the effect of gamma irradiation by using several in vitro methods including for. methods: platelets were collected from healthy donors (n = 12) using apheresis technique. the pc from each donor was divided in 2 units, one served as control and the other was gamma irradiated with 25 gy. the pcs were stored on a flatbed agitator at 22°c for 7 days. samples were taken on day 0 (= day of collection) for analysis of blood gases, metabolic parameters (glucose and lactate), platelet count and swirling. samples taken on day 1, 5 and 7 were also analysed for hrs, cd62p (p-selectin) and cd42b (gpib) expression utilising flow cytometry. evaluation of coagulation by for was performed on day 1, 5 and 7. the maximum elasticity (g'max) and the time to g' were evaluated from the for elasticity curves. results: there was no difference between irradiated and nonirradiated pcs regarding any of the tested parameters during the storage period. swirling, hsr, platelet count and percentage of cd42b expressing cells were well maintained for 7 days of storage. glucose decreased and lactate increased significantly during the storage period, from 2.5 mmol/l to 16.2 mmol/l for lactate and from 16.5 mmol/l to 8.9 mmol/l for glucose. the percent cd62p expressing cells increased significantly during storage from 15% on day 1 to 32% on day 7. po 2 was well maintained but ph increased and pco 2 decreased significantly between day 0 and 1 whereafter ph decreased and pco 2 continued to decrease. the for parameters g'max and time to g'max increased significantly between day 1 and 5 and the time to g'max continued to increase significantly between day 5 and 7. the results indicate a well preserved platelet quality after storage for 7 days. gamma irradiation did not affect the platelet quality. cytokine release during storage of buffy coat platelet concentrates produced manually and automatically background: transfusion reactions following platelet transfusion are still a problem even when leukoreduction is included in the production process. platelet derived cytokines released during storage upon activation or lysis, accumulate in the platelet products and have been suggested to be involved in transfusion reactions. rantes (regulated upon activation, normal t cell expressed and presumably secreted) is a chemokine playing an important role in the inflammatory immune response and causes degranulation of eosinophiles and release of histamines from basophiles, which again can cause allergic reactions. tgf-b1 (transforming growth factor b1) has been shown to be immunosuppressive, inhibits the proliferation of t-and b-lymphocytes and decreases the secretion of igg and igm from b-lymphocytes. aims: as part of our quality control program, we aimed to quantify the amounts of rantes and tgf-b1 released during storage in platelet concentrates produced from pooled buffy coats by our manual routine method (m-pcs) and by an automated method using the orbisac system (gambro) (a-pcs). methods: pcs were produced from 5 buffy coats. following overnight storage at 20-22°c, buffy coats were pooled with 300 ml t-sol (baxter). forty-two pcs were produced either manually (n = 21) using the imugard iii s-pl set (terumo) with integrated soft leuko-reduction filter or by the automated procedure (n = 21) using the orbisac validation bc set (gambro) equipped with the lrp6 leuko-reduction filter (pall). swirling was scored visually, platelet count and mpv were measured on a cell counter (cobas argos, roche), and blood gas analyses, glucose as well as lactate were measured on an abl700 series analyser (radiometer). samples for testing of cytokines were centrifuged for 15 min at 4800 g, 20°c; supernatants were harvested and frozen at -86°c until analysis. cytokines were quantified using quantikine human rantes immunoassay (r&d systems) and human tgf-b1 elisa immunosorbent assay (bender medsystems gmbh). all analyses were performed on days 1, 4 and 7. results: platelet concentrate volume (mean): m-pcs: 316 ml, a-pcs: 328 ml. platelet yield was found to be 3.15 ¥ 10 11 for m-pcs and 3.43 ¥ 10 11 for a-pcs (p < 0.0001). in all pcs ph levels were between 6.9-7.2. glucose consumption and lactate production from days 1-4 and days 4-7 did not differ significantly. rantes levels (pg pr 10 9 plts) were significantly higher in a-pcs than in m-pcs (p = 0.04, repeated measures analysis of variance), but no significant difference was found in tgf-b1 levels (pg pr 10 9 plts). summary and conclusions: preparation of buffy coat platelet concentrates by the automated orbisac system improves platelet yield compared to our manual processing procedure, but the levels of the chemokine rantes were significantly highest in the automatically produced products. the clinical importance of these findings is still unclear, but may be related to the shear stress the platelets are subjected to during the automated production process. the quality of cryopreserved vs liquid stored platelets: a comparative study table 1 . the mismatches can be divided into the two categories. the first of them is characterized by differences in allelic groups, i.e. at low-resolution level. 22 allelic group differences were detected in the group with one mismatch, most of them in hla-c locus (this locus was not concluded in primary donor search). in the other category there are differences in alleles within the same group, i.e. at high-resolution level only. 12 differences within the same group in all tested loci were detected in the group with one mismatch. the mismatches described above were heterogeneous and a correlation of specific mismatch with transplantation outcome was not possible in this group. conclusion: the use of high-resolution dna methods makes the identification of hla match/mismatch more accurate and can affect the outcome of unrelated hsct. this work was supported by the grant iga mz, no. nr/7984-3. pre-freezing and post-thawing quality controls in umbilical cord blood assigned for transplantation p bergamaschi, c perotti, g viarengo, c del fante, c parisi, a marchesi, l bellotti and l salvaneschi irccs policlinico 'san matteo', pavia, italy background and aims: nowadays umbilical cord blood (ucb) represents a well established source of haematopoietic stem cells for unrelated transplantation in children affected with haematological and inherited diseases. thanks to the large-scale banking of unrelated units and the preliminary encouraging results, ucb employ in adults is quickly growing up. in this context, total nucleated cells (tnc) count of the graft is considered the main predictor for clinical outcome; however, other indicators of the haematopoietic potential, such as cd34+ cell content and short-term culture clonogenic assay, are recommended in accordance to netcord-fact stan-dards. in order to guarantee the safety and the prompt availability of a ucb unit assigned to a matched recipient, a pattern of rigorous quality controls should be carried out not only at the time of cryopreservation but also before the release for transplant. we report the results of the quality controls performed on thawed cryovials referring to the 26 units delivered by our ucb bank compared to the pre-freezing values. methods: every ucb unit stored in our bank is accompanied by 3 satellite cryovials available for subsequent controls. for each unit issued for transplantation, one cryotube was thawed in 37°c water bath with gentle agitation without washing out dmso. tnc and mononucleated cells (mnc) were estimated by an automated cell counter; viability and cd34+ cell count were evaluated by flow cytometry with a no-wash, single-platform technique and 7aminoactinomycin d. cfu assay was performed using commercial reagents (methocult gf h4434, stemcell technologies) and colonies were counted after 14 days. the same tests were performed before cryopreservation, taking a sample from each fresh unit. moreover, before the delivery for transplant, a second cryotube was thawed to investigate the bacterial contamination by direct microbial culture, whereas the sterility test before freezing was performed by inoculum into 30 ml media (bact/alert®fa/fn, biomérieux inc). results: the ucb characteristics before freezing and after thawing are detailed in the tables 1, 2 and 3. post-thawing tnc and mnc, as well as cd34+ cells, showed no significant difference in comparison to the pre-freezing values. despite of the expected decrease of the overall viability after thawing, we observed a highly satisfactory viability referred to the cd34+ cells. the colony forming units (cfu) growth after thawing was documented and was always lower as respect to the pre-freezing assay. finally, the results of microbial cultures were negative for all the 26 units on both fresh and thawed specimens. conclusions: in our experience, well standardized evaluation of ucb content could be obtained with regard to tnc, mnc and cd34+ cell. concerning the results of short-term cultures, the presence of dmso as inhibiting factor may be advocated to explain the discrepancies between fresh and thawed samples. finally, rigorous quality controls documented that the procedures of manipulation and cryopreservation did not affect the quality of ucb to be infused for transplant and provided to the physician all the parameters necessary for a safe transplant in a close and appropriate time. bone marrow transplantation or bmt transplantation of progenitor blood cells to regenerate blood normal cells in patients with blood disorders. bone marrow has an organized and structured architecture in which close relationships exist between a regulatory microenvironment and primitive hematopoietic cells. in fact, normal hematopoietic cells depends on critical interactions that occur between stem cells and their microenvironment. this microenvironment is a complex meshwork composed of growth factors, stromal cells, and extracellular matrix. marrow injury can occur as a consequence of a variety of diseases. some diseases could be due to a microenvironment that fails to support hematopoiesis. a possibility is that aplasia and leukemia share a common etiology such as drug, chemical, radiation, virus or other environmental hazards. we can say that microenvironmental abnormalities in interactions between stromal cells and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans. background: intrauterine growth retardation, with associated low birth weight, represents one of the most important cause of baby mortality and morbidity. understanding the genetic bases of this adverse event is still an open goal. there is evidence that motherchild hla compatibility and hla-drb1 foetal genotype are associated with a reduced placental growth and a low birth weight. the recent institution of cord blood banks, with their huge amount of hla types, offers an unique opportunity to look inside the molecular bases of normal birth weight. aims: we investigated whether the baby-linked immunogenetic profile, i.e. hla gene frequencies and homozygosity rate, affects the physiological variance of the size at birth. methods: 1868 cord blood units (961 from males and 907 from females) were hla typed with pcr-ssp and/or reverse pcr-sso techniques and recorded in the cord blood bank database of pavia-italy. all were defined at low resolution level for hla-a and b genes and at high resolution for hla-drb1. 686 blood units were also randomly typed for hla-dqa1 and dqb1 at high resolution. results: mean birth weight was 3430 g and mean relative birth weight (i.e. corrected for gestational age according to the gender) was 0.19 g. 70 babies were <5th centile (2782 g) and 70 were >95th centile (4068 g). comparing the hla allele distribution in these extreme bands we found that hla drb1*08 was significantly associated with high relative birth weight: 5.1% in 95th centile vs 0.7% in 5th centile, p = 0.032. on the contrary, hla-drb1*14 and dqb1*05 were associated with low relative birth weight: 7.9% and 30.4% respectively in 5th centile vs 2.2% and 9.6% in 95th centile p = 0.035 and p = 0.019. all infants were analysed as to the effect of the above mentioned alleles. we confirmed the positive association of hla-drb1*08 and higher relative birth weight (mean 0.17 vs 0.08; p = 0.033) as well as the association of hla-drb1*14 with lower relative birth weight (mean 0.03 vs 0.27, p = 0.04). no significant association was found as far as hla homozygosity was concerned. conclusions: the present findings confirm the role of foetal hla-drb1 gene in the intrauterine growth. about the specific involved alleles, one possible explanation comes from the studies of crystallography and amino acid sequencing of hla-dr binding groove. it has been demonstrated that hla-drb1*08 and hla-drb1*14 genes encode for different amino acid sequences in the pocket 4 of the molecule (aa 70, 71, 74). this implies distinct functional restriction patterns. the sequence motif of hla-dr14 is characteristic of some autoimmune conditions, such as hashimoto's thyroiditis, and preeclampsia which is associated with intrauterine growth retardawhich provides a high yield and excellent purity without lymphocyte and erythrocyte contamination. in a 3 month period, we studied blood samples from bone marrow transplant patients and from normal subjects. the extraction of leukocyte polymorphonuclear was obtained with a 6%-7% dextran solution in 0.8% saline. after incubation at room temperature with lymphopre solution, the mixture was centrifuged. two clear and separate rings of mononuclear and pmn leukocytes were obtained. to eliminate any red blood cells, pmnl ring was separated and washed three times with cold ammonium chloride. after a short period of incubation 4°c, mixture was centrifuged and the pmnls were isolated. the purity and viability of total leukocyte population was counted and the percentage of pmnl obtained was established. the total blood samples studied were divided in two groups, i.e., bone marrow transplant patients and normal subjects. in both cases the pmns isolated were of high purity and viability. the overall percentage of pmnls obtained from both groups under study was 98% to 99% when stained with gimsa or wright staining method. the viability of isolated pmnls was also 98% too, which is excellent for numerous immunological or molecular studies. the pmnls isolated by this method were highly pure and viable in comparison with standard methods used to isolate human pmnls. generation a high amount of pmnls is another advantage of the suggested method. this method to separate pmnls is recommended for in vitro studies of different subjects. et al. 1950 .) the object of exchange transfusion (et) is to remove bilirubin already present in the plasma or remove alloantibody which can cause hemolytic disease (in order anti-d, anti-c and anti-k are easily the most important) or remove anti-d positive red cells. we have studied exchange transfusion in our hospital in neonatal intensive care unit, during 1997 to 2004. at delivery cord samples were taken for determination blood group, rhesus, hb and ht have been counted. also direct antiglobulin test (dat) has been performed. in cases of positive dat, hb and bilirubin levels were monitored. newborn body-weight were weighted (ranged 710 g to 3100 g). the blood for et was of group o, d negative and kell negative and was compatible with the serum of mothers; it was less than 7 days old. the blood was screened for hbs and for anti-cmv as well as being submitted to all usual tests. the method has been determined by using the umbilical vein; the multi-way tap makes it possible to draw blood from the infant into the syringe, to discard the blood into a sterile empty vessel, then to draw blood from a donor unit into the syringe and inject this into the infant. results: 17 exchange transfusions were carried out. four out of 17 et due to abo incompatibility mother-newborn. five out of 17 due to rhesus incompatibility mother-newborn and eight out of 17 due to jaundice undetermined origin and immaturity. the last two years only three et were carried out due to immaturity. conclusions: phototherapy, when applied early enough and with sufficient intensity, can avoid the need for exchange transfusion in many infants. phototherapy alone or phototherapy plus high dose igg therapy has been used to minimize exchange transfusions in this population. detection abo blood group system antibodies of neonatal using fully automated column agglutination technology (auto-vue) background: the abo blood group system remains the most important in transfusion practice. this is because of the regular occurrence of the antibodies anti-a, anti-b and anti-a, b reactive at 37°c, in persons whose red cells lack the corresponding antigens. the regular presence of anti-a and anti-b is used in the routine determination of abo blood groups; in addition to testing red cells for a and b antigens, the group is checked, in serum or reverse grouping, by testing the serum against red cells of known abo groups. methods: 100 samples were taken from 100 newborns at first 24 h of life for abo blood group typing during 2001-2004. simultaneously the presence of anti-a and anti-b antibodies has been studied using fully automated column agglutination technology (auto vue ortho diagnostic systems) with bio-vue cassettes aborh/reverse. the column agglutination technology is based upon the ability of glass beads to form a physical barrier between agglutinated and unagglutinated red cells. to determine the abo serum group, test serum and abo reagent red cells were added to the top of column containing diluents. the abo grouping columns were centrifuged and examined for agglutination. the presence or absence of agglutination has been recorded. results: in 68 out of 100 newborns were found detected antibodies anti-a, anti-b. in 24 out of 100 newborns no antibodies were found. in 8 out of 100 were found antibodies maternal origin. the automated reader detected all positive reactions. positive results were recorded on a scale from +0.5 to +4. conclusions: the technical performance of device allows objectivity and precision to detect abo blood group antibodies of newborn. the origin and the type of antibodies and also factors that influence their presence are to be studied. introduction: diagnosis, management and prevention of red blood cell immunization have improved, so hemolytic disease of the newborn (hdn) has changed from a common to a rare pathology. aim of the study. in this study we have retrospectively evaluated the benefits of the immunohematological screening for the management of pregnancies with alloimmunization. methods: in the last 3 years, we have performed an immunohematological screening on all pregnant women assisted by our hospitals. ab0 and rh typing, antibody screening and, eventually, identification and titration were performed on maternal specimens by microcolumn technique. results: not considering ab0 incompatibilities, we have discovered 64 alloimmunized women with the following specificities: 10 anti-d, 9 anti-c, 7 anti-c, 9 anti-e, 3 anti-jka, 1 anti-d + anti-jka, 1 anti-d + anti-s, 3 anti-d + anti-c, 1 anti-d + anti-c + anti-k, 5 anti-s, 10 anti-k, 2 anti-m, 1 anti-c + anti-e and 2 anti-d + anti-c + anti-g. the most severe hdn were the 2 d + c + g, the c + e and 2 out of 7 c newborns, with mean hemoglobin between 5 and 6 g/l, bilirubin = 20.5 g/l, reticulocyte count = 10%. in these exchange transfusion needed at the delivery. other newborns were only treated with phototherapy. conclusion. thanks to the immunohematological monitoring, the diagnosis of alloimmunization, the correct management of pregnancy and the adequate neonatal therapy were possible. in fact all newborns survived and showed no neurological lesions in the following controls. conclusion: in order to provide a highly specialized perinatal care, immunohematologist, obstetric and pediatric should provide a good antenatal and perinatal screening. this is an interesting case of rhd immunisation in rhd negative woman despite the application of rhd immunoprophylaxis. case report: a blood sample of pregnant woman, 30 years of age, in 12th gestation week, was sent to our laboratory for serological analysis. her blood group was o, ccddee and she had an anti-d antibody reactive only with enzyme treated panel of test erythrocytes. her husband was a, ccdee, and two children were both a, ccddee. on the next visit, she gave the data of one arteficial and one missed abortion before the 12th gestation week covered with 50 mg of rhd immunoprophylaxis, but without the measurement of fetomaternal haemorrhage (fmh). both of abortions were after the deliveries. until the end of the pregnancy, detailed serological analysis showed anti-d specificity of antibody in her sera which remained reactive only with enzyme treated red blood cells. the fetus was under permanent ultrasound control. she delivered a mature, rhd positive, ccdee male child, without any sign of haemolytic disease. the proper personal history, measurement of the size of fmh, distinguishing the anti-d and anti-g specificity of the antibody, administration of rhd immunoprophylaxis and cooperation between transfusion medicine specialists, gyneacologists and neonatologists still remain major principles of prenatal and perinatal care concerning haemolytic deisease of the fetus and newborn caused by anti-d antibody. anti d antibody, reactive only with enzyme treated red blood cells is usually harmless for fetus and the newborn. introduction: red blood cell (rbc) transfusion is widely used in neonatal intensive care units for acute or chronic pathological conditions. clinical indications for rbc transfusion are shock, sepsis and/or anemia with the following laboratory criteria: a) hematocrit (hct) < 20% or hemoglobin (hb) < 7 g/dl and reticulocytes < 4%; b) hct < 30% or hb < 10 g/dl in these conditions: o2 required < 35%, recurrent apnoea and bradicardia, cardiac rate > 180 bpm and respiratory rate > 80 bpm for more 24 h; c) hct < 35% or hb < 12 g/dl with severe respiratory distress. aim of the study. aim of this study has been to evaluate the effectiveness of rbc transfusion therapy in premature and at term newborns independently of initial pathological conditions. methods: our therapeutic objective has been to achieve an hct of 50% after the whole cycle of transfusion therapy. for each little patient, the volume of transfused rbc unit has been calculated, according to international guide lines, using the following criteria: weight (kg) ¥ blood volume (100 ml per kg if premature or 80 ml per kg if at term newborn) ¥ (hct desired -hct observed)/hct of unit transfused. particularly we have considered that premature infants (with a gestation age of 26-36 weeks) show a range of weight from 600 to 1.800 g, while at term newborns from 1.800 to 4.000 g. in order to avoid a circulatory overload, the indicated hemocomponent has been always packed rbc with higher possible hematocrit. for the same reason, the rate of infusion has been always 3-10 ml/kg/hour. methods: this is a descriptive study. the name of the neonates who received transfusion was obtained from the blood bank of beheshti hospital. information concerning the type of blood product, frequency and indication of transfusion, sex, gestational age and weight of infants was recorded in questionnaire and analyzed. results: out of 541 neonates admitted during one year, 118 (68 male, 50 female) received blood components. fifty four percent received one, 35% two and 11% received three types of blood components. the frequency of transfusions were 311 times. the most common used blood products were fresh frozen plasma (49%), red blood cell (33%), whole blood (14%) and platelets (4%). all the blood products except whole blood were used more common in premature and low birth weight infants. appropriateness of transfusion of red cells, fresh frozen plasma, platelets and whole blood were 92%, 88%, 100% and 91% respectively. (hdn) . in accordance to current regulations, this study is carried out in all pregnant women attending in our service. according to our protocol, when an alloantibody of any specificity is detected through the liss-coombs gel technique, the same determination is made using papain-treated screen cells to detect any association with other antibodies which could be of clinical relevance for a hdn. there are scientific evidences that the use of enzymatic techniques increase the test's sensitivity, though clinical relevance of 'enzyme only antibodies' may be questioned. aims: demonstrate the importance of a routine identification of irregular antibodies in pregnant by two methods (liss-coombs -enzymatic) and the prevalence of anti-d associated antibodies, only detected by using enzymatic technique. analyze the need to carry out a follow up of sensitized patients to determine if the associated antibodies only detected with in an enzymatic medium can be detected with a liss-coombs medium during pregnancy, thus acquiring clinical significance for hdn. materials and methods: between january 2000 and december 2004 we studied 1970 d-negative pregnant women. the studies performed were: abo grouping, d and weak d, rh phenotype, direct antiglobulin test and irregular antibody detection (iad) against commercial screen cells with liss-coombs (diamed) ® gel-medium technique, according to the manufacturer's specifications. when iad were positive, an antibody identification using two commercial cell panels with gel techniques (liss-coombs-enzymatic) was performed. along the pregnancy, periodic controls were carried out to determine the exact moment when antibodies, previously only identified in an enzymatic medium, could be detected in a liss-coombs medium (clinically significant antibodies). results: out of 1970 d-negative studied samples, 278 (14.1%) had a positive dai and it was only showed anti-d specificity in a liss-coombs medium. after analyzing this specificity against enzymetreated erythrocytes, it was possible to determine that 79 patients (24.8%) had in their serum other anti-d associated alloantibodies: 5 anti-k (1.8%), 63 anti-c (22.7%), 10 anti-e (3.6%) and 1 anti-c + e (0.3%). during the immunohematologic follow up, it was determined that in 4/79 patients some of the antibodies which were previously only detected in an enzymatic medium, could be identified in a liss-coombs medium and later they were identified in the red cell elution of the newborn. conclusions: these results confirm the relevance of a screening for irregular antibodies of clinical importance by means of a conven-tional technique and one of increased sensitivity in all pregnant women. the detection of an association of antibodies provides information for the undertaking of diagnostic and therapeutic measures, both by the obstetrician, as for any eventual transfusional requirements for hdn. it was also concluded that, although antibodies detected in an enzymatic medium are considered of low clinical significance, its investigation and follow up is suggested in pregnant women to determine the moment in which they can be detected in an antiglobulinic medium, thus revealing their clinical significance. background: fibrin glue is one of the most complex human plasma derivatives both in terms of composition and clinical applications. this product mimics the last step of coagulation cascade through activation of fibrinogen by thrombin, leading to the formation of a fibrin clot in the presence of factor xiii. in contrast to synthetic adhesives, the significant advantage of this plasma-derived sealant is its biocompatibility and biodegradability as well as the fact that it does not induce inflammation, foreign body reaction or extensive fibrosis. readsorption of the fibrin clot is achieved during wound healing within days/weeks following application, depending upon the type of surgery, the amount and type of product used or the proteolytic activity of the treated site. the risk of virus transmission by commercial fibrin glue products is still debated and investigators are looking for alternative fibrinogen sources. many of these studies rely on autologous on single donor cryoprecipitate as source of fibrinogen. aims: the aim of this study was to compare single and double methods of cryoprecipitation of fibrin glue. the influence of different plasma preparation methods and plasma storage temperatures (-30°c and -80°c) on the quality of fibrinogen concentrate was examined. methods: whole blood was collected by standard phlebotomy technique and centrifuged at 4000 ¥ g for 5 min within 2 h of collection. plasma was removed. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts (aprox. 219.7 ml) and immediately frozen. two of these units were stored at -30°c and 2 units of ffp at -80°c. after one month the plasma was thawed at 4°c during 16-18 h. the fibrinogen concentrates (21-25 ml) were received by single and double cryoprecypitation. to compare single and double methods of cryoprecipitation, the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined. results and conclusion: the levels of fibrynogen were significantly higher in fibrynogen concentration obtained by double cryoprecypitation from plasma stored at -30°c. there were no significant differences in the level of plasminogen in both tested groups. double cryoprecipitation of a single unit of plasma (stored -30°c) is an efficient, simple and safe method of obtaining fibrin glue. background: talking about professional risk we generally consider the risk of acquiring some kind of infective disease through accidental injury. in this article i would like to point out another side of the problem-the risk of making preventable medical error. with all its consequences. aim: how blood transfusion errors are among the most serious types of medical errors, the final goal is to initiate nationwide, regular, mandatory error reporting. information obtained, distributed and openly discussed at professional meetings will contribute to improving the patient safety. at the same time it would contribute to avoidance of blaming and shaming of many health care providers. prevent what preventable could be! method: retrospective analyze was conducted at the middle size blood transfusion center (10 000 donations per year and utilization of approximately 25 000 of components). results: after clearly distinguishing adverse events due to underlying patient condition from preventable medical error we fined out that: -great majority of adverse events resulted from medical error -every part of blood transfusion center, from blood donation ward, through laboratory testing to component issuing has it' weak points' or vulnerable places -any educational level is equally liable to error there is no significant difference about occurrence time: -working day/holiday -emergency/routine request -routine h/out of routine h -main error cause were as follows: -donor sample misidentification -rhd typing error -abo typing error -incorrectly performed cross match -recipient misidentification -wrong component prepared -sample confusion during freezing preparation conclusion: the truth incidence of transfusion medical errors is underestimated. mandatory report of fatal or 'only' harmful errors to the referent institution and its periodical announcement is the step ahead in preventing errors. those reports should be discussed at professional meetings (not at the 'yellow pages') and served as educational tool. but, as the most of the errors are system related, the key to reduce them is to focus on improvement of the system and nil for plasma. wastage rate was highest for plasma components. the influence of local practices on such discarding and whether avoidable shall be discussed. audit for blood discarding and corrective actions to minimize discarding is essential for all transfusion services and blood centers. designed technical and economic support. options include importing finished products and/or procuring products made from locally collected plasma. one approach is to consider local fractionation of plasma by building and operating a plasma fractionation facility, which may produce, finished products, or may produce intermediate products that are further manufactured in another facility. an alternative approach is the implementation of a plasma fractionation program where local plasma is sent to an established fractionator, and the plasma is fractionated following preagreed terms. the end products are returned to the country of the plasma supplier. in 1954 the national center for the production of blood products was established, under the direction of elias politis and 9 years later in 1963 begun the production of dried plasma from greek donors. by the year 1965 the center started the production of fibrinogen and by the year 1967 the production of antihaemophilc factor. in 1992 all the activities of the center settled down due to administrative aspects. at the beginning of 1990s a contract fractionation program was instituted (under the direction of k. sofroniadou) concerning the fractionation of liquid plasma and production of albumin, which by the end of year 2000 stopped and was replaced with a new contract for the fractionation of source plasma and the production of albumin. the challenge of adapting to the new and more stringent regulations governing the manufacture of blood products was great and brought a lot of changes in the structure of our center. a new bar-coding system ensuring the traceability of blood donations was instituted together with complex software for packaging and preparation of plasma shipments to the fractionation center together with all necessary paper work. a close collaboration with the medicines regulatory authority in order to be able to fulfill all the requirements that regulate issues associated to the quality and safety of human derived medicinal products. collaboration with blood collection establishments was promoted in order to increase the amount of plasma produced. there is a continuous effort from all the implicated parts in order to follow defined quality assurance procedures as highlighted by international guidelines for the blood donor selection, collection procedures, testing methods, donation handling, storage and transportation of plasma. the plasma contract fractionation program may serve, as an initial step prior to switching production to a locally built facility. this lapse of time may be used to expand the plasma collection potential, and to permit appropriate design, qualification and validation of the facility as well as training of local personnel. background: fibrin glue became a reality in the early 1970s, when techniques for the isolation and concentration of clotting factors were improved. in 1972, matras et al. described successful application of fibrin glue for peripheral nerve repair. this encouraging report prompted the use of fibrin glue in wound closure, skin grafting and bone union of osteotomies. the fibrinogen component of fibrin glue is produced from single unit donations of fresh frozen plasma. such procedure helps to reduce the risk of transfusion transmitted infections encountered by exposure to pools from large numbers of donors or by use of fibrinogen prepared from autologous blood prior to surgery. the second component, a mixture of thrombin and cacl2, is commercially available. thrombin is applied to the operation site simultaneously and in equal volume to the fibrinogen but from a separate syringe. there are many methods of fibrinogen concentrate preparation but none of them has been described in detail. aims: the aim of this study was to choose/select the most effective, simple and safe method of obtaining fibrinogen concentrate (basic component of fibrinogen glue) which would also be easy to prepare in blood transfusion centers. methods of precipitation of fibrinogen by polyethylene glycol (peg), ammonium sulphate, ethanol or cryoprecipitation were compared. methods: plasma was obtained after centrifugation (4000 ¥ g for 5 min) of whole blood. four units of plasma were pooled into a 2000 ml bag, mixed, divided into 4 parts and immediately frozen. one of them was stored at -30°c and after one month the plasma was thawed at 4°c during 16-18 h. fibrinogen was obtained by cryprecipitation and each of the three remaining units was precipitated with ethanol, peg and ammonium sulphate. the levels of fibrinogen, fibronectin, plasminogen and factor xiii were determined in each fibrinogen concentrate. results and conclusion: the level of fibrynogen ratio in fibrinogen concentration, obtained by peg and ammonium sulphate was significantly higher. cryoprecipitation is a simple, economic and reproducible procedure with the advantage of being performed in a closed system. plasma fractionation program in greece: an unknown history the provision of safe and sufficient plasma derivatives to meet the needs of local population requires special consideration and a well(light cycler, roche diagnostic systems, nj) was used for the identification of the c282y, h63d and the s65c point mutations of the hemochromatosis gene, and were based on protocols developed, for c282y by the unidad de medicina molecular (ingo, santiago de compostela, espanha), and for the other two mutations by bolhalder m et al. the primers and probes were designed by tib molbiol (berlin, germany). results: the analysis of the percentages of genotypes and allele frequencies of the hemochromatosis gene mutations are described in the table. no differences were found between the patients and the controls. when we compared subgroups of patients based on their hepatitis c genotypes, a higher value for the c282y allele was obtained (9.0%) in individuals with genotype 1, however without statistical significance. discussion: hereditary hemochromatosis is a common disorder and is associated, in some studies, with a worst prognosis in patients with viral hepatitis. follow-up studies are necessary in order to evaluate if the presence of these mutations can cause a more severe course of the illness (greater risk to develop fibrosis or cirrhosis) and a different outcome when treated with antiviral drugs. also, it will be important to evaluate if aggressive phlebotomies will modify their clinical evolution. introduction: portugal has a higher prevalence of viral hepatitis, with probably more than 250.000 patients chronic infected with hepatitis b and/or c. hereditary hemochromatosis (hfe) is one of the most common causes of known hereditary illnesses with hepatic repercussion. hfe mutations are also found in linkage desiquilibrium with particular hla haplotypes, conferring, eventually, a different response to viral agents and antiviral drugs. in this study we evaluated the prevalence of the main mutations c282y, h63d and s65c for the hfe in a population with chronic hepatitis b and/or c and in a cohort control. background: haemolytic disease of the newborn (hdn) is the destruction of the red blood cells of the fetus and neonate by antibodies produced by the mother. although postpartum rhig prophylaxis reduced the incidence of alloimmunization from pregnancy from 16% to 1-2%, the doubt subsists if it is appropriate to use it as routine antenatal prophylaxis. material and methods: a total of 1836 samples (918 mothers and 918 newborns), from 01/05/2003 and 30/04/2004, were studied. all abo, rh typing, antibody tests and dat were carried out in column agglutination tests. results: from the 918 cases studied it was found 778 cases without incompatibility (85%). from the 140 incompatibilities, 88.6% were abo, 8.6% were rhd, 1.4% were other rh incompatibilities, and 1.4% were due to auto-antibodies. 80% of the mothers were rhd+ and 20% rhd-. conclusion: of the 918 pregnant women studied, only 184 were rhd-. from this group 83 (45%) delivered rhd-newborns, what revealed that the antenatal prophylaxis they were submitted was unnecessary. from the 184 pregnant women rhd-, 13% had incompatibility abo, which decreases to near 2% the risk of development of rhd immunization. being anti-d immunoglobulin a product that has the potential risk of infection transmission, is it appropriate to use indiscriminately as a routine antenatal prophylaxis? the introduction of molecular methods to determine the fetal rhd genotype could rationalize the use of antenatal anti-d immunoglobulin prophylaxis. introduction: the frequency of hla a201 haplotype expression has been found about 44-46% in caucasian and in greek population particularly, 44.3%. because of the high frequency, it is used widely in anticancer immunization. the immune system plays an important role in the defense against neoplastic disease and immune response show temporal chances related to circadian variation of antibodies and total lymphocytes in the peripheral blood. aim: the probable difference in the frequency of hla a201 expression and their lymphocyte phenotype into a group of cancer patients and a group of healthy donors, during screening of immunization with hla-combined peptides. materials and methods: 323 healthy donors who proceeded in the department of transfusion medicine, university hospital of heraklion crete were tested for the hla a201 expression. in 11 of these donors the expression of cd3, cd4, cd8, cd2, cd19, cd20, hla dr, cd3+cd4+, cd3+cd8+, cd16-cd56+, cd3+cd16-cd56+, cd3-cd16-cd56+, cd3-cd16-cd56+ was examined. meanwhile, 132 patients with metastatic cancer who were hospitalized in the department of medical oncology, university hospital of heraklion crete, were tested for their hla a201 expression, while in 50 of them for their lymphocyte phenotype. the antigens expression was examined in flow cytometry. the hla a201 expression in healthy donors was 44.8% and in cancer patients 45% (p > 0.05). in table 1 the mean, standard error, t-test and p of the two groups are included. (see table 1 ). the two groups (healthy donors and cancer patients) revealed no statistical significant difference on lymphocyte phenotype, except of the cd20 expression, which was higher in cancer patients. summary and conclusion: the expression of hla a201 in cancer patients and in healthy donors was comparable. also, the lymphocyte phenotype among the two groups has not statistical significant difference, except of the cd20 (total b-cells). the significance of this result has to be investigated. in the course of original documents research i found out that dr. kalic, head of the first organized blood transfusion institution in the balkan region (at beograd, serbia, in 1936), set himself a professional goal: blood should be awaiting all patients and transfusion should not be a privilege of large city inhabitants only. dr. kalic's idea was that blood transfusion should be administered according to clearly given instructions and using simple blood sets. encouraged by the conclusions of the congress held in paris in 1937, dr. kalic started preparations for the transport of blood to the inland. he concluded bravely that citrated blood could be sent by regular mail, as an ordinary parcel, without particular protection from the outside temperature. he advised his colleagues to use blood as an intravenous injection. blood was taken from voluntary female donor in belgrade (capital), march 1938. after keeping it for 10 days at storage, blood was forwarded on a two-day journey to a small town, 150 kilometres away from belgrade. there it was kept on a room temperature before its final use for a treatment of a patient suffering from secondary anaemia. the patient underwent the procedure without side effects and responded to the transfusion with blood sent in this manner much better in comparison to earlier methods of direct blood transfused. reminding ourselves of the courage of our ancestors to implement their professional knowledge and personal original ideas in a new way with the desire to help the patient as successfully as possible, we pay them the deserved respect and gratitude for inspiring and encouraging us in this way to try the same. conclusions: automation leads to increased standardization, faster specimen processing and reporting, elimination of manual specimen identification, uniform interpretation of serological reaction patterns and objective reading of haemagglutination endpoints. using auto-vue allowed the staff uninterrupted time to perform quality assurance duties, extended antibody identifications, preventative maintenance, inventory control. the instrument allowed us to leverage current staff to a more productive, less stressful level. introduction: exosomes are 60-100 nm secreted vesicles produced by antigen-presenting cells (apcs). the finding that exosomes from dc pulsed with tumor-derived peptides elicited potent antitumor tcell responses and tumor regression in mice has led to the proposal that human exosomes could be effective vectors for antigen delivery in the context of cancer immunotherapy. aim of the study: to establish the method of producing a new kind of tumor vaccine -exosomes secreted by dc, pulsed with tumor peptides. methods: exosomes used in this study were generated from monocyte-derived dc pulsed with peptides from k562 tumor cell lines. exosomes were purified by the methods of ultrafiltration and ultracentrifugation. the methods of dynal magnetic beads, flow cytometry and western-blotting were used to determine the surface molecules of the exosomes. the function of the exosomes was deterobjective: to develop an immunoheatological technique for the study of erythrocyte hyaluronic acid sodium salt (cd44) receptor expression in red blood cells (rbcs) from adults and newborns. materials and methods: samples of anticoagulated blood from adults (n = 30) and umbilical cordon (n = 30) were used. several dilutions oh hailuronic acid sodium salt solution 2% (sigma l-77h0544) were confronted with 2% erythrocyte suspension in phosphate saline buffer (pbs) ph 7.4. the rbcs were previously treated with an enzymatic solution of 5% bromeline in pbs ph 7.4 (sigma l60h0168). agglutination readings' were been by slow sharking after of 12 h incubation at 4°c. the results were expressed through the sensibility parameter which involves titer and score. this is defined by a mathematical expression a = à6 si. di-1. 10-3 (i = 1, 2, 3 . . .) where si represent the score and di-1 is dilution inverse. the adult' rbcs showed a = 26 ± 8, while en the newborn the parameter was a = 3 ± 1. our results showed significant differences between both groups. conclusions: in this work, we present a simple immunohematological technique for the hyaluronic acid sodium salt (cd44) receptor expression in red blood cells, which could be a useful tool to evaluate the alterations of the receptor's expression in rbc. a new technology for crossmatching tests adapted to a fully automated system l gaillard, v desvigne, a boulet, l fauconnier and jm pelosin diagast, loos, france we have developed a new automated technology for crossmatching (compatibility) test suitable for automation and high throughput. the method does not require centrifugation steps thanks to the use of magnetised red blood cells (rbc). all the steps described are performed on the fully automated qwalys system. this methodology requires washing steps under magnetic field and is based on the fixation of sensitised rbc on the surface of a well coated with monoclonal anti-human globulins. in a first step, the red blood cells from target blood bags were magnetised during 10 min. then the patient plasma is distributed on a microplate and incubated with the previously magnetised rbc during 20 min at 37°c. excess of unbound immunoglobulins is removed by washing steps. in a third step, sensitised magnetised rbc were transferred in the antiglobulins coated plate and placed 5 min on a magnet plate. wells in which antigen-antibody interactions have occurred display a confluent layer of rbc (positive reaction). the negative reaction appeared as a pellet in the middle of the well. the test can be read by an automatic reader or by naked eye. the patterns in the well are stable for at least 24 h at room temperature. the plasma samples are provided by the laboratory of haematology of the chru of lille. the red blood cells are collected from segment of tubing of blood bags coming from the laboratory of blood donors of the efs (french blood services) nord de france-lille. the results are obtained in 40 min. comparative studies showed that our new technology, without any centrifugation steps, is reliable and sufficiently sensitive and specific enough to perform cross matching tests using a high throughput automated system. the mechanisms of p53-dependent apoptosis involve a set of genes that possess the ability to modulate oxidative stress. one of them pig3, is induced by p53 through a microsatellite in its promoter region. this microsatellite has been proposed to represent an evolutionary adaptation of tumor suppressor mechanisms. microsatellite instability and genetic constitution, comprising the presence of the low repetition allele (10 tgycc repeats), at this locus have been hypothesized to provide an increased risk for cancer development. aim: in the present analysis we examined this polymorphism in blood samples from voluntary health donors and compared it with human lung cancer samples, employing two different ethnic groups, greek and british. results: analysis of this locus in both types of samples showed: (i) the homozygous presence of the 10 repeats allele only in the samples from healthy blood donors; (ii) a very low frequency of microsatellite instability (<1%) and no loss of heterozygosity in matched normal-tumor tissues; and (iii) a non-significant increase of the most frequent allele (15 repeats) in the cancer groups as compared to samples from healthy blood donors. the last two observations were found in both greek and british populations. conclusion: taken together, these data do not support the notion that this pig3 polymorphism is associated with an increased risk for cancer susceptibility. background: blood group determinations are routinely performed by the sensitive technique 'gel test' for the last few years. many weak d and partial d phenotypes which react as d negative or weak d by slide test, are assigned the rh d + status by gel test. this is most desirable in the case of blood donors but creates concern in case of patients and antenatal women with a partial d phenotype. case report: we report a female patient (blood group o, c+, c+, e-, e+) whose red blood cells gave a positive reaction of different strength and speed with different anti-d antibodies in slide tests. we were asked to type the patient and provide the appropriate blood units. the patient's cells gave a 4+/3+ reaction in the standard screening procedure for the rh d in gel test micro-typing system that contains a polyclonal reagent of human origin (which allows a direct detection of most weak ds), a 4+ reaction in a test with monoclonal anti-d and a 4+/3+ reaction in the gel test micro-typing system destined to detect du and which contains polyclonal anti-d of human origin. however, since the slide test gave a rather slow onset of agglutination with one commercial reagent (made up of a blend of polyclonal and monoclonal anti-d) we tested the patient's red cells against 6 anti-d reagents in the id-partial d typing system. one of these (number 4) gave negative reactions and the remaining five gave positive reactions (ranging from 3+/2+ to 4+), indicative of a partial d category vii phenotype. the patient's red cells were also tested in the id-card 'diaclon abo/d' . this card provides the complete profile for abo/rh d in one single procedure step, including the confirmation of rh d. it contains two different anti-d reagents within the gel matrix in two consecutive microtubes. the first anti-d (polyclonal human) is expected to give a positive result with d+ red cells and partial d category vi, while the second (monoclonal rabbit) is expected to give a negative result with dvi+ red cells. our patient's cells gave a negative reaction with the first and a 3+/2+ reaction with the second anti-d in this system, indicating a d variant other than dvi. finally the patient was assigned the partial d category vii phenotype (according to the pattern of the reactions obtained with the id-partial d typing set) and rhesus d negative blood units were issued. this case illustrates the diversity of reagents used for rhesus typing in different laboratories. failure to disclose some d variants is a disadvantage when typing patients. a combination of techniques is often needed to reveal the real rh d phenotype. the only single system that could have revealed a d variant in our patient from the beginning, is the id-card 'diaclon abo/d' with two different anti-d reagents in two consecutive microtubes as described above. a cost-benefit analysis should be undertaken to show whether it should replace other screening tests for abo and rh d when typing patients. who cares about the quality of life of the chronic patients treated with blood products? d ilcenco*, e hanganu-turtureanu † , c burcoveanu † , c vartolomei ‡ and d azoicai § *blood transfusion center, † hospital 'sfantul spiridon', ‡ institute of hygiene, § university of medicine, iasi, romania quality of life is one of the methods used to appreciate the quality of the health system. romania is going to join soon the european union, so there must be a concern regarding the improvement of the national health system. blood receiver's life quality never been researched before in romania. we have been chosen a batch of chronic ill patients who have been received blood transfusion with blood or blood components, and asked them to complete two types of questionnaires regarding their life. we used nottingham health profile and beck's depresion index. results shows that this kind of patients need special care, because they all (with one single exception) feel frustrated and feel like a burden to the other normal persons. evolution of the pain index, mobility index, energy index, emotions index, sleep index and social isolation index was in concordance with the depression index. in conclusion, this type of patients needs special attention and medical authorities should make more efforts to assure their life quality support. transfusion medicine practice in surgically treated urology patients: our experience il ilincic*, bm bozovic* and ts tadic † *clinical center dr dragisa misovic, † natio. blood transfusion inst., belgrade, serbia objective: multiple studies demonstrate that the use of blood/blood products in patients undergoing elective urology surgeries, as well as the actual needs assessment, present the issue of numerous debates. method: using the retrospective method, utilization of blood/blood products was analyzed, as well as the ratio of prepared/used blood units in urology patients in the surgical ward, in the intensive care unit (icu) and at the urology center within the cc dr dragisa conclusion: due to a rather liberal use of primarily ffp in certain cases (cystectomiae in the first place), and a discrepancy between the prepared and actually used blood units, hospital transfusion committees should be an imperative in order to solve current dilemmas regarding justified use and proper administration of blood and blood products. background: the safe collection, production, distribution and application of blood and blood products in a high quality needs logistic on a high level. since the seventies computers, special software and barcode are used in transfusion medicine and improved the safety of processing data. in the last years a new technology was developed for industrial use, the radio frequency identification (rfid). aim: the aim of our studies was to check whether rfid can use reasonable in transfusion medicine. methods: at first we developed a flow chart, where we can use the technology and where are the problems by introduction. so we tested in the red cross donation centre in saxony about 1000 passive rfid smart label under real conditions. in cooperation between the akh vienna and novatech research a new handheld pc software 'labelview' for all steps around the transfusion was developed, including the identification of the patient and the processing of the haemovigilance data, and tested in first time. results: passive and semiactive (with temperature control) rfid labels survives all hard steps during the working up of the whole blood (e.g. centrifugation by 5000 g, separation, etc.). as a result of the contactless identification they are help to make easier the documentation of all processing steps according good manufacturing practice. in clinical practice they are a good supplement to bed side transfusion software. conclusion: for all lot of problems by the logistic and the safe identification around the transfusion existing various single point solutions such as patient-wristband, bed-side test, double check of blood group typing and donor -donation registry in software, etc. the lecture will deal with new developments in logistics and data management, which can help to reduce the problems associated with documentation, safe identification and reporting of haemovigilance data. our experiences with the immunohaematological analyser olympus pk 80 applied conventional and no conventional (hemolytic medium time) techniques in 23 sera of patients with ascariasis. results: the ai and hk tests showed: b epithopes in 4 ae from b patients and in 3 ae from ab patients; a epithopes in 1 ae from ab patient and in 3 ae from a patients; p and p1 epithopes in 18 ae and only p1 epithopes in 3 ae. these 21 patients had both epithopes in their erythrocytes. the hemolytic techniques showed: anti b immune antibodies in 8 sera and anti a immune antibodies in 8 sera. the presence of abo and p epithopes in ae and immune antibodies in patients with ascariasis show a relation about blood groups and ascariasis. the fact of to find the same abo and p antigens in a. umbricoides and in its hosts suggests that the parasite might absorb them during its life cycle. these epithopes would be involved in the molecular mimicry. the use of filters for leucocyte depletion in anemic patients on maintenance hemodialysis g poposki*, s kovaceski*, b krstanoski*, s mena* and n solaz † *institute of nephrology, struga, macedonia, † ankara university, faculty of medicine, ankara, turkey introduction: renal anemia is one of the major chronic complications in end stage renal disease. it is caused by reduced production of erythropoietin (epo) due to uremic toxin effects, reduced halflife of rbc, iron deficiency, aluminum intoxication, blood loss during hemodialysis, gastrointestinal hemorrhage, epistaxis, infections etc. allogenic blood transfusion is transplantation of certain or all cell types. however, allogenic blood transfusion can contribute to many immune system disturbances with clinical side effects. besides erythrocytes, mononuclear, t and b-lymphocytes, are also transfused, which cause immunomodulatory disturbances in immune system of recipient. leukocytes are responsible for frequent febrile non-hemolytic transfusion reactions, alloimmunization toward leukocytes and hla antigen and transmission of cmv. anti-le antibodies, forming of immune-complexes, complement activation with pirogenic c3a and c5a immunoinflamatoric citokines cause febrile reactions. commercial use of filters for leukocyte depletion with removal of leukocytes and degraded products of microagregates and cytokines, cause minimum harmful immunomodulatory effects and prevent transmission of cmv. aim: the aim of the study was to present the effects of transfusion of erythrocytes with residual number of leukocytes in anemic patients on chronic hemodialysis at institute of nephrology in struga. matherial and methods: during 1997-2004 period all anemic patients on hemodialysis were divided in 4 groups. the first group 34 pts with febrile non-hemolitic transfusion reaction. the second group-25 pts immunized toward leukocyte and hla antigen. the third group 57 young candidates for kidney transplantation for prevention of hla immunization. the fourth group 16 pts with sle (for immune-complexes and autoantibodies). total 132 patients (65 males and 67 females) received 319 units of rbc with residual number of leukocytes. commercial filters of baxterô (lekostop 4 lds) and terumoô (imugard iii rc) of second and third generation with microagregate filter and synthetic polyurethane fibers, with 20-40 microns pores that remove leukocytes, platelets, microagregates and fibrin were used. erythrocyte concentrates are filtered until 5 days of collection. result: aabb permits maximum <5 ¥ 106 wbcs/unit for prevention of febrile non-hemolytic reaction. the filters we used reach residual leukocyte number of 2 ¥ 10 5 the le reduction of 99-99.99%. the number of rbc after filtration is minimum 90% -40 g hb per unit. in none of the patients who have received the leuco-filtered blood, no adverse post transfusion reactions were noticed. conclusion: the used filters for leucocyte depletion are characterized with superior biocompatibility, excellent elimination of all types of leucocytes and high 'recovery' of erythrocytes. the use of filters for le depletion reduces and minimizes the side effects of allogenic blood transfusion in patients on chronic hemodialysis who are alloimmunized, in patients with sle, and particularly in young patients candidates for kidney transplantation. background: fv leiden, prothrombin g20210a, mthfr c677t are three most common and important prothrombotic inherited mutations. aims: the aim of the case-control study was to assess the prevalence of mutations and their single or combined effects as risk factors for thrombosis. methods: the study included 103 thrombotic patients (venous thromboembolism, chronical venous diseases, different etiology) and 200 asymptomatic healthy individuals as control group. extraction of genomic dna was followed with genotyping of fvl by pcr-ssp, prothrombin and mthfr mutation by pcr-rflp. results: a statistically significantly higher prevalence of fvl mutation was found in thrombotic patients (32.0% heterozygous, 1.9% homozygous) compared to controls (3.0% heterozygous), p < 0.0001. the or for heterozygous carriers was 15.24 (95% ci 6.13-37.94), confirming the association of fvl mutation with the risk of thrombosis. there was no statistically significant difference in the prevalence of the prothrombin mutation in patients (5.8%) and controls (3.5%), or 1.71 (95% ci 0.56-5.21), p = 0.3441. although the group of thrombotic patients showed a higher prevalence of homozygous carriers of c677t mthfr than the control group (17.5% vs 10.5%), or was not significant (1.81, 95% ci 0.91-3.57), p = 0.0859. analysis of combined effects of mutations showed an additional thrombotic risk for carriers of fvl mutation and both mutated alleles of c677t mthfr gene (tt and ct) (or 9.40, 95% ci 3.41-25.80), p < 0.0001. conclusions: fv leiden mutation was detected as significant single risk factor for thrombosis in studied patients group. additional prothrombotic risk have carriers of fvl mutation and c677t mthfr gene mutation. a female patient in a high fever due to urinary tract infection does not respond being given antibiotics. on the contrary, leukocytes rose (to 30 ¥ 10 9 /l), anaemia became even deeper, as well as thrombocytopenia. hemocultures were negative. hematologist decided to search for hematological disease. the first citology results of bone marrow aspirate suggested lymphoproliferative disease (15% atipical plasma cells). to treat heavy anaemia (hgb 53 g/l) hematologist asked for red blood cell concentrate. pretransfusion testing revealed warm autoantibodies in the patient serum and on red blood cells. antibodies had no apparent specificity. biochemical parameters (bilirubin, ldh, haptoglobin) suggested mild hemolitic process. electroforesis revealed polyclonal hypergamaglobulinaemia. the 9th day of hospital treatment, the therapy with corticosteroids was introduced (solu-medrol 125 mg per day). coagulation parameters were tested: pt 1.75 inr, aptt 41s, fibrinogen 0.5 g/l, trb 66 ¥ 109/l, d-dimer 251 mg/l, atiii 52%. dic was suspected. liver enzymes showed mild liver dysfunction (normal ast, alt, elevated ggt, low che). substitution therapy started with 1 dose of cryoprecipitate, 1 dose of fresh frozen plasma, 1000 iu atiii, 2 doses of red blood cells and vitamin k 10 mg. two days after the substitution therapy we saw pt 1.28 inr, aptt 31s, fibrinogen <0.5 g/l, trb 75 ¥ 10 9 /l, atiii 71%. during the next few days erythrocytes and thrombocytes rose, but due only to corticosteroid therapy and not to substitution therapy. the patient had neither signs of con-sumptive coagulopathy, nor hypoproduction of coagulation factors, except for fibrinogen. till 17th day of therapy, fibrinogen was below 0.5 g/l. there was no hemorrhagic diathesis. after that, fibrinogen rose, and on the 21st day the patient was recovered, in both clinical and laboratory terms. the results of immunological tests, collected later, confirm the diagnosis of systemic lupus erythematosus. we did not have any specific test to confirm antibody mediated hypofibrinogenaemia, but in the setting of sle, without any specific treatment except corticosteroids, fibrinogen recovered. we assume it is quite enough for highly suspected immunological hypofibrinogenaemia. results: twenty-four-year-old male patient with severe hemophilia type a suffering from low incoercible digestive bleeding secondary to ischemic colitis caused by autoimmunity (vasculitis) without response to current management. treatment was initiated with 90 mg/kg/dose of rfviia (*) for 7 days, after which there was clinical and endoscopic recovery, and an inh decrease to 1.0 ub/ml (fviii dosage 19%) . he began to take meprednisone (1 mg/kg/day) for 45 days, after which the inh titre was 12.0 ub/ml. (table 1a ,b) the patient underwent surgery the following year (correction of equinus foot). he entered the operating room with an inh of 26.7 ub/ml and was treated with 120 mg/kg/dose of rfviia (*) for 10 days, obtaining an excellent hemostatic response. he had two autologous blood units, but it was not necessary to be administered. the inh titre decreased again (down to 5.7 ub/ml) during the intratreatment stage. thirty-five days after rfviia, the inh titre was 7.5 ub/ml. (table 2a ,b) the presence of high titre inh against fviii is a critical problem in cases of bleeding or surgery need due to the inefficacy of the available therapeutic options and the severity of the events. in this case, we have observed that, apart from inducing hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. we have introduced the case of a 24-year-old patient with hemophilia complicated by a high titre inh against fviii. in this case, we have observed that, apart from inducing an effective hemostasia through the activation of the coagulation extrinsic path, rfviia could reduce the inh titre in sequential dosages. there was also a decrease in the inh titre concomitantly with an increase of the plasma fviii level during its use. this phenomenon suggests that rfviia could produce a modulation in the immune response. evaluation of an automated blood collection system with standard ratio of anti-coagulation and integrated filter for whole blood leucodepletion l dadiotis, a kolokytha, m dimou, a perdiou, c alepi, p spyropoulou, e igoumenides, c velidou, v panagopoulou and s matsagos tzaneion general hospital, pireas, greece automated blood collection system (abc) is a device manufactured by macopharma which collects by gravity a preset volume of blood and mixes it with anticoagulant (ac) in standard ratio (7 : 1). this is managed by passing the ac, which is stored in a special bag, anti-erythrocyte antibodies are immunoglobulins that belong to the igg, igm and iga classes. their common characteristic is a specific reaction with antigens that are located on the erythrocyte surface. they can emerge as auto antibodies and alloantibodies. the blood transfusion in patients may induce a post-transfusion hemolytic reaction (pthr). in order to avoid or reduce the danger of the pthr it is necessary to examine whether there are irregular anti-erythrocyte antibodies in the patient's serum as well as in the serum of the voluntary blood donors. all the irregular anti-erythrocyte antibodies are not clinically relevant. the experience shows that the clinically significant antibodies most often belong to abo, rh, kell, kidd, duffy and ssu blood groups. in the period from april, 2003 to november, 2004, we monitored and examined, at the institute for blood transfusion, clinically significant antibodies in the serum of the patients who are treated with blood transfusions as well as in the serum of the voluntary blood donors. we used the following tests for detecting irregular anti-erythrocyte antibodies: enzyme test, indirect coombs test, screening test by the commercial test erythrocytes and gel filtration method. the detected irregular antierythrocyte antibodies are identified by means of the commercial test erythrocytes for identification. our results are the following: voluntary blood donors: anti d, anti c + d and anti-leb antibodies. patients: anti-d, anti-k, anti-fya, anti-c and anti-e antibodies. in nine patients, anti-erythrocyte antibodies were discovered, namely, those that react at the temperature higher than 30°b ut whose specificity we could not discover with the existing techniques. improved predictive factors of response for myelodysplastic syndrome patients treated by the combination of erythropoietin and g-csf s park*, c kelaidi † , s grabar ‡ , v bardet ‡ , d vassilieff ‡ , f picard ‡ , m guesnu ‡ , mc quarre ‡ , p fenaux § and f dreyfus ‡ *service hématologie, hopital cochin, † hématologie, hopital avicenne, ‡ service hématologie, hopital cochin, § hématologie, hopital avicenne, paris, france it has previously been shown that serum epo level and number of previous red blood cell transfusions are predictive factors of response to epo + g-csf treatment of myelodysplastic syndromes (mds). in a subgroup of patients with mds having sepo <500 ui/l, known to be good responders to epo + g-cscf, the gfm group wanted to refine the model predicting the response to epo + g-csf, especially with cytology (who classification with dysplasia and percentage of erythroblasts and blasts). in a population of 64 patients (ra, rars and raeb <10% blasts) receiving epo ± gcsf between 2000 and 2004 and having serum epo <500 ui/l, the response rate at week 12 (iwg criteria) was 60%. six variables were associated with response to epo ± g-csf for mds: age >70 years (p = 0.02), number of prior red blood cell transfusions <2 packs/months (p = 0.005), serum epo level <200 ui/l (p = 0.02), percentage of blasts <5% (p = 0.05), percentage of erythroblasts >30% (p = 0.02) and low ipss score (p = 0.001). we did not found any influence of dysplasia, type of rhepo (darbopoietin alfa or epoietin alfa) and karyotype on response rate. in multivariate analysis, age through a rotating pump. the abc can be used with all types of p-417 pan-european blood safety alliance the pan-european blood safety alliance is a unique alliance of patient organizations, formed to promote the highest level of blood safety for all in europe. it was formerly established on 11 february 2005, during the course of the first general meeting of the pbsa, which comprised of 8 founding patient organizations. the objectives of the pbsa are: 1. to promote the fundamental right and duty to safety of all patients in need of blood transfusions and blood products. 2. to ensure the availability of sufficient amounts of safe blood, to meet all treatment need through: -the education of all staff handling blood components, to reduce human error. -the implementation of and access to, proactive blood safety technologies, for each patient across europe. -haemovigilance -the adequate access to blood transfusion services, which should be provided free of charge to the patient. other objectives are to raise awareness on a local and european level regarding blood safety, to promote eu legislation that improves safety standards of blood transfusion services, including stem cell preparation and storage across europe and to lobby for increased patient influence on eu health policy makers. very importantly, the alliance aims at providing a forum for patients, healthcare professionals, health policy makers and relevant industry, as well as acting as a point of reference to the national health authorities, the european commission and other european institutions, when seeking the opinions of patients on blood safety. cerns of insertional mutagenesis and the safety of some viral vectors that randomly insert genes through the genome have been recently resurfaced following the development of a haematological malignancy in a child treated with a retroviral vector. particularly questions also remain as to, whether gene therapy and the production of ectopic factor viii and ix will be a risk for inhibitor development or indeed whether it might promote tolerance in those patients with inhibitors. w-pl4-11 gene therapy for thalassemia: will it become reality? university of washington, seattle, wa, usa experiments aimed to develop gene therapy approaches for the beta chain hemoglobinopathies, sickle cell disease and beta thalassemia started about 25 years ago. in the beginning results were dismal because of the extremely low and variable expression of globin genes contained in the therapeutic vectors. a major development occurred in 1987 with the discovery of powerful regulatory elements that could guarantee high level of globin gene expression. these elements when incorporated into viral vectors allow expression of therapeutic levels of the transferred globin genes. a second major progress was achieved with the development of safe lentiviral vectors that can efficiently infect the human pluripotent repopulating hemopoietic stem cells. as a result of this progress, today beta thalassemia and sickle cell disease can be cured in murine models of these disorders. considerable effort is already being devoted into further improvement of lenti viral vectors with emphasis on incorporating elements which will decrease the probability of insertional mutagenesis and leukemogenesis. the major challenge for the clinical application of stem cell gene therapy of thalassemia is the need for genetic correction of large numbers of mutant stem cells. in vivo selection of corrected stem cells is being investigated but there are questions about its safety because of the possibilities of clonal expansion of stem cell lines carrying undesirable integrants. other major challenges have to do with logistics: production of therapeutic vectors, infrastructure required for stem cell gene therapy delivery, and sponsoring and funding of the clinical trials. gene therapy trials on limited number of patients are expected to be initiated relatively soon. if these trials are successful and cures of beta thalassemia ensue, the major challenge will be the delivery of this molecular therapy in the context of medical practice. w-pl4-10 gene therapy for haemophilia haemophilia is an ideal target for gene therapy because only a small rise in factor levels to 1-2 u/dl would achieve the goals of prophylaxis without regular infusions of concentrate and deliver a substantial improvement in lifestyle for patients with severe haemophilia. gene therapy for haemophilia today relies upon addition of normal factor viii or ix genes. with present technology gene therapy can offer the prospect of a true 'cure' for haemophilia in animal models, although this may not be currently realizable in man. more than 30 patients with haemophilia have now been treated in phase 1 gene therapy protocols. all studies have failed to conclusively show that therapeutic levels of factor viii and ix can be reliably obtained. the first trial reported used im injection of a factor ix containing recombinant adeno associated virus (raav) in adult patients with severe haemophilia b. only very modest increases in factor ix level, <2 u/dl rise, in 4/8 patients enrolled were observed, although less factor ix concentrate was needed in 3/6 subjects. a similar study using the same raav vector via intrahepatic artery infusion has been conducted. this has been complicated by the observation of aav vector in the semen of subjects. in six patients enrolled, no durable levels of ix above 1 u/dl were seen. further development of this raav vector is suspended. for haemophilia a, three systems are have been tried. the first study was an ex vivo addition of factor viii gene to autologous fibroblasts and then laparoscopic reimplantation. preclinical assessments demonstrated durable expression of factor viii (>5% of normal) for >1 year in mice following a single treatment. in 4/6 patients treated repeated factor viii rises (0.5-3.5 u/dl) were seen, but no improvements lasted beyond 10 months. the second protocol used a murine leukemia retrovirus containing factor viii, injected intravenouslya development of preclinical data in rabbits and haemophilic dogs. 0/13 patients enrolled sustained levels of factor viii >1 u/dl. the third study, using a modified, 'gutless', adenovirus containing factor viii gene has recruited one patient. this patient demonstrated transient liver toxicity and thrombocytopenia at doses lower than those that cause toxicity in primates. sustained levels of factor viii of ~1 u/dl have been observed over a number of months. accrual to the study has been poor. haemophilia remains a prime target for gene therapy. however, haemophilia is no longer a life threatening disease with current therapy that is both safe and efficacious. a balance between the benefits and theoretical risks must be borne in mind when considering gene-based approaches to therapy. conreference: petz ld, garratty g. immune hemolytic anemias. 2nd ed. philadelphia: churchill livingstone, 2004, pp 375-400. w-pa-114 autoimmune neutropenia introduction: autoimmune neutropenia (ain), a granulocytic disorder due to the presence of anti-neutrophil antibodies, may present as neutropenia of varying degree with or without recurrent infections un previously healthy individuals (primary or idiopathic ain) or in patients with a known underlying disease such as lupus erythematosus, lymphoid malignancies, etc (secondary ain). the condition affects more frequently infants of small ages while it is rare in adults [1] [2] [3] [4] [5] [6] [7] . in some patients, diagnosis is established in occasion of a respiratory, urinary or cutaneous infection, but in many cases is simply a finding of cell blood counting performed for unrelated reasons [6] . clinical and laboratory findings: in general, physical examination is negative. laboratory investigation reveals the existence of isolated neutropenia. association of the disorder with autoimmune hemolytic anemia or autoimmune thrombocytopenia is rarely seen [8] . blood biochemistry is normal while serologic tests for bacterial, viral or other pathogens may be positive depending on the underlying infection. bone marrow is hypercellular without maturation arrest of granulocytic series. hemopoietic stem cell reserves and function are normal or increased, and stromal cell function is within the range of the normality [9]. methods for the detection of granulocyte-specific antibodies: serology for the detection of granulocyte-specific antibodies has been marred, compared to erythrocyte serology, because the target cell here, the granulocyte, is short-lived, fragile and becomes easily activated. the former two of these difficulties require absolutely freshly (<6 h old) isolated neutrophils from a panel of donors to be used every day to run the tests with sera from patients, while the third difficulty is more important since spontaneous cell clumping in vitro is very common and nay mimic the specific aggregation caused by cross-linkage of surface bound antibodies in the granulocyte agglutination test (gat). in order to overcome these problems, the second international granulocyte serology workshop [10] recommended a combination of two tests as the best screening procedure for the detection of granulocyte autoantibodies in patient sera, gat and granulocyte immunofluorescence test (gift). gat is mainly mediated by igm antibodies and is positive in about 2% of cases. gift detects igg antibodies and is positive in about 98% of cases. it is to be noted that flow-cytometry fluorescence may arise not only from the surface but also from the cytoplasm of neutrophils, necessitating assessment of membrane fuorescence by microscopy. a good direct anti-granulocyte test is not available today. this is due to the fact that too few neutrophils can be obtained from the blood of neutropenic patients, and also to the observation that neutrophils are often activated in vivo because if an underlying infection or other inflammatory process, thus expressing fcgrii and fcgriiib to which nonspecific binding of w-pa-113 practical approach to transfusion in autoimmune hemolytic anemia (aiha) g garratty american red cross blood services, pomona, ca, usa a major problem when transfusing patients with aiha is that often all units are incompatible. this may be due to autoantibodies (autoab) and/or alloantibodies (allo-ab). if the incompatibility is due to only auto-ab, then transfusion of incompatible blood will not usually result in a clinically significant reaction, but if due to alloab, the result may be similar to that seen in any other patient (i.e. a hemolytic transfusion reaction ranging from mild to severe). thus, it is essential (as in any other patient) to exclude the presence of allo-ab. it is wise to phenotype all patients, for as many antigens as possible, before the patient receives transfusion. there are two popular approaches to determine if allo-abs are present but being masked by 'warm' auto-ab activity. the preferred method is to remove the auto-ab by adsorbing the patient's serum with autologous rbcs treated with enzymes, or preferably, with zzap reagent. the latter reagent contains an enzyme leading to optimal adsorption of auto-ab, and dtt. these two chemicals will destroy significant antigens other than rh and kidd (e.g. mns, duffy, kell, lutheran, dombrock, cromer, lw, some yta and ge, inb, jmh, ch, rg, pr antigens), thus will not adsorb alloantibodies to these antigens. if autoadsorption is not possible (e.g. patient has been transfused recently or there are too few rbcs), one has to perform adsorption with enzymes or zzap-treated allogeneic rbcs. one does not have to be concerned with covering any antigens destroyed by zzap (e.g. kell and duffy systems). we use a rough guide relating the strength of the indirect antiglobulin test to the number of adsorptions needed to remove auto-ab (1+ = 1 adsorption; 2+ = 2 adsorptions; 3+ = 3 adsorptions; 4+ = 3 or more adsorptions). if there is no activity left after the adsorptions, then one can suspect that the incompatibility was due to auto-ab, but on rare occasions one can be wrong and an allo-ab to a high-frequency antigen has been removed. this is a major disadvantage of using allogeneic adsorptions, and is why adsorptions with autologous rbcs are preferred. if time (2-8 h) does not allow for adsorptions, one can dilute the patient's serum (e.g. 1 in 4, or 1 in 5) and test the dilution against a panel to see if any alloantibody specificity becomes obvious. another approach is to select units matching the patient's phenotype as closely as possible. when dealing with cold agglutinin syndrome one can usually exclude allo-ab activity by testing strictly at 37c. this can be helped by performing adsorptions with enzyme-treated autologous rbcs at 4c, but it is difficult to adsorb all of the powerful cold autoagglutinin activity. it is reported that 30-40% of aiha have allo-abs; the incidence is even higher in patients who have received multiple transfusions. thus, we feel that procedures such as those discussed above must be performed before transfusing incompatible blood if time allows. one should always negotiate with the attending physician regarding the time it will take to perform adsorptions. a decision may be made not to perform adsorptions if the patient has life-threatening hemolysis, and especially if the patient has never been transfused, or pregnant. serum igg may occur (naig). the presence of immune-comlexes in the serum, such as in patients with felty's syndrome, lupus erythematosus and other diseases, as well as the presence of immune aggregates formed in sera stored frozen for long time, may give false-positive tests given that they may bind to fcgriiib molecules expressed on the surface of neutrophils. elimination of immunecomplexes and immune aggregates can be easily obtained by ultracentrifugation [11] . another cause of false-positive results may be the presence of anti-hla antibodies because of allo-immunization. these allo-antibodies react with hla molecules found not only on neutrophils but also on the surface of many other cells including lymphocytes. these allo-antibodies can be eliminated by platelet absorption. it seems that the best method in the search of true antigranulocyte antibodies is the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) [12] . with this method one can specify anti-granulocyte antibodies using a panel of known granulocyte antigenic specificity. finally, it is notable that the levels of serum antibodies to neutrophils may vary considerably over the time. one negative test does not exclude ain. usually, two to three tests have to be run over a period of months [13] . antigenic specificity: human neutrophil antigens (hna) are classified according to an international granulocyte antigen working party [14] . three glycoproteins have been found to be involved in the determination of antigenic specificity, fcgriiib, gpnb1 (cd177) and gp70-95. the respective antigens, frequencies and alleles are illustrated in table 1 . antigenic specificity can also be studied by using methods applied in molecular biology. a promising approach is transfection of mammalian cells by cdna derived from granulocyte antigen specific mrna. cell lines have been established with cells expressing the respective human granulocyte antigen, making the detection of anti-granulocyte antibodies more easier. genotyping of hna antigens can also be stydied with the pcr technique [15] . references are available from the author upon request. w-pa-115 a rare case of 'coombs negative' autoimmune haemolytic anaemia due to red cell autoantibodies of iga class warm autoimmune haemolytic anaemia (waiha) is usually associated with red cell auto-antibodies of the igg class, which can be detected by polyspecific direct antiglobulin test (dat). routine polyspecific direct antiglobulin tests contain anti-igg and anti-c3d components, and are not standardized to react with iga-or igmsensitized red blood cells. haemolytic anaemia caused by warmreacting auto-antibodies solely of the iga class is exceedingly rare. those cases of autoimmune haemolytic anaemia can be difficult to diagnose because of the negative polyspecific coombs' test, which is a standard in investigation of possible causes of haemolysis. we present a case of severe warm autoimmune haemolytic anaemia caused by iga class autoantibodies. a 40-yr old male patient was admitted with anaemia, haemoglobinuria, and other signs of severe haemolytic disease. he received multiple transfusions but haemoglobin level did not rise above 7 g/dl. the initial polyspecific direct antiglobulin test, containing an anti-igg and anti-c3d antiserum, was negative. tests for cold agglutinins and other possible causes of haemolysis were negative. only by using a monospecific, anti-iga antiserum could we show that the warm iga auto-antibodies against red blood cells were present on patient's erythrocytes. we have not detected signs of complement activation by iga autoantibodies in this patient. the patient received corticosteroids with good initial effect. his haemoglobin level stabilized and he did not require more transfusions. anti-iga direct antiglobulin test became negative about to weeks after the therapy was initiated. however, in spite for the initial effect of steroid therapy haemolysis continued, and splenectomy was performed 6 months after diagnosis was made. it has been shown that human lymphocytes, granulocytes and monocytes contain specific fc receptors for iga, and both monocyte-mediated phagocytosis and antibody-dependent cellular cytotoxicity due to iga auto-antibodies has been demonstrated. there is also increasing evidence that iga auto-antibodies can activate complement, both via the classical and the alternative pathway. a phenomenon of 'reactive haemolysis', which involves c3-independent binding of c5b6 complexes to 'bystander' red blood cells, has also been described. we emphasize the importance of performing additional testing in cases of apparent 'coombs' negative' haemolytic anaemia due to iga, igm or 'low affinity' igg autoantibodies, and serological aids that are available for that purpose. described. both siblings were born on term, in good general clinical status, free from any signs of infection, and with isolated severe neutropenia (170 and 123 neutrophils/ml). the diagnosis of annanti hna-2a was made upon exclusion of other possible causes of neonatal neutropenia, and confirmed by serological testing of granulocyte antigens and antigranulocyte antibodies. in both cases, the course of the disease was mild, with bacterial omphalitis on day 15 and 5, respectively. omphalitis was successfully treated with 7-day antibiotic therapy according to antibiotic sensitivity report. the first neonate received standard dosage of intravenous gammaglobulins for 5 days without success. this was followed by an attempt at neutrophil count increase with 2-week corticosteroid therapy, also without response. the second neonate received no specific therapy for neutrophil count increase. the children were discharged for home care with clinical and laboratory control examinations at 2-week intervals. in spite of prolonged neutropenia (2 and 6 months, respectively), no other infections were recorded. discussion and conclusion: in our patients, the therapeutic approach to ann was individualized, based on standard antibiotic therapy, intravenous gammaglobulins, corticosteroids, available literature data, and our own clinical experience. although in the last few years rh-gcsf is successfully used in patients with neutropenia, we decided to postpone its use in case the neonatal sepsis developed. the reasons for such decision were: (1) the fact that both neonates were in good general clinical status, with a mild course of the disease with only short-term umbilical infection successfully managed with antibiotic therapy; (2) literature reports suggesting the unexpected failure to respond to rh-gcsf therapy in patients with neutropenia induced by anti hna-2a immunization, and (3) the unknown effect of rh-gcsf on developing tissues of the neonate. the choice and efficacy of specific therapy for neutrophil count increase in the management of alloimmune neonatal neutropenia have not yet been fully defined and require additional evaluation in the majority of cases. male donors for the production of fresh frozen plasma: a special issue for trali patients trali is a significant cause of transfusion associated morbidity and mortality, and has been reported as the third most common cause of fatal transfusion reactions. there is no good evidence on which to base transfusion support policy for patients who have experienced trali. the hypothesis that there may be patient associated factors that contribute to the risk of trali is generally accepted. for this reason it seems reasonable to try to avoid further transfusion during the period of illness. if this is unavoidable the next best solution to reduce the risk of recurrence seems to be the avoidance of using plasma containing blood components (especially ffp) as there is a high chance of positivity for leucocyte antibodies especially for those coming from female donors. as fresh frozen plasma transfusion accounts for up to half of all trali cases and as our center is the only in greece responsible for the testing and processing of blood from blood donors representing military recruits, the last two years we tried to set up a project in order to provide components from male donors on request. our donor base consists predominantly from males donors (98.7%), aged between 18 and 25 years old, with a small chance of having a positive history for transfusion the difficulty of the project consisted on the fact that these donors are assigned to military camps throughout greece, which makes difficult the on time arrival of the units to our establishment in order to be processed for the production of ffp conform the european council quality requirements. this was the main reason why, till now, all plasma produced from these donations was regarded as plasma for fractionation. the first step for implementing the new project was to evaluate the number of donations that, by minor changes on the time of arrival, could be processed for ffp production. the next step was to re-schedule the shifts of the personnel for the on time production of ffp. during 2003, 25% of donations were fulfilling the specifications for the production of ffp and with the flexibility of the schedule the 97% of them were successfully processed to ffp. during 2004, 30% of the donations were fulfilling the specifications and 97.8% of them were processed to ffp. so it is feasible to increase the proportion of male ffp by organizing better the transportation of blood from the donation sites to the blood establishment and by retaining available specialized personnel to cover the extra shifts. maximising the blood supply chain in times of shortage shortages in the blood supply chain may occur for a variety of reasons. they may be temporary e.g. due to a flu epidemic or prolonged e.g. due to the exclusion of a high proportion of donors due to new pre-donation tests or because of a lack of volunteer donors. increasing awareness of the possibility of blood shortages mainly related to increased precautions associated with the possible transmission of vcjd by transfusion has been the driver for the development of blood shortage contingency plans in the uk. in england and north wales, hospitals and the national blood service (nbs) have worked together to develop an integrated blood shortage plan (ibsp) designed to ensure that hospitals and the nbs work together within a consistent, integrated framework giving patients equal access to available blood on the basis of need. an essential element of the plan is the principle that shortages can, in most cases be avoided by reducing the current usage of blood through appropriate use programmes. the impetus for hospitals to implement these programmes was a government circular (hsc 2002/009). hospitals have embraced the circular and have recruited specialist hospital transfusion practitioners, introduced lower hb triggers, cell salvage and hospital transfusion teams and are participating in the blood stocks management scheme (bsms). audits of compliance with the circular have taken place, and a web based tool kit is available. the demand for blood has declined for the last three years, with a decrease of about 5% during 2004-05, suggesting that the drive for improvement has been successful. the shortage plan introduced in england and north wales has two key aims: that the national pool of blood is available for all essential transfusions for all patients and that overall usage is reduced to ensure the most urgent cases receive blood. the plan is structured to provide actions for the nbs and hospitals in three phases, 'normal' circumstances, reduced availability and severe prolonged shortage. hospitals should have documented emergency blood management arrangements for each of the phases. the national plan is activated when the nbs red cell stock level falls to pre-defined levels, hospitals are informed by fax that they should reduce their normal stock holding levels according to guidance in the ibsp and comply with the daily hospital usage budget. the bsms has used its knowledge of hospital inventory levels and demand to provide guidance on appropriate inventory levels for normal and reduced status, it also provides the daily hospital budget. to monitor progress against the recommendations in hsc 2002/009 hospitals will be benchmarked against a number of performance indicators. these include the presence of emergency blood management arrangements, median red cell usage for a number of surgical procedures and percentage wastage of blood. there have been no shortages within the nbs for more than six years, it is hoped that the implementation of the ibsp will help to ensure that in the unlikely event of reduced availability blood will be available to the maximum number of patients requiring a blood transfusion. w-pa-120 transfusion during disaster g klein nih, bethesda, md, usa publicity given to blood donation during wartime has created a powerful association between the need for blood and occurrence of a disaster. blood is rarely needed in excessive quantities at the moment a disaster occurs. the outpouring of blood donors, especially at the site of a disaster, often proves counterproductive. the terrorist attacks on the world trade center on september 11, 2001, with almost 3000 deaths and more than 4000 injuries, provides an instructive model. more than a million potential donors contacted blood-collection centers. hundreds of thousands of prospective blood donors crowded collection facilities and many waited for hours, often to be turned away. qualified staff were in short supply and screening errors occurred as minimally qualified staff were recruited and as collection personnel fatigued. supplies and storage capabilities were pushed to their limit. some blood was inadequately processed and stored. resources were diverted from needed apheresis collections and component preparation to whole blood collection. in the aftermath of the disaster, blood outdated and volunteer donors became disillusioned as their 'gift of life' was refused or unused. similar responses have occurred numerous times over the 60-year period since blood-donor programs were introduced. in virtually every civilian disaster in the u.s. during the past century, all the blood that was needed was immediately available from blood inventory. in only four cases were more than 100 units of blood used in the first 24 to 30 h. in 2001 in new york, the five hospitals closest to the disaster site admitted only 139 disaster victims. the new york blood center, which supplies 80 percent of blood for the city's hospitals, added 600 units to routine inventory at hospitals. the center received 12 000 telephone calls and collected more than 5000 units of blood in the first 12 h. in the area of the pentagon, the chesapeake and potomac red cross blood center supplemented hospital blood inventories within h of the disaster. meanwhile, spurred by well-meaning media and federal officials, lines of blood donors were being processed at local hospitals, makeshift collection centers, the small research hospital at nih, and at a building next to the white house. in the week after september 11, america's blood centers collected 167 000 more units of blood, and the american national red cross collected 287 000 more units than in the same period the previous year. more than 475 000 units were collected for the disaster victims, but only 258 units were used. u.s. blood collectors and federal agencies have created a disaster plan that acknowledges the need for altruistic people to volunteer for blood donation in the time of disaster and speaks with a single voice to avoid needless collection activity while harnessing the good will of well-intentioned people to supplement the ongoing need for volunteer blood donation. rehabilitation of blood transfusion service in azerbaijan cd asadov, ga huseynov and ab hagiyev institute of hematology and transfusiolo, baku, azerbaijan at the end of 80th years of the last century in azerbaijan as well as in other republics of the former ussr began process of progressive deterioration of blood service parameters. in result there was an essential reduction of prepared blood and blood components quantity, manufacture of preparations from blood's plasma has completely stopped. it is connected by that our republic experiences a heavy transition period from scheduled to market economy. after reception by azerbaijan of the sovereignty on development of a national policy the big work has been lead to areas blood transfusion and development of national rules and the standards regulating functioning of establishments of blood service. in 1996 the law about ' the donorship of blood and its components in the azerbaijan republic' has been accepted, instructions on physical examination of donors and preparations of blood and its components are authorized, and also the new speciality transfusiology has been entered into the nomenclature of medical specialities. now the national program of blood service development is developed. at drawing up of the program social and economic conditions of the country, ethnic both cultural traditions and a mental potential of the nation are considered. within the framework of this program is planned to refuse gradually a paid blood donation during the certain period of time to reach 100%s' voluntary unpaid blood donorship. however in connection with limitation of resources, the state is not capable to allocate enough of means for its realization. the big work on attraction of the international organizations has been carried out. now the project of the united nations development program (undp) 'rehabilitation of blood transfusion service in azerbaijan' is carried out at sponsor's support of the norwegian government. realization of this project will lead to reorganization of blood transfusion service in our country according to practice of the european countries. within the framework of project realization it is planned to make changes and additions to a existing law about a blood and its components donorship to bring it into accord with recommendations of the europe council. updating of the russian law 'concerning the donation of blood and blood components' on june 9, 1993, a law, 'concerning the donation of blood and blood components, ' was signed by the first russian president, boris eltsin. now, after more than ten years of market economy and democratic evolution in russia, this law was significantly changed on august 22, 2004, as shown in the following sections: 1. the development of a voluntary blood donor system. 2. removal of the upper age limit for blood donors. 3. funding for blood donations. from january 1, 2005, each level of the state power budgets for a blood donor service to supply blood products for federal, regional, or municipal hospitals. costs of these drugs and the need of prolonged growth factor treatment in these disorders. w-pa-127 can iron administration reduce peripartum blood transfusion c breymann university of zurich, zurich, switzerland the prevalence of iron-deficiency anemia in different regions of the world ranges from 12 to 43%. the increased iron requirement in pregnancy and the puerperium carry with it an increased susceptibility to iron deficiency and iron-deficiency anemia and perioperative or peripartal blood transfusion. however, if ever possible administration of blood transfusion should be avoided for several reasons which will be pointed out in the talk. infections: it is well known that various pathogens such as bacteria and virus can be transmitted by administration of blood. around 0.03% of are contaminated by bacteria such as yersinia or pseudomonas species but are not screened routinely for bacteria. in addition there is no donor screening for hepatitis a, herpes species (cmv, ebv, hhv 7, hhv 8), parvovirus b19, hepatitis g (3.4%) and tt (transfusion transmitted) virus (1.9%). numbers for positive testings for 'classic' virus such as hiv, hep. b and hep. c vary from country to country and lie around 1 : 30 000 to 1 : 5000 000 depending on quality of donor screening programs, pcr sensitivity etc. recently there is increasing evidence that even prions which cause the jakob creutzfeld disease variation ('mad cow disease') might be transmitted by transfusions. therefore the fda has determined that blood donors from countries with high prevalence of prion positive persons are not permitted to give blood in the us (e.g. donors from uk). beside infections, other well known effects of transfusion are problems due to incorrect blood or components transfused, post transfusion purpura, acute and delayed lung injury, graft versus host disease and other acute and delayed allergic reactions. beside these negative effects it was also shown that patients who receive blood transfusion liberally after operations or in icu show higher morbidity and mortality compared to patients with restrictive transfusion policy. this might be due to negative effects on immune functions and inflammatory reactions and lack of stored blood to efficiently improve organ oxygenation. for example it is known that stored blood has worse capillary perfusion and worse viscosity properties compared to fresh blood. taken together there is increasing scientific evidence that blood transfusion is not the gold standard for anaemia management and alternatives such as endogenous blood pooling and efficient treatment of any anaemia must be enforced in the clinical settings. prevention and correction presuppose reliable laboratory parameters and a thorough understanding of the mechanisms of iron therapy. in order to correctly diagnose the type and degree of anaemia, a prerequisite for selection of the proper therapy, one must first of all correctly differentiate between the relative, i.e. the physiological anaemia of pregnancy due to the normal plasma volume increase during pregnancy, and 'real anaemias' with various different pathophysiological causes. when defining the hb cutoff value for anaemia in pregnancy, the extent of the plasma volume changes with respect to the gestational age must be taken into consideration. it has been found that haemoglobin values <11.0 g/dl in the first and third trimesters, and <10.5 g/dl in the second trimester may point to an anaemic situation which should be further clarified. the first important steps for diagnosing anaemia in a pregnant patient include a thorough check of her medical w-pa-126 impact of epo treatment on transfusion requirements in myelodysplasia c gardin and p fenaux hopital avicenne, aphp, university of paris 13, bobigny, france myelodysplastic syndromes (mds) are clonal disorders of hematopoeisis, associated with bone marrow failure and an increased risk of evolution to acute myeloid leukemia (aml). despite an normal or increased bone marrow cellularity in most cases, cytopenias worsen with time due to increased apoptosis and defective differentiation of blood lineage precursors. incidence of mds increases with age and reach 15/100 000 above 70 years of age. bone marrow cytogenetics number of cytopenia and percentage of bone marrow blasts are strong predictors of survival and evolution to aml. a composite international prognosis scoring system (ipss) is used in everyday practice to guide the management of these diseases. these disorders are heterogeneous and include 'low risk' patients (less than 10% bone marrow blasts) with a prolonged evolution marked by chronic anemia, and 'high-risk' patients (excess of bone marrow blasts >10%) evolving in a short timespan with severe cytopenias, and to aml in approximately 50% of cases. at diagnosis, 80% of mds patients are anemic, with an hemoglobin level less than 100 g/l, and 80% of them will require chronic blood components transfusion, during the evolution of their disease. chronic anemia and multiple blood products transfusions are associated with an altered quality of life, clinical iron overload, and important health care costs. although transfusion practices and patient's transfusion need are variable, elderly mds patients require a mean of 10-20 units/year of follow-up, in recent surveys. therapies able to diminish or abolish the need for rbc transfusion have therefore a major role in the management of mds, as allogeneic bone marrow transplantation, the only curative therapy of these diseases, is limited to a small subset of mds patients. high-doses of recombinant erythropoetin (epo) (150-300 u/kg tiw, or a 40 000-60 000 u as single weekly dose) are typically used in low-risk mds. the response rate to epo is 25-40%, including major responses (suppression of rbc dependency or rise of hemoglobin level of more than 20 g/l). absent or low rbc transfusion needs and a serum epo level less than 500 u/l are strongly predictive of response to epo, in patients with low-risk mds. the duration of response is variable (12-20 months) in most studies, with some long-term responders. the use of higher doses of epo or its prolonged administration may be associated with higher response rates, although no randomized studies are available combination of epo and low-dose granulocyte-colony stimulating factor (g-csf) increases the response rate to 40-60%, including in patients not responding to several weeks of treatment with epo alone. two randomized trials published in 2004, compared g-csf-epo to rbc transfusions and confirmed the efficacy of this combination, and a longer survival of epo-g-csf responding patients. studies are ongoing in mds, including with darbepoetin, a modified erythropoetin with longer half-life, administered once a week. two such studies have been recently reported, (darbepoetin 150 or 300 ug/week) with response rates varying from 35% to 60% in low-risk mds. in both studies, a response to darbepoetin was observed in some patients, who failed to respond to previous treatments with alpha or beta epoetin. further assessment of the optimal dosage, administration schedule of these drugs, and validation of their likely impact on qol are required, in order to epo and its derivatives to gain acceptance in mds, due the high history and a medical examination. this procedure often lays the basis for a correct diagnosis. the current gold standard to detect iron deficiency remains the serum ferritin value. to be reliable, this requires the ruling out of an infection (chronic or acute) as a cause of the anaemia. we recommend a complete laboratory test for the exact haematological status as well as the assessment of specific chemical laboratory parameters. these should the hb level alone is insufficient to guide management. a complete work-up (ferritin, transferrin saturation) is essential, preferably with haematological indices such as hypochromic and microcytic red cells and reticulocytes, classified by degree of maturity, in particular, before parenteral therapy is given. since ferritin acts as both an iron-storage and acute-phase protein, it cannot be used to evaluate iron status in the presence of inflammation. a high ferritin level thus requires the presence of an inflammatory process to be eliminated before it can be taken at face value. if the c-reactive protein level is also raised, the soluble tfr concentration can be used, since it is unaffected by inflammation. inadequate understanding of the complex chemistry of parenteral iron administration was previously responsible for serious side effects, such as toxic and allergic reactions, and even anaphylactic shock, in particular with dextran preparations. however, the current type ii iron complexes that release iron to the endogenous iron-binding proteins with a half-life of about 6 h are not only effective but carry a minimal risk of allergic accident and overload, especially after a comprehensive pretreatment work-up. after correct diagnosis, major emphasis should be put on safe and effective treatment of anemia which again depends on severity of anemia, time for restoration and patients characteristics. today effective alternatives to oral iron only or blood transfusion such as parenteral iron sucrose complex and in selected cases also recombinant erythropoietin have been investigated and show promising results concerning effective treatment of anemia during pregnancy and postpartum. our departmental data collected over 10 years and backed by postmarketing experience in 25 countries indicate that iron sucrose complex therapy is a valid first-line option for the safe and rapid reversal of iron-deficiency anemia. w-pa-128 iron therapy in orthopaedic surgery surgery of the vertebral column, hip or knee is considered a bloody procedure (blood loss >1 l) and as a consequence represents the main indication for red blood cell transfusion in orthopaedics. because of the non-negligible residual risk of transmission of infectious agents by transfusion, but mainly because of immunologic complications induced by the administration of foreign proteins and cells, an alternative solution has been actively sought. studies have clearly shown that in patients undergoing such surgery, transfusion risk correlates inversely with pre-operative hemoglobin level. correction of even slight preoperative anemia is thus mandatory. in the elderly, iron and vitamin deficiency (b12 and/or folic acid) should be looked for as a matter of routine. we recommend the use of iron + epo whenever a rapid correction (<4 weeks) of the anemia is desirable in cases with transferrin saturation <30% and ferritin levels <200 mg/l. with this regime it is possible to collect up to 6 autologous blood units in cases of increased perioperative blood loss (e.g. double hip replacement). in the post-operative period, anemia worsens because of the existing inflammatory state. this inhibits iron absorption from the intestine and iron release from the macrophages while it affects epo function and production. there is increasing evidence that i.v. iron combined with epo induces a rapid correction of post-operative anemia. it is thus recommended to stimulate erythropoiesis by i.v. iron and epo starting on the first post-operative day and to avoid transfusions in asymptomatic patients even in cases with hb as low as 70 g/l. background: hereditary hemochromatosis is one of the most common inherited disorders in which an excessive amount of iron is absorbed from the diet and then deposited in organs. the effective treatment is the regular whole blood removal which causes erythropoesis activation and leads to decrease of iron stores. red cell apheresis is an optional method for removing of higher amount of erytrocytes in one session. we performed 262 red cell apheresis in 15 patients with diagnosis of hereditary hemochromatosis (14 ¥ c282y homozygotes, 1 ¥ c282y + h63d heterozygote) using haemonetics mcs 3p cell separator (protocol tae) in which red cells are removed from patients in 2-5 cycles; plasma and buffy-coat are reinfused. collection time, donor convenience, side effects and red cell yield were recorded and analysed. samples for hematology and iron studies in patients were drawn, analyzed and compared to baseline levels. background: the collection of 2 units of red blood cells by apheresis (drbc) has been reported to be safe and effective in increasing the yield of rbc units from a donor population. however several reports demonstrated the risk of inducing iron depletion when the interval between a drbc donation and a subsequent rbc donation is shorter than 180 days. aims: to evaluate the recovery from anaemisation and iron stores depletion after drbc donation. methods: donors who underwent drbc donation between december 01, 2002 and february 28, 2003 have been enrolled in a follow up program to monitor haemoglobin (hb), htc, serum iron and ferritin values. these parameters have been assessed on the day of donation and, thereafter 7 and 180 days after drbc procedure. donors suitable to drbc apheresis had to have: age between 18 and 60 years, weight > 70 kg, hb > 15.5 g/dl and serum ferritin between 50 and 400 ng/ml. a written informed consent about the collection procedure and the follow-up program has been obtained from all the enrolled donors. drbc collection procedures have been performed by using a mcs + (haemonetics) cell separators. results: out of 130 donors who donated drbc during the study period, only 14 males completed the follow up program and have been analysed. baseline haematological values and iron metabolism parameters were: mean hb 16.3 ± 0.5 g/dl, ferritin 85 ± 31 ng/ml, serum iron 121 ± 42 microg/dl. on day 7 mean hb was 14.3 ± 0.5 g/dl (p < 0.001). on day 180 mean hb was 15.3 ± 0.8 g/dl (p < 0.001), ferritin 53 ± 22 ng/ml (p < 0.05), serum iron 111 ± 27 (p ns). only 6 out of 14 donors (43%) had a ferritin value > 50 ng/ml. in the studied donors the collection of 2 units of rbcs induced an expected reduction of about 2 grams of hb, however only 50% of this reduction was recovered after 180 days (p < 0.001). similarly, also iron stores have not been restored after 6 months from donation, as shown by a 38% reduction in mean serum ferritin value. according to these data it appear that the amount of iron 'lost' with the donation of 2 units of rbcs (approximately 320-420 mg of elemental iron) could not be completely compensated by iron absorption from the diet intake. further data are necessary to define the risk of iron depletion after the donation of a drbc, however, at least in areas where iron intake by diet is not very high, the opportunity to prolong the interval between a drbc and a subsequent rbcs donation beyond six months or to provide adequate iron supplementation therapy should be carefully considered. background: increased transferrin saturation and/or serum ferritin have been observed in italy in approximatively 5% of subjects at first blood donation and, in these subjects, hfe mutations prevalence was 0.11 for c282y and 0.26 for h63d (velati et al., 2003) . aims: the role of the c282y mutation is well known in the patho-genesis of iron overload, whereas the role of the h63d mutation remains uncertain. the aims of the present study were first to study the main hfe mutations prevalence in a random group of repeat blood donors and second to evaluate iron parameters and iron depletion in repeat blood donors heterozygous for the h63d mutation in comparison to a population of blood donors wt/wt for the h63d mutation. methods: a total of 1165 repeat blood donors were examined in 10 italian transfusion centers (6 in northern italy and 4 in southern) for c282y and h63d mutations. out of those, 50 blood donors heterozygous for the h63d mutation and 31 wt/wt for the same hfe mutation, both groups wt/wt for the c282y, were enrolled to evaluate iron parameters and iron depletion. these two groups were similar for number of blood donations (expressed as iron loss) and for sex distribution. serum ferritin (sf) was the iron index recorded at first and second observation. results: table 1 summarizes the allelic frequencies in the 900 blood donors. table 2 reports the haematological evaluation in the 50 subjects heterozigous for h63d mutation and the 31 wt/wt for the same mutation. conclusions: these data suggest that subjects with h63d mutation of the hfe gene have, at first observation, a higher ferritin levels than subjects wt/wt. this seems to be more evident in blood donors of southern italy than in northern. blood donation induces significant reduction of the iron stores both in h63d heterozygous and in wt/wt subjects. although our observation is preliminary and restricted to a limited number of subjects, it seems worthwhile to extend the follow-up of blood donors h63d heteroxygotes or even homozygotes when available, in order to get further insights on the h63d role in iron metabolism. background: cd 43 is a sialylated glycoprotein expressed on the surface of most hematopoietic cells and has been implicated in cell adhesion and signaling. consequently the levels of soluble cd43 as well as the expression on the cell surface is a marker of cell activation. furthermore, downregulation of this molecule has been correlated with increased susceptibility to infections. the myelodysplastic syndromes (mds) are a group of stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenias and an increased risk of leukemic transformation. the mds patients are often introduced to transfusions for anemia improvement and present increased susceptibility to infections. aims: we studied cd43 expression in transfusion-dependent and non-transfused mds patient in an effort to investigate mechanisms of regulation of this molecule. we also studied other activationassociated antigens in the absence of manifest infection. material and methods: forty-two patients were included in the study suffering from refractory anaemia (ra). thirty-one were males and 11 females aged 33 to 85 (median 75). twenty of them had never been transfused (group a) and 22 were regularly transfused (group b). nineteen age matched healthy individuals were used as controls (group c). cell surface antigens were detected by direct immuno-fluorescence evaluated by flow cytometer. the following mouse monoclonal antibodies were tested: anti-cd11b, anti-cd18, anti-cd43, and anti-cd45. leukocytes were gated according to cd45. we used a sensitive sandwich enzyme linked immunoassay to measure the level of soluble vascular adhesion molecule as an indicator of endothelial cell activation. the r&d elisa kit was used according to the manufacturer's instructions. results: the cd43 was found down-regulated in the transfusiondependent mds patients compared with the non-transfused ones (p < 0.001) and controls (p = 0.012). this downregulation concerned the proportion of cd43 + cells, that was lower in the transfused patients than the non-transfused (p < 0.01) and controls (p = 0.006), and the rfi (relative fluorescence intensity) value that was also lower in the group a compared to the group b (p < 0.01) and group c (p = 0.001). negative correlation was observed between the cd43 expression and cd11b (p = 0.001) and cd18 (p = 0.001). cd11b was found up-regulated in the transfused patients. the rfi value was significantly elevated in the transfused patient compared with the non-transfused and controls (p = 0.001 and 0.001 respectively) while the percentage of cd11b cells did not differ significantly between the various groups. increased expression of cd 18 was also found in the group a compared to group b (p < 0.01) and c (p = 0.008). the proportion of cd18 + cells did not differ between the various groups. the levels of immuno-reactive svcam-1 as determined by elisa were found 512.02 + 35.56 in group a, 3.42 + 65 in group b and 194.11 + 45.48 in the control group. conclusions: activated hemopoietic and endothelial cells are found in mds that may be associated to the vascular disorders found in these patients. cd43 downregulation may also be associated to increased susceptibility to infections in these patients. despite improved safety of the blood supply, allogeneic blood transfusion continues to be associated with risks that can be eliminated or reduced by autologous transfusion. preoperative autologous blood donation (pad) prevents transfusion-transmitted viral infection, red cell alloimmunization, and some adverse transfusions reactions. it may decrease the risk of postoperative wound infection because immunosuppression as a result of allogeneic blood transfusion is avoided. pad also supplements the blood supply, provides compatible blood for patients with alloantibodies and rare red cell phenotypes, accelerates erythropoiesis, and provides peace of mind to patients. as any medical intervention, pad has both advantages and disadvantages. with proper patient selection and dedicated attention to process control and quality assurance, the advantages outweigh. background: prestorage pooling of whole blood derived (wbd-pc's) buffy coat platelet concentrates (pc) is common practice in europe event-free survival was significantly better in patients who responded to epo + g-csf. we have reviewed data in 2 centers and the gfm has the intention to extend the study to a larger population in at least 10 centers in france blood components and preparations. the new law prohibits the mixing of different blood products, i.e. blood components and blood fractions. different methods are necessary for the quality control of blood components and blood preparations privileges for blood donors include: -a paid day off work on the day of blood donation and medical examination for blood donation additional paid day off work after blood donation an extra paid day off work if blood is given during vacation or on a holiday this award will be given to non-remunerated donors after 40 blood donations or 60 plasma donations. before 2005, each 'honoured donor of russia' or 'honoured donor of the ussr' had three privileges: free use of public transportation, receipt of certain pharmaceuticals free of charge, and a discount on apartment utilities previously, municipalities also could have their own blood establishments. this resulted in more than 1500 blood establishments in the russian federation. from both administrative and financial points of view, many of these are too small to be costeffective, and should be discontinued. services, and wider implementation of modern technology for blood collection, testing, processing, storage, and distribution acknowledgements: we thank ksw microtec ag, dresden/ germany for sponsoring the rfid-labels and novatech research gmbh, vienna/austria for developing the clinic-software. background: the national preparation human immunoglobulin g 5% for intravenous use (ivig) that is produced at the serbian institute for blood transfusion is used in therapy of neurological, heartand haemolytic diseases and on patients that have undergone surgery. aims: it is our aim to prove the impact of this national medical preparation human immunoglobulin g 5% for intravenous use on patients that have been infected with sepsis as a consequence of surgery. material and methods: human immunoglobulin g 5% for intravenous use (ivig) has been used in the study. the preparation is liquid, 5% stabilised with glucose of a ph value of 4.25 ± 0.25. it is used in those cases where sepsis developed after surgery. both an ivig group (n = 40) and a control group (n = 40) were viewed; the control group not being treated with ivig. the number of specimens with the ivig therapy cholecystitis is (n = 3), and the control group (n = 2); pancreatitis (n = 8) control group (n = 7); intestinal obstruction (n = 7) control group (n = 8); abdominal organ perforation (n = 5) control group (n = 8); abdominal perforate injuries (n = 12) control group (n = 13); serious abdominal interventions (n = 5) control group (n = 6). the period of hospitalisation of the patients in the ivig group was 25 ± 9 days while the period of hospitalisation in the control group was 28 ± 12 days. the mortality rate in the ivig group was 40% counter 63.7% in the control group. summary: toxic gram -negative bacteria caused synergistic damage of human tissues and generalized inflammatory responsesepsis. by using human immunoglobulin g 5% for intravenous use, in cases of severe disease, the mortality rate is significantly lowered, depending of course on the anamnesis of the patient prior to surgery and the presence of other diseases such as diabetes mellitus, neoplasma, cardiac diseases etc. background: manual production pc from buffy coats (bc) is a procedure with some consecutive manipulations. the orbisac system (gambro bct) automates the steps and we assessed its performance. material and methods: 160 pc were produced by this device and some parameters were studied. for the preparation of pc, 5 bc were pooled using the orbisac set, with an integrated filter (pall lrp6). bc pool was resuspended in the additive solution t-sol in order to obtain a final ratio plasma/t-sol 35/65. the pc was stored in a gambro elp bag. results: the average platelet count per unit was 3.41 ¥ 10e11. the platelet recovery from pooled bc was 85.12% (range 79.75%-93.21%). all products of the tested pc containing <1 ¥ 10e6 wbc (by flow cytometry). the values of ph on day 1 and 5 of storage were 7.45 and 7.43. the swirling phenomenon was good until day 5°. the average loss of haemoglobin per bc was 6.8 g.conclusions:the orbisac system is very suitable for routine pc preparation and it allows increased productivity and better standardization method for pc preparation. platelet concentrates met the requirements for leucodepleted product. increased production of plasma components from male donors background: we routinely separate whole blood (wb) after hard centrifugation into a red cell concentrate (rcc), a buffy coat (bc) and plasma (pl) by an automated expresser (compomat, fresenius). the bcs are subsequently processed into platelet concentrates (pcs) by soft centrifugation and an additional (manual) expression step. the atreus 3c system (gambro bct) eliminates several of those hand-on steps by combining them into one integrated process. a processing 'circular' bag is placed in the device and filled with the wb. while the bag is centrifuged, the system expresses pl, pc and rcc into separate containers. the rccs are subsequently leukoreduced (manually) with a filter (lr-rccs). this study was designed to evaluate the storage characteristics of the rccs obtained with a prototype of the atreus system in comparison to rccs obtained by routine procedure. methods: whole blood (500ml) was collected in top-and-bottom bags, and randomly selected to be processed by either (1) current routine or (2) atreus 3c. rccs were leukoreduced with the integrated inline filter: fresenius (routine group) or pall rc2d (atreus). lr-rccs were stored at 4°c and sampled until day 42. various in vitro measurements were performed (n = 20 per group).results: see table (mean ± sd). the lr-rccs contained significantly more leukocytes in the atreus group. despite the rbc loss in the bc, hemoglobin (hb) content was 6% lower in the atreus group, but met the requirements. in vitro storage characteristics for the rccs were similar in both groups. the atreus pcs contained 73 ± 28 ¥ 10 9 platelets in 78 ± 6 ml. although plasma volume was higher in the routine group, subsequent preparation of pcs would have resulted in an additional loss of 50 ml per unit in the control group. atreus plasma had extremely low levels of residual wbc and rbc. 0.02 ± 0.02 4.0 ± 4.2 <30 <0.001 aims: the aim of this study was to examine platelet quality of prestorage pooled prp-derived pc's for up to 7 days storage. methods: pc's were manufactured from wbd-pc's using in-line filtration of prp on day 0. on day 1, either 4, 5, 6, or 8 pc's were pooled into an elx® container using a sterile connecting device. studies were performed on days 1, 5 and 7 for the following measure of platelet quality. ph, morphology score (ms), extent of shape change (esc), hypotonic shock response (hsr), percent in surface expression of p-selectin (p-sel), phosphatidyl serine (ps), glycoprotein 1b (gp1b) and by thromboelastography of the prp (maximum aplitude, ma). results: a total of 20 pools were studied, 5 each of 4, 5, 6 and 8 pc's. the mean platelet yield was 4.3 ¥ 10e11 with a range of 2.4-7.0 ¥ 10e11. the five 8 pc's had a mean yield of 6.0 ¥ 10e11 and all maintained a ph > 7.0 on day 7. all products had less than 1 ¥ 10e6 residual wbc. platelet quality data is presented in the table. data are the mean ± 1 sd, n = 20. conclusion: platelet pools manufactured from pc's produced by inline filtered prp and stored in elx® containers show good quality preservation to day 7 over a range of platelet yields. introduction: the big progress in treatment of critically ill children significantly increases the need for blood and blood products. loss of blood (lowering of the total erythrocyte mass), as well as decreasing of oxygen capacity of blood that can influence cardiovascular function, is main indication for the erythrocyte transfusion. aim of the study: to present the number of erythrocyte concentrates (ek) that were issued to the pediatric clinic in skopje, as well as to point out how they were distributed. material and method: this is a retrospective study performed in nitm-skopje from january till may 2004. the following criteria were followed: hemoglobin (hb), hematocrit (htc), as well the clinical evaluation, and then final decision for transfusion was made.results: there were 254 blood units (ek) issued for the mentioned period to pediatric clinic for 73 pediatric patients (~3, 5% units/per child). the biggest consumers are children at intensive care unit and at the hematology-oncology unit. one unit of leukodepleted erythrocytes (er) was split equally to 3-5 bags. for small and prematurely born children and for some other selected patients er unit was filtered and irradiated. the dosage was 25-100 ml er (depends on age and body weigh). 173 ek was issued as washed concentrates, 15 ek were filtered and 25 ek were resuspended in ab plasma. distribution among abo system was the following: conclusion: gynecologic patients consumed rbc more than 4 times than obstetric ones (159 vs 44) and the number of given transfusions is high. the a blood group is the most needed one. we should insist on using the who guidelines for the proper clinical use of blood and try to minimize the percentage of given transfusion. and z. cermakova university hospital, ostrava, czech republic background: fully automated system olympus pk 80 is an immunohaematological analyser for detection of red blood cells antigens of ab0, rh (d, c, c, e, e) and kell systems without centrifugation by mam (microplate agglutination method) on unique terraced microplate olympus. in the czech republic analyser pk 80 is used only in blood center ostrava. aim: to evaluate the validity of results, sensitivity of microplate agglutinaton method, cause of abortive tests, requirements for analyst, capacity and reliability. methods: 95 880 blood samples of donors were tested between july 2002 and january 2005. all samples were analysed for ab0 blood group. 69 052 samples were tested for rhd antigen and 15 220 ones for rh (c, c, e, e) and k antigen. the validity of the results was evaluated for ab0 with parallel testing antigens and antibodies, while for rh (d, c, c, e, e) and k using two diagnostic serums. sensitivity of mam i.e. occurrence false negative or positive results were found out when results were confronted with previous ones in our data bank acquired testing classical manual tube or microplate methods. requirements for analyst were evaluated in according to demands for needful knowledge for new analyst, necessity of control pk during testing and maintenance. capacity were evaluated as a number of samples tested per day. reliability determine by occurrence disorders. results: ab0, rh (d, c, c, e, e) and k were investigated truly by first testing at 99.5% samples. two diagnostic serums anti-d olymp igm and totem differentiate directly rhd negative and rhd positive donors. false negative or positive results were not founded out due to mam or quality of diagnostic serums. about 0.5% samples with abortive tests were analysed next time the same testing or manual technique. causes of abortive tests were microagglutination several samples except for anticoagulative edta, weak solution of red blood cells prepared by analyser, damage of microplate, hemolysis due to impurity of microplate. in one case analyser evaluated false ab blood group due to hemolysis. analyser has friendly software, simple maintenance and sound control during testing, capacity about 400 samples per day and minimal occurrence of weighty disorders. conclusion: analyser olympus pk 80 is an effective alternative full automation for medium serological laboratory and together with mam easy and truly proves blood groups of majority samples with minimal necessity repetition due to abortive tests. introduction and aim of the study: the purpose of this study is to establish nested-pcr for the detection of hepatitis b virus (hbv) in blood and blood products. methods: the primer pair set was designed to amplify 513 bp in sregion of hbv genome in the first pcr and 233 bp of first pcr amplicon with rubisco (internal control) in the second pcr. to assess the specificity of pcr results, all the samples were tested cross-reactivity or interference in the assay. results: in case of hbv spiked blood products such as immunoglobulin and coagulation factors, this method could detect hbv dna up to 62.5 iu/ml. nested-pcr was compared with pcr-elisa and hybrid capture ii (hc-ii), the pcr-elisa showed a sensitivity of 96% (hc-ii; 77%) and a specificity of 98% (hc-ii; 100%) (p < 0.005). the results of the study show that nested-pcr and pcr-elisa could be used equally in the management for hbv detection in blood and blood products. p-398 blood component therapy: slow improvement a mrdja health center subotica, subotica, serbia background: transfusion department at general hospital was founded in 1953. since that time till now it has answered to all demands in blood and blood components. aim: the aim is to present development of the transfusiology department in the last 20 years, so that we could see how much of scientific knowledge we have adopted and in which direction our department goes at the moment. method: retrospective analysis of blood/component utilization in period from 1. january 1984 to 31. december 2004. results: in 1984 whole blood participated in the consumption with 84.18%, packed red blood cells with (rbc) only 5.94%, washed rbc were used in 9.87% of the cases. in 2004 whole blood participated in the consumption with 31.5%, packed rbc with 61.65%, washed rbc with 0.14% and rbc in additive solution with 6.71%. as far as plasma preparations are concerned, there has been, since 1984, a great consumption of plasma -witch was separated from whole blood in period up to five day in 97.1% cases, and small consumption of fresh frozen plasma (ffp) only 2.9%. since 2003, there has completely been cancelled the production of five day old plasma, only ffp is being used. from 1984 to 2002 for the patients who needed platelets, platelet rich plasma (prp) was prepared and applied right after preparation. the consumption rate was from 35 units to 103 units per year. in 2002, after the purchase of platelet shaker, began the production of platelet concentrates (pc) and consumption suddenly rose from 58 units in 2002 to 462 units in 2004. conclusions: it is obvious from the analysis that irrational consumption of whole blood was reduced to more acceptable values and therefore the use of component therapy got increased. variation in blood consumption and its slight increase is obvious though application red blood cells was conducted according to strict indications. in 2003 the production of plasma old up to five days was cancelled and instead we produced only ffp. pc we prepared for patients only in agreement of treating physician. although very slow progress in development of transfusion therapy in our department can be seen in accepting scientific knowledge. transfusion specialist are active participants in patient treatment and by accepting scientific achievement are able to set standards and help our colleagues, clinics, in successful hemotherapy. introduction: blood groups may act as receptors of parasites, bacteria and viruses. there is evidence that they perform a function and play a biological role. objective: the aim was to detect abo and p epithopes in ascaris lumbricoides extracts (ae) and to study the presence of immune antibodies in patients with ascariasis. materials and methods: 50 ae were prepared by refrigerated mechanical rupture of adult specimens. agglutination inhibition (ai) and haemogglutination kinetics (hk) tests were made with the ae. the patients´ abo and p blood groups were determined. we 1998 1999 2000 2001 2002 2003 2004 total febrile non haemolitic 2 3 2 3 2 4 2 5 2 4 2 4 2 9 4 34 16 male transfusion reaction 2 1 1 2 3 2 2 5 1 8 f e m a l e alloimunisation on le/hla 3 1 2 2 1 3 1 3 3 4 1 5 2 25 6 male antigens 2 2 1 2 3 3 3 3 1 9 f e m a l e kandidates of renal 4 3 4 4 5 4 6 4 6 5 10 8 12 8 10 7 57 43 male transplantation 1 1 2 1 2 4 3 14 female lupus nephritis 3 3 2 3 2 1 1 1 1 6 male 3 3 2 3 2 1 1 1 1 6 f e m a l e total 12 12 12 16 16 18 21 25 132 65 female 67 male introduction: irradiation of blood product has been in routine use to prevent graft-versus-host disease (gvhd) in certain recipients for many yeas. gamma irradiation can abrogate the ability of lymphocytes to proliferate in vitro, 1500 cgy of gamma radiation reduce lymphocyte response to mitogens by 90%.the aim of the study: 1. to estimate potassium level increment in stored irradiation blood units. 2. to compare the increment in potassium level between leucodepleted and non leucodepleted, irradiated stored blood units. 3. to evaluate the expiratory date of blood units post irradiation. the study included 40 units of blood collected in cpd-adsol (as-1). in twenty units the blood collection bag was with inline leucodepletion, while the other 20 units were non leucodepleted. all the 40 units were irradiated using caesium 137 as a source of irradiation, with a dose of 1500-2500 cgy. baseline samples from the bags were obtained for measuring of extra cellular potassium (k+). control samples included. results: there is statistically significant increment in potassium level in the irradiated samples compared to the non irradiated samples starting from 1st day post irradiation and continues to day 35 post irradiation. comparing the group of irradiated leucodepleted, with irradiated non leuconondepleted, for potassium level estimation during the days of storage post irradiation. there is no statistically significant difference between the two groups during all the days of storage, starting from base line samples and other samples post irradiation until day 35, p value of more than 0.05. 1. gamma irradiation of bloods units can cause cell damage that the use of such components needs to be modified. 2. there is a significant increment in the extra cellular potassium level in irradiated blood units that shows doubling value within 48 h post irradiation. 3. there is no significant difference in extra cellular potassium level increment post irradiation when prestorage leucodepleted units are compared with non leucodepleted units. 4. an out date of 28 days post collection (unless they expired before) for irradiated red blood units seems reasonable to ensure transfusion of irradiated units without serious complications, except in neonates and massive transfusion cases where irradiated blood units should be fresh and used within 48-72 h post irradiation. 5. the percentage of irradiated blood units requested by our physicians (0.09%) is very less that reflects the needs of physicians awareness of the indications for requesting irradiated components that can prevent serious post transfusion complications. the use of whole blood and blood components in treatment of surgical patients in ten years period was analyzed in iran. in accordance with world trend of using blood component therapy, in medical centers throughout the country in ten years period, there are decreasing trend of using whole blood from 26% (1994) to 9.37% (2003) and increasing trend of using packed red cells component therapy from 70.3% (1994) to 88. 54% (2003) . there is also increasing trend of using fresh frozen plasma (ffp) from 26.4% (1994) to 55. 2% (2003) . comparing 1994 and 2003 year, in use of blood therapy related to hospitalized patients at surgical department who received blood and patients who did not received blood; it appears that there is statistically significant difference between these two years. results: during 1 year period, a total of 5310 units of blood and 867 units of f.f.p were used. more specifically, the results can be shown in the following table 1 . a high rate of f.f.p usage is observed both in surgery and pathology clinics. the main causes of its usage are: haemodynamic disorders -volume depletion, and coagulation disorders and low blood protein, for the two clinics respectively. conclusion: the only way for rational usage of f.f.p is the regular reminding of plasma transfusion indications to the clinical doctors, so that undesirable side-effects caused by plasma transfusion will be reduced and the percentage of plasma used for fractionation will increase. acquired factor v inhibitor is extremely rare and is associated with diverse clinical symptomatology that varies from asymptomatic forms of the disease to very severe hemorrhagic episodes with a potentially lethal outcome. it may occur spontaneously or as a result of various clinical conditions. a 50-year-old man was admitted to our hospital with a diagnosis of left-sided periscrotal abscess and scheduled for an incision procedure. during the routine preoperative procedure screening coagulation tests showed pathologic values: aptt 41s, pt 35%, fibrinogen 2.8 g/l, fv 15% (other factors were in normal range), platelet count 189 ¥ 10 9 /l. factor v inhibitor was detected by a modified bethesda assay. the assay showed a low level of inhibitor of about 1.30 bethesda units (bu). the patient's medical history showed no major morbidity except appendectomy performed 22 years ago. the patient was prepared for operative procedure, with preventive preoperative administration of fresh frozen plasma (ffp) in a dose of 15 mg/kg (~1500 ml). upon ffp transfusion, repeated determination of the factor v plasma was unchanged from the initial finding (15%), indicating a failure of therapeutic response. as the measured level of factor v activity was at the borderline hemostatic level, and the operative procedure was not associated with a high risk of hemorrhage, the patient underwent abscess incision. the procedure and postoperative course were uneventful and without major hemorrhage. laboratory testing for the possible systemic autoimmune disorder produced normal findings. control examination performed two years later revealed no major clinical or laboratory variation, while a low factor v level persisted (17%) along with the presence of factor v inhibitor at a level of 1.15 bu. we have evaluated two groups of rcc's, one we routinely use (quadruple leucoflex lcr5 t/t cpd/sagm) (macopharma) and one using an automated collection device which gives the ability to collect whole blood in cpd with a rate of 7 : 1 respectively during the whole donation. the mentioned system has been evaluated using the suitable, quadruple leu-coflex lcr5 t/t cpd/sagm (macopharma). whole blood 450 ml in cpd was collected from random donors. in both groups the whole system was stored at scaled r.t. 20 ± 2°c for 2 to 5 h. after component separation (beckman coulter j6mi-optipress i-baxter), the red cell concentrates were filtered immediately at r.t. 20 ± 2°c. sampling was done after filtration and wbc measurements were determinated using nageotte champer (bright line-detection limit 0.05 wbc/ml) with leucoplate solution (sobioda). the other parameters were measured with (celldyne 1700 abbott). conclusion: all products met the accordance of national and european norms for blood components quality. leucodepletion with leucoflex lcr5 and abc leucoflex lcr5 (5.040 and 5.015) is highly efficient. the use of abc leucoflex lcr5 showed better scaled donation in terms of collection (statistical analysis mann-whitney, minitab p-value = 0.0392 < 0.05). additionally less hb-loss occurred, due to the filtration process, most probably due to the total absence of clots (analysis man-whitney, minitab, p-value = 0.0382 < 0.05). this new generation of collection gives the ability in blood services to collect well calibrated donations, indoors or outdoors. smaller quantities of donations theoretically can be valid because of the stable 7 : 1, rate of donation. the abc system gives the ability of fully traceability during donation. macopharma's blood collection bags, with or without integrated filters, provided they have this modified system of storing the ac. the device can keep records for many parameters and can transfer them to the data base server of the blood center. to evaluate the performance of the abc, we conducted a comparative study between abc leucoflex lst1 system and leucoflex lst1 system we currently use. the later is a well known macopharma's system for collection of whole blood with integrated whole blood filter and the final production of one unit of leucodepleted crcs and one unit of leucodepleted plasma. the abc was handled with the same system modified with the storage bag for the ac. issues of comparison were the accuracy in donation volume, the duration of filtration, the loss of blood in the filter and the residual wb cells after filtration. we performed 24 donations with the lst1 system and 28 donations with the abc lst1 system. all the donors were random male volunteers and they were meant to donate 450 mls of whole blood. our results analysed by mann-whitney, minitab statistical analysis have as follows:1. no significant difference was found between the two systems concerning the whole blood volume, but there was broader distribution of the values in the lst1 system compared to the abc lst1 system. 2. the duration of filtration has been found without statistically significant differences between the two systems. 3. the loss of volume in the filter of lst1 is higher than in the abc lst1 (p = 0.007 < 0.05, which is statistically significant). 4. there was very good leucodepletion with the lst1 systems (median reduction of the wb cells 4.7 log) but there was a superiority of the abc lst1 over the lst1 concerning the leucodepletion per litter and per unit (p: 0.0368 and p: 0.0398 respectively). 5. the personnel after a very short period of training accepted fully the abc procedure.in conclusion abc is an easy to handle device which provides with high quality blood products in combination with leucoflex lst1. evaluation of a post-storage filter for wbcs with an incorporated waste bag for washing rccs leucolab lcg4 is a system manufactured by macopharma for poststorage filtration of a rcc unit (with or without an incorporated waste bag for further washing of the filtered product). to evaluate the efficiency and reliability of the above system we conducted a study with twenty three random units of rccs stored in cpd/sag-m, aged 2-6 days, filtered and consecutively washed with 200 ml of normal saline, span down in the regular way and the supernatant extracted in the waste bag. issues for evaluation were:1. the duration of priming and filtering the rccs. 2. the loss of volume in the filter.3. the efficiency of the filtration. 4. the acceptance of the personnel of a new (to them) filtration system.our results have as follows:1. we counted the duration of priming and filtration. median time of priming was 90 s (range 40-300) and of filtration was 12 min (range 8-20). 2. the median volume lost in the filter (as calculated) was 23.4 ml (ranging from 22.4 to 28.1). this narrow range is apparently due to the non-flexible cell of the filter. 3. the efficiency of the leucodepletion was counted by flow cytometry. the median wbc counted per lt was 0.9 ¥ 106 (range 0.3-2.1 ¥ 106) and per unit was 0.17 ¥ 106 (range 0.05-0.5 ¥ 106). the median reduction of the wbc count was 4.2 log (range 3.9-4.7). 4. the personnel involved in the procedure found the system easy to handle, even without specific training. in conclusion the lcg4 is a reliable and easy to handle system, for leucodepleting (and washing) rccs, very efficient in removing wbcs with negligible loss of volume. objective: standardization of blood banks and establishing quality assurance are important landmarks in the new era of transfusion medicine. as the number of blood banks grows and the capacities of them changes, centralization need arises and trends to nationally coordinated blood services eventually appear. aim: to investigate the types and capacities of blood bank in the country and evaluate the statistics of them. material and method: blood banks and transfusion society of turkey conducted a nation-wide survey of a comprehensive questionnaire. this is a preliminary report of this investigation and illustrates the capacities of the most blood banks in turkey. it also guides the nationally planned renewing structure of blood banks. results: there are nearly 340 blood banks and blood stations in whole country. the overall blood collected at those centers is about 1.800.000 units. nearly one in third is being collected at government hospitals (31.5%), nearly the same is collected at university hospital blood banks (29.6%). the third major group is the red crescent society blood centers-rcsbc (23.5%), followed by the social security hospitals (12.9%). blood collecting capacities are not appearing in the same order. the major blood banks belong to the rcsbcs, whereas the small ones are mostly government hospitals. the only donor recruitment organization is run by the rcsbcs. yearly blood collecting capacity blood banks (%) >50.000 0.4 20.000-50.000 4.8 10.000-20.000 8.7 5.000-10.000 13.8 <5.000 72.3conclusion: there are many steps for improving blood safety in a country, and the prior ones are structuring the blood transfusion system and donor organization on a national basis, and then establishing good manufacturing practices. these are only possible after centralization of all existing blood banks. in our country, we should first arrange all small capacity blood banks and standardize them. controversial clinical questions considered in a medical opinion forum by physicians for the advancement of transfusion medicine (patm) patm is a newly formed group of close to 300 physicians drawn from pathology and hematology, transfusion services, hospital blood banks and blood centers. its mission is to address the concern that patient oriented medical opinion and influence has been diminished in transfusion medicine (tm). they believe that a patient oriented voice should be distinct from institutional, commercial or regulatory weight and have a common focus on patients and the therapeutics of transfusion and related therapies. therefore, their first objective is to create a new medical, patient oriented voice that weighs in on national policy pertaining to treatments related to tm. as such, patm held its first medical opinion forum where members of the group debated important questions pertaining to tm clinical practice. the forum was held just prior to the aabb annual meeting and attracted 58 physicians. the questions debated were preselected by patm membership via an email survey. respondents were asked to select their top four preferences from among 17 topics in five broad categories. the top two topics were selected for the forum from the 80 completed surveys. the subjects selected and debated were (i) what are the medical considerations for reducing the rate of mistransfusion? and (ii) what are the medical considerations for managing a limited blood inventory? the participants were divided into four groups, with each topic assigned to two groups. all the groups were given two h to debate and arrive at consensus on their topic. each group then presented a summary of their discussions along with specific recommendations for addressing these clinical practice issues. there was remarkable consensus between the groups debating the same issue. the conclusions and recommendations on these two topics will be presented in detail. patm is a new organization that will add an important medical voice and opinion on current topics in tm. at its first meeting, two topics were successfully discussed and debated with broad consensus achieved on current issues confronting the field. key: cord-010092-uftc8inx authors: nan title: abstract of 29th regional congress of the isbt date: 2019-06-07 journal: vox sang doi: 10.1111/vox.12792 sha: doc_id: 10092 cord_uid: uftc8inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner 0 s fate illustrates the brain drain beginning in the early 30 s escalating in 1939 with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early 80 s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in 1995. as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu28. levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of 3% r&d intensity was first met in 2014 and is 2018 the sixth highest among oecd countries and the second highest in the eu28. austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon 2020 and the preceding 7th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd19 targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd19 directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd19car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about 30% of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to 75% of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in 4% of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il-1rap is expressed by the leukemic but not by the normal cd34 + /cd38-hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il-1rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il-1rap mab (#a3c3 clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with 3rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp9 (inducible caspase 9) and a monitoring/selection cell surface marker δcd19. we demonstrated in-vitro and in an in-vivo xenograft murine model that il-1rap car t cells can be activated in the presence of il-1rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a 4-year period does not affect transduction efficiency of cml patient t-cells by il-1rap car vector and that autologous cart-cells are able to target il-1rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il-1rap expression on a tissue macroarray comprising 30 normal human tissues (3 donors) , with #a3c3, detected various il-1rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a3c3 flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il-1rap. as expected, monocyte subpopulation is targeted by autologous il-1rap cart cells (ratio e: t = 1:1), but at a lower level that il-1rap cml cell line. in-vivo investigation of specific toxicities of autologous il-1rap cart-cells against hsc and/or immune cells on a human-cd34 + cord blood cell engrafted/nog murine model, but also by an in-vitro cd34 + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd34 + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp9/ rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than 90% of cart-cells, after exposure to ap1903. in conclusion, based on cml model, we demonstrated that il-1rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from 20 to 30% for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about 20% when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of 5% for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of 2 h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within 15 min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at 4°c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an 1/1/1 ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the 6th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and 24 h organ failure. this trial will be recruiting 200 patients in 6 french trauma centers and is planned to be initiated second half of 2019. local/neighbours day: innovation in germany 1c-03-01 mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct1: eudract number: 2011-005441-13; ortho-ct2: eudract number: 2012-002010-39; maxillo1: eudract number: 2012-003139-50) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. 2018 introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using 50 ml stirred flasks. cells were differentiated into mks using tpo, scf and il-3 in apel2 medium for a period of 22 days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il-2rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an 8-fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of 18.7 ae 6.8 9 10 7 in microcarrier-assisted bioreactors in comparison to 4.9 ae 1.3 9 10 7 mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il-2rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than 12 years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september 30, 2016 . it has travelled billions of kilometers, just to study a small (4 km diameter), black boulder named 67p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd19 expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. 1d-04-03 cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb2) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from 450-1200 ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between 3-28 days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation (450/470 ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard 450 ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline 28 days after blood donation, but endurance performance was normalized already after 14 days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, 4 -5 weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" (1970) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby 2004) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease 2a-02-01 immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be 29-36% and 53-72%, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be 8.4-14%, 12.5-27.7%, 3.3-3.5%, respectively, and the alloimmunization rates were 17.6%, 14.3-30%, 7.1%, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of 2-6% is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from 7% to 76%, the highest figures being established when an abo/rh1-only matching policy is implemented. in a meta-analysis of 24 publications, the overall alloimmunization rates were around 20%. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a 61% increased risk of producing additional antibodies versus 30% in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to 70% of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; 1) which antibody specificities are clinically relevant 2) which antigens are most immunogenic 3) what is the availability of specific antigen typings in the donor population 4) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over 50 years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to 15-30 min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first 72 h of life, and late thrombocytopenia, occurring after the first 72 h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet-2/matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of 50 9 10 9 /l had a significantly higher rate of death or major bleeding within 28 days after randomization than those who received platelet transfusions at a platelet-count threshold of 25 9 10 9 /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from 1% to 8%, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> 60 years or < 3 years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within 12 h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: 1) using normal pediatric values published in the nelson textbook of pediatrics; and 2) using patients as their own controls and comparing pre-and post-transfusion values with either a 10% or 20% difference threshold. we monitored for taco up to 24 h post-transfusion. a total of 136 patients were included. taco incidence varied from 1.5% to 76%, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of 96 samples. from november 2017 to january 2018, a total of 10,141 blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas 6800 platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than 5.00e+04 iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was 7.05e+03 iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence 1:1,268) , whereas 17 hev rna positive donations were identified by id-nat (incidence 1:597); all id-nat only positive donations had vl < 25 iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately 50 % higher if id-nat was used. however, vl were below 25 iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last 25 years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b 2 ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost 20 years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p1 and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p 1 and p 2 phenotypes. the system (isbt no. 003) currently includes three different antigens, p1, p k and nor. the p1 antigen was discovered already in 1927 by landsteiner and levine while p k and nor were described in 1951 and 1982, respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p1/p k carbohydrate structures. anti-p1 is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p1 have been reported to react at 37°c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p 1 k /p 2 k . the rbc of the fetus as well as of the newborn express low amounts of p1, p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p1. recently it was clarified that the same a4galtencoded galactosyltransferase synthesizes both the p1, p k and nor antigens and in addition the p 1 and p 2 phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p 1 allele in the 5'-regulatory region of a4galt, which enhance transcription of the gene. it has been debated whether the p k and p1 antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p1 antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p1, has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata1 nor klf1 represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf1 gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd44) and p1, among others. since the first description of klf1 variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf1 table of blood group alleles. other than klf1, a mutated gata1 gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata1 allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in 1995, when the disruption of a gata motif in the ackr1 gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata1 binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata1 binding to a control element located 3.7 kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata1 binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p1 antigen has been known for a long time to be determined by the a4galt gene but the molecular basis underlying the common p1/p2 phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon 2a, which showed a very good correlation with p1 antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx1 tf in the expression of p1 antigen, by selective binding to a regulatory site present in p1 but not in p2 alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p1 and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. 2c-06-03 clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of 1) guiding targeted rh prophylaxis in non-immunized rhd negative women and 2) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in 6-7 european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of 99.9%, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified 837 studies, but only 10 was eligible for full text review and 5 met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. 2d-07-02 applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in 1976. during the 1994 genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from 1995, the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs 4, 5 and 6. today, rwanda has an ambitious vision to put all 12 million citizens within 30 minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in 2016, the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in 15 -45 minutes depending on a hospital's location. the average duration was between 2 -4 hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to 25 health facilities throughout the country and are set to reach 90% of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of 3.1 hours per delivery and a total of 10,115 hours of lost time on road pick up they could instead dedicate to patient care. by march 2019, over 11,000 deliveries have been made, with 30% of those being emergency deliveries. a total of more than 19,500 blood units have been delivered. in february 2018, zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to 100 km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. 2d-07-03 scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna-1b, fcgriiib and hna-2 have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna-1a, -1b, -1c, -1d, hna-2, hna-3a, -3b, hna-4a, -4b and hna-5a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna-3a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna-1a, -1b, hna-2 and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna-1, -2, -4, -5 and hla class i antibodies are clearly detectable in the gift while hna-3 antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna-3 with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna-3, can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna-2 in the gift because the molecular reason for the hna-2-null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd177*787a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. 2d-08-02 norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa-1, -2, -3, -5 and -15), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa-4, -6 to -11 are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd109 and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the 19th international platelet immunology workshop of isbt (2018) . the investigations also include measurement of the anti-hpa-1a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa-1 typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. 2d-08-03 molecular basis of hna-2 expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen 2 (hna-2) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd177 (also known as nb1). hna-2 is absent on the neutrophil surface of 3-5% of the healthy individuals that divided the population to hna-2 positive and hna-2 null individuals. exposure of hna-2 null individuals to hna-2 positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna-2 and consequently the production of iso-antibodies. the hna-2 iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd177 on a neutrophil surface of hna-2 positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna-2 positive and negative subsets. the cd177 gene contains 9 exons encoding a protein of 437 amino acids. the lack of hna-2 (in hna-2 null individuals) is associated with the presence of a missense mutation, cd177*c.787a>t in exon 7 of cd177 gene inducing a premature stop codon in codon 263. this mutation alone or in combination with cd177*c.997delg has been introduced as the main reason for the absence of cd177 in hna-2 null individuals. a pseudogene (cd177p1) highly homologous to exons 4-9 of cd177 gene is located downstream of the cd177 gene. conversion of exon 7 of cd177p1 into cd177 gene is responsible for the generation of cd177*c.787a>t missense mutation. in addition, the heterozygosity or homozygosity of cd177*c.787a>t is accounted for regulation of hna-2 negative and positive neutrophils subpopulations. genotyping has revealed the hna-2 null individuals, heterozygous for cd177*c.787a>t mutation without cd177*c.997delg, indicating the presence of a complementary mechanism regulating cd177 expression. newly in hna-2 null individuals and individuals with atypical cd177 expression a cd177*1291g>a polymorphism in combination with cd177*787a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd177 expression on the neutrophil surface. this presentation will summarize recent findings on cd177 expression and highlights the potential genotyping methods for genetic assessment cd177 expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, 100 transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding 1) blood product ordered, 2) whether the patient experienced a reaction, and 3) the start and end times of the transfusion. for each of these fields across all 100 sampled notes, the claritynlp tool reproduced these data points with 100 percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than 1 h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and 24-h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid-1980s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:-1,2,3,4, (5),6,7. genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel*02.03/ 02.06 (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 5'and 3'-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than 1000 human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked 30 years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" 3a-s02-03 background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns1 proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at 650 nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of 10 -10 2 tcid 50 /ml for zikv and denv (serotypes 1/2/3/4) after a detection phase of around 5 min. the first results obtained on 16 zikv (+) clinical samples previously tested by commercial real-time pcr (ct < 36, altona) showed an 88 % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than 10 min (sampling + detection) instead of 2 h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during 2016, with up to~2% of blood donors reactive for zikv rna in id-nat testing at the peak in june 2016. aims: perform a serosurvey for anti-zikv igg using six panels of 500 donor specimens each collected in march 2015, at the beginning, peak and end of the 2016 epidemic, and from march 2017 and april 2018. methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns1 antigen from bio-techne to characterize zikv seroprevalence in the 6 9 500 cross-sectional sample sets (anonymized with selected demographic information). results: 500 pr donor samples collected in april 2015 were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative (3 positive, 3 equivocal) despite the high dengue virus seroprevalence (>90%) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected 1-3 months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > 99%, and an area under the curve (auc) of 0.999, demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional 5 sample sets (2500 samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in 6,636 female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, 17b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo-3am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp9 (a selective trpc6 activator). results: sex hormone intake by menstrual status was higher in premenopausal women (25.3%) than in postmenopausal women (7.4%). female hormone intake was significantly (all p < 0.0001) associated with reduced storage hemolysis in all females (0.32 ae 0.16% versus 0.35 ae 0.29% in controls), enhanced susceptibility to oxidative hemolysis (37.9 ae 9.3% versus 35.8 ae 9.9% in controls), and reduced osmotic hemolysis in postmenopausal women (23.1 ae 10.2% versus 26.8 ae 12.0% in controls). in vitro, supraphysiological levels of progesterone (10 or 20 lmol/l), but not 17b-estradiol or testosterone, inhibited spontaneous or hyp9-induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after 30 day cold storage (0.95 ae 0.18% versus 1.85 ae 0.35%, progesterone 10 lmol/l versus solvent control (dimethyl sulfoxide, 0.1%), p < 0.0001). co-incubations (2.5 h, 37°c) of rbcs in the presence of progesterone and a trpc6 activator (hyp9, 25 lmol/l) suggested that progesterone protected against hyp9-induced hemolysis (1.45 ae 0.13% and 1.01 ae 0.09% versus 2.63 ae 0.19%; hyp9 + progesterone at 10 or 20 lmol/l versus hyp9 alone, p < 0.05 by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc6 channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. 3a-s05-01 international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid-1970s, the red cross was active in the national blood programs in approximately 95% of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly 50% of all worldwide donations take place (home to only 17% of the population). who data shows that the median annual blood donation rate in high-income countries is 3.21% of the population compared to 0.46 % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last 30 years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their 2018 global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by 25 % in lebanon; vnrbds by 71% annually in kirgizstan, and from practically zero to 3'588 in south sudan. the importance of rc societies was also underlined in the 2018 published global mapping of gap, which showed that 36 (19%) of them provide level a (full blood service), 75 (39%) are level b (systematic blood donor recruitment) and 47 (25%) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ 132420/d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in 500 pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: (1) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. (2) corresponding educational materials for students and teachers were developed and distributed. (3) blood donation clubs were established in 500 pilot schools. (4) trainings were conducted for 688 personnel of moh and trc on blood donation regarding their responsibilities. (5) cascade trainings were conducted for 3218 personnel of transfusion centers and 4399 school principals in 81 provinces. (6) information seminars were delivered to 251.475 students and 15.739 teachers and family members of students during school campaigns. (7) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). (8) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. (9) media spots were produced and broadcasted 3.851 times in 33 different tv and radio channels. (10) billboard posters and brochures were prepared and distributed to 81 provinces for raising public awareness. (11) advertisements about the project and the importance of vnrd were displayed 386 times on national and local newspapers, 1.117 times on online news, and broadcasted on 6 national tv channels. (12) during the campaigns, 28.310 units of whole blood were collected in pilot schools. (13) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. (14) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to 32.07 % and 35.99 % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from 73.66% to 82.54 in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite 70% of pakistan's population being under 29 years, only 10% of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p0 checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with 3-10 walk-in donors and an average of more than 20 telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june 2019. the statistics generated since january 2019 are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to 31 patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since 01.01.2013, whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from 01.01.2013 until 01.03.2019, an extended determination of rbc antigens was performed in 352 subjects presenting for blood donation. twenty-nine rare donors (8%) were identified and included in the rare donor file: 25 fy(a-b-), 1 lu (a-b-), 1 lu(b-), 1 fy(a-b-) and s-, 1 ccddee (r'r'). overall, these 29 donors provided 105 rbc units (range . to date, all donors are still active and 14 are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately 100. -chf, resulting in a total financial effort of around 35,200.-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: 1.) avoidance of transfusion transmittable infections; 2.) quality of the blood product with a strong focus on component therapy; 3.) prevention of severe transfusion reactions; 4.) avoidance of clerical errors; 3.) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. 1.) sufficient availability of blood and proper utilisation; 2.) avoidance of transfusion transmittable infections; 3.) avoidance of clerical errors; 4.) prevention of severe transfusion reactions; 5.) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at -20°c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from 51 samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with 2654 probes covering exons of 64 genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, 51 samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > 2509, with 94% of target at a quality of q30. optimal sample plexity for a standard run was determined to be 14 samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted 99.5% of snp based red cell genotypes. script predictions were 100% concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna2 genotypes defined by cd177 could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p1pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to 14 samples can be reliably sequenced in a single run. our script correctly predicts over 99% of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type 600,000 uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > 1:20,000 identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = 7,871) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was 99.9% for 95,022 comparisons; 29 of the 89 discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across 48 antigens for which serology was available, genotyping provided a 3.6-fold increase in the number of typing results available per donor (47.9 vs 13.2). furthermore, genotyping provided data on an additional 224 clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from 3,146 patient referrals with > 3 alloantibodies between 2014 and 2018, 772 unique alloantibody profiles were identified. we found that there was a 2.6-fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the 4,721 nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from 293 to 246, representing an additional 164 patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. 3c-s06-04 biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near 1500 wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including 2 transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by 4 dna samples with targeted sequencing on illumina miseq, and specific variants were verified by 40 dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, 1) m, n: insufficient sequencing reads, 2) c, c: identical rhce exon 2 with rhd exon 2 for c allele, 3) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, 13 systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a2, has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. 8:14750, 2017) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine 27 (suppl.2) :42, 2017). here we have used it to analyse and document bel-a2 blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a2 cell-line. methods: bel-a2 cells (day 196) were cultured in expansion medium and genomic dna (gdna) was isolated from 1 9 10 6 cells on day 5. for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for 41 genes encoding all 36 known blood group systems and 12 further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a2 genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c4a/c4b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a2 did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr1, cdan1 and tmx4, these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a2 genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, 2017) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a2 blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from 16 countries were enrolled (67% in north america, 17% in europe, 7% in oceania, 5% in asia, and 4% in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = 360) differed significantly on a regional basis (p = 0.04) and ranged from 12 (8-41) x10 9 cells/l in the middle east to 45 (20-66) x10 9 cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = 0.08), nor did the tpc prior to therapeutic transfusions (n = 189) differ on either a regional (p = 0.16) or national (p = 0.57) basis. for children supported by ecls (n = 90), there were no regional (p = 0.06) or national (p = 0.40) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = 0.02) and national (0.04) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [28 (16-81) x10 9 cells/l]. for children with an underlying oncologic diagnosis (n = 233), no differences were seen in the tpc for prophylactic transfusions (n = 175) on a regional (p = 0.19) or national (p = 0.20) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional (0.86) or national (p = 0.33) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < 0.001) and national basis (p < 0.001). the median (iqr) dose based on volume ranged from 8.4 (5.0-11.2) ml/kg in north america to 12.6 (9.8-16.0) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < 0.001) and national (p < 0.001) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, 2015) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ 10 9 10 11 /l and a platelet dose of 1.1 9 10 11 per square meter (sm) of body-surface area (bsa) in inpatient and 2.2 9 10 11 /sm in outpatient setting. aims: in january 2018 we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of 1.1 9 10 11 /sm and outpatients a dose per transfusion of 2.2 9 10 11 /sm. platelets were transfused when the count was ≤ 10 9 10 11 /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january 2018 to december 2018 a total of 9839 platelet pediatric aliquots were transfused: 5534 (56.2%) were obtained from apheresis platelet concentrates and 4305 (43.8%) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots (7505-76.3%) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, 6365 aliquots were transfused in inpatient setting: 1675 (17%) in the hematology unit, 2772 (28.2%) in the oncology unit and 1918 (19.5%) in hsct unit. a total of 1140 (11.5%) aliquots were transfused in outpatient setting: 840 (8.5%) to patients affected by hematological malignancies and 300 (3%) to patients with solid tumors. five major bleeding events (who grade ≥ 3) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade 3 bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = 3, 1.2%) developed intracranial bleeding (who grade 4). the platelet count at the time of the event was 22 9 10 9 /l, 25 9 10 9 /l and 8 9 10 9 /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade 4 bleeding has been observed in inpatients setting only (1.2% vs 1% of plado trial, sj slichter, nejm, 2010) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ 3) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell 3000 elite (best theratronics, canada) at a dose of 25 gray. all rbcs were used for transfusion for 14 days from the harvest day. 70 pediatric patients with various oncological and hematological diseases were randomized to 2 groups of 35 members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. 3-5 days after the transfusion, the direct antiglobulin test (dat) was performed and after 14-21 days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = 0.2), as well as the concentration of potassium (p = 0.44) and haptoglobin (p = 0.25) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = 1). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = 0.39). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for 14 days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama 2018) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over 5 years duration from 2012 to 2016. methods: using 5 years data (2012) (2013) (2014) (2015) (2016) perioperative transfusions decreased steadily per year from 6.4% in 2012 to 5.5% (14% cumulative decline) in 2016 for children of all ages (or 0.966; 95% ci 0.957-0.976; p trend < 0.001). the cumulative change in elective procedures was 9.2% versus 27.2% decrease in urgent/emergent procedures (p trend < 0.001). summary/conclusions: in this large prospective registry study of > 350,000 children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across 5 years from 2012 through 2016 with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk 3c-s08-01 transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of 2-20% of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ 100 iu/ml and being id-nat (procleix-ultrio plus tm [95% lod: 3 iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < 6 iu/ml (n = 44) or nat repeat reactive (rr) with vl < 6 iu/ml (n = 32). french samples initially tested nat nr/nrr with procleix-ultrio (lod 95%: 11 iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: 6 iu/ml]). hbv dna was purified from 5 to 12 ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in 79% of all samples with undetectable or vl < 6 iu/ml. hbv genotypes were a1 (33.3%), a2 (18.4%), a3 (3.3%), b (3.3%), c (1.7%), d (25%), and e (15%). all samples were anti-hbc positive and 73% of ultrio-negative samples tested positive with ultrio plus. unusual 1-2 nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = 16) or deletions (n = 4) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of 30 samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. 3c-s08-03 background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since 1998, hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of 15 and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i1000 analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas 8800 platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p22cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in 69 blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, 59/69 (85.5%) donors were anti-hbc positive, and 10 (14.5%) negative. 18 donors had both anti-hbc and anti-hbe reactivities. a group of 11 young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and 9 patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g600 automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is 2 logu/ml and the lower limit of quantification (loq) is > 3 logu/ml, due to nonlinearity results between 2 and 3 logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss 19.0.0. results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: 3.0 logu/ml, range 2.0-4.9), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in 34/69 obi donors (49.3%), with a mean value of 2.21 logu/ml (range 2.0-3.20). hbcrag could be measured only in 2 obi donors (3.0 and 3.2 logu/ml), being below the loq of the test in the majority of obi (32/34). considering the presence of anti-hbc, hbcrag was detected in 29/59 (49.1%) anti-hbc+ and in 5/10 (50%) anti-hbc-obi, with no significant difference in their mean levels (2.21 ae 0.32 vs 2.14 ae 0.26; p = 0.44). interestingly, the presence of anti-hbe (18/62) was independently associated with higher hbcrag levels (2.38 ae 0.42 vs 2.14 ae 0.22; p = 0.03). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. 3c-s08-05 hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss 20.0 statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < 0.05 was considered statistically significant. results: 1. proliferation characteristics of t cells. the proliferation of cd4 + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group (3.0%, 3.3% vs. 1.7%), with significant difference (3.0% vs. 1.7%, p = 0.016, 3.3% vs 1.7%, p < 0.001). 2. the frequency of specific ifn-c secreted t cells. the response intensity of the obi group (25 sfc/10 6 pbmcs) and chb group (25 sfc/10 6 pbmcs) was higher than that of the hc group (5 sfc/10 6 pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group (64.0%). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term (15-20 years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under 10 themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = 2.11; 95% ci: 1.03-4.31). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for 72,000 participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = 150,000), were calculated for all dbds participants. 12,903 female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of 10 depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile 1 contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = 1 from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between -0.42 and 0.50 (mean 0.01). a total of 12,523 female donors were classified as "not tired" and 380 (2.9%) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: 0.00; not tired mean prs: 0.01). an age-adjusted logistic regression model found this to be insignificant (or: 0.74, 95% ci: 0.33-1.66), p = 0.47). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile 6) had or = 1.02 (p = 0.93) of being tired when compared to those in quantile 1 (or set as 1). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv-1 subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv-1 infected blood donors. methods: from 2016-2018, 177 blood donations collected from 24 blood centers, covering almost the whole of china, were confirmed as hiv-1 positive by national centers for clinical laboratories using abbott realtime hiv-1 assay or cobas taq-man hiv-1 test, version 2.0. then hiv-1 gag (973 bp, hxb2: 1074-2044), pol genes (1454 bp, hxb2: 2068-3521) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv-1 subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv-1 subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among 177 donations, gag and pr-rt regions of 160 samples were sequenced successfully. the distribution of hiv-1 genotype was as follows: crf_07bc = 66 (38.8%), crf_01ae = 58 (36.3%), b = 9 (5.6%), crf_08bc = 2 (1.3%), crf55_01b = 4 (2.5%), crf59_01b = 1 (0.6%), crf65_cpx = 1 (0.6%), crf67_01b = 2 (1.3%), crf79_0107 = 1 (0.6 %), crf85_bc = 1 (0.6 %), urf_0107 = 6 (3.8%) and urf = 9 (5.6%). 26 of 160 hiv-1 isolates were identified to have drms. there were 4 (15.4%, 4/26) protease inhibitors (pi) accessory drms, 3 pi major drms and 20 (76.9%, 20/26) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf01_ae and crf07_bc (61.5%, 16/26). 3 of 4 pi accessory drms were q58e. the pi major drms included m46l, m46i and n88s. n88s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v179d/e is main nnrti drm (70.0%, 14/20). a combination of v179d and k103r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k103n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf07_bc (38.8%), crf01_ae (36.3%). besides, other rare crfs and several urf_0107 and urfs were also found in these hiv-1 isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in 16.3% donors in the study, which may result in resistance to pis and nnrtis, especially the hiv-1 variants with n88s mutation in pr gene and k103n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv-1 in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv-1 among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in 100% plasma were biotinylated at day 1 and day 7 of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, 50 ml of pcs was washed twice and incubated with 5 mg/l biotin, dissolved in phosphate buffered saline-pas-e (1:9), for 30 min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd62p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in 65% pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, 98.4% ae 0.9% of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd62p expression was increased in biotinylated platelets 48.4% iqr(41.7-56.2%) compared to the control samples 12.3% iqr(9.5-12.7%) on day 1 of storage. however, biotinylated platelets were not more activated compared to sham samples 50% iqr(41.7-56.2%). thus only the procedural steps led to increased cd62p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day 7, a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd62p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd62p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs4 insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs4, these insulators are small-sized (119-284 bp vs 1.2 kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a1, one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il-3-dependent 32d cells, which upon transduction with oncogenic vectors become il-3-independent, leading to transformation. 32d cells were transduced with sin-lvs: the b-globin-τνs9.3.55-, the insulated b-globin-a1-tns9.3.55and the oncogenic sffv-gfp-vector. transduced cells were expanded in 10% il-3 and transduction efficiency was determined by vector copy number (vcn). transduced 32d cells were seeded in methylcellulose with 10% or 0-1% il-3 to detect the il-3-independent and potentially transformed clones. the il-3-independent clones were further expanded in 10% il-3 and infused in partially myeloablated and il-3-treated c3h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a1 insulator did not negatively affect vector titers (τνs9.3.55, a1-tns9.3.55, sffv-gfp: 2.8, 1.8, 2.5x10^8 iu/ml, respectively). 32d cells were successfully transduced with all vectors (%vcn positive colonies: 40-100%) and expanded up to 400-fold. the a1-insulator decreased the number of il-3-independent colonies by 78-93% over the uninsulated vectors. the uninsulated vector-transduced, il-3-independent colonies, were greatly expanded in culture with 10% il-3 over the a1-transduced colonies (sffv, τνs9.3.55, a1-tns9.3.55: 48, 40, 10 fold change, respectively). il-3 independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il-3-independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a1 insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a1 may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including 46 missense variants reported in rhd exons 6 and 7, was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published 3d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c.1065c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c.1154g>t (gly385val; d-negative) disrupts totally normal splicing, while c.1154g>c (gly385ala; weak d) and c.1154g>a (gly385asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a 3d model suggests that gly385asp, but not gly385ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons 6 and 7 by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in 15/46 (32.6%) variants, which correlates well with the quepasa-like prediction (sensibility = 0.93, specificity = 0.94). additionally, while normal exon inclusion is affected by c.1012c>g (weak d type 70), the associated leu338val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in 2014, participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of 6.210 compensated and 68.205 non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either 25 eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after 24, 36, 48 and 72 months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups (51.9%), refreshments (50.0%) and free (testing) laboratory parameters (38.3%) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by 28.6% and 25.3%, respectively. the lowest acceptance was for release from work (17.8%), complementary items (13.9%) and additional cash reimbursement (11.8%). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received 25 eur as a monetary reimbursement, the proportion of regular donors after 48 months (19.9%) was significantly higher than among comparable non-compensated donors (9.2%). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice (1-5 prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand 1) how deferral reasons and donor experience jointly affect donor lapse, and 2) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between 2013 and 2015 (n = 343,564). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included 25% new donors, 41% novice donors, 30% experienced donors, and 4% reactivated donors. deferral reasons included travel, hb, medical short-term (<28 days duration), medical long-term (>28 days duration), and miscellaneous. next, we interviewed 31 temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, 2012) to identify key topics and underlying themes. results: of target donations, 11% were deferred, mostly for travel (40%), medical short-term (23%), and hb (19%). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < 0.75, p's<0.001). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: 8.4 mmol/l (135 g/l) for men and 7.8 mmol/l (125 g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at 8 weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us 35% -75% of deferrals are due to low hb, especially in women (editorial, transfusion, 2012) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from 2007 to 2016. we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of 1000 new entrant donors (500 males and 500 females). prior information from the clinical literature (boulton, vox sanguinis 2004) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of 58 days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of 100 to 419 days, while the estimated recovery time for females varies between 54 to 503 days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of 23% reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, 2019) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (<30 years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: 330 ml water drink, 500 ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within 400 days after the index donation and we analysed their return for a donation attempt within 421 days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the 8199 episode participants who had received an invitation, 6538 (79.7%) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or 1.2, 95% ci 1.06-1.4 and 1.3, 1.12-1.5 respectively). return was slightly lower in women (or 0.88, ) and lower in first-time donors (or 0.86, 0.77-0.96) than after a 2 nd -4 th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or 0.47, 95% ci 0.37-0.60 and or 0.53, 0.46-0.61 respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, 79.7% returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > 50 years contributed 41% of all donations made in 2017. however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's 45 and up study baseline data collected between 2006 and 2009 to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up 22,058 active whole blood donors for 200,403 eligible person-years (average age at recruitment 56.4 years, 56.3% female, average follow up 9.1 years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the 2 years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made 1066 (95% ci 1056-1075), 987 (981-993), 900 (891-909), and 710 (690-730) donations per 1000 person-years, respectively. iron-deficiency was identified in 8.9% of donors in the study (n = 1964, 95% ci 8.5-9.3) . sixty percent of those with iron deficiency (n = 1,175, 95% ci 57.6-62.0) visited their general practitioner (gp) within 60 days of the identification of irondeficiency, and 48.4% (95% ci 45.5-51.3) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = 0.004). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting 25% of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to 30% of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp01-02 the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: 1) optimising the patient's own blood, 2) minimising blood loss and 3) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. 3d-s11-01 department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c3bc/c3d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c5 to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd163 and cd91-mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase-1 (ho-1) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho-1 has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. 3d-s11-02 understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. 3d-s11-03 cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il-12p70-producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il-12 production. in diseases characterized by local th1 inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band 3. other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd47. a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by 90%. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. 3d-s12-02 treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, 60,000 children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately 20% patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is 88% in paediatric and 65% in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss6 inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since 1987, include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with 204 national thalassemia associations in 62 countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive 2004/33/ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive 2005/61/ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to 15 days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers 95% of total blood supply. data on ttis in 3,067 patients with thalassaemia and scd-thalassaemia in 2010-2017 are analysed. results: tti prevalence in thalassaemia syndromes was: hbv 1.8% (occult type 1.3%), hcv 54%, hiv 0.3%, htlv 0.8%, wnv 0.5% and hev 3%. most frequent adverse reactions in 2015-17 were allergic (incidence 1:854), non-haemolytic febrile reactions 1:2,631, "other" 1:5,883, alloimmunisation 1:6,667, taco 1:100,013, tad 1:50,006, tt-hev 1:100,013. hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in 2010-2017 show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs10421768, rs117345431, and rs142126068 with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of 102 samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs10421768, rs117345431, and rs142126068. statistical analysis was performed on ibm*spss* statistic 23 using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.-582a>g variant (p = 0.02). for rs117345431 statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = 0.058). all samples were homozygous for allele t of rs142126068. summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to 20 years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by 2030 might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to 12 months in july 2016. since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s1. deferral of msm during the 4 months preceding the donation; s2. deferral of msm who have had more than one sexual partner in the 4 months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july 2016-december 2017 which is the baseline rr with the current 12month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤180 days) since all hiv-1 antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s1, from msm blood donors with the current deferral policy (12 months) and for s2, from monogamous msm of the general population. results: from july 2016 to december 2017, 8/28 (29%) hiv-1 positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at 0.16 in 1 million donations [95% ci: 0.04-0.34], or 1 in 6,380,000 donations. for s1, the number of additional msm donors was estimated at 733 and the number of additional hiv positive donations at 0.09, resulting in an hiv rr of 0.16 in 1 million donations [95% ci: 0.04-0.35] or 1 in 6,300,000 donations. for s2, the number of additional msm donors was estimated at 3,103 and the number of additional hiv positive donations at 4.92, resulting in an hiv rr of 0.23 in 1 million donations [95% ci: 0.05-0.56] or 1 in 4,300,000 donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by 1.5, the risk would be 1 in 6,225,000 donations for s1, and 1 in 3,000,000 for s2. summary/conclusions: for both scenarios, the hiv rr remains very low. for s1 (4-month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s2 (no more than one sexual partner, 4 months), the risk is 1.5 higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s1, since the risk could be 2 times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. 3d-s13-03 background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering 8 of 9 provinces in south africa. controls were frequency matched at a 3:1 ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < 1.5. eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the 6 months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november 2014 to january 2018, we enrolled 323 incident hiv cases and 877 controls; 202 (62.5%) cases and 544 (62%) controls were ≤ 29 years old. there were significantly more female cases 230 (71.2%) than female controls 439 (50.1%) (p < 0.0001). significant hiv risk factors (all p < 0.0001) reported within the 6-months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from 2000 to 2016, among male blood donors (mbds) found hiv-1 positive at blood donation screening, 31% did not disclose any risk factor for hiv infection during post-donation interviews, while 38% reported having sex with men (msm), and 24% and 7 % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv-1 infection in mbds, we performed an hiv-1 genetic network analysis, including hiv-1 positive mbds and patients included in the french primary hiv infection anrs co6 primo cohort (pc). methods: 284 mbds, who donated blood between 2000 and 2016, and 1340 pcs, included between 1998 and 2014, were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: <6 months). a partial transmission network was computed based on tamura-nei 93 nucleotidic distance (threshold for hiv-1 s/t b = 1.5%; for non-b s/ t = 0.8%) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv-1 strains from 81 mbds and 126 pcs were linked into 54 clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger (30 vs. 36 year-old; p < 0.01) and were more likely to have a recent infection (48% vs. 34%; p = 0.03). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < 0.001) and to have the same risk factor for hiv infection (p < 0.001) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: 38% vs. 49%, hts: 24% vs. 21%, other: 7% vs. 6% and unknown: 31 vs. 24%. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to 18% (31/176) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv-1 associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in 2002. monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in 2013 and 2015. aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every 2-3 years. results: in the uk 2002 -2017, 254 htlv-infected donors were identified. prevalence among new donors was steady around 5/100 000 donations. prevalence among repeat donors peaked in 2002 (2.7/100 000 donations), with most in previously untested. from 2004 to 2016, prevalence of 0.07 per 100,000 donations (average of one positive/year) was recorded. in 2017, prevalence among new donors increased to 9.9/100,000 donations (17 positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women (182/254, 72%), uk-born (125/254;49%) and htlv-1 infections (228/254;90%). mean age was 43 years. almost all positive donations were from previously untested donors (240/254), with seroconversion within a year of previous donation confirmed for only 5 of the 14 previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of 114 htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over 800-person years follow-up, none had developed atll or ham. summary/conclusions: over 16 years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in 2017 nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the 60°and 70°n latitude. the length of the country is 1150 km and width 550 km. by surface area it is the fifth largest country in eu. the population of the country is 5.5 million resulting in the lowest population density in eu (15.8 inhabitants/km3). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as 10-20% of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since 1948. frc bs collects annually about 200 000 whole blood units of which 50 % are collected in 10 fixed sites and 50 % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by 8 am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the 1930s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in 2007 and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice (4 courses) -pbm: general (7 courses) -pbm: medical (6 courses) -pbm: acute care and surgical (4 courses) -pbm: obstetrics and maternity (3 courses) -pbm: neonates and paediatrics (7 courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from 1 july 2007 to 31 january 2019: -489,600 people registered as learners -1,072,299 courses were completed -these learners came from 182 countries, with 11,141 (2.3%) of them from outside of australia. analysis by profession shows that: -82.4% are nurses and/or midwives -11.2% are medical -6.4% are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = 2,885) from 1 april 2015 to 31 january 2018 shows that these courses have a positive impact, with 88.7% of respondents stating they gained additional knowledge, 65.2% able to make changes to clinical practice, and 88.2% reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with 93.5% gaining knowledge, 76.4% able to change their clinical practice and 91.9% believing this will improve patient outcomes. analysis of red cell usage in australia shows that since 2012 there has been a 21.1% reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in 2012 and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. 3d-s14-03 prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a 12-month period (january 2018-december 2018). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > 50,000/ll, ≥2 doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy1) were compared to subsequent years (pgy > 1). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $500 for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < 0.05 considered statistically significant. results: there were 1446 platelet doses requiring approval with 670 (46%) routed to the pgy1 group and 776 (54%) to the pgy > 1 group. there were 847 (59%) ordered doses that were in compliance with hospital transfusion policy and 599 (41%) that were not in compliance with hospital policy. of the 847 appropriately ordered doses, the pgy1 group declined release of 7 necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > 1 group. when paged by the blood bank, pgy1 physicians approved product release not in compliance with policy for 191/670 (29%) doses while pgy > 1 physicians approved not indicated products for 79/776 (10%) of doses (p < 0.01). products not indicated by hospital policy were held from release by pgy1 physicians for 113/670 (17%) doses and 216/776 (28%) doses by pgy > 1 physicians (p < 0.01). the ordered doses not in compliance with hospital policy had an estimated cost of $299,500. of this cost, there was a calculated $164,500 savings of products not released due to prospective auditing. there was an additional potential savings of $135,000 for products not indicated but released ($95,500 from the pgy1 and $39,500 from the pgy > 1 group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > 1 group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy1 group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy1 physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. 3d-s14-04 what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in 2015 we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in 2017 and 2018 were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of 188 adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty (10.6 %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected 39 (20.7 %) events and formal internal or external reviews detected one event. seven (3.7 %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of 16 wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of 22 events related to blood ordering, two of seven errors in testing, six of 17 events where incorrect blood had been transfused, and eight of 64 events related to donor selection. in 80 reports human error was listed as the cause of the event and 27 of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average 1.2 g/l (0.9%) higher than from the venous blood samples. the range of the difference was -21 -+22 g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in 1.4 % (12/845) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + 9 g/l. 92 % of the hemoglobin results from the finger prick were in the range ae10 g/l compared to the venous hemoglobin results. 63 % of the results from the finger prick were between ae5 g/l (the precision of the device) compared to venous hemoglobin results. in 13 cases the difference between finger prick and venous measurements was outside 2 standard deviations from the mean i.e. 2.5 % from the bottom (n = 9) or top (n = 4) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about 8% (men) to 81% (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of 56 days, a donor needs to absorb 4.5 mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of 3-4 mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every 5th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < 15 lg/ l, or ≥ 15 and ≤ 30 lg/l (for 12 and 6 months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from 2020 onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. 3d-s15-02 superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (<7.8 mmol/l [<12.57 g/dl] for women and < 8.4 mmol/l [<13.54 g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since 2010 with more than 110,000 participants. extensive genotyping has been performed on approximately 72,000 dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than 150,000 icelandic individuals (an independent discovery cohort), we constructed 9 different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of 2017 was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at 5, 10, 25, 40, 60, 75, 90 , and 95 th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was 6.9 donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the 40-60 prs percentile group, donors below the 5 th percentile had lower (-0.23 hb mmol/l (95% ci: -0.25; -0.21)) and donors above the 95 th percentile higher (+0.19 hb mmol/l (95% ci: 0.18; 0.21) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the 40-60 prs percentile stratum as reference, donors below the 5 th percentile and donors above the 95 th percentile had odds ratios of deferral of or = 2.58 (95% ci: 2.27; 2.93) and or = 0.46 (95% ci: 0.39; 0.54), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, 2012) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles (58 features, 3,162 donations from 1,025 repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < 20 ng/ml for women and < 30 ng/ml for men), or absent-iron donation (ferritin < 12 ng/ml for men and women). we trained multiple machine learning models on 2,543 of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of 620 donations, which were not used to train or select the model. we then used our model to generate risk estimates for these 620 test donors as a function of days since their last donation, which varied from 56 days to 250 days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes 250 days after the last donation was lower than the risk 56 days after the last donation for most donors (risk of hemoglobin deferral decreased for 84% of donors; risk for low-iron donation decreased for 61%; and risk for absent-iron donation decreased for 94%). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, 2011) and from the danish blood donor study (rigas, transfusion, 2014) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for 15-24 month follow-up of donation frequency and iron status. a brief checklist of 8 food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of 2425 enrolled donors, 1406 were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was 7. 2, 13.1, and 22 .0 mg of heme iron weekly. these values are equivalent to approximately 3, 6, and 9 servings of beef per week, or alternately twice as many servings of chicken or pork. across 2236 follow-up visits with iron outcomes assayed, almost 33% of donor visits were associated with intermediate iron depletion (serum ferritin < 26 ng/ml) and 8.5% with complete depletion of iron stores, representing serum ferritin < 12 ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than 2fold higher risk for complete iron depletion during all follow-up visits (rr 2.40, 95% ci 1.51, 3.81, compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately 250 mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included 2,552 donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = 292). hb levels were measured in edta whole blood samples using a hematology analyzer (xt-2000, sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci8200, abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, 2,260 (1,186 female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) (95% confidence interval (95% ci)) in men and women respectively: 0.147 (0.069 to 0.225) and 0.094 (0.013 to 0.175) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (-0.015 (-0.026 to -0.004) and -0.018 (-0.032 to -0.005) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: 0.073 (0.046 to 0.107) and 0.065 (0.031 to 0.100) lg/l for heme and -0.003 (-0.008 to 0.000) and -0.007 (-0.013 to -0.002) for non-heme). more mvpa was negatively associated with hb levels in men only (-0.004 (-0.008 to -0.001)), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd20 moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. 4a-s16-02 thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple 1 , r aslam 2 , e speck 2 , j rebetz 1 and r kapur 1 1 lund university, lund, sweden 2 st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last 10 years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately 30% of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp4, amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood 2010) . methods: platelet glycoprotein (gp) iiia (cd61) knockout (ko) mice were immunized with cd61 + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra (1, 10 and 20 ug/kg) had significantly higher platelet counts by 72 h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after 2 weeks and were fully rescued to control levels after 3 weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: 112 itp sera were investigated in this study. 35 (31%) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): 3.23, range: 1.76-13.61, p = 0.0001). in addition, 23 (21%) sera caused higher ecl binding to test plts (ecl-fi: 2.31, range: 1.54-5.7, p = 0.0001). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between 2014 and 2018. the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from 64 patients (52% women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was 63 years (range 22-89 years). the mean hg level at diagnosis was 68.60 g/l. dat results were positive mostly with igg+c3d (59%) or igg (31%). most patients had warm aiha (70%). other types of aiha diagnosed were mixed aiha (15%), cad (11%), pch (1.5%) and dat negative aiha (1.5%). in 6 cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as 1st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of 80% of patients were transfused at a mean hemoglobin level of 67.88 g/l. during this period we detected 136 dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november 2017 to march 2018. completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented 42% (58/137) of english acute trusts. median number of adults with aiha diagnosed annually was 4-6. in the preceding 5 years, 31% (18/58) recalled at least one patient who had died due to aiha. although 7% (4/57) undertook a bone marrow biopsy in all patients, 93% required additional features, mainly: neoplasia, age over 60 or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, 59% (34/58) would not organise confirmatory tests, either because it was considered unnecessary (29/34), or because clinicians were unsure how to access tests (5/34). when determining aiha subtype, 29% (17/58) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with 12 considering this unnecessary and 5 unsure how to access tests. in 4 clinical scenarios of patients with aiha and dat positive to c3d ae igg ae cold associated symptoms, up to 87% (47/54) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone 1 mg/kg given before judging the patient refractory and reducing the dose was 3.5 weeks (sd 1.70, range 1-19 weeks). second line treatment of choice was rituximab for 82% (45/55) of respondents and splenectomy for 5%. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for 88% (49/56) but single agent steroid for 9%. we also explored the potential for future audit and research. 64% (37/58) of respondents were able to identify patients with aiha who previously required transfusion. 96% (55/57) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c3d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although 44% (25/57) would continue prednisolone at 1 mg/kg beyond the recommended 21 days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. 4a-s17-02 low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < 8.0 g/ dl, 5 mmol/l) vs. high-trigger (hemoglobin < 9.7 g/dl, 6 mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed (1) severe adverse transfusion reaction, (2) acute myocardial infarction, (3) stroke, (4) new-onset renal replacement therapy, (5) vascular reoperation, and (6) amputation of the lower limb. results: the primary outcome, mean hemoglobin within 15 days of surgery, was significantly lower in the low-trigger group: 9.46 g/dl vs. 10.33 g/dl in the hightrigger group (mean difference 0.87 g/dl; p = 0.022, longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november 1, 2017, standard double-unit rbc transfusion was indicated with a hb threshold ≤ 8.1 g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to 7.3 g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ 8.1 g/dl and ≤ 9.7 g/dl applied. we evaluated rbc blood product utilization over a 6 month period starting december 1, 2016 (liberal protocol) and december 1, 2017 (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ 7 days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, 137 patients were admitted who in total received 164 therapy cycles, including 56 acute myeloid leukemia (aml) induction cycles and 69 autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in 303/402 (75.4%) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from 29/112 (29.0%) to 81/111(73.0%) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units 4.0 (interquartile range (iqr) 2.0-8.0) during the liberal versus 2.5 (iqr 0.0-9.0) during the restrictive period (p = 0.36)). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: 0.25 (iqr 0.11-0.33) versus 0.15 (iqr 0.00-0.32) units per day during the liberal versus restrictive period, respectively (p = 0.06). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from 2.0 (iqr 0.0-2.0) to 0.0 (iqr 0.0-1.5) units (p = 0.06), corresponding to a reduction from 0.13 (iqr 0.00-0.21) to 0.0 (iqr 0.0-0.13) (p = 0.01) units per neutropenic day. moreover, 14/38 (36.8%) patients during the liberal versus 21/31 (67.7%) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. 4a-s17-04 assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain 1 , n marwaha 2 and s sachdev 2 1 transfusion medicine, aiims, new delhi 2 transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from 350 ml and 450 ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of 900 prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group 100 collections were done in double 350 ml, triple 450 ml and quadruple 450 ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) 9 volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = 0.007). hemoglobin concentration of prbc ranged from 14.2-29.6 g/dl; mean hb was 21.02 ae 2.90 g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = 0.0001) and rtvd (p = 0.008). the hb content of prbc units prepared from 450 ml collection ranged from 35.19-87.36 g and from 350 ml collection ranged from 30.77-65.78 g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = 0.730, p = 0.0001).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from 155 ml to 370 ml (including both 350 and 450 ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. 4a-s17-05 nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a 639 bedded tertiary care oncology centre with approximately 18,000 to 20,000 rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of 4413 rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < 0.05. results: total 4413 rbc transfusion events for 2012 patients were analyzed. there were 1877 (43%) events in 628 patients of medical oncology and 2536 (57%) in 1384 patients of surgical oncology. maximum transfusions were received by patients in age group of 41 to 60 years (47%). total 83% of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < 0.05. inappropriateness was more 53%(396/735) and significant in daycare setup (p < 0.05). anemia was the most common indication of rbc transfusion observed in 90% of events (3971/4413). total 62% rbc transfusions were given as planned and 38% as urgent transfusions. most common adverse transfusion event observed was allergic reaction in 0.3% of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. 4a-s18-01 paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed 0.38% of donors were positive for babesia dna or antibodies (moritz, nejm, 2016) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â 6800/8800 systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the 4 babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october 2017 under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of 6 (mp6), plus 3 idt replicates with cobas â babesia. reactive index donations were also tested with 2 validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, 256,802 valid donations have been screened with cobas â babesia, and 15 (0.006%) were reactive. 13 of 15 (87%) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. 1 of 13 (8%) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and 1 (8%) was collected in a state where babesia is not considered endemic (iowa). 11 of 13 (85%) confirmed positive donations were collected in states with high endemicity. 8 of 13 (62%) confirmed babesia-positive donations were detected in late fall or winter. all 13 (100%) confirmed babesia-positive donations were reactive in mp6. serology results are available for 12 of 13 confirmed-positive donations: at index, 6 of 12 confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; 3 were positive for both igg and igm. 3 of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of 99.999% (256,787/256,789; 95% exact ci: 99.997%>100%). summary/conclusions: the cobas â babesia test successfully identified 13 babesiapositive donations, including 3 confirmed-positive donations with no igm or igg reactivity. 2 donations were collected in states considered low-or non-endemic for babesia. 8 confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a 2013 study of~14,000 donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november 2018, 50,752 blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of 14,758 tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the 50,752 donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of 14,758 samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october 1, all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only 1 tma-confirmed-positive donation of 50,752 tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. 4a-s18-04 background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september 2015 and july 2017, 520 blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the 520 blood donors (mean age 38.55 ae 11.14; range 18-65 years old), 38.1% were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence (55.1%) followed by the north (37.2%) and the south (25.3%). blood donors living in rural areas had a significantly higher seroprevalence (p = 0.001) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence (60.2%) found in the age group of 46-55 years old (multiple logistic regression [mlr]: or = 7.68; ci: 4.08-14.46; p < 0.001), and decreased with educational level (p < 0.001). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = 0.02). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = 2.72; ci: 1.27-5.86; p = 0.001). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. 4a-s18-05 who is syphilis testing excluding? c reynolds 1 , c pearson 2 , k davison 2 and s brailsford 1 1 nhsbt/phe epidemiology unit, nhs blood and transplant 2 nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the 1940s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from 12 to 3 months in november 2017 and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between 2009 and 2018. methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within 12 months for regular donors. results: between 2009 and 2018 there were 153 recent syphilis cases, 121 hiv and 32 acute hbv infections identified by donation screening. recent syphilis rates per 100,000 donations increased from 0.66 to 1.64 whereas hiv decreased from 1.04 to 0.19 with less than 5 positive donations in 2018. acute hbv rates rose slightly from 0.28 to 0.38 in 2018. males outweighed females accounting for 71.9%, 63.6% and 59.4% of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below 25 years old. of the male donors with recent syphilis, 58.2% reported sex between men and women (sbmw), 19.1% sex between men (sbm) and 22.7% did not report a risk. this contrasted with hiv where 45.5% of male donors reported sbm, just 2.6% not reporting a risk. overall 19, 32 and 3 males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in 2018, 26 donors with recent syphilis aged 23-65 years (median 36 years) were excluded from the donor pool, including 3 non-compliant to the sbm deferral. there were fewer than 5 hiv cases identified in 2018, all over 40 years old, all compliant, reporting sbmw. of the 6 hbv acute cases in 2018, 5 were male, all but one in the 45 and over age-group. summary/conclusions: over the 10 year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a 3 month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated 113 million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last 10 years. plasma donors are bled up to 104 times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in 2014, the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into 6 categories (16 subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in 2018, with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between 1/1/2017 to 9/30/2018. severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade 2 or higher were individually evaluated by a physician for grading accuracy. results: in 1,511,758 needle-in collections, 31,320 dae were graded for severity. the majority (16,143, 51.5%) were vasovagal reactions (vvr), followed by 8,570 apheresis-related (27.4%), 6,572 needle-related (21.0%) and 35 allergic (0.1%) events. the majority of dae were grade 1 accounting for 98.6% of all dae, followed by grade 2 (1.2%), and grade 3 (0.1%). there were 2 grade 4 and no grade 5 dae. among the vvr, 98.1%, 1.6%, 0.2% and 0.01% were grade 1, 2, 3, and 4 respectively. grade 3 vvrs included 14 concussions, 11 fractures, 1 dental injury, and 2 pre-faint and 8 fainting events requiring hospitalization for work-up. two grade 4 vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only 6 and 5 grade 2 reactions respectively, and no grade 3 or 4 events. needle-related dae included 98.3% grade 1, 1.6% grade 2, 0.1% grade 3, and no grade 4 events. of the six grade 3 needle-related dae, 4 were nerve irritations lasting > 6 months, and 2 were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since 2006. aims: we analysed the data collected in 2006-2016 in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from 2008 the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in 2015. reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per 1000 donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was 7, iqr 2-8). the total number of country years (cy) was 138, covering 155 million donations. the overall complication rate was 6.3/1000 donations and the median country rate was 3.2 complications/1000 donations (iqr 1.1-10.1). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall 4.6/ 1000 donations, median country rate 3.1/1000 donations (iqr 0.6-7.7). rare and apheresis-related types of complications such as generalized allergic reaction (0.10 per 100,000, 40 cy), and major blood vessel injury (category available since 2015; overall 0.12 per 100,000, 6 cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total 89 cy, 101.6 million wbd and 26.3 million aphereses, total 128 million donations). for these hvs the median rate of vasovagal reactions was 3.4/1000 wbd (iqr 1.0-9.1) and 1.5/1000 apheresis procedures (1.0-4.2) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was 0.39/1000 wbd (iqr 0.31-1.2) vs 4.2/1000 aphereses (0.69-5.6); rates of arm pain and/or nerve injury (not separated in 2006-2013) also tended to be higher: median 0.09/1000 with wbd, iqr 0.03-0.34, vs 0.39/1000 with apheresis, 0.05-0.57. summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. 52000 registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in 8000 control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each 4 th (women) or 8 th (men) donation, and with yearly testing of young adult blood donors below 25 years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that 20% of female and 9% of male applicants cannot be registered because of low hb (125 and 135 mg/l respectively). adding ferritin testing, a preferred cut-off level of 30 lg/ml (male reference level), would result in additionally 24% female and 1.3% male applicant donor loss. as this would threat the blood demand, cut-off was set to 15 lg/ml for women, above the female reference 10 lg/ml, with an acceptable 6% loss of female applicant donors. for registered blood donors, 4000 mg of extra iron tablets were offered at low ferritin (10-29 lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above 30 lg/ml. donors with ferritin below 10 lg/ml (in 0.6% applicant donors, 0.8% registered donors) or above 600 lg/ml (0.5% applicant donors, 0.1% registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from 3 to 5 months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from 50% to 70% approved hb at return. the team of nurses processing ferritin and blood count results (1½ nurse fulltime weekdays) reacted to approximately 40 donor results daily, representing 20% of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november 2017 a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at 30 and 15 ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors 46% (44% of 16,433 male donors, and 48% of 13,525 female donors) have ferritin levels below 30 ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of 43 ng/ml. in contrast, we found that only 27% of new female donors (n = 13,283) and 1.7% of new male donors (n = 6,334) have a ferritin levels below 30 ng/ml. the average ferritin level in new donors was 148 ng/ml for males and 60 ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between 1.5 and 2.0 was observed in female donors and between 3.2 and 4.4 in male donors. both ratios increased with donor age. at the end of december 2018 2884 donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by 13 ng/ml per year whereas average ferritin levels in male donors increased by 34 ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from 1.5 to 2.0 in female and from 3.2 to 4.4 in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~7000 different rare diseases and the genes for half have been identified. approximately 3.5 million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts 2.2 years. the main aims of the 100,000 genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in 2013 and dna samples from 100,000 nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the 100,000 genomes project comprising phenotype and genotype data from 13,037 individuals recruited at 83 hospitals using approved eligibility criteria for 15 rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over 165 million unique variants in the 10,258 genetically independent samples, with 47% of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in 2,000 unique diagnostic-grade genes for the 15 domains. over 1,000 reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from 0.5% to 55%, while the proportion of novel causal variants ranged between 25% and 73%. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying >30 novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the 100,000 genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped 7588 donors from england and the netherlands with this new array and observed a 99.92% concordance when comparing 92,387 blood centredetermined antigen typing results with genotype-determined ones. for the 48 red cell and hpa antigens that were available for 7,473 donors, the array typing provided a 3.6-fold increase in typing results per donor (13.2 vs 47.9) and 38 rare donors were identified. using the genotyping data we identified 2.6 times more compatible units among this cohort of donors when blood demand was modelled using referral data from 3,146 english patients with more than three red cell alloantibodies. in conclusion the 100,000 genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another 500,000 dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early 1990s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found 4 amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons 266 and 268 are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than 250 abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a1,3-gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. 1: our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); 2: identification of novel abo alleles by others; 3: more snp data from genome sequences and potential problems for abo genotyping; 4: findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; 5: a1,3-gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and 6: the potential causes of generation of abo polymorphism and of species variations of the gbgt1 gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. 4c-s20-01 a large deletion spanning xg xg and gyg2 gyg2 constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd99. the cd99 gene resides within pseudoautosomal region 1 on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first 3 exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly 11% of women and 34% of men are xg(aà). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs311103c variant disrupts a gata motif between xg and cd99. this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from 13 archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the 1000 genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg2. database searches indicated that the most likely deletion was the infrequent genomic structural variant esv2662319 reported in the 1000 genomes project. further analyses with a short (714 bp) and a long (3555 bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately 114 kb and corresponded well with esv2662319. this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the 13 cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific 714-bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr6b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the 1000 genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg2 genes accounts for the xg null phenotype underlying the majority (10 of 11) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c.143c>t, p.thr48met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c.230c>t (p.thr77met) causing partial exon skipping and designated gybp*03n.01 (gypb*ny) and c.270 + 5g>t, an intron change causing complete skipping of exon 5, designated gypb*03n.03 (gypb*p2). aims: samples from three individuals, a previously transfused african american sickle cell patient (p1), a blood donor of unknown ethnicity (p2), and an african american patient (p3) (lapadat r. 2014 aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p2 were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p1 and p2. p1 was also tested by gypb*s/s as-pcr, exon 5 pcr-rflp for c.270 + 5g>t and as-pcr for c.230c>t. p3 was tested for gypb*s/s and c.230 c>t and c.270 + 5g>t changes by a real-time pcr-fluorogenic 5' nuclease taqman chemistry. for all, gypb exons 1-6 were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c.143t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c.230c>t and c.270 + 5g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sàu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c.143c/t, c.230c/t and c.270 + 5g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c.59t/g and c.60a/g (p.20leu/trp), c.67a/t (p.23thr/ser), c.71a/g and c.72g/t (p.24glu/gly), c.143c/t (s/s), c.208g/t (p.70val/leu), c.230c/t (p.77thr/met), and c.270 + 5g/t. all samples were also c.251g/g (p.84ser) and heterozygous for several previously recognized silent changes in exon 2, c.87t/c, c.96t/c and c.102a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sàs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c.230t or c.270 + 5t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c.270 + 5t change is on a gypb*03n.02 [gyp*he(ny)] background. c.230c>t (rs79492560) and c.270 + 5g>t (rs139511876) have a frequency of 0.0053 respectively 0.032 in the african population (exac). although we identified 3 samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of 25 antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin-5a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of 15 exons located on chromosome 19q13.2. a number of rare lutheran phenotypes have been previously recorded in israel, including lu:-6,9 observed among iranian jews, lu:-20 in one thalassemia patient and one case of lu:-21. in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with 2-aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:-5, lu:-6, lu:-8, lu:-13, lu:-21, lu:-22, and lu:-23 cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: -1,2,8,12,13,-19,21 . bcam sequence analysis confirmed the patient to be lu*02, lu*18 and revealed a novel homozygous mutation c.1351a>c in exon 11, encoding p.lys451gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c.1351a>c (p.lys451gln) in exon 11 of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises 25 antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome 19. currently, isbt lists 20 high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: -1, 2, 3, 5, 6, 8, 13, 20, 21 . bcam sequencing revealed a novel homozygous mutation c.121g>a in exon 2, encoding p.val41met in the lu glycoprotein. the c.121g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of 3.98 9 10 -6 and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val41met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema7a exon-specific primers and sequenced. results: the proband was a 32-year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at 27 weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted 1 + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge2 was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge2 and anti-ch1 could be ruled out. the serum was nonreactive with two jmh:-1 and positive with two jmh:-3 samples. the patient was found to be jmh1 positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg4. overall, these results were consistent with a probable jmh variant and prompted us to perform sema7a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon 7, c.709g>a (p.asp237asn, rs140707085, maf < 0.01, sift score = 1); a common synonymous change in exon 12, c.1545a>g (p.gln515gln, rs741761, maf = 0.5); a rare non-synonymous change in exon 14, c.1865g>a (p.arg622his, rs140128092, maf < 0.01, sift score = 0.36). the analysis of surface accessibility of asp 237 and arg 622 using the 3d structure of sema7a (rcsb pdb-3nvq https://www.rcsb.org/structure/3nvq) showed that only arg 622 was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg4 antibody showing a similar pattern of reactivity, and with the same three changes in sema7a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg622his substitution in sema7a. we propose to provisionally assign the name jmh7 for this antigen. interestingly, our two unrelated jmh:-7 individuals were from north african ancestry. background: the abo system was discovered almost 120 years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca3(fuca2)gal and gala3(fuca2)gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, 1985) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca3(fuca2)galb3galnaca3(fuca2)galb4glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a3-specific gh110 family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, 40% of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px2 but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a 1 b individual with the p1 k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p 1 k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen 500-litre reference preparation and confirmatory preparation from 24 freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bà human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a 1 b p 1 k plasma contained anti-px2 and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px2 was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex-4hexnac-hex-4hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type 2 hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala3(fuca2)galb4glcnacb3galb4glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 1990's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p2y12 antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. 4c-s21-02 university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between 0.05 -15% of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day 100 (33%) compared with those who had normal elpi at baseline (7%) (p = 0.00034). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < 50 ml plasma). until, in 2011, we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in 2017, we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under 50 years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (>24 days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (<10 days storage) red cells from ever-pregnant donors (3-year cumulative incidence of death 15.4% versus 4.8%). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. 7.2% versus 4.7%). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy (6-9 months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s-303) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (>5.3 log reduction) and plasmodium falciparum (>7.5 log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase 1 clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase 1 clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase 1 clinical trial in africa, 20 clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade 2) within the first 24 hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within 59 (ae3) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment 24 hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase 3 clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase 1 clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's (2014) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every 4 weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from 2 months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions 1 and 2, donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition 3) or the revised email (condition 4) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every 4 weeks, and a prompt to forward-book appointments. results: the final sample (n = 6788) comprised 3859 women (57%) and 2929 men (43%) aged 18-70 (mean = 32). after two months 37.2% of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition 4 (revised email + phone call), or = 1.305, ci = 1.128-1.510, and bau. donors assigned to the two telephone conditions (condition 3 and 4) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c282y mutation), but also carriers of other hfe mutations (p.c282y/p.h63d or homozygous h63d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< 200 ng/ml in females, < 300 ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis (2rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ 18-60 years, total blood volume ≥ 5 l, bmi < 35 kg/m 2 , hb ≥ 140 g/l, elevated sf levels and no end organ damage due to iron overload. 2rbcaph (360 ml rbc) were scheduled every 14 days and wbph (450 ml) every 7 days until sf was < 100 ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up 8 weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: 30 subjects (5 females; mean age 47 years) were randomized to wbph (n = 16; 1 female) or 2rbcaph (n = 14; 4 females). hfe mutations were p.c282/ p.c282y in 17 subjects, p.c282y/p.h63d in 9, and p.h63d/p.h63d in 4. at baseline, mean hb was 149 g/l (sd 7.8) and median sf was 504 ng/ml (iqr 406-620 ng/ml). 222 procedures (wbph n = 146, 2rbcaph n = 76) were completed; 9 were interrupted (local hematoma, insufficient flow); 35 (16 wbph, 19 2rbcaph) were postponed because of low hb and 15 for non medical reasons. there were 2 drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < 130 g/l in males, <120 g/l in females) occurred after 15 visits in 8 wbph subjects and after 5 visits in 3 2rbcaph subjects. fatigue was reported after 37 phlebotomies and 31 aphereses. only 5 participants (17%) completed the study per protocol. 136 blood components (94 rbc concentrates and 42 plasma units) for transfusion were obtained. overall, a median of 7.5 wbph (iqr 6.2-9.8) was needed to reach sf < 100 ng/ml, corresponding to 1.8 times of 2rbcaph (median 4.0, iqr 3.0-5.8) (p = 0.0001). analyzing separately p.c282/p.c282y and p.c282y/p.h63d carriers, the relation wbph to 2rbcaph was 1.6 and 1.8, respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = 0.0001 and 0.007, respectively). summary/conclusions: 2rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately 200 ml red blood cells (rbc) and 200-240 mg iron are lost with wb donation. double unit rbc (2rbc) collections of 360 ml (ca. 40 ml less than the rbc amount of two wb donations) lead to a loss of about 400 mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every 6 months for 2rbc and once every 3 months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and 2rbc at our institution. methods: we included 294 wb and 151 2rbc donors (n = 445) who donated with the maximal allowed donation frequency over 48 months between 2008 and 2013, yielding 4,704 wb and 1,208 2rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were 135 g/l for wb and 140 g/l for 2rbc donation. with 2rbc apheresis 360 ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was 53 (wb) and 48 years (2rbc), respectively. at the first donation, mean hb was 153 g/l (sd 13) in wb and 159 g/l (sd 8) in 2rbc donors; mean sf was 44 (sd 52) and 73 lg/l (sd 56), respectively. on average, hb and sf were higher in 2rbc donors (5.1 g/l and 26 lg/l, respectively; p < 0.001). there were 137 subjects with sf < 30 lg/l in wb and 19 in 2rbc group, and 85 with sf < 50 lg/l (but > 30 lg/l) and 40, respectively. in 2rbc donors, between the first and the last donation, mean hb declined from 159 g/l to 157 g/l (p < 0.05) and mean sf from 73 lg/l to 66 lg/l (ns). in wb donors, mean hb dropped from 153 g/l to 152 g/l (p < 0.05) and sf from 44 lg/l to 35 lg/l (p < 0.001). similar results were found when adjusting for age and season. hb values dropped from baseline until the 11 th donation for wb donors and until the 4 th donation for 2rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the 4 th donation in both wb and 2rbc donors (37 lg/l and 60 lg/l) and increased thereafter in 2rbc donors. in wb donors, sf followed a parabolic trend that peaked at the 10 th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and 2rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15% or 30% mfg (gelafundin 4%, b. braun melsungen ag) or hes (hespan 6%/450/0.7, b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd11b, cd62l and cd66b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < 0.001 respectively), whereas only the higher concentrations (15% and 30%) of mfg showed lower relative migration distances (p < 0.001 respectively). track straightness was reduced with both sedimentation agents at 15% and 30% to the same extent (p < 0.001 respectively). hes resulted in lower cd11b expression (p = 0.028) and higher cd62l expression (p = 0.007) compared to the controls, whereas the differences for cd66b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, 2001) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to 12 plateletpheresis donations per year, with a minimum 4 weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between 01/01/2014 and 08/31/2018 from the french civilian blood donors' base and then selected a cohort of donors with at least 24 donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. 8 measures for each donor. results: the cohort includes 2,186 donors (384 women and 1,802 men). mean platelet counts fluctuate between 276.4 and 278.6 platelets/ml. analysis of variance does not show any statistically significant difference (f = 0.462), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the 24 donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than 20,000 described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the 1950s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb1, -dqb1), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb3, -drb4, -drb5, -dqa1, -dpa1). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa1a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa1a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa 1a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa-1a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n162a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd61 (c17) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa 1a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n162a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, 166 plasma samples with anti-hpa1a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, 2016) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n162a-fccriiia correlated with the level of fucosylation of the hpa1a antibodies, as measured by mass-spectrometry (r = à0.524; p < 0.0001). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n162a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa 1a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa1a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa-1a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa-1a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa-1a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa-1a screening program in poland. aims: our aim was to assess the frequency anti-hpa-1a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa-1a screening of 24 244 pregnant women in 8-40 gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa-1a negative/hpa-1b/1b women were tested for hla drb3*01:01 and for anti-hpa-1a antibodies by maipa (followed up at week 17-20, 28, 32, 38-40 and 6 weeks after delivery). if anti-hpa-1a were detected, quantitative maipa was performed. all hpa-1a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa-1a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: 554 hpa-1a negative women were identified (2.3%). anti-hpa-1a was antibodies were detected by maipa in 44 women (two delivered tweens). in addition, anti-hpa-1a antibodies were later detected by paklx in further 3 women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was 47 (8.5%). they delivered 49 babies; 35 were boys. three women were treated by ivig: two by 4 and 8 injections since 33 th and 26 th gw respectively. the anti-hpa-1a concentration in the 1 st one was 0.4; 0.1; 5.88 iu/ml in 17, 28, 32 gw respectively and in the 2 nd <0.05 iu/ml in all examined samples. the decision on treatment was based on the low plt count~50 g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the 3 rd treated woman entered the program in 35 gw (anti-hpa-1a concentration was high 22.64 iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in 5/49 newborns: in 3 with anti-hpa-1a detected in paklx only and in 2 with antibody concentration in maipa -1 st : 6.86/12.91/ 23.36 at 28/32/38 th gw respectively; 2 nd : 8.85/17.58 at 32/38 th gw respectively. ich was observed in all of them; plt count was < 50x10 9 in four, 56 9 10 9 / in one. summary/conclusions: 1/ the severe thrombocytopenia due to anti-hpa-1a alloimmunisation in our prospective study occurred in 2/10 000 pregnancies 2/ the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative 3/ the hpa-1a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in 2014 that the diagnosis of alloimmunization was carried in only 33% of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by 3 different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all 2017 and 2018 data were collected using the laboratory information management system. results: 544 patient files were analyzed. no incompatibility is demonstrated in hpa-1 to -9, -11 and -15 systems in 19.3% (n = 105). hpa-1 and / or 3 and / or 5 incompatibilities were found in 271 cases (49.8%), hpa-2 and / or 15 in 87 cases (16%). platelet alloimmunization was globally confirmed in only 10.6% of the cases. 58 platelet alloabs were identified regardless of clinical manifestations: 24 anti-hpa-1a (41.4%), 20 anti-hpa-5b (34.5%), 8 anti-cd109 (13.8%), 2 anti-hpa-5a and anti-hpa-15b (3.4% respectively) and 1 anti-hpa-1b and anti-cd36 (1.7% respectively). 40 alloabs were found in the context of neonatal thrombocytopenia, 6 in ich and 10 in fdiu, and 1 in a follow-up of pregnancy. even if no anti-hpa-3 alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = 55, n = 32 and n = 17 on 114 cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa-3 and hpa-5 incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa-3 and hpa-15 systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa-1a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the 2% hpa-1a negative women. at present, hpa-1a typing is mostly done by genotyping. for costeffective implementation of anti-hpa-1a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa-1a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa-1a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg1 anti-hpa-1a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, 20 ll of the uppermost plasma of 3 -6 days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < 0.500 in the hpa-1a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, 506 consecutive samples were tested. in phase ii, the hpa-1a elisa was performed in another 62,171 consecutive samples, with confirmatory q-pcr in 1,825. the two phases combined, samples from in total 1,585 hpa-1a negative and 823 hpa-1a positive pregnant women were genotyped. the assay reached a 100% sensitivity with a cut-off od between 0.075 and 0.200, leading to a specificity of 99.6%. summary/conclusions: a quick, low-cost and reliable assay for hpa-1a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa1a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of 1047 pregnant women in 2002 found that 30.4% of irish women were cmv seropositive in comparison to 56% from western europe and 92% eastern europe and 97% from africa. an internal study carried out by the irish blood transfusion service (ibts) in 2010 indicated the rate of cmv seropositivity in irish blood donors was 21.97%. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in 2018 the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing 48 confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = 54). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing 6127 donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be 100%. the seroconversion sensitivity reported 42 out of 54 samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be 1.12 iu/ml. the validation reported 65 discordant results from 6127 donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. 60 discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified 27 discordant results as positive, 12 as negative and 21 as indeterminate. 5 discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be 99.80%. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity (99.80%) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of 6127 donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: 1) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, 2) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, 2014) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: 1. to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa 2. to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: 900 negative new donor samples and 100 confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was 0.56%, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than 750.000 blood and plasma donations are tested, on average 3.000 samples per day. for more than 10 years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july 2018, serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from 6 months before and 5 months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june 2018 (prism) and august to december 2018 (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: 1) hbsag p 0.01 % (42/390.736) versus a 0.03 % (87/322.394); 2) hiv p 0.07 % (262/390.735) versus a 0.06 % (201/322.470); 3) anti-hcv p 0.11 % (427/390.737) versus a 0.03 % (109/322.476). the rate of anti-hbc reactive samples was not significantly different between prism (0.39 %) and alinity s (0.42%). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s (0.39 %) and prism assays (0.34 %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to 0.16 %, which was significantly lower than for the three prism assays (0.33%). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by 0.17 % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on 2,748 blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag 6.0, enzygnost anti-hcv 4.0, and enzygnost hiv integral 4 assays and on the pk7300 with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i1000 system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas 8800 system) were available. results: based on the results from testing 2,748 blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around 90% of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e 801 analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e 801 analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e 801 analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using 30 preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar (99.81-100.00% versus 99.79-99.98%; n ≥ 5195). in specificity analyses, there were 14 discrepant results for hiv testing, 27 for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay (83.33%; 95% ci 65. 28-94.36) was higher than the prism hiv o plus assay (76.67%; 95% ci 57.72-90.07), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv (85.19%; 95% ci 67.33-95.97), hbsag (70.00%; 95% ci 50.60-85.27), anti-hbc (100.00%; 95% ci 85.75-100.00), and syphilis (100.00%; 95% ci 88.43-100.00); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e801 (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june 2018, close to 10,000 aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = 10%) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of 7,389 plasma and 2,242 serum samples, 41 test results representing 37 individual donations were found rr on one or both instruments. two samples were confirmed positive (1 9 hbsag, 1 9 hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e801): hbs ag: 0.04/0.03 % in a total of 9590/9589 samples tested; hcv ab: 0.01/0.09 % in 9620/9585, p < 10%; hiv ag/ab: 0.03/0.09% in 9362/9329, p < 10%; syphilis ab: 0.1/0.07% in 2971/2987; hbc: 0/0% in 1531/1549. no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd34 + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type 1 (hiv-1), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd8 + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd8 + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered 50 years of soviet occupation. we held hands in a 600 km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june 1 st , 2004. the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in 1991, the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in 1992. a lot of advice came from finnish colleagues. in 1997, it was decided to move towards non-paid voluntary donations. the process took 6 years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than 15 years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in 2006-2015. it resulted in 51% of the donations being unpaid. the second program initiated in 2016 is still ongoing, aiming towards 100% non-remunerated donations by 2020. by the end of 2018, they had reached 99.2%. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: 13.8 ae 0.01 vs 15.5 ae 0.01 g/dl; p < 0.000). percentage of females with low hb < 12.5 g/dl were 7.1%, 4.9%, 5.2%, 6.7% and 4.2%, percentage of males with hb < 13.5 g/dl were 0.5%, 0.6%, 0.7%, 1.7% and 1.2% for the age groups 1-5 respectively. ferritin values were higher in males compared to females (median; 25 th -75 th %>tile: 51;31-79 vs 157;106-231 lg/l; p < 0.000) and in older age groups compared to younger age groups (median; range in age groups 1-5 in females: 40;3-702, 52;6-619, 60;6-480, 60;8-725, 98; 10-604 and in males: 99;8-661, 161;18-1080, 206;15-1090, 220;26-1430, 221;33-981 respectively) . percentage of females with ferritin ≤ 15 lg/l were 8.6%, 3.8%, 3.7%, 6.7% and 2.0%, while percentage of males with ferritin ≤ 15 lg/l were 0.2%, 0.0%, 0.1%, 0.0% and 0.0% for the age groups 1-5 respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: 7.2 ae 0.02 vs 6.6 ae 0.03; p < 0.000). percentage of females with wbc > 12x10 9 /l were 1.8%, 1.3%, 1.7%, 1.4% and 0.3%, while percentage of males with wbc > 12x10 9 /l were 1.4%, 0.9%, 0.5%, 1.2% and 0.9% for the age groups 1-5 respectively. none had wbc < 2x10 9 /l. platelet counts (plt) were higher in females compared to males (meanaesem: 257 ae 0.6 vs 222 ae 0.6; p < 0.000).percentage of females with plt < 150x10 9 /l were 1.0%, 1.5%, 2.2%, 2.4% and 1.0%, while percentage of males with plt < 150x10 9 /l were 2.6%, 3.6%, 2.7%, 2.4% and 1.6% for the age groups 1-5 respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with 75% younger than 30 years of age and the female/male ratio was 58/42. of the 16 583 first time donors with data on ferritin available, 14% had low ferritin (≤ 30 lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb (6%<12.5 g/dl) and low iron stores (23%≤30 lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period (2017-2018), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november 2017 was assessed by comparing results in two study years. results: a total of 491 pdi were recorded: 266 in 2017 (1/396 donations) and 225 in 2018 (1/452 donations) with the following distribution: nonsexual risk as tattoo and piercing (17.5%), surgical procedures (17.1%), travel history (16.1%), infections/ contact (15.3%), other medical reasons (13.4%), endoscopy/invasive diagnostic procedures (12.2%), malignancy (4.3%), autoimmune diseases (3.7%) and sexual risks (0.4%). majority (76.4%) were late pdi, revealed on the future donation(s): 58.7% on the first next donation, 28.9% on the second and 12.3% after more than 2 subsequent donations. the mean age of blood donors associated with pdi was 40 ae 12 years (median 39 years), while the mean age of all donors in 2017/2018 was 38 years (median 37 years). of all pdi, 81.1% were related to male donors (84% in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > 0.05). the median number of all donations preceding pdi was 6 for female donors and 19 for male donors. implementation of education leaflet for blood donors resulted in 15.4% reduction of pdi in 2018 compared with 2017 (p > 0.05). the effect is more pronounced (p < 0.05) when comparing second and first half of 2018 (-25.6%). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from 21.1% in 2017 to 26.7% in 2018, which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. 45 % of omani populations are àa/àa gene carriers, 44% àa/aa, and 11% of the population are aa/aa. around 10 % of omani nationals carry the gene for hbs, and 2 -3 % carry the gene for b-thalassaemia. recent statistics show that there are around 400 patients with thalassaemia major and 3000 with scd in oman. the other rbc abnormality that is common in oman is g6pd deficiency which is found in 28 % of males and 12 % of females. omanis are known to have the highest frequency of a thalassaemia and g6pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get 40 new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: 418 patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the 418 scd patients, 49% of the patients were male and 51% female, mean age was 17 years, in the range of 1-72 years. 38 % of the scd cases were positive for the alloantibodies, 72% were female and 28% were male, the age range was from 6-68 years. 78% of the positive were scd, 19% s trait and 3% were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. 51% of the patients were with single alloantibody, 30 % of them with two antibodies, 10 % with three antibodies, 7 % with four antibodies and 2 % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority 23%, followed by anti-d 13%, anti-k 17%, anti-c 14%, anti-c 8%, anti-jk a 5%, anti-jk b 5%, anti-fy a 5%, anti-e 3%, anti-s 3%, antis 1%, anti-kp a 0.7%, anti-fy b 0.3% and igm being 2%. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and 19% of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in 2018, a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ 1, 2-5, 6-9 and ≥10), previous pregnancy, number of transfused units (2, 3-5 and 6-10 and > 10), and years after last transfusion (<1, 1-2, 2-5, >5y) with presence of alloantibodies results: 226 patients (100 males and 126 females, median age 17 years, range 1.7-66) were included. the median number of transfusions was 3 (range 2-40). the median years after last transfusion was 2 (range 2 weeks-55.5 years). in 56 patients, anti-rbc antibodies were detected. in 14 of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least 34 patients (15.0%) alloimmunization was demonstrated or suspected; in 11 patients the alloantibodies were 'enzyme only'. besides, the 36 alloantibodies of known specificity (8 anti-d, 2 anti-d+c, 16 anti-e, 1 anti-c, 3 anti-e, 1 anti-k, 1 anti-s, 1 anti-le a , 1 anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients (3 had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated (2 dau-alleles and 1 diii type 4 or diva-2). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the 34 patients. immunobiology -red cell alloimmunity 65 fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion (12 patients had dark urine). adverse reactions were associated with the number of units received (or 1.71 (95% ci, 1.25-2.33; p = 0.001), but not with the presence of antibodies (p > 0.7) summary/conclusions: in at least 15% of multi-transfused patients with scd alloimmunization could be demonstrated, mainly (80%) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of 12 patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately 1% of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman /3 rd pregnancy, 1 st delivery, 2 abortions in 1 st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd1) by biorad and anti-d duo igm+igg, clone: th28 + ms26 by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons 5,7,10) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon 1 to 9 revealed presence of a nucleotide deletion in position c.147dela, which is specific for allele rhd*01el.04. this nucleotide change results in the amino acid change p.val50leufs*5 causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the 2000 level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd*01el.04 allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt 00023736 and rvo-vfn 64165. 5a-s29-05 ea scharberg 1 , s rothenberger 1 , a st€ urtzel 1 , n gillhuber 1 , s seyboth 1 , e richter 1 , g rink 2 and p bugert 2 1 institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden 2 institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di6) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc4a1*c.1643c>t (p.pro548leu; isbt allele name: di*02.06) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of 125,742 sequenced genomes (allele frequency: 0.000004). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di6 in 1,700 blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in 3,652 patients and in 964 blood donors was performed in the gel technique (biorad ahg id-cards) using a 3 cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di*02.06 allele in 1,700 blood donors revealed no single positive individual. within the first 2 weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell 84 patients with anti-rb a were found. it was 2.3% of 3,652 patients tested in 21 laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in 18 of 964 randomly tested blood donors (1.9%) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di*02.06 allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around 1% of patients and donors. 5a-s30-01 national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since 1935. real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in 2003 in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in 2002, who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in 2017, national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since 2016, reporters to shot have been asked to score(0-10) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between 2010-2017(inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between 2016-2017 was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between 2010-2017; majority (56/67, 83.6%) were red cell transfusions but aboi plasma (9/67) and platelet transfusions (2/67) were also seen. most errors occurred in the clinical area (45/67, 67%), and could have been detected at point of administration. in 21(31%) cases, the error could not have be detected at the point of administration with a primary laboratory error in 10/21(48%) incidents. reviewing data from hfit for cases in 2016-2017 (13 aboi cases), the total score for staff culpability was 100, compared to a total score of 99 for all the other three organisational and system factors. this disparity is most obvious for the 4 aboi red cell cases, all of which scored the maximum 10 for staff culpability, i.e. 40/40 compared to 8/120 as the combined total score given to the other factors. in the preceding years (2010 to 2015), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by 37 cases that included an outcome of the local case review and 14 (37.8%) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with 2/56(4%) that resulted in death, 14/ 56(25%) major morbidity and 40/56(71%) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between 2015-2018 to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = 27 were possibly related to treatment, n = 2 trars were probable, and n = 2 were definitely related to treatment; n = 37 trars were grade 1, n = 4 were grade 2, and none were grade 4. in recipients of conventional wb, there were n = 13 (1.12%) ars, n = 32 (2.75%) fnhtrs, n = 1 (0.09%) taco, n = 0 trali, and n = 16 (1.38%) unclassified transfusion reactions. of the confirmed trars, n = 55 were possibly related to treatment, n = 1 trar was probable, and n = 8 were definitely related to treatment; n = 54 trars were grade 1, n = 1 was grade 2 and n = 1 was grade 4. there were 21 mirasoltreated wb transfusions in pregnant women and 2 trars (9.5%), both grade 1 and probably related. there were 80 transfusions of mirasol-treated wb and 84 transfusions of conventional wb in patients < 18 years old resulting in n = 7 (8.33%) trars in recipients of mirasol-treated wb and n = 10 (11.90%) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of 2181 wb transfusions in routine use in ghana, there were 4.02% trars in recipients of mirasol-treated wb and 5.59% in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january 2008 to october 2018 at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified 310 patients who received fludarabine (n = 52, 17%), bendamustine (n = 29, 9%), cladribine (n = 17, 5%), dacarbazine for hl (n = 164, 53%) and alemtuzumab (n = 48, 15%). the median age of patients was 46 years (range 8-92) and 171 (55%) were male. median follow-up was 30 months (range 0-132). post-exposure, 42 patients (14%) received transfusions with 33% correctly receiving irradiated rbcs. the remaining 28, all from haematology/oncology, received a total of 192 unirradiated rbcs. in 8 patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of 14.5 months (range 0-75) from first rbc transfusion. after medication administration, 99 patients had g&h requests after a median of 3 months (range 0-129). only 23% of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only 20% asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march 2017. for audited patients, these were written from 38 days prior to 104 days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february 2018 to february 2019. ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after 15 minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version 23.0. results: a total of 500 transfusions forms were analyzed. over all compliance rate was 18%. out of 500, 115(23%) forms were available in source notes and of 115, 88 (76%) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = 0.000). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion(3%) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion(89%). a total of 02(0.4%) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was 2 hours and 30 minutes for red blood cells and for platelets it was 1 hour and 13 minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after 6 hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. 5a-s31-01 as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately 15 cases per 100,000 but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of 2-4 weeks (range, 5 days to 5 months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and 130 (4.6%) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of 8.7 9 10 3 , 8.0 9 10 3 and 7.2 9 10 3 , respectively. mean cci decreased in a linear fashion between day ≤ 2 and day 7 pcs (9.0 9 10 3 , 9.1 9 10 3 and 8.5 9 10 3 at ≤ 2 days; 6.7 9 10 3 , 5.8 9 10 3 and 4.9 9 10 3 at 7 days, respectively), although the number of pc transfused on day 7 to autohsct patients was small (n = 71). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late 1990s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from 4 bcs (35% plasma in additive solution ssp+) were spiked with virus suspension (10% v/v). pcs (n = 2, 375 ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses (0.05, 0.1, 0.15 and 0.2 (standard) j/cm 2 )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid 50 ) by endpoint titration in microtitre plate assays on vero 76 cells (atcc â crl-1587 tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of 6.2 (bag no. 1) and 6.5 (bag no. 2) log 10 tcid 50 /ml was received in the pcs. at a uvc dose of 0.10 j/cm 2 and higher niv was inactivated down to the detection limit of the system (1.9 log 10 tcid 50 / ml), resulting in log 10 reduction factors of ≥4.3 (bag no. 1) and ≥4.6 (bag no. 2). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. 364 ae 20.6 9 10e11 platelets/unit, p < 0.001), whereas the platelet content of apheresis pc did not change (305 ae 9.9 vs. 300, ae16.1, p = 0.57). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. 1478 first-time donors has attended to the study in 18 regional blood centres in 29 cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = 14). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = 3.55, close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: 0.47, 0.28. 0.19, 0.17, and 0.5, respectively). donation anxiety was the negative direct predictor of intention (-0.05). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy (0.90 and 0.08). paraphernalia anxiety was the negative indirect predictor of intention (-0.03). descriptive norm did not show any significance. our model accounted for 75.3% of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from 16 countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp1 coordination, wp2 dissemination, and wp3 evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp4 inventory of donor selection & protection practices; -wp5 development of risk-based guidelines for donor selection and protection; -wp6 development of a standard donor health questionnaire (dhq); -wp7 training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september 2017 and will complete in spring 2020. wp4 has completed its work in october, wp5 will complete its work in june 2019, and wp6 and wp7 have recently commenced. results: with the use of the deliverables created by wp4, we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp5's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from 16 countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to 1) provide a comprehensive inventory of current systems for selection and protection of donors and donations, 2) critically review them and 3) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of 163 experts (40 blood; 40 plasma; 27 tissues; 47 stem cells; 9 art) and 39 (24%) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of 24 stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years 2014-2017, both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by 77%), arterial puncture (77%) and nerve damage (54%). vasovagal reactions were also frequently included (75%); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma (86%), citrate reactions (86%) and arm pains and nerve damage (both 57%, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl-03-01 modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg1/2) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a 2 c 2 ). we have previously identified bcl11a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas9 editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas9:sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + 58 bcl11a erythroid enhancer results in highly penetrant disruption of gata1 binding motif, reduction of bcl11a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl11a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl11a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m101 was developed in the medical device named hemo 2 life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on 60 patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo 2 life â and grafted on recipients. in 2018, a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo 2 life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m101 making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since 2012; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at 9-11 march 2018. aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from 25 countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations 4 different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: 1. should have university degree preferably in marketing and business administration field. 2. should have a certificate and/or professional experience in public relation 3. should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team 4. should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter 5. should be a permanent staff 6. should have basic salary and performance bonus might be given 7. is eligible to monitor and modify mobile team working period at blood drive 8. should participate the mobile blood drive which he/she has organized 9. should participate the group who will create promotional materials for national blood service 10. number at each blood establishment should be defined based on annual blood collection such as 5 staffs for 50,000 whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga 1 and j emmanuel 2 background: africa is a large continent with 55 independent states and a total population of 1,275,710,034 (february 2018) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the 2 who regional offices; eastern mediterranean regional office (emro) supporting 8 arabic speaking countries and the african regional office (afro) responsible for 47 sub-saharan countries. population distribution is approximately 40.6% urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise 3 level accreditation program. in 2016 emro held a consensus meeting developing a "strategic framework for blood safety and availability for 2016-2025" with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in 2001 all 54 member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least 15% of the national fiscal budget. in 2013 ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha63.12 on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's 2016 global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the 2016 report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of 4 staff on the morning (am) shift (from 8:00 to 17:30) and 5 staff on the afternoon (pm) shift (from 13:00 to 22:00) on weekdays and 3 staff on the am shift and 5 staff on the pm shift on weekends. bdt lab has a staff strength of 15 to be rostered for the 2 work shifts. each staff is on a five-day work week and has to work 11 pm shift and 9 am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving 5 staff on am shift and 4 staff on pm shift was conducted. the total number of pm shift per month was reduced from 124 to 112 using the re-defined process. the 10% reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having 2760 licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of 70,000 blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of 6 months. after overcoming all the challenges we minimised hard copies to 6 registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by 20%. waiting time for attendant decreased by 10%. traceability of all the units became 100%.supervision of the activities being carried out was 90% accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by 50% thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p-007 ct smit sibinga 1 , y abdella 2 and f konings 2 1 iqm consulting, zuidhorn, netherlands 2 who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in 2013 blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the 22 member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only 9/22 countries are put in force by governments [1960 (egypt) till 2017 (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in 2008-2017 including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in 21 regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from 170 in 2008 to 133 in 2017 in favor of mobile collections, which increased from 8,672 to 13,189, the former is still the number one location for donating blood. on average 47.36% of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar (583,908 in 2008 and 588,184 in 2017); over 99% of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from 237,658 in 2008 to 143,038 in 2017). the total number of donations increased from 1,076,655 in 2008 to 1,249,655 in 2017; most frequent were whole blood collections (from 1,016,411 in 2008 to 1,171,302 in 2017) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs (996,678-1,154,239 units per year), fresh frozen plasma -ffp (1,090,369-1,289,021 units) and platelet concentrates -pcs (81,692-129,143 units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs (52-57.6% in respective years irradiated, 75.18-92.07% leukocyte-depleted), than to rbcs (4.46-8.46% irradiated, 7.65-20.7% leukocyte-depleted). in 2010, the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in 2017 approximately 11.5% of pcs and 8% ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland 80% of hospitals depend on blood for the treatment of patients; over 1.5 mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. 1 st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the 20 year-period in two stages. goals at 1 st stage: 1. data digitalization; scanning of paper documents. 2. development of a uniform template for collecting digital data from various sources. 3. standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. 4. selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) , b) digital stored in two file types (.doc and .xls, for the years 2005-2010 and 2011-2017, respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: 1. 1344 pages of paper documents were scanned. 2. models developed for data from 3 different sources: a. paper-data were rewritten and ascribed to its model; outcome -3 tables, 272 columns, 10,400 rows. b. .doc and .xls. filesdata were ascribed to 2 other models; outcome -6 tables, 1656 columns, 788 rows. 3. the 3 models were merged into 1 analytical table to create a 588 mb database (comparable to approx. 784 min of music). 4. the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. 5. selection of data for analysis at 2 nd stage. summary/conclusions: the 1 st stage provided a set of selected data for analysis in the 2 nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far 8 workshops were organized and each year around 30 countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. 6. supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from %96 whole blood (at 1996) to 5%. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management 7. around 11,000 blood bankers attended national courses, 7,000 attended national congresses, 18,000 attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in 2016 an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages 4.3% of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages 122.8% (2018) based on a 5-day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated (2016/2017) to be at 3.2% of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october 2018, an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level 6. prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the 12 months prior to emr implementation was compared with 3 months post. ntr numbers reduced from 4220/month to 2019/month (52% reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of 27/mth to 6/mth. wbit numbers have increased slightly: before having median 1 (range 0-2), after with median 2 (range 0-3). although it was hoped that wbit incidence may reduce with the new emr, 4 of the post implementation 6 wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa 1 , k teo 2 , s tsai 1 , p heng 1 , r sagun 1 and m wong 1 1 laboratory medicine 2 khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a 761-bed general and acute care hospital, opened in 2010, serving more than 800,000 people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase 1: an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase 2: the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase 3: elimination of the witness step for blood collection. specimen collection and rejection data from 2016 to 2018 was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january 2016 and march 2017, before the implementation of the e-t&s phase 1, the average rejection rate for blood bank specimens was 0.16% and 1.14% for identification and clerical errors respectively. during phases 1 and 2 of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february 2018, with the implementation of the final phase of the e-t&s system the specimen rejection rate was 0.38% and 0.12% for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after 15 min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on 30 patients admitted to the outpatient clinic for 45 red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january 2017-december 2017) 76 patients, 40 females and 36 males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was 12.0 months (interquantile range-iqr 26). the median length of stay was 16 days (iqr 30). in total 400 blood components were transfused in which of 227 red blood cell concentrates, 114 apheresis platelet concentrates, 6 granulocyte concentrates, 51 fresh frozen plasmas, and 1 cryoprecipitate and 1 whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < 5%, 5-10%, 11-15% and > 15%. most of the patients (63.2%) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were 5478.76 euros (iqr = 11280.02) and 130.57 euros (iqr = 354.86), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: 0.674, p < 0.01). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: 0.247, p = 0.032) there was also a significant weak positive correlation between the age and transfusion associated cost (p = 0.048, r: 0.227). a significant difference was found between the patients with and without hematological malignancies (p < 0.01) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < 0.05). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately 55.7% of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january 2016 to december 2018 were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total 177,370 ordered prc unit, 166,910 (94.1%) were subjected to pretransfusion testing and 63.1% (105,260) of ordering prc unit which are pretransfusion testing were transfused. this means that 5.9% (10,460) of ordering prc unit were not subjected to pretransfusion test. this showed savings of 1,098,300,000 rupiah. c/t ratio was 1.6 which demonstrate a good ordering pattern. however, 16.4% (27,451) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of 2,882,355,000 rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of 19 questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, 2014 to february, 2015 . results: the overall mean percentage of 'correct' responses for 19 questions in the baseline study was 61.75%, whereas post sensitization it was 77.21%. the mean percentage increase in general awareness related questions was 21.49%, 13.95% for storage of blood/blood components related questions, 17.37% for pre-transfusion checks and bedside transfusion practices related questions, 21.33% for testing and blood component preparation related questions and 27.27% for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < 0.001). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in 2014 to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online 24/7, complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during 2018, tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from 3/5 international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been 2,376 paediatric and neonatal courses completed from 6 march 2018 to 28 february 2019 with 89.6% of learners being nurses and/or midwives. analysis of course evaluation data (n = 33) showed that these courses: -provide knowledge (96.9%) -improve patient safety and outcomes (84.9%) -result in change to clinical practice (69.9%) -are relevant to clinical practice (70.9%) -are easy to use (67.7%) -are readily accessible (58.1%). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of 1 month [1 st april-30 th april] 2017. we selected two groups of care staff: the 1 st group consists of 50 students at the end of their training at the higher institute of nursing sciences. the 2 nd is made up of 50 nurses working in 5 university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of 15 simple or multiple choice questions, 7 were related to theoretical knowledge of labile blood products and 8 to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was 100%. the 2 nd group participants had an average seniority of 9 years . more than half of them (64%) had seniority of less than 10 years. only 12% had more than 20 years of experience. the rate of correct answers for all items combined was 44.4% for students versus 42.5% for practicing nurses. the theoretical knowledge part was more mastered in the 1 st group than that of practicing nurses (44.8% vs 33.4% of correct answers). on the other hand, the control of the transfusion act was better in 2 nd group (44% vs 50.5%). the overall "dangerous" response rate was 47% for students and 41.7% for practicing nurses. false practical knowledge was more common in group 1 (59.5% vs. 41.5%). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive 2004/33/ec on blood donor selection criteria is 15 years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than 24 stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive 2004/33/ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in 2018, we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive 2004/33/ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified 64 risks considered to be significant, distributed between donors and recipients. for 35/64 (55%) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for 9/20 criteria concerning general health and medication, 12/22 for transfusion transmissible infections, 10/13 for high-risk behaviour and travel, and 4/9 for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive 2004/33/ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of 5 possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since 2007, showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after 10 years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all 27 states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since 2007, when the assessment covered 109 blood services, marpsh reached 1218 blood services in 2017 (57% of the blood services registered) what corresponded to almost 100% of the inspection cover in this year. over this period (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from 26% to 9%. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over 10 years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = 2), training coordinator (n = 1), medical doctor for donor selection (n = 2), hemovigilance officer (n = 1), laboratory technicians performing donor sampling, blood qualification and production (n = 7), biomedical scientists (n = 3), microbiologist (n = 1), clinical biologist (n = 1), nurse (n = 3). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is >8 h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: 19.3 million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab0, rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version 4.2; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than 8800 blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over 1000 blood donors from more than 20 non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of 600 migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of 1.6%. amongst 509 hla-typed potential stem cell donors, we found 28 (5.5%) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund 2014-2020 (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of 1 year starting from june 2017 to may 2018 at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ 16) that were admitted within 24 h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period 885 patients (83.5% males) were enrolled. mean iss scores was 21.89 . mean time to hospital admission after injury was 9:03 (iqr 3.38-13:48) hours. mean time to first rbc transfusion following admission was 2:09 (iqr 0:27-2:45) hours. approximately 49.3% (436) patients were in shock (sbp < 90 mm hg &/or pulse rate > 110/min). whereas, 160 (18.1%) patients were coagulopathic (pt ≥ 1.5 times of normal). during initial 24 h of admission, these patients were transfused with 2929 (69.7%) rbc, 1986 (51.8%) ffp, 2327 (42.9%) rdp and 384 (81.5%) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ 5 units/4 h) was given to 190 (21.4%) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of 9% per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on 78 patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in 2016. pc consumption was examined from january 2016 to december 2018. the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched (10-8/10) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of 78 patients aged from 3 to 74 years were analyzed; 48 (62%) were male and 30 (38%) female; 35 (45%) were cmv-negative and 43 (55%) were cmv-positive. out of a total of 78 transplants, 14 (17.9%) were haploidentical and 64 (82.1%) hla-matched. according to the stem cell source, bm was transplanted in 22 cases (28.2%), and pbsc in 56 cases (71.8%). two patients also received a cd34 + stem cell boost. our analysis showed that, with a mean follow-up of 652 days, the number of pc transfused to our patients treated by hsct ranged from 0 to 383 units, with an average of 37 and a median of 15, illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = 0.034) higher number of pc transfused for patients treated with a haploidentical (89) versus hla-matched (26) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused (2016: 20% (371/1813), 2017: 19% (322/1742)). moreover, approximately 8 products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october 2017 to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july 2014 and december 2016 to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ 25% of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ 25%: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took 2.5 min using the pneumatic tube system (maximum of 2 units per tube) and 4.5 min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december 2017. one year following implementation of the change in policy 940 rbc units were issued to the or (a 54% reduction); 38% (361) were transfused, compared to 19% in 2017. wastage rates decreased from 8 products a month to 1 per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, 34% of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from 1 may 2018. the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired 5-days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july 2018 set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of 704 daily blood bank statements which were submitted between may and december 2018 from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was 130.4% (sd +/-101.6), the available stock was 142.3%: (sd +/-118.8) and the demand versus supply was at 95.6% (sd +/-11.3).the overall bsms was 122.8%; (sd +/-77.2) for the study period. gweru and masvingo nearly supplied all the demanded blood with 99.2%, overall bsms of 118.1% and 99.7%, overall bsms of 144.3% respectively. bulawayo supplied 98.3% of the blood demanded with an overall bsms of 99.1%. mutare supplied 97.8% with a bsms of 180.1% and harare 85.6% and a bsms of 78.0%. there were monthly variations but the service could supply above 90% of the blood demand. in the month of may the service met 91.6% of the demand and a bsms of 87.8%. in november and december it supplied 92.6%, bsms of 100.8% and 92.8%, bsms 131.8% respectively. august also had a below average supply of 93%, bsms -98.2%. june, october and september recorded above the average values; 97.3%, bsms of 126.8% and 98.7%, with a bsms of 117.2% respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in 2008. subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in 2016 and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where 4 or more red cell units are transfused in <4 h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: 65 from 10 treating units including cardiothoracic surgery (18) hepatobiliary/gastrointestinal/colorectal surgery (24), vascular surgery (6), neurosurgery (4), orthopaedic surgery (3), endocrine (2) and "other" (encompassing general surgery, urology, general medicine and oncology -8). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. 18 of 65 reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at à40°c, and irradiation. biolog-id tags are passive hf (13.56 mhz) tags. they are compliant with is0 15693, iso 18000-3 and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, 2010). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt 128 format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with 450 ml water. centrifugation at 4,500 rpm for 10 min. 270 tags processed, 15 tags per kit affixed at different positions. -shock-freezing at à80°c: shock-freezer (angelantoni, sf40), 50 units processed, reading immediately after removal from shock freezer. water, 30 tags irradiated at 30 gy and 30 tags at 50 gy results: all biolog-id tags were encoded and read with a 100% success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at à40°c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an 18-year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh34). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd*01/rhd*03n.01 and rhce*cevs.01/ rhce*cevs.03. aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion 2013 53(2s):174a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier 1 donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier 2 donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier 3 donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: 102 tier 1, 357 tier 2 and 100 tier 3 donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier 2 unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from 7 to 1.9 g/dl. at that time, she was transfused the only compatible units available -2 of the rare -dphenotype and her hb increased to 3.8 g/dl and eventually to 7 g/dl. the tier 2 rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform (1) assuring the patient identification with barcode techniques; (2) designing a structured order entry; (3) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; (4) automatically reminding physicians the happening of reaction and suggesting relevant test; (5) building a complete traceability log system; and (6) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected (0% after implementation, p < 0.01), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency 30-min blood crossmatch (98.1% after implementation, p < 0.05). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply (0% after implementation, p < 0.01). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow (2004 moscow ( -2011 . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during 7 days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in 7 days after were compared. results: in 2004-2011, 5 terrible ta occurred in moscow: 141 people died and more than 629 were injured. with the explosion in subway in 02/2004 42 people died, 250 were injured. the number of d-rbcus increased by 10% on ta-day, and by 30% during next 7 days. dbdn in the 1st day after ta increased 6,7 times, and in the next 7 days -1,6 times. second explosion in subway in 08/2004 resulted in 10 died, 50 injured. the number of d-rbcus increased by 80% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 1,1 times, and in the next 7 days -2,2 times. in 2006 (explosion on market) resulted in 14 died, 61 injured. d-rbcus delivery increased by 20% on ta-day, and by 10% during 7 days. dbdn in the 1st day increased 2,0 times, but decreased to 0,6 times during the next week. with subway explosion in 2010 40 people died, 88 were injured. the number of d-rbcus increased by 10% on ta-day, and by 0% during 7 days. dbdn in the 1st day after ta increased 6,5 times, and in the next 7 days -1,6 times. with the explosion in airport in 2011 35 people died, 180 were injured. rbcus delivery increased by 50% on ta-day, and by 40% during next 7 days. dbdn in the 1st day after ta increased 6,1 times, and in the next 7 days -2,0 times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for 7 days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of 70,000 blood units. 2000 patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. 1000 patients were taken as control and the other 1000 were taken as cases. 2482 blood units of healthy donors were chosen (2442 were males and 40 were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to 1000 patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of 6 months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in 5 cases (0.5%) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common (99.44%) followed by d (95.45%), c (89.65%), c (53.1%) and e (17.65%). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of 12.5 million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab0, rhesus and kell systems were analyzed retrospectively in 352362 blood donors for 14 years (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab0, rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih-1000" (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) 35.8%, 0 (i) blood group 34.3%, b (iii) 21.3%, ab (iv) 8.6% (n = 352362). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was 82.8% and 17.2%, respectively. donor's phenotype detection was routinely conducted from the 2013 year, therefore the number of donors was 152883. the most common phenotype among donors ccdee (32.61%), the second in frequency ccdee (19.33%), the third in frequency rhesus negative phenotype ccddee (15.42%) in the studied population. the ccdee and ccdee phenotypes were 14.04% and 12.17%, respectively. the most rare are ccdee (2.65%), ccdee (1.86%), ccddee (1.54%). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of 0.37% (n = 152883). cw antigen was tested in 104230 donors and was detected in 5.28%. cw is most commonly found in donors with ccddee phenotypes (2.36%), ccdee (2.03%) and ccdee (0.79%), with other variants of the data phenotype, the antigen was detected in 0.1% of the examined (n = 104230). antigen k was detected in 6.8% of donors, in 93.2% of this antigen is absent (n = 352362). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in 2014 the number of outdated units at the hospital side dropped considerably. results: since 2008 when the issued number of red blood cell units (rbc) was 5170 the amount of issued units rose to 5678 in 2011 and then dropped more or less steadily to 4850 in 2018. in this period the hospitals' programmes rose for 10% in all areas. number of donated units declined from 6330 in 2011 to 4820 in 2018. after reorganization in 2009 the number of outdated units fell from 3% of stocked units to 1.5%. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from 397 to 41 in 2018. for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens 4 times a day; at 10 a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high (99%). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of 78% blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve 100% voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january 2013 to january 2017 were collected from donor management software. the data includes socio demographic data. data has been process with spss version -17 results: during 4 years study period, total of 276,290 blood donation happened from both mobile blood collection and in-house blood collection. out of 276,290 collection, 48351 (17.5%) are from 18-24 age group; 106924 (38.7%) are from 25-31 age group 53324 (19.3%) are from 32-38 age group; 34536 (12.5%) are from group 39-45 age group; 23761 (8.6%) are from age group 46-52 and 9394 (3.4%) from age group above 53 respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal 18-38 years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in 2018 to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by 80 of the 81 blood banks authorized nationwide between january and december 2018. results: sihevi-ins© received information of 854,462 accepted donors, 33% of them with more than one donation in the same blood bank in a period of 12 months. a total of 72 abo or rhd discrepancies were identified in 69 people, who made donations in 20 blood banks (estimated risk: one discrepancy per 11,876 accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of 6,086 ae 3,785 units, representing 3.6% of the national collect). the remaining blood banks are distributors (average collection: 31,389 ae 25,704 units per year, representing 44% of the national collect). 75% of blood group typing discrepancies (n = 36) were related to the abo group. the most common discrepancy was between a typing group and ab typing group (67%) . in 25% of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = 10) or b type (n = 4). rhd typing discrepancies account for 25% (n = 18) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of 4417 edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, 1 anti-s, 1 anti-jka and 6 anti-k. in one case ih-1000 failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples (0.045%) false-positive result were observed both on ih-1000 system and neo iris and in two cases (0.09%)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih-1000 system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from 2017 to 2018 in taiwan. aims: the proficiency testing (pt) held semiannually from 2017 to 2018 were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into 4 groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of 11 kinds of routine work categories of 841 blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the 11 categories were as follows: 1. characteristics of institution 2. the equipment of blood bank 3. the kinds of tube in blood bank 4. the present kinds of blood bank tests 5. abo and rh type tests 6. the cross-match tests 7. the irregular antibody tests 8. hemovigilance system 9. other blood bank tests 10. massive transfusion protocol 11. quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of 3rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum 7 days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since 2001 year with 844 collections of stem cells (686 in patients and 159 collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt 128 standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, 2017 to dec, 2018) qc data from archi-tect i2000sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of 6532 data points (3523 data points in 2017 & 3009 data points in 2018) were obtained for 10 different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% (5.70-12.05) were within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2018. five out of six positive quality control levels cv% (5.72-15.80) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ 10%, anti-hcv ≤ 15%, syphilis ≤ 15%, hiv ≤ 14%) in 2017 except syphilis tp positive control (15.8%). all four negative quality control levels showed the sd values within 0.009-0.41 in 2017 and 0.009-0.41 in 2018 respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: 1. to assess the existing transfusion practices in the institute with specific quality indicators 2. to introduce corrective reforms to improve the existing practice 3. to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, 295 transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within 30 min of release from the blood bank (iii) close observation of transfusions for the first 15 min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within 30 min of issue. transfusion practices were once again monitored by following up 306 transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. 57.3% transfusions were verified in phase i while 73.6% were verified in phase ii (p < 0.001). 69.5% transfusions were started within half an hour of issue while in the second phase, it rose to 83.1% (p < 0.001). 47.8% transfusions were observed in the first 15 min in phase i and 88.6% were observed in the second phase (p < 0.001). in phase i, 54.6% transfusions were completed within right time while the same in phase ii was 73.9% (p < 0.001). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a1 & b) antibodies were selected. the titers of 126 samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, 12 of these samples were tested in the 2 automated systems for 5 times (120 datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured (3 expert technicians) in 2 different sample workload (1 and 10 samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, 2 different sample workload (1 and 10 samples testing) were assessed in manual method (3 expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ 1 titer: 73% ≤0.5 titer). the results proved that both instruments were equivalent in performing titration (differences ≤ 1 titer; 85% ≤0.5 titer). the precision results showed no difference between titers obtained through the 100% of the runs performed with the grifols analyzers (differences ≤ 1 titer: 75% ≤0.5 titer). the manual hands-on in automated system was reduced in a 15% compared to manual method for 1 sample. when the number of samples was increased (10 samples), the difference in hands-on in was even more reduced (65%). in addition, the walk-away was 46% higher in automated system compared to manual method. furthermore, when the number of samples was increased (10 samples), the walk-away difference was increased even more (78%). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least 15% the hands-on, increasing at least 46% the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by 30%>50% of users and "easy" and by 50-80% of users; 90%>100% of the users considered "sufficient" the design to ease the interaction; and 60%>80% of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), 40% of users considered tasks "very easy" or "easy"; 40% of users considered "sufficient" the design to ease the interaction; and 40% of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), 100% of users considered tasks considered "very easy" or "easy"; 90% of users never found any situation of not knowing how to proceed; and 100% of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno (1 and 2) and correspondent two mp-readers lyra (1 and 2) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a1, b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of 4 + 2 tubes of whole blood and 2 tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno 1/lyra 1 system. ward to ward. methods: a prospective observational pilot study was done for around 140 prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to 90 min, with highest time taken 795 min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be 10 min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to 70 min, maximum being 310 min (jan 2019), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p-059 hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: 204 healthy blood donors of greek origin (18-24 years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < 0.05. results: b-thal-het represented 9% of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch (11% p = 0.039, 26% p = 0.008 and 30% p = 0.006, respectively) and b) increased rbc count (20%, p = 0.042) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = 0.053) and serum total protein concentration slightly reduced (p = 0.054) in the same group. a trend for higher plasma antioxidant capacity (p = 0.052) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by 13%, p = 0.022) and hemolysis (by 41%, p = 0.001) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = 10 per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > 0.05). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of 23) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding 100,000 per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years 2010-2017 obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from 400 in 2010 to 716 in 2017 (+79%). in the group of male donors this trend remained rather stable with a slight decrease: from 181 in 2011 to 151 in 2017 (à16.6%). in the group of female donors the growth was more prominent: from 219 in 2010 to 565 in 2017 (+158%). in terms of the recorded confirmed hcv infections a downward trend can be observed: from 63 in 2010 to 16 in 2017 (à74.6%). in the group of male donors from 43 in 2019 to 12 in 2017 (à72%), in the group of female donors from 20 in 2010 to 4 in 2017 (à80%). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last 6 months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a 6 month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, 18.3% of donors who received a reminder message attempted to return compared to 12.8% of donors in the control group (p < 0.05). looking at each time point, donors who received the message 1 week before their deferral ended were 64% more likely to attempt to return compared to the control group (p < 0.05). the 1 week prior reminder message was particularly effective with males, with 30.3% attempting to return to donate, compared with 25.2% of females (p < 0.05). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide 100% voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of 1000 interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: 1. provide 100% voluntary donation for safe blood. 2. establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with 60 questions in total was divided into 6 sections out of which 14 questions on blood practices were answered by all 1000 interviewees. 416 people answered 9 questions on the blood transfusion service. 5 questions on blood knowledge were answered by 270 people. 5 questions on the knowledge of the blood transfusion were answered by 220 people, 17 questions on blood donation were answered by 420 people and 10 questions on the communication channels were answered by 500 people. results: out of 1000 interviewees, 19% have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, 40% did not donate, but expressed the readiness to donate in the future, 10% have donated voluntarily only once, 24% were family donors, 3% regular volunteer donors, and 4% have donated voluntarily several times and did not want to donate anymore. from those who have donated, 59% have donated for one of their relatives, 28% have donated for thalassemic children, 10% have donated to benefit free check-up and 3% have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, 57% have answered yes, 22% no and 21% were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around 60% of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with 3 adults of 10 overweight and 1 of 10 obese in 2017 with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < 25 kg/m 2 , overweight with a bmi ≥ 25 and < 30 kg/m 2 , and obesity with bmi ≥ 30 kg/m 2 . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least 3 donations in the last 2 years, and were eligible to donate. results: of the 2468 blood donors enrolled between july 2017 and january 2018, 1390 were regular repeat donors, 1102 did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and 288 accepted, of which 83 were deferred from blood donation and were excluded from the analysis. among the 205 included participants 68.3% (n = 140) were male, age ranged from 19-61 years with a mean age of 39.8 ae 11.1 sd and 31.7% (n = 65) were female age ranged from 20-62 years with a mean age of 37.7 ae 11.0 sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found 50.7% in men and 16.9% in women. our survey showed that 84.4% of the participants evaluated their health as "good", without gender difference (men, 86.4% vs women, 80.0%). besides, 14.6% reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged 18-25 attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics 22.0. categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < 0.05 was considered statistically significant. results: a total of 600 students were surveyed (300 squ, 300 non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, 73% of the squ and 25% of non-squ students reported the university as the main source for information (p < 0.001), while 56% of squ and 45% of non-squ students reported that the social media was the main source respectively (p = 0.048). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = 0.868). about 84% of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation (44.3% vs 29.7%, p < 0.001). squ students reported greater influence of peers (84% vs 60.7%, p < 0.001), personal knowledge (82% vs 74.7%, p = 0.029) and personal experience (79.3% vs 69%, p = 0.005) when compared to non-squ students. they also reported more feeling of commitment to the society (90.3% vs 78%, p < 0.001). squ students reported lower influence of parents (53% vs 65%, p = 0.005), lower rates of fear from needles (18% vs 32%, p < 0.001) and lower rates of fear from blood (16% vs 29%, p < 0.001). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about 15% of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately 85% of the expatriate population (and 71% of the emirate's total population) are asian, chiefly indian (51%) and pakistani (16%). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in 2019. the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of 1080 donors were surveyed and asked about the languages which they can read and understand and 1860 responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = 760; 41%), arabic (n = 272; 14.6%), hindi (n = 268; 14.4%) and malayalam (n = 233; 12.3%). the donors come from different countries, most common 591 (54.7%) donors are indian and 73 (6.8%) are from uae. it was found that 45% donors can read and understand only one language. majority 884 (81.9%) donors can read and understand either of the official languages arabic or english. however, 196 (18.1%) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, 1290 responses were obtained. the most common mode of communication were sms and telephone (85% together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as 18.1% donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type 2 diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a1c (hba1c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period 2015-2017 and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was 83.7%, plasmapheresis donors -13.2%, blood cell apheresis, including plateletapheresis, donors -3.1%. for the period 2015-2017, the percentage of repeated and regular whole blood and apheresis donors increased from 66.2% to 72.2%. the percentage of first-time donors ranged from 27.8% to 33.8%. the largest proportion of plasmapheresis donors was observed in the volga fd (22.0%). about 28.9% of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from 1.9% to 2.3%. the largest percentage of plateletapheresis donors was observed in the central fd (2.9%), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to 69.9% in 2017. actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between 20 to 23 years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january 2016 to december 2018. the data were retrieved from the official reports of each mobile blood donation. results: a total of 828 young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is 25.6%. the main causes of deferral are low haemoglobin (hb) level (20.8%) followed by low blood pressure (16.0%), upper respiratory tract infection (11.3%) and sleep less than 5 h (9.4%). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than 5 h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is 12.7% and in non-pregnant women is 30.2%. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was 454913. repeated blood donors was 44.8% of total population and had the highest number of referrals, followed by first and lapsed donors with 31.2% and 24% respectively. in terms of gender distribution, 6.2% were female and 93.8% were male. the highest rate of hemoglobin level less than 12.5 g/dl was found in first-time donors with 51.6% and the lowest prevalence was observed in lapsed donors, followed by repeated donors with 24.7%. 44.8% of the repeated blood donors have hemoglobin higher than 16.5. there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit 50,000 blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of 10% (500,000) of the finnish population. (11.58%), dental examination 10(3.32%) and medication history 6(1.98%). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were 5(1.65%) and 8(2.64%) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years (2016-2018). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold (120 g/l in women, 130 g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies (15.9% versus 3.4%) or in continental france (2.9% and 0.8%). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march 11 and march 28, 2019. results: this study ferridon has been approved by ethical research committee. nine thousand (9000) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, 150 donations should be included in the french west indies and 120 in reunion island. additionally, 1500 whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than 26 ng/ml and iron overload is defined by ferritin higher than 300 ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may 2019 to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around 10 million people, only 3.8% are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of 95% in 2018, instead of 80% in 2016, which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the mean interval between donations is shorter for former regular donors (4.9 months, p < 0.01) whereas donors with an interval of 9 to 12 months are more likely to be regular (aor 16; 95% ci 1.6-164). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least 60 9 10 9 cells per dose), and in kazakhstanin therapeutic doses (at least 200 9 10 9 cells per dose). in 2017, the estimated consumption of platelets in kazakhstan exceeded the russian indicator by 28.0%. 86% of platelets in kazakhstan and 69.9% in russia are harvested by the apheresis method. inactivation of pathogens is performed in 92% of platelets in kazakhstan and in 15.4% in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in 2017 exceeded the russian indicator by 141.4%. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in 10% of plasma in kazakhstan and in 3.6% in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. 0.7% of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p-091 finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: 1. no bacteria growth near the puncture spot (result 0 cfu) in ≥ 90% of the samples. 2. total amount of bacteria on average is at most 2 cfu/25 cm 2 . at most 10% of samples are allowed to have 2-10 cfu/25 cm 2 . methods: microbiological samples were taken with contact plates from 30 voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from 20 to above 500 cfu/25 cm 2 in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean (0 cfu/25 cm 2 ) in 93.3% of the samples and 86.7% of the samples had 0 or only 1 cfu/25 cm 2 . the average number of bacteria after disinfection was 0,6 cfu/25 cm 2 and the maximum number was 4 cfu/25 cm 2 . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the 2018 meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb201 + (hemocue ab) point-of-care (poc) device. donors with hb < 125 g/l for females or < 135 g/l for males or with a decrease > 20 g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = 305) and control (n = 331) groups, the proportion of male donors (27% and 26%) and the mean initial skin-prick hb (122 g/l and 123 g/l) were similar. significantly less donors were deferred from donation in the study group (40%) than in the control group (51%; chi-square test p = 0.004). the mean difference in venous hb with the poc device versus the hematology analyzer was à3 g/l (range à12 to + 6 g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only 1 g/l below the limit of donation eligibility (124 g/l for a female and 134 g/l for a male). interestingly, a further 33 donors (27% of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above 60 years of age. this age group has been reported to use up to 50% the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged 60 years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ 60 years from blood centres of four countries. the estimated proportion of blood donors aged ≥ 60 years old for each country was 0.81% in south korea (sk); 2.57% in hong kong (hk), <3.18% indonesia (indo) and 9% in japan (jap). the minimum hb criteria varied between each country and ranged from 11.5-12.5 g/dl for women and 12.5-13.0 g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe-2100d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are 6 medication questions on the donor history questionnaire (dhq), including any medication use in the last 3 days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in 2017 were included in the analysis. donors' answers to each of the 6 medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were 975,067 donation attempts with a completed dhq. overall, 34% of donors answered yes to medications in the last 3 days, 8% to vaccination, and less than 0.1% to others (38% any). slightly more were female (42 vs 36%) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from 24% of 17-19 year olds to 57% aged 60 + (p < 0.0001 for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel 7 and tomes software j schreier 1 , a davison 1 , j gambarte 2 , y l opez 2 , c calonge 2 and e herranz 2 1 terumo bct, lakewood, united states 2 centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than 4000 apheresis platelet collections were completed in 2018, of which almost 3000 were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel 7 was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel 7 devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel 7 with tomes compared to trima accel version 6. methods: this was a retrospective study analyzing 1372 apheresis procedures on trima accel version 6 during the control period from 2 january 2018 to 10 september 2018 compared to 541 apheresis procedures on trima accel 7 during the test period from 11 september 2018 to 28 december 2018. this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a 2-proportions test, whereas donor demographic data were analyzed using a 2-sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = 3.0 or 3.5 9 10 11 ) or double (target platelet yield = 6.0 9 10 11 ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = 5148 ml, test = 5124 ml, p = 0.545), hematocrit (control = 43.7%, test = 43.6%, p = 0.233), or platelet pre-count (control = 249 9 10 3 / ll, test = 253 9 10 3 /ll, p = 0.091). females represented 21% of donors in the control arm compared to 23% of donors in the test arm. platelet split rate (platelet products per procedure) increased from 1.15 with trima accel version 6 to 1.18 with trima accel 7; procedure time decreased from 55.7 min to 53.3 min for single collections and from 63.0 min to 60.9 min for double collections with trima accel 7 (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from 62.4% to 84.8% (p < 0.001) and the rate of completed apheresis procedures increased from 89.8% to 92.4% (p = 0.083) with trima accel 7. manual transcription of data during the procedure was discontinued with the implementation of trima accel 7 with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel 7 significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the 19 th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec 2017, distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of 36 respondents, 24 bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering 2,888,390 plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per 10,000 plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to 16% of the estimated total blood volume but maximally 750 ml, including anticoagulant. respondents had differing practices and scale of collection program 15 be were aligned or lower, and 9 be had higher collection volumes. altogether 14 reported the immediate vvr with loc rate, which mainly was lower than 10/10000 collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by 16 respondents. the retention rate banded in the range of 50%>80% (mean = 60%, min = 25%, interquartile range = 14%, max = 90%). the association between maximum allowed yearly plasma collection (25 l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide 0 s limits for collection volume of maximum of 750 ml per donation and 25 l per year per donor. methods: serum ferritin concentrations were established from sera stored at à30°c from repetitive platelet donors between 2014 and 2016, using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn 1800 â . sixteen samples were obtained from women (age: 35.3 ae 12.4 years, range: 18-61) and 35 samples from men (age: 36.2 ae 9.4 years, range 20-61), corresponding to 55.2% and 37.2% of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, 43.8% of women and 40% of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was à38.4 ae 52.2 ng/ml when the donations had an interval less than three months, vs 3.3 ae 13.0 ng/ml when the time between donations was higher (p = 0.046). in men the change in the ferritin levels was à38.5 ae 61.2 ng/ml with donation times less than three months vs © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 à3.3 ae 38.9 ng/ml with prolonged donation times (p = 0.042). in women, the change in platelet count was à2 ae 46.2 9 10 3 /ul, when the donations had an interval less than three months vs à22 ae 45.3 9 10 3 /ul when the time between donations was greater (p = 0.43). in men, the delta of the platelet count was à12 ae 37.0 9 10 3 /ul in donation times less than three months vs à13 ae 26.6 with higher donation times (p = 0.93). no correlation was found between the concentrations of serum ferritin and the platelet count (r = 0.05, q = 0.75 for males, and r = 0.34, q = 0.22 for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version 7 (ta7) by comparing its routine performance with that of the previous software version 6.06 (ta6). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta6 and ta7 sequentially. data were collected from december 2017 to april 2018 on ta6 and from june to july 2018 on ta7. simple and double doses of platelets (respectively 5.0 9 10 11 , 5.5 9 10 11 and 8.0 9 10 11 , 9.0 9 10 11 ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from 1.06 (ta6) to 1 (ta7). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety (590) collections with ta6 and 607 with ta7 were recorded, with 92% and 95% complete procedures respectively. mean duration of procedures was 70 min on ta6 against 72 min on ta7, p < 0.05. the mean alerts number per procedure on ta6 was 4.1 against 0.5 on ta7, p < 0.05 whereas the maximum alerts number per procedure was 57 and 12 respectively. on ta7, 76% procedures did not require operator's intervention against 40% on ta6,. with ta7 the inlet flowrate was automatically adjusted in 97.2% procedures. the inlet flowrate was increased in response to access pressure in 45.6% of procedures, for 3% of the procedures the inlet flowrate was decreased and for 48.6% of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta7 autoflow system. summary/conclusions: ta7 with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel 7 (ta7) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta7 leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta7 software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version 6 (ta6) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta6 from 4th january 2016 to 10th october 2017 were compared to 995 procedures, started on ta7 from 18th october 2017 to 31 st december 2018. procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta6 vs. ta7 respectively) were comparable and were characterized by: tbv -5207 vs. 5344 ml; platelet count pre-procedure -252 9 10³/ll vs. 252 9 10³/ll; hematocrit pre-procedure -44% vs. 44%. gender distribution was 11% female with ta6 vs. 2% with ta7. venous access pressure alerts were significantly improved by ta7 with an average of 0.2 alerts per procedure as compared to 2.0 with ta6, i.e. 92% decrease. this decrease went down to 91% if only male procedures were analyzed. the maximum number of pressure alerts went down by 84% from 90 alerts in one particular run in the ta6 cohort to 14 alerts in one ta7 procedure. procedure time for single platelet products was reduced from 51 to 49 min and for double platelet products from 59 to 54 min (ta6 and ta7 respectively). donor qualification possible was 52% of procedures yielding single products and 48% of procedures yielding double products with ta6. the percentage of procedures qualifying for doubles increased to 91% with ta7. in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from 1.48 to 1.91 from ta6 to ta7, respectively. in fact, the observed split rate rose modestly from 1.01 to 1.05, as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta7 decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta7 has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel 7 to trima accel version 6 august 11 2017 to october 17 2017 or trima accel 7 during the test period from november 28 2017 to january 9 2018. this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: 63 donors completed the survey during the control period whereas 86 donors completed the survey during the test period. the mean number of previous donations for the control period was 3.0 (min 1 max 6) and for the test period was 3.2 (min 0 max 7). there were no first time donors during the control period and 20 first time donors during the test period. 91% of donors rated their overall donation experience as good on trima accel 7 compared to 90% on trima accel version 6. zero (0) donor rated their experience on either trima accel device as poor. 100% of donors who responded to the question said they would donate on trima accel 7 again. summary/conclusions: no significant difference was observed in donor experience between trima accel version 6 and trima accel 7 as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in 2014, a higher number of the platelets was consumed compare to 2011 by 7.8%. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in 2018, 635 (8.6%) of the 7,371 patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is 14,292, which is 668 higher (by 4.9%) comparing to 2017. platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + 9000 (haemonetics), upp and trima accel (terumo bct) version 6.0 protocols. methods: the data for 4868 protocols of platelet donations performed in 2018 were analyzed: 2773 on trima accel and 2095 on msc + 9000. all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was 32 years old, height -175 cm, weight -76 kg, platelet count before donation -254 9 10 9 /l, hematocrit -42%. detailed data are presented in table 1 . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than 0.05 was considered as significant. results: the data obtained showed significant difference (p < 0.01) between average number of platelets collected on trima accel (6.3 ae 1.06 9 10 11 /l) and on mcs + 9000 (5.09 ae 0.78 9 10 11 /l). while cost of consumables are comparable, trima accel demonstrated 23.7% higher efficiency. procedure duration also was comparable and averaged within 94 min for both devices. detailed data are presented in table 2 . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during 2018 and reached 56, 7% in total (2017-19.4%). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from 7.64% up to 43.7%. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by 23.7% compared to mcs + 9000 while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from 2010 till 2019. all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ 3.0 9 10 11 equal to 12 random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was 150.000/ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was 273.000/ll, with range from 182.000/ll to 397.000/ll. male were 77% of the donors and females were 23%. the single procedure usually took 45-70 min. the median platelet count collected was 4.0 9 10 11 , range 2-6.5 9 10 11 . the median processed blood volume was 3215 ml and median used acd-a was 352 ml. mean total volume of collected product was 312 ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso4) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso4 solutions with specific weight (y) 1.053 can to detect value of hemoglobin above or same with 12.5 gr/dl. but, cuso4 solution specific weight 1.053 can not to detect and elimination value of hemoglobin above 17 gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso4) with a specific weight (y) 1062 to detect value of hemoglobin below 17gr/ dl and determine value of hemoglobin above 17 gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above 17gr/dl with the test of 35 samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso4 solution with specific weight 1.062 in to detect and elimination value of hemoglobin donors above 17 gr/dl. methods: used the method cyanmethemoglobin and cuso4 (y) 1.062 determination value of hemoglobin donor. test results were analyzed with spss software version 23 using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso4 solution with specific weight (y): 1.062 in detecting and eliminating hemoglobin value donors above 17gr/dl. from data processing using spss with the wilcoxon test p value 0.02. summary/conclusions: it was found that the cuso4 solution (y): 1062 can detect hemoglobins value above 17gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version 23 with the wilcoxon test p value < 0.05. it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, 8194 whole blood donors (20,864 whole-blood donations) were included in the zinc study, 63% being female donors. previous zpp showed a statistically significant association (p < 0.001) with hb levels in females, but the size of the association was quite small (regression coefficient, b = à0.17, 95% confidence interval à0.22 to à0.11). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was 66 years until the end of year 2013 and since beginning of 2014 donors between 66 and 70 years have been able to donate blood. blood donation after the age 66 is possible if the donor has donated within the last 24 months. the upper age limit was raised up based on adverse event data from frcbs donors up to 66 years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from 2013 with upper age limit of 71 years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than 66 years have more adverse events compared to other donors. the most common donor adverse event for donors over 66 years, haematoma, was registered 211 times (0.05%). in the other age groups haematoma was registered 4408 times (0.56%) and the difference between the oldest age group compared to all other donors was statistically not significant (chi2, p = 0.012). vvrs with loc were registered 21 times (0.05%), vvrs without loc 25 times (0.06), and the total number of all daes was 293 (0.65%) in the age group 66 years or older. the respective numbers in the other age group were: 1152, 0.15%; 11055, 1.41%; 17550 (2.23%). the number of vvrs with and without loc, and total number of all daes in the age group 66 years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi2, p < 0.0001). summary/conclusions: donors over the age of 66 years have less donor adverse events than other age groups. decision to raise the age limit from 66 to 71 seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in 2016-2018 years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last 3 years (2016-2018 inclusive) were reviewed to look for reports of dvt post donation results: a total of 135 saeds were reported in uk from approximately 5.8 million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for 2% of the saeds reported and rate of dvt of 1 in 1.9 million donations collected. -case 1: a regular male whole blood donor in his early 30s reported worsening arm pain 12 days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case 2: a female donor in her mid-40s gave her sixth whole blood donation without event. 11 days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case 3: a female donor in her 20s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february 2013 to march 2015. the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study 6,955 whole blood donors were included, during the period of 2 years, 105 (1.50%) adverse donor reactions were reported. majority 89 (84.76%) of adverse donor reactions were mild in nature such as, sweating; 27 (25.72%), light headedness; 19(18.09%), nausea and vomiting; 15(14.28), allergy and bruises; 11(10.47%), sore arm; 9(8.58%) and hematoma; 8(7.62%) while 16 (15.24%) were severe adverse reactions similarly, anaphylaxis; 11(10.49%), loss of consciousness; 3(2.85%) and convulsive syncope; 2(1.90%). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with 18-23 years old at higher risk compared to 24-55 years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th1/th2 cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, 2008; ellertsen & hetland, clin mol allergy, 2009). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for 7 weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm (82%), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v 1-induced basophil activation in whole blood determined by cd63 expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct03198455, clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v 1 and anti-t3, were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for 2 months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in 20/9/2014. the data presented in this abstract is till 28/2/ 2019. data is collected from total of 114475 blood donors (86.67% male donors and 13.33% female donors). repeat donors accounted for 59.34% against 40.66% of first time donors. of the total number of donor adverse events recorded, 2.73% (2633) reported for male donors and 6.97% (1063) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group 18-24 years (male 5.45% and female 13%). among age group 25-40 years, (male 2.4% and female 4.02%), whereas in age group 41-65 years, (male 1.52% and female 2.11%) data analysis of total 6226 reported and registered donor adverse events, are categorized as hyperventilation (864), sweating (1458), dizziness (pre-syncopal 3160), loss of consciousness (416), vomiting (108), convulsions (220), hematomas with re-bleed (255), nerve irritation (8) and off-site reactions (333). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january 1 st 2016 to december 31 st 2018 and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january 1 st 2016 to december 31 st 2018 was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into 8 categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < 0.05 was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, 193,228 donors were registered from january 1 st 2016 to december 31 st 2018 and 41,037 (21.24%) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from 24.07% (15302/63563) in 2016 to 20.79% (13180/63394)in 2017 and further to 18.74% (12419/66271) in 2018 (p < 0.05). the specific deferral rate due to low hb also significantly (p decreased during these three years (86/1000 in 2016, 68/1000 in 2017, 58/1000 in 2018), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june (6/1000) and then increasing with a peak in october (19/1000) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of 4/1000 in june and increasing to 10/1000 in october (p < 0.05). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels 2017 thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of 28 days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from 2015 to 2018 on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the 7 days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in 2015-2016 and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in 2015-2018, 16,400 donation attempts led to onsite deferral for wnv risk; 87% at whole blood donation attempts, 13% at new donor examinations. in total 19,624 offsite deferrals could not be traced directly to a donation, but based on the next donation more than 90% were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from 920 in august 2015 to 1711 in august 2018. this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred (92% versus 95%); for wnv deferred new donors the return rate is 75% (versus 87% for no deferral). thus wnv deferral resulted in approximately 400-500 extra lapsing donors during these 2 years. however wnv deferred donors, that are older (odds ratio (or) 1.03; 95% confidence interval (95% ci) 1.02-1.03), of male sex (or 1.16; 95% ci 1.03-1.31) and whole blood donors as opposed to new donors (or 2.01; 95% ci 1.70-2.38) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since 2012 with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given 14 days of one tablet of iron supplementation (100 mg elemental iron) since 2012. an electronic questionnaire was sent to blood donors in 2017 to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: 3212 donors (male to female was 1:1.9) replied to the questionnaire. of them, 594 received iron supplementation (male to female was 1:1.6). most of the respondents (94%) had one or more donations in the preceding 12 months. of the 594 donors received iron tablets, only 246 (41%) took all; 69 (12%) took more than 50% but not all; 130 (22%) took some but less than 50% and 149 (25%) did not take any. gastrointestinal upset was reported in 113 (25%) donors and constipation seen in 193 (43%) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than 50%) to those who did not (took less than 50%), significantly more donors in the former knew vitamin c could enhance iron absorption (p < 0.05). on the other hand, no difference was seen when they were asked if 1) iron can only be absorbed from meat; 2) tea and coffee consumed during meal can enhance iron absorption; 3) everyone can take iron supplementation on their own; and 4) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by 27% for whole blood donors and 22% for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with 36 whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor 10,000 study in 2015. we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may 2015 and december 2017. participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer 2018. we included the 1435 participants (596 men and 480 premenopausal and 359 postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = 31), good (n = 483), very good (n = 684) or excellent (n = 237) health tended to report improved (58%), similar (63%), similar (55%) or poorer (55%) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal 51%, post-menopausal women 38%), or = 1.37 95% ci 1.02-1.85). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in 2018. methods: the data of volunteers from january to december 2018 were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were 9081 blood donors in 2018, 69 (0.76%) of whom had adverse reactions and 9 causes were induced, among which mental stress was the most common factor that accounted for 78.26% (54 cases). there was no significant difference in the incidence of adverse reactions between men and female (p > 0.05). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < 0.01). when it comes to age, the incidence was different and the 18-25 age group was the highest (1.61%). among different blood group donations, there was no significant difference (p > 0.05). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least 8 weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to 24 times per year (if spaced more than 14 days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥4 whole blood donations or 16 plasmapheresis or plateletpheresis donations, and female donors with ≥3 whole blood donations or 12 component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past 2 years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ 2.07. the repeat donors took iron supplements for 4 weeks and the same tests were repeated after 2 and 4 weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of 53 male and 57 female repeat donors were recruited, and 30 each male and female ft-ra donors were recruited to the control group. after 4week iron supplementation, among male donors, the prevalence of: low hb level (<13.0 g/dl) decreased from 24.5% to 1.9%; low ferritin level (<15.0 ng/ml) decreased from 58.5% to 3.8%; high tibc level (>380 lg/dl) decreased from 66.0% to 37.5%; low transferrin saturation (<20.0%) decreased from 58.5% to 35.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 28.3% to 2.3%. among female donors, the percentage of: low hb level (<12.0 g/dl) decreased from 43.9% to 9.8%; low ferritin level (<15.0 ng/ml) decreased from 78.9% to 11.8%; high tibc level (>380 lg/dl) decreased from 68.4% to 24.5%; low transferrin saturation level (<20.0%) decreased from 70.2% to 22.4%; and ide (stfr/ferritin ≥ 2.07) decreased from 94.7% to 44.4%. in total, 15 male (28.3%) and 29 female (56.9%) blood donors reported undesirable side effects related to iron supplementation. a total of 38 male (71.7%) and 36 female (70.8%) blood donors were administered iron supplementations for 28 days. 89 participants (85.6%) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, 4-week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than 3 mg/l has been independently associated with a 60% excess risk in incident of coronary heart disease (chd) as compared with levels less than 1 mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci4100 (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl 180, erba mannheim). data was analysed using stata version 15 (stata corp) statistical software. results: in total of 313 blood donors, 278(90.8%) were males and 28(9.2%) were females, the mean age was 32.3 ae 9.3 years. two hundred and thirty four (74.8%) were first time donors and 79(25.2%) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors (0.95 ae 3.7 vs 0.35 ae 1.7 mg/l, p = 0.06) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors (87.56 ae 23.9 vs 46.44 ae 31.4 ng/ml, p = 0.02). interestingly, levels of serum hs-crp were significantly higher in male than female population (0.52 ae 2.4 vs 0.17 ae 0.2 mg/l, p < 0.03) and smokers than non-smokers (1.4 ae 4.6 mg/l vs 0.4 ae 1.9 mg/l, p = 0.02). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes 250 mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = 13.5 g/dl for male and > or = 12.5 g/dl for female • inter donation interval = 56 days • 5 donations/year for male and 3/year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: 1056 first time and regular donors accepted to enroll in this study. only 38 female donors (3.6%) participated to this study. 11.3% of the participants were first time donor. 21% of male and 26% of female frequent donors are iron deficient 173 out of 1018 male blood donors were iron deficient (17%) with serum ferritin < 30 ng/ml. 98.8% were repeat donors. 7 out of 38 female donors were iron deficient (18.4%) with serum ferritin < 13 ng/ ml, all were repeat donors. 19.2% of repeat donors were iron deficient 3/180 of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a 12-month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years 2014-2018 in the group donors of aged 18-24. we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years 2014-2018 the total number of 1904 adverse reactions among the donors was recorded which is 0.39% of the total number of collected donations. 50% of the adverse reactions occurred in the group of donors aged 18-24. vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled 50.2% of all adverse reactions. in the group of donors ages 18-24 it totalled 27% of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled 29.8%, in the analysed group of donors 15.96%. vascular reactions (bruises) totalled 10.2% of all adverse reaction, in the analysed group 5.6%. the remaining adverse reactions totalled 9.8%. summary/conclusions: 1. vasovagal reactions (with and without fainting) were proved to be 2 most common adverse reactions in the group of donors aged 18-24 i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. 2. it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. 3. it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" (2 nd day) and "old" (42 nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd235a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd235a, cd44, cd47, cd55, cd59, and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0.5 lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units (201 ae 139 mvs per 1 ll), as compared to the microvesicles in the "fresh" (67 ae 53 mvs per 1 ll). at day 2, the microvesicles had elevated expression levels of cd47 and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd55 and cd59 molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of 42-day vesicles. the phagocytosis index was significantly higher (28.44%) for the microvesicles isolated from 42-day stored rbcs than for microvesicles from the 2background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of 5-7 days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to 7 days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in 30% plasma/70% ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into 3 units (n = 6 of each) on day 2 and stored either at room temperature (rt; 20-24°c with agitation) for 5 days, cold stored for 21 days (2-6°c no agitation) or cryopreserved (à80°c with the addition of 5-6% dimethyl sulfoxide) for 33-109 days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at à80°c. the mean size and concentration of microparticles were measured using nanosight ns300 nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < 0.05 was considered significant. results: at day 2, sheep pcs had a microparticle concentration of 3.77 9 10 10 ae0.84 9 10 10 microparticles/ml with a mean size of 156.6 ae16.7 nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration (4.39 9 10 10 ae0.81 9 10 10 microparticles/ ml; p = 0.0087) and the mean size (170.9ae 20.5 nm; p = 0.0008) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day 21 was comparable to room temperature pcs stored for 5 days (165.4 ae17.5 nm vs. 163.6 ae20.9 nm; p = 0.4081 and 4.11 9 10 10 ae 0.54 9 10 10 microparticles/ml vs. 3.82 9 10 10 ae0.71 9 10 10 microparticles/ml; p = 0.16 respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the 21 days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and 2,3-dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p50: the oxygen tension (po2) at which 50% of the hemoglobin is saturated with o2. an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po2. this relationship is dependent on temperature, ph, pco2 and 2,3-dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p50 is at a po2 of about 26 mm hg. not much is known about p50 of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. 2010;50:2386-92). methods: rbcs were prepared in sagm (n = 3) or pagggm (n = 3). pagggm is designed to better maintain both atp and 2,3-dpg during storage. rbcs were stored at 2-6°c and sampled on day 1, 35 and 56 for (internal) ph, atp, 2,3-dpg and p50. p50 was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment (2%) using n2 gas. p50 was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. 2,3-dpg content of sagm-rbcs decreased during storage and was below the detection limit after day 14. 2,3-dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day 7 on. at day 56, pagggm-rbcs still contained 2.3-dpg (2.2 lmol/g hb). p50 values decreased during storage from 26 mmhg at day 1 to 20 mmhg at day 56 for sagm-rbc and from 31 mmhg to 26 mmhg for pagggm-rbc. p50 values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p50 decreased in all rbcs. the p50 was higher for the pagggm-rbcs during the whole storage period. the higher p50 in pagggm-rbcs seems to correlate with the higher 2,3-dpg content in these cells. background: in belgium 100% of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of 3.0 9 10 11 per platelet concentrates (pc). therefore routine pools are produced with 6 buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from 6 to 5 bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with 5 bc and 250 ml platelets additive solution (pas) instead of 280 ml for 6 bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ 3.0 9 10 11 platelets with a ratio plasma/pas between 32 to 47% required for pi. after validation, deploy a dual pooling strategy (5 or 6 bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce 44 ml bc with 40% haematocrit (htc) and > 90% platelets recovery with average platelets content of 1 9 10 11 . 5 random bc are pooled with 250 ml or 6 bc are pooled with 280 ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl 80; horiba). results: during the study 45594 bc were processed into 8225 pools (3756 (45.7%) with 5 bc and 4469 (54.3%) with 6 bc). before tacsi separation, bc mixture with pas-e had volumes of 421 ae 9 ml (5 bc) and 491 ae 8 ml (6 bc) with respectively htc of 19 ae 1% and 20 ae 2%. the plasma/ pas ratio was 37 ae 1% in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was 3.8 9 10 11 ae 0.5 with 5 bc and 4.7 9 10 11 ae 0.5 with 6 bc (average ae standard deviation). pools below the limit of < 3.0 9 10 11 were 139/3756 (3.7%) with 5 bc and 1/4469 (0.02%) with 6 bc. the platelets concentrations (10 3 /ll) were 1395 ae 167 (5 bc) and 1491 ae 155 (6 bc). platelets recovery was 85% ae5 for 5 bc and 86% ae 6 for 6 bc. summary/conclusions: 45594 bc could theoretically produce 7599 pools of 6 bc or 9118 pools of 5 bc. this means a maximum potential gain of + 20% pc. in practice during shortage periods we switched from 6 to 5 bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods (626 pc, +8%). the disadvantage of pooling randomly 5 bc is that 139 pools contained less than 3.0 9 10 11 platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the 5 bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica 2018, pmid: 30115655). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and 4°c in the presence or in the absence of caspase-3 inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: 13 ae 1% vs. 5 ae 1% p = 0.018). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: 6.13 ae 1.89 vs. 18.53 ae 3.64, p = 0.038). accordingly, a decrease of the procaspase-3 level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase-3 inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: 74 ae 3% vs. 22 ae 3%, caspase 3 inhibitor vs. ionomycin, p = 0.005). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o 2 , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o 2 (so 2 )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within 24 h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for 3 h on an orbital shaker (50 rpm) at 22°cae2 and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at 4°c. a g-irradiation treatment (25 gy, gammacell 3000 elan, theratronics) was applied at day 2 or 14 and the rccs (expiry dates at day 16 or day 28, respectively) were stored until day 43. hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so 2 values were of 63.7%ae18.4 (n = 12) in control and of 20.8%ae9.8 (n = 12) in treated bags, and reached 90.8%ae9.1 and 6.6%ae5.9 at day 43, respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around 70 mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day 2 and the values stayed equivalent through end of storage (at day 16, hemolysis (control) = 0.37%ae0.24, hemolysis (treated) = 0.34%ae 0.15, p-value > 0.9999). when irradiated at day 14, hemolysis was lower (p-value = 0.033) in treated rccs at the end of storage (day 28, 0.67%ae0.16) compared to control (1.06%ae0.33). seven days post-irradiation, two-third of the control rccs were above the limit of 0.8% whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o 2 content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day 2 and was an advantage when irradiated at day 14. importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o 2 depleted rbc. in summary, the reduction of o 2 level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, 2017] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as 0.5 ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a 3d printed support. the accuracy of flow rate measurements between 0.5 ml/min and 1.5 ml/min was evaluated as well as the optimal measurement time. for 4 different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under 8% was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under 3% for a measurement time of 2s. the mean error is still under 5% for a measurement time of 0.5s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags 2, 3 and 4. hematocrit estimation for bag 1 diverges from reference for values above 6%. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under 8% without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood (450 ml) was drawn into compoflow blood bags containing cpd and sag-m from 10 healthy controls and 10 newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing 20 ml prp (range 270-320 9 10 9 platelets/l). platelet quality tests were performed on days 0, 1, 3, 5 and 7 of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd41, cd42b, and cd62p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd40l and scd62p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included 7 males and 3 females. the mean age was significantly lower in the control group, 35 years (22-55 years), than in the hh group, 55.7 years (29-77 years) (p = 0.004) while ferritin levels were significantly higher in hh patients (median 847.5, range 498-1889) than in controls (median 45.8 ng/ml, range 9.28-97 ng/ml) (p < 0.001). in the hh group, 5 each had the c282y/c282y and c282y/h63d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < 0.05) with the exception of glucose (higher in hh patients on all time points, p < 0.05). platelet aggregation and the expression of activation markers (cd62p and cd42b) on platelets and in the supernatant (scd62p and cd40l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd42b expression decreased and cd62p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = 16) were collected using a trima accel platform in 40% plasma/60% pas (ssp+). after resting for 1 h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (<6 h; t0), and the others remained within the shipper, without agitation. the second component was removed at 6 h post-collection (t6), and the third was removed at 23.5 h post-collection (rounded up to 24 h; t24). platelets were tested on day 1, 5 and 7 post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < 0.01 was considered significant. results: platelets held without agitation for 24 h consumed significantly more glucose than those removed at 6 h or immediately upon arrival (p < 0.0001), even on day 1 post-collection. this was accompanied by increased lactate production (p < 0.0001), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t24 platelets (p < 0.0001), and on average it was 0.4 ph units lower than in platelets held in the shipper for 6 h or less. however, the ph remained above 6.9 in all components. mean platelet volume was also reduced in t24 platelets (p < 0.0001), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd62p and microparticle generation were significantly higher in the t24 platelets throughout the storage period (all p < 0.0001), suggesting platelet activation. release of scd62p was also increased in t24 platelets (p = 0.0006), whereas extended storage in a shipper did not affect release of rantes (p = 0.4488). adp-induced activation of glycoprotein iib/iiia, measured by pac-1 binding, was decreased in t24 platelets (p < 0.0001), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = 0.0001) and adp (p = 0.003) were significantly lower in t24 platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for 24 h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to 5 days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after 5 days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, 9, 2018) . our new target is to extend this research in 2 extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from 5 to 7 days. methods: in this study, platelets were collected from 11 normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of 450 ae 10 ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc 1200). samples were drawn aseptically with a needless access coupler (cair-lgl) on days 1, 5, and 7. platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day 5 and day 7, maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days 5 and 7 of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of 7 days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at 4°c or room temperature (rt) over 10 days for 4 apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with 5% or 10% dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either 5% or 10% dmso. results: for pcs standard quality assurance tests did not show a major difference between 4°c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day 1-7, p < 0.001 and day 10, p < 0.02). for red cells, we found by rt-dc no impact of gamma irradiation with 30 gy over the entire storage period of 42 days assessing 3 different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation (0.052 for 5% dmso versus 0.034 for the control without dmso; p < 0.00004). however, this did not differ to high extent whether 5% or 10% dmso were used for cryopreservation (0.035 and 0.041, respectively; p < 0.02). hpsc viability was lower after cryopreservation using 10% dmso in comparison to using 5% dmso. overall, blood cell regeneration is comparable between 5% and 10% dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight (16 h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day 0 was separated into plasma, bc and red cell concentrates either at day 0 or at day 1. bcs were then stored until the pooling step at 20°c without agitation and pcs were prepared at day 1 by pooling 5 isogroup bcs. seven "16 h-pcs" were prepared from bcs stored for 16 h (whole blood separation at day 0) and six "90 min-pcs" from bcs stored for 90 min (whole blood separation at day 1). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd42) and of activated platelets (marked with cd62 the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: 32 dd-bc-pc were prepared with 8 bc and 280 ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a 600 ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within 1 h of preparation and after storage for 8 h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po 2 , pco 2 , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = 32) was on average 6.9 ae 0.3.10 11 in a volume of 393 ae 6 ml. the mean of plasma ratio was 42% [min: 39.3max: 43.9]. all pc contain <1.10 6 wbc [min: <lq; max: 0.38]. po 2 decreased from 166 ae 12 mmhg after separation (1 h) to 44 ae 18 mmhg after 8 h, ph (+22°c) and pco 2 was stable during the same time period. glucose decreased from 9.0 ae 0.3 mmol/l (1 h) to 8.7 ae 0.3 mmol/l (8 h) while lactate increased from 6.07 ae 0.46 mmol/l (1 h) to 6.57 ae 0.54 mmol/l (8 h). the vpm was stable with 7.6 ae 0.2 at 1 h and 8 h. ldh increased from 120.4 ae 8.6 u/l at 1 h and 125.8 ae 10.3 u/l at 8 h as p-selectin 45.7 ae 7.6 ng/ml at 1 h and 57.7 ae 7.0 ng/ ml. all units have maintained swirling. the changes observed during the 8-h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of 8 h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in 2018 the ibts collected 130,183 whole blood units and performed 9,654 platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september 2017, the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years 2016 to 2018 were analyzed. in 2016, the ibts prepared 5,609 orbisac platelet pools of which 5,477 were qc tested. in 2017 5,031 orbisac and 1,625 tacsi platelet pools were prepared and tested. in 2018 6,783 tacsi platelet pools were prepared of which 4671 were qc tested. qc data comprised pc volume, platelet content (10 9 /unit), residual leucocyte content (10 6 /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the 10,508 o-pcs tested in 2016 and 2017, mean pc volume was 292 ae 9 ml. initially the t-pcs mean volume was 273 ae 13 ml but more recently the volumes were increased by addition of a larger volume of additive solution to 303 ae 13 ml. platelet content (10 9 /unit) was significantly improved in t-pcs (384 ae 47) compared to o-pcs (356 ae 50) [p < 0.01] as well as residual leucocyte content (10 6 /unit) which was lower in t-pcs (0.05 ae 0.04) compared to o-pcs (0.1 ae 0.2) [p < 0.01]. ph at expiry was lower in t-pcs (7.32 ae 0.07) compared to 0-pcs (7.38 ae 0.06) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately 48 t-pcs per hour compared to a lower throughput of 20 o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a 10% increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga 1 , f puente 1 , a aranda 1 , j domingo 1 , l call en 1 and a bah 2 1 banco de sangre y tejidos de arag on, aragon, spain 2 terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes 44,000 whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november 2012 and routine use started in july 2013. in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november 2012 to improve transfusion safety. in november 2015, the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in 2012, 369 whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from 2013 to 2018, qc data of rccs (n = 3025), plasma (n = 660) and pcs (n = 660) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from 2015 to 2018 were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: 296 ae 21 ml vs 251 ae 19 ml; hb: 59.9 ae 6.2 g/ unit vs 49.2 ae 7.8 g/unit; hct: 59.5 ae 2.7% vs 56.1 ae 6.3%). control plasma units had higher volume (280 ae 17 ml) and lower residual platelet content (9 ae 7 10 9 /l) compared to reveos plasma units (volume: 249 ae 23 ml; residual platelet content 19 ae 13 10 9 /l). for pcs, volume was higher for units processed with reveos compared to control (350 ae 20 ml vs 310 ae 10), but no statistical difference was observed in platelet content (reveos: 361 ae 60 9 10 9 /unit; control: 386 ae 80.10 9 /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the 5 years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from 4% to 0% between 2013 and 2018 and platelet content in pcs increased from 370 9 10 9 /unit (first six months in routine) to 390 9 10 9 /unit. plasma volume increased from 249 ml during validation to 267 ml in 2018. survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from 10% in 2015 to 59.7% in 2018 with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations (450 ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm (100 ml) and then filtered by gravity in average 2-3 h after blood collection to obtain leukoreduced rbc. average temperature during filtration was 26°c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = 27) were performed. rbc characteristics post-filtration and separation were as follows: volume (266.8 ae 17.1 ml), hematocrit (57.8 ae 2.1%) hemoglobin (19.8 ae 1.1 g/dl) and hemoglobin per unit (53.0 ae 5.4 g); residual platelets (11.7 ae 2.2 x10³/ll); residual wbcs (0 à 0.004 9 10³/ll). the mean filtration time was 19 ae 3 min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between 2 and 8 h from collection using the 3c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day (42 nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate 4 whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in 2018. the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to 8 h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, 1005 therapeutic units of lpcs were produced after overnight hold: 38 pools of 4 ipus and 966 of 5 ipus were made using platelet pooling set (terumo bct), using 250 ml ssp+ solution (macopharma). results: 42 therapeutic units of lpc were analyzed. the mean volume of lpc was 387 ae 13 ml, platelet content was 3.67 ae 0.46 x10 11 , and white blood cells (wbc) count was 0.15 ae 0.13 x10 6 , 100% all lpcs had wbc below 1 9 10 6 . ph measurements were made on the fifth day of storage, the ph was determined in 15 units of lpc and was 7.2 ae 0.04. summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for 5 days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term (10 days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about 250 ml) were obtained from a healthy blood donors of type o blood group and divided into a 30 ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd62p, gpiba (cd42b), annexin v), degree of reactive oxygen species and sialidase activity on the 7th, 10th and 12th day, and phagocytosis using the hepg2 cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the 2nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day 12 and sialidase activity was maintained lower than that of control group on day 12. the degree of phagocytosis by the hepg2 cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for 2 h and 24 h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos 3c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of 2 h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within 8 h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately 4 to 6 ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool (4 units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately 19 units of whole blood using the reveos system and approximately 4 units of pooled platelet products. a total of 19 units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the 19 units, 10 units have been processed on the reveos device using the fresh procedure (within 8 h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a 22-min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn 2000 cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than 1 million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = 4) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax 20 (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including 40 units in 450 ml quadruple bags (450q) with/without integrated leucofilter and 18 units in 350 ml quadruple bags (350q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into 2 groups, mixed at 2 revolutions per minute (rpm) for 2 min (n = 30) and 30 min (n = 28), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both 450q and 350q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for 2 or 30 min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for 30 min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as 3, 5 or 10 min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year 2000, universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs2 for rcc in comparison with sepacell tm r-s11 and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over 40,000 whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from 5 (routine in italy) to 4. the 4-bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the 4-bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from 4-bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory (2.0 9 10 11 cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february 2018 to february 2019. study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version 23.0 results: a total of 2880 blood products were available at our institute including 960 each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was 3.5%. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. 102(10%). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with 02 common causes, including the expiry of unused products 60(59%) followed by broken bags 30(29%). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early 2000s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of 1 liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of 2010-2017. results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than 200 blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are 18 blood centers in the republicone for each of the 18 administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since 2012, the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in 100% of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group 0 rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis 30 units of pooled platelet concentrate derived from buffy coats and 30 units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl 80 analyser. 1. pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: 4.12 9 10 11 /unit ae 0.50 the number of platelets after the reconstitution: 3.40 9 10 11 /unit ae 0.50 the loss in the number of platelets: 17.5% 2. platelet concentrates derived from apheresis the number of platelets before the reconstitution: 3.68 9 10 11 /unit ae 0.55 the number of platelets after the reconstitution: 2.91 9 10 11 /unit ae 0.49 the loss in the number of platelets: 20.7% summary/conclusions: 1. reconstitution results in the loss of 17.5% of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. 2. the average number of platelets after the reconstitution is 3.4 9 10 11 /unit, which fulfils the quality requirements. 3. reconstitution results in the loss of 20.7% of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. 4. the average number of platelets after the reconstitution is 2.9 9 10 11 /unit, which slightly deviates from the quality requirements. 5. the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf4 was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf4 antibody on the column. hereafter, pf4 was purified with concentration tubes having 5 kda molecular weight cut-off and dialyzed via dialysis tubing with 3.5 kda cut-off. pf4 concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf4 specificity. molecular weight of obtained pf4 was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf4 had 30 kda molecular weight which is corresponding with a tetramer pf4. in addition, elisa showed that the pf4 qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf4. summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf4. nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf4 is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf4. unused human pcs and specific anti-pf4 antibody can considered as an alternative to separate the pf4 particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first 2 weeks of storage at 4°c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from 7 days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: 34 units of whole blood were leucodepleted within 24 h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for 7 days at 4 ae 2°c. on the 8 th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of 35 days at 4 ae 2°c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, 2.3 dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units (0.23 ae 0.2.10e6/u)) while maintaining 80% of the initial platelet content (85 ae 20.10e9/u). mean volume (464 ae 24 ml), hemoglobin content (54 ae 6 g/u) and hematocrit (36 ae 3%) was within conformance range. after 7 days of storage, 50% of the initial platelet content at collection is maintained and mean hemolysis is 0.27 ae 0.15%. rcc processing at day 8 was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume (266 ae 20 ml), hematocrit (56 ae 3%), hemolysis (0.18 ae 0.19%) were in conformance with mandatory standards at day 8. after 7 days of storage (i.e. day 14 after collection), rcc quality parameters were maintained with however an increase in hemolysis (0.34 ae 0.36%) which was above usual values routinely observed with rcc produced from day 1 processed units. 3 rcc out of 34 were above the maximum limit of 0.8% at day 14 after collection such increased hemolysis was more pronounced at day 28 after collection (0.70 ae 0.45%) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold (20% of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after 7 days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within 21 days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland 1,2 , f mahmood 3 , l nissen-meyer 1 and i nentwich 1 1 immunology and transfusion medicine, oslo university hospital 2 immunology, university of oslo, oslo 3 immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, 2005) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were 1) to assess sensitization profiles against an array of 55 allergens in 100 randomly selected blood donors and 2) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from 100 blood donors randomly selected on one donation day outside the pollen season. we used 300 ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for 55 analytes (euroline atopy screen, euroimmun, germany), which comprised 28 food allergen extracts and single allergens, 24 inhalant allergen extracts, 2 insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of 44 serum samples for ige against 55 allergens in less than 3 h. results are expressed as east (enzymoimmunoassay) classes: class 0 (no sensitization), 1-2 (weak sensitization), 3 (moderate sensitization), 4-5 (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class 5none detected. class 4, sesame seed: 1. class 3, apple 1; hazelnut 2. class 2, apple 1; almond 2; hazelnut 1; sesame seed 1; soybean 1; cow milk alpha-lactalbumin 3. class 1: 26 donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class 5, 4 and 3 none, class 2, wasp venom 7; honey bee 2. lower classes (0-1) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor (1%) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to 5 days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = 53) were collected, stored at 22°c with agitation, and tested at day 1, day 5 and 7 post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd61 + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a 2 (txa 2 ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin-1 (rca-1; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to 4-fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to 4-fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only 27% of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa 2 was also higher in the arachidonic acid responders (p = 0.027). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day 5 was weakly associated with donor age (p = 0.009, r 2 =0.2613) but not bmi (p = 0.9143, r 2 <0.001), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than 10%) are b-thal-het that meet the criteria for blood donation (hb > 13.5 g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from 10 healthy non-smoker donors (5 b-thal-het carriers and 5 controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca 2+ and proteasomal activities were determined. for statistical analysis, significance was accepted at p < 0.05. samples from the red cell units were collected aseptically, processed (dual centrifugation at 3,000 g for 15 min) and stored at à80°c. processed samples were thawed, and then analysed using the nanosight ns300 nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d2, red cell units contained an average of 8.1 9 10 9 ae 4.0 9 10 9 mvs/ ml. the mean size of these mvs was 112.8 ae 10.5 nm and the mode size was 79.9 ae 10.3 nm. the concentration of mvs increased gradually throughout storage (p = 0.0276), reaching a maximum at d42 of 32.4 9 10 9 ae 1.8 9 10 9 mvs/ml. both the mean (p < 0.0001) and mode (p < 0.0001) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d2 vs. d7: p < 0.0001 for both mean and mode). by d42, mean and mode size of mvs was 176.1 ae 4.8 nm and 171.8 ae 5.6 nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than 400 nm in red cell units. both the concentration and size of mvs present in red cell units increased during the 42 days of routine storage. the concentration of these mvs was approximately 100-fold higher than we had previously detected using flow cytometry (aung, pathology, 2017) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than 24 h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six 14 day old leukocyte depleted red blood cells (ld-rbc) and six 4 day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated (25 gy). a volume of 350 ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for 6.5 min at 2888 9 g at 4°c (hettich roto silenta 630 rs) before removing the supernatant using compomat g5 extractor. wash procedure was repeated twice using 450 ml sagm solution, and after removal of the last supernatant, 100 ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after 24 h, 7 days and 14 days storage at 4 ae 2°c. results: all washed ld-rbcs met european specification for haematocrit (0.50-0.70) and all but one for hb content (≥ 40 g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than 0.8%) 14 days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration 14 days after washing and irradiation did not exceed those levels found at the end of shelf life (day 35) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than 0.05 mg/dl (0.5 mg/l). our iga method 0 s lower limit for detection is 0.05 mg/l and all results were below this level. total albumin were well below finnish specification (<250 mg/unit). background: room temperature has been the standard storage condition for platelets since the 1970s, when it was shown that this improved in vivo survival compared to when stored at 4°c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at 4-9°c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from 5 buffy coats and 300 ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for 23 days at 4-9°c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d1-d16: 7.05-6.77 with platelet count 400 9 10 9 /u and glucose still at 2 mm at least until 16 days of storage. platelet activation maintained acceptable levels with cd62p expression < 30%, while ps exposure increased rapidly; >20% after 6 days of storage. aggregation tests showed functional platelets until 13 days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at 4-9°c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at 4-9°c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets 0 rhd negative were frozen in 5-6% dmso and stored at à80°c for 6 months. cpp were thawed at 37°c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was 24%. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount 194 9 10 9 /unit, concentration 0.73 9 10 9 /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value 3 in fp and on value 1 in cpp. the average plasma content in fp was 31% compare to 3.22% in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low (0-1:2). cpp had faster clot initiation (rotem clotting time (ct) in cpp 69.2 s, fp 81.8 s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp 39.8 mm, fp 53.2 mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of 24 ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, 250 ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept 1 h on bench in room temperature followed by 1 h on agitator at 22 ae 2°c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of 420 ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of 7 days. results: during validation of the method, 12 pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po2, pco2 and lactate on day 2, 5 and 7 of storage. the platelet count was 2.27 ae 15 9 10 11 per unit on day 2. the ph value was 6.9 on day 2, 7.1 on day 5, and 6.9 on day 7. the glucose concentration decreased from 5.8 to 3.5 and 1.6 mmol/l on day 2, 5 and 7, respectively. the mean po2 level was 25.3, 25.0 and 28.7 kpa while the mean pco2 was 3.8, 1.7 and 1.8 kpa and the lactate concentration was 5.9, 9.6 and 12.9 mmol/l on day 2, 5 and 7, respectively. since routine implementation of the method in april 2017, regular quality controls showed an average of platelet count of 2.63 ae 34 9 10 11 (n = 161) with a volume of 204 ae 7 ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of 7 ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from 11 published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested 4-10 ml sample volume per culture bottle. the 11 studies classified results based on aabb bulletin 04-07 definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta 3d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of 217,932 platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day 3, 4, or ≥ 6, and represented 8.9%, 44%, and 47% of the units tested, respectively. true positives were detected in 174 platelet units representing 0.08% of the total platelets tested. the majority were reported from platelets tested on day ≥ 6 with a total of 128. data showed the bta 3d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤27 h with the majority detected in ≤24 h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in 5 of the 11 studies for platelets tested at 3 days and ≥ 6 days with a total false positive rate of 0.27% (range of 0-1.1%). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, 1,136 culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at 3 to 8 days post collection provided evidence that the bta 3d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert 3d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day 3 and up to day 7 when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts (0.5-2 mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with 2 mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~2 nm, medium 30 nm and long~100 nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at 0.5-1.0 mg/ml hsa coated particles with 0.57 ae 0.36 and 0.54 ae 0.39 pg/platelet, respectively. however, the 1.0 mg/ml hsa coating resulted in~100-fold higher binding affinity to platelets than particles coated with 0.5 mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of 80, 220, and 360 pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day 3, by the increased expression of cd36. the percentage of cd36-positive cells among the population of large platelets did not change during storage. on the day 3, increased expression of cd42b and cd62p was detected, but only on large platelets. small and medium-sized platelets had increased cd62p expression only on day 5. the expression of cd42a on total platelets increased significantly on day 3, and stayed unchanged until day 5. the same pattern of cd42a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd41-positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the 5-day storage period. increased annexin and pf4 concentrations were detected on day 5. the concentration of b-tg increased on day 3 of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february 2009, the transfusion service in our hospital has managed 3 autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of 3 types of autologous blood components in a single institution over an approximately 9-year period. methods: between february 2009 and december 2017, we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of 9,193 patients (9% of whom received operations) received blood transfusion, of which 5,080 (55%) received autologous blood transfusion alone, 2,101 (23%) both autologous and allogeneic blood transfusions, and 2,012 (22%) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was 78%. pacs units were transfused to 5,830 patients (60%), pad units to 2,845 patients (30%), and anh units to 982 patients (10%), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was 98.6%. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were 12 and 0.1%, respectively, of which the most common was febrile non-hemolytic transfusion reaction at 9 (75%), followed by allergic reactions at 2 (17%), and hypotensive transfusion reaction at 1 (8%). the severity of adverse reactions to pad transfusion was grade 1 (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was 0.55%, that to frozen pad units was 0.05%, and that to autologous ffp units was 0.10%. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was 78%, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp215 (haemonetics corp.), is approximately 1%. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: 3-40%) for up to 24 h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for 48 h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in 100% plasma were washed on day one after blood collection using the automated closed-system cell processor acp215 (n = 6). wpc, which were rested 1 h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at 20-24°c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was 110.9 ae 1.9 (910 10 /l) and the volumes of the control and test units after the division were 120 ae 11 and 123 ae 12 (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than 6.85 during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco 2 , po 2 , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd62p expression were significantly higher in the test units than in the control units on days three (52.6% ae 6.0 vs 40.9 ae 5.7, p < 0.01); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd42b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a 2-month field trial over 10,000 data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < 0.0001) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ 0.3246). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > 90% of ld components have less than 1 9 10 6 rwbc/unit. for ld failures (n = 14) there was a good correlation (r 2 =0.9848) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around 6 and 8 rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < 0.0001) in rrbc contamination, with lower levels in apheresis platelets (median = 15 rbc/ll, n = 1820) compared to those produced from buffy coats (median = 783 rbc/ll, n = 77) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately 2-3 h for 60-90 samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was 60 to 220 nm (without oxidation). the heights (h) of dips were 4 to 10 nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only 10% to 15% of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from 8 donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn 2+ (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to 600 nm. the young's modulus e = 10 ae 5 kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn 2+ ) significantly increased the absolute value of the young's modulus of rbc membranes up 2-3 times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from 200 nm to 600 nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january 2016 to december 2018, in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january 1st 2016 to december 31st 2018. results: a total of 54,599 whole blood units and 3,166 apheresis platelets were collected during the study period. of the 66,289 blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of 2,891 (4.4%) blood components were discarded, of those 75.4% due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in 2016 was 5.6%, in 2017 was 3.8% and 0.2% in 2018. similar tendency was shown in the pooled platelets for 2 consecutive years with 4.8% (2016) and 3.8% (2017), but with an increase in 2018 (7.7%). the rate of apheresis platelets had a more variable behaviour from 2016 to 2018 with rates of 1.3%, 0.6%, and 0.9% respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from 1 buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, 31 apheresis pcs and 66 pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba1c from red cells. triglyceride levels > 2.5 mmol/l and hba1c levels > 42 mmol/mol were defined as high. results: twenty two percent (21/97) of the donors were marked as 'rapid acidifiers' and 71% (15/21) of these donors had health issues. 'rapid acidifiers' were of age 57, 31-69 (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type 2 diabetes, indicated by high cholesterol and/or triglycerides, high hba1c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' (29%) did not have high triglyceride or hba1c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, 2012). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than 10 visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, 2016). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis (1246.8 ae 90.1/ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from à600 mv to +1100 mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse 80i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + 500 mv and + 800 mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than 10%) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from 4 healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph 7.4. we used ultraviolet (uv) irradiation of blood or nano 2 as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico 2800, usa) to measure the absorption and scattering of light (0.5 nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo2,l c hbo2 l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno2 -,l c methbno2 -l+ e methbno,l c methbno l+k+s/k 4 l (1), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano 2 action (up to 90%). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r 2 = 0.98). incubation of rbcs with cytoflavin leads to reduction of methb to hbo 2 . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr5, the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr5 leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl2 and mgcl2) in ph 7.2 containing 2 mm edta and 4%(w/w) dextran 40 without additive amounts of triton x100 was the most optimized buffering system for lcr5 filter back flushing. total cell content was also determined as 6.28*10 8 granulocytes, 4.27*10 8 lymphocytes and 0.8*10 8 monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis (2019) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of 5.2 g ig (70% intravenous -privigenand 30% subcutaneous -hizentra) and 26 g albumin (alburex) per kg plasma fractionated, corresponding to 998.000 g ig and 4.992.000 g albumin; based on present demand, this represents 97% and 100% self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted 6.000 g fibrinogen (riastap) and 6.000.000 iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of 5.000.000 iu at and 3.000.000 iu pcc, capable of ensuring self-sufficiency for naip regions until 2020. summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in 2017 in italy 923,944 litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, 151 of the 176 italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of 3 parts: a) "surrogate" bags (check-bags) of 250 and 700 ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period 2016-2018, at the pievesestina be of emilia romagna region 210,871 plasma units were frozen so as to allow complete freezing within 60 0 to a temperature below à30°c, in 8,349 freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be 108 tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of 8,349 freezing sessions carried out at the pievesestina be, 27 (0.3%) were detected to fail to reach à30°c at the core of the check-bags within 60 0 . of the latter, in most cases (70%) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (<24 units), ii) labelling time (<8 0 ), iii) optimal storage t (à40°c vs. à30°c), iv) optimal time between two openings of storage sites (>30 0 ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january 2018 -december 2018) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check (6 months) and data with double check (6 months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of 6818 from the first six months a 38 mismatch was found which equals 5573 dpmo and 4.1 sigma metric. and in a sample size of 5854 from the second six months a 25 mismatch was found which equals 4277 dpmo and 4.2 sigma metric. out of the whole 12672 pooled units 63 were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a 4.1 sigma metrics obtained from the first 6 months, were shifted to a sigma metrics of 4.2 in the second 6 months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from 150 ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (à20°c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced 7 mc; 1 mc as the control, 3 mc were lyophilized with excipient and 3 mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at 30 days at room temperature (30-32°c) and blood bank refrigerator temperature (2-8°c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher (9.8 iu/ml) than without excipient (4.5 iu/ml) and the storage at blood bank refrigerator (2-8°c) is better than at room temperature (30-32°c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature (2-8°c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from 2016 to 2017. they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of 27 patients were included (12 patients as control group) and (15 patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph 2.5 after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was 6.99 d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of 4.2 million habitants. annually, it collects about 100,000 whole blood and 3,500 apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for 7 days at 22°c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of 4 bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in 33% plasma and 65% pas and mcd were subsequently evaluated also in 38% of plasma and 62% pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days 1, 5 and 7. bacteria sterility test was performed at day 7 for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = 10) and ad pcs (n = 8) stored in 33% plasma showed at day 7 an average ph ≥ 6.9; swirl ≥ 2.5 and yield = 3.3 9 10 11 . their untreated counterparts showed average values for ph ≥ 7:0, swirl ≥ 2.9 and yield 3.3-3.4 9 10 11 . mcd stored in 33% plasma (n = 6) that underwent prt showed at day 7 average values for ph = 6.7, swirl = 1.2 and yield = 3.05. control mcd showed average values for ph = 7.0, swirl = 2.8 and yield = 3.1 9 10 11 . mcd stored in 38% plasma (n = 8) that underwent prt showed average values for ph = 6.6., swirl = 1 and yield = 3.1. their untreated counterparts had average ph = 7.1, swirl = 2.9 and yield = 3.2. total protein content in pcs derived from wbd (n = 12), ad (n = 15) and mcd (n = 27) was 22 g/l, 20 g/l and 20 g/l, respectively. while the coefficient of variation of wbd and ad ranged from 3% to 4%, plasma products 129 respectively, the one of mcd reached 14%. all prt-pcs were negative for bacterial growth at day 7. summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day 7 and passed the requirements of the eu guidelines (19 th edition). quality of mcd units met eu criteria at day 5; swirl decreased significantly at day 7 which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~25% of the annual produced buffy coat platelet concentrates (bcp) (~40.000 bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~25% pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period (2017) . this cost was offset by substituting a semi-automated production method for bcp, which was used in 2017 to produce 28.7% of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by 89.2%. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in 2018 by 0.19%. the extension of max. storage time from 5 to 7 days for 24% of the bcp-units that were pr resulted in decreasing our overall outdating rates by 29% (versus 2017). this reduction in outdating rates reduced our opc in 2018 by 2.2%. in 2018 we gamma-irradiated 25.9% fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~25% pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to 100% if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day 14 of storage then stored for a further 14 days. rcs were also irradiated on day 5 of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into 4 arms on day 1 of storage, with 10 units in each arm. all units received an irradiation dose of 25.9-48.3 gy. two arms remained as standard volume rcc and were either cor x-irradiated on day 14 of storage. the other two arms were both split into 6 rcs on day 4 of storage before being irradiated on day 5 (early arm) or day 14 (late arm) of storage. for each replicate in these arms, 3 splits were c-irradiated and 3 splits x-irradiated. all arms were tested a day prior to irradiation and 1, 7 and 14 days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and 2,3 dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, 1 split was tested on each testing day post-irradiation. a 2-way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (>75% of units with < 0.8% haemolysis) and 100% of units had atp levels above the recommended minimum for acceptable post-transfusion survival (2.3 lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz 1 , c coello de portugal 2 , m morales 3 , f solano 4 , c perez parrillas 5 , a rodriguez hidalgo 5 , t diaz rueda 5 and m flores 5 1 direccion, regional transfusion center toledo-guadalajara 2 transfusion service, toledo hospital complex, toledo 3 transfusion service, general university hospital of guadalajara, guadalajara 4 transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera 5 regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves 3 general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of 943,000 inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since 2008, rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from 5 to 7 days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last 8 years divided into four periods ( results: pc were predominantly obtained from whole blood collections with 85% of bc platelets/15% of apheresis platelets. 77% of the available bc were used in production for period a and 88% for periods b, c and d. after wastes of approximately 1.9%, the distribution of pc was stable for the 4 periods studied. 7075 pc were distributed for period a, 7047 pc for b, 7467 pc for c and 7083 pc for d. the % of pi platelets with 7-day shelf life available in the 3 hospitals was limited to 13% during period a. it was then increased to 14.1%, 20.9% and 26% for periods b, c and d respectively. the percentage of wastes was stable at 0.2-0.3% but the discards due to expiry went down from 24.24% (period a) to stabilize at 10.9% in periods b and c and 10.3% in period d. in the 3 general hospitals the expiry went down from 20% to 5.89%(hct), 29.4% to 14.5% (ughg) and 33.36% to 17.68%(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with 7-day shelf life allowed reducing discards for expiry from 24.24% to 10.3% in the last period analyzed at rtc and the 3 major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in 100% donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day 5. the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh-800, beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of 2-3. the average volume per unit of the pre-inactivation was 288 ml (278-302 ml) and post inactivation was 269 ml (254-284 ml), with average volume loss during inactivation was 36 ml (24-43 ml), corresponding to 13% (8-15%). the average platelet yield per unit pre-inactivation was 3.7 9 10 11 (3.0-4.4 9 10 11 ) and post inactivation 3.2 9 10 11 (2.8-3.9 9 10 11 ) with an average platelet loss of 13% (3-20%) . the average rbc contamination per unit (0.01-0.02 9 10 9 rbc/ml). the culture tests were negative, the average ph at day 1 was 7.4 (7.1-7.6), average ph at day 4/5 was 7.3 (7. 2-7.4 ). the average residual amotosalen concentration post treatment was 0.30 lm (0.23-0.35 lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below 2 lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of 106 independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of 2 ml pre-inactivation and another sample post-inactivation (16 h after pi). the platelet viability of each sample was evaluated by demonstrating the cd62p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of 106 independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd62p marker, was 18%, while the percentage of functional platelets post inactivation was 19%, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a 95% confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, 2016" in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than 10%. for the intermediate precision, the coefficient of variation was less than 15%, but for the pcs after treatment, this result rose up to 21%. the r² value for the linearity was 97.7%. the detection limit corresponded to a result of 6 edel and the loq (equal to 10xsd) is 19 edel. concerning roc curves, the men apcs threshold was 58.5 edel compared to women apcs with 62.5 edel. the threshold for bcpc was 54 edel. all of these results had 100% of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, 404 apcs (182 women and 222 men) and 263 bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, 14.0% of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was 89 edel. our study showed a threshold with 100% specificity and 90% sensitivity at 58.5 edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at 62.5 edel compared to 66.5 edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold (54 edel) was lower than abonnenc threshold which was at 59.8 edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a 3-ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to 1 lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: 1) complete treatment, 2) plasmas with mb without illumination, 3) plasmas without mb with illumination and 4) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol 1 where n = 20, were realized during the viro-inactivation process, t1 corresponds to a dosage of plasmas before treatment, t2 the plasma after the mb dry tablet passage, t3 is the time after illumination and t4 corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than 50%, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series 1 and 3, the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series 2 was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t4 (238 ae 16 edel at t3 and 209 ae 17 edel at t4). however, in the absence of mb (series 3 and 4), the results at t1 and t4 were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis (2019) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ 2.00 g/l was 94% (>70% required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were 75% and 90%, respectively. residual platelets were < 25 9 10 9 /l, leucocytes < 1 9 10 4 /l and red blood cells < 4 9 10 9 /l. all units had a protein content > 50 g/l. residual amotosalen was below 2 lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c3a and c5a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at 37°c was consistently short (6-7 min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine 8-10 to 6-7 min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included 5 experiments. for each experiment 5 male-donor, abo-compatible whole-blood derived plasma units (≥ 270 ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the 5-unit pools were split into 2 equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into 3 (≥200 ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from 5 single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, 2018) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ 18 lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, 2017) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ 18 lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade (0 to 70 lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to 2 different fluorochromes: phycoerythrin (pe) or brilliant violet (bv421). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and 4 populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): 0 (mfi 0.4), 4 (gmp mfi 12; non gmp mfi 9.5), 18 (gmp mfi 44; non gmp mfi 42), 70 (gmp mfi 174; non gmp mfi 180). streptavidin-bv421 brighter than streptavidin-pe is a promising tool because it amplifies by 1.7 the signal of fluorescence and allows a good differentiation of the 4 populations of rbc treated with only 0, 1, 4 and 18 lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ 18 lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases 2 (timp2) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, 2016) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = 9) were centrifuged within 3 h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = 9) were used as adult counterpart. platelet concentration was adjusted to 7-10 9 10 11 / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at à30°c/37°c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of 50,000 au spot density was defined as cut-off. data were analyzed by graphpad prism 8 using two-way anova. results: semi-quantitative evaluation of 105 analytes per array revealed significant differences. in plasma samples and platelets 63 and 48 analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (>200,000 au spot density) compared to adult plasma (6/63 vs. 3/63). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor 15 (gdf 15), platelet derived growth factor aa (pdgf-aa) and serpin e1 (p < 0.0001). more highly prevalent proteins were detected in npl (10/48) compared to apl (1/48), and 20 proteins were significantly elevated including vascular cell adhesion molecule-1 (vcam-1), platelet factor 4 (pf4/cxcl4), epidermal growth factor and lipocalin-2 (p < 0.0001). in adult samples only 10 proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < 0.05 to p < 0.0001). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, 2018) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, 1999) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are 1) to describe a procedure to label injectable human platelets with 2 densities of biotin, 2) to evaluate the impact of biotinylation on platelet functions, 3) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with 1.2 and 10 lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap (60 lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid (1 mm), amplitude of aggregation is 81.7 ae 2.5% (bio 0); 82.6 ae 2.3% (bio 1.2 lg/ ml); 79.2 ae 1.6% (bio 10 lg/ml). using collagen (2.5 lg/ml), amplitude of aggregation is 65.6 ae 4.2% (bio 0); 61.4 ae 4.0% (bio 1.2 lg/ml) 40.8 ae 6.6% (bio 10 lg/ml). the 2 biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than 48 h. after 4 h, the mean fluorescence intensities are 0.13 ae 0.02 for unlabeled circulating mouse platelets, 1.01 ae 0.03 and 8.2 ae 0.15 for circulating human biopl covered respectively with 1.2 and 10 lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, 10 ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, 9 ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to 30%. auto seds were administered in the form of 20% eye drops. results: auto seds have been applied to 17 ophthalmological patients (10 men and 7 women), previously resistant to standard therapy. in total 44 treatments were performed (each lasted 20 days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were 71.3 and 19.6 respectively). also, objective progress was present in 83% of all patients (p < 0.001). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. 1 platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of 2,3-dpg while reducing storage lesions stemming from oxidative stress. 2, 3 on the other hand, effects of steady hypoxia (pco2~10-20 mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for 3-week storage at 1-6°c. methods: 11 units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into 70 ml cp2d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o2-free bag. 5 ml of wb were collected from each unit at day 1, weeks 1, 2, 3. plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < 0.05. results: h-plt counts declined to~60% by the o2-reduction process, while similar decline was observed after 1 week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac-1 showed large increase after 1 week, then remained steady (h << c). plt activation by trap or adp declined modestly over 3 weeks (~15%) while h-plt showed additional~10% reduction for all time points. collagen activation for c-plt increased after 1 week (74%) and gradually increased to 100% after 3 weeks (~20% reduction with h compared to c). plt-derived mv (cd61 and cd61/annexin v) increased~4-fold over storage time; day 0 mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd42a) in wb supernatant increased 17-fold after 3 weeks for c, while h suppressed increase to 7-fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over 3-week period when stored at 1-6°c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated 500 ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted 1:1 by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, 19 patients were enrolled, 4 of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): 1) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); 2) autologous serum eye drops (sed). the creation of both types of platelet gel started in 2014 but only in 2017 we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from 2018, is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. 1) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between 800-1200 10 3 /ll and negative blood cultures, required by the italian law; the units are frozen at à80°c and last 5-year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. 2) the ophthalmologist's patients, with dry eye syndrome, donate 120 ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution (30%) are divided in 2 boxes containing 30 single-dose vials each: they are stored at à80°c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc 10231 strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. 10% calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of 1 9 10 3 cfu/ml and 1 9 10 4 cfu/ml were inoculated on sabouraud dextrose agar at the 1 st , 2 nd , 4 th , 8 th and 16 th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after 18-24 h of incubation at 30°c. chemokines and kinocidins (platelet factor-4, interleukin-8 and thymosin-b4) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp-10 3 group showed that the antifungal activity was still going on at the 8 th hour. the difference in colony production between the two groups at 8 th hour was statistically significant (p < 0.05). it was observed that the antifungal activity continued at the 4 th hour, decreased at the 8 th hour in the group prp-10 4 group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp-10 3 group. although there was an increase in il-8 levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb4 values results from the antifungal activity on the advancing hours in the prp groups. whereas pf-4 did not act an antifungal activity on prp-10 3 and prp-10 4 . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that 4% of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions (883 ml of red cells) in the preceding 10-day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of 225 ml, the hemoglobin decreased from 8.6 to 5.8. anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant (68% reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as 7-10 units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over 27,000 responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over 3,500 samples from 75 blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered (0.06%); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for 3-5 days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for 20 h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected 0.5 ml of ipm (2 mg/kg) intravenously between 15 to 30 seconds into each 5 health mice, weighing 17-22 grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during 24 h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after 24 h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother 0 s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the 1980s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than 6 years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since 2 years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab0-identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: 31 patients were identified receiving allogeneic sed, 15 patients had been treated autologous previously. in total, allogeneic sed have been produced 54 times since june 2017. indications were ocular gvhd (n = 15.48%), neurotrophic keratopathy (n = 9.29%), mucous membrane pemphigoid (n = 2.7%), sj€ ogren syndrome (n = 1.3%) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = 4.13%). contraindications for autologous donation were underlying disease (n = 19.61%), poor venous access (n = 12.38%), low haemoglobin (n = 8.25%), low body weight (n = 8.25%), very young age (n = 7.22%), circulatory disturbances (n = 3.10%) and lack of response to auto-sed (n = 2.7%). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = 2.4%), high donor haemoglobin (n = 2.4%) and unspecific positive serological findings (anti-hbs n = 2.4%). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a 8 year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood (20 ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for 1 h at 37°c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into 0.3 ml segments (capsules)and the artificial tears applied to the left eye 8 9 daily. results: ulcer healing was reported after 4 weeks of therapy with artificial tears. the dosage was reduced to 4 9 daily. no recurrence of corneal ulceration was observed after subsequent 8 weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > 80% of ab+/rnadonations since 2016 testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ 1.5), longstanding (ab+/rna+, lag odn > 1.5) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit 0.02 lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during 2017, 1671 donors tested hiv-positive of whom 1315 had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was 9.3% (n = 122) with efavirenz most frequently detected (115), followed by lopinavir (5) and nevirapine (2) . potential elite controller cases had the highest proportion of detectable arv (68/80; 85%) (p < 0.0001) followed by longstanding (37/741; 4.9%) and recent (17/366; 4.6%) infections. none of 82 acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older (35 to 64 years) hiv-positive donors (49/305; 16.1%) were significantly more likely to test positive for arv than younger (15 to 34 years) donors (73/1010; 7.2%) (p < 0.0001). arv use was more frequent among first time (101/ 716; 14.1%) than in lapsed (13/277; 4.7%) or repeat (8/322; 2.5%) donors (p < 0.0001). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites (10.4% vs 5.0%; p = 0.0051). summary/conclusions: the 9.3% prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the 4.7% prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year 2018. methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of 11 yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than 160.000 samples annually from whole blood and apheresis donations, testing on average around 600 samples per day. for the last 5 years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of 2019. although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the 4 mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to 2.000 unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: 19/33 anti-hcv, 19/21 hiv ag/ab and 04/22 hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: 1) hbsag aly 100. 00% (1994 00% ( / 1994 00% ( ) vs arc 99.95% (1993 00% ( /1994 ; 2) hiv aly and arc 99.95% (1992/1993); 3) anti-hcv aly 99. 90% (1994 90% ( /1996 90% ( ) vs arc 99.75% (1991 90% ( /1996 . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of 2 years from january 2016 to december 2017. blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. (78) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of 0.09%, thereby total reactivity being 2.39%. 55% (22/40) of these were hcv reactive & 45% (18/40) for hbv. the nat yield was 1 in 1086 and the viral loads of nat reactives ranged from 1-9 x10 4 iu/ml for hcv & all the hbv yields had an extremely viral load of < 06 iu/ml. 12/40 nat reactive showed sero-conversion after 4-8 months with follow-up eclia screening, and 7 of these were hcv reactive and 4 hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. (1/190,298) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect 283 infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e801 equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv-1/2, hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv-1/2 or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than 900 plasma samples collected in 7 sub-saharan africa countries (2011) (2012) and the amazon region of brazil (2016) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv-1 (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b19, chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv-1 genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in 2014 during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i2000sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m2000 realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a (5.78 log iu/ml), b (5.90 log iu/ml), c (5.62 log iu/ml), crf01 (5.61 log iu/ml), crf02 (6.19 log iu/ml), and urf (6.33 log iu/ml), as well as single donor high viral titer hcv genotypes 1a (6.84 log iu/ml), 1b (6.73 log iu/ml), 3a (6.64 log iu/ml), 2 (6.10 log iu/ml), 2q (6.65 log iu/ ml), and 4t (6.07 log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately 1 ml of residual sample volume, was then tested using the 0.6 ml abbott realtime hiv assay (lod 40 copies/ml) or 0.5 ml abbott realtime hcv assay (lod 12 iu/ml) and an hcv ag immunoassay (lod 1.24 fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = 24 per platform per virus schema) run on alinity s, alinity i, and architect i2000sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< 0.20 s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< 0.20 s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i2000sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in 2015 per 2.083.914 donations, 54% % of confirmed positive hiv, 87% of hcv, and 93% of hbv cases has been reported among first blood donors. in the end of 2015 a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after 3 months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in 2015, about 0.156% of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in 2017 was 0.082%, which shows a significant decreasing the ttis prevalence among blood donation from 2015 to 2017. the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in 2017 compared with the 2015. prevalence of hiv among donations reduce from 0.0032% in 2015 to 0.0024% in 2017, for hcv and hbv the same results have been experienced, respectively from 0.040% and 0.113% in 2015 reduce to 0.026% and 0.053% in 2017. it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from 80.19% in 2015 to 86.97% in 2017. it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from 0.0156% in 2015 to .082% in 20117. abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 61 samples were tested (19 for hcvab, 24 for havab igm and 18 for havab igg) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hcvab ranged from 0 to 4.474%. 5 samples were tested for havab igm in a total of 55 essays and the %cv ranged from 0 to 11.475%. havab igg was tested in 3 samples during 33 essays and the %cv ranged from 0 to 4.861%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 85 samples were tested (10 for hbsag, 20 for hbsab, 18 for hbcab, 14 for hbeag and 23 for hbeab) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 30 essays we found the %cv hbsag ranged from 0-4.375%. 3 samples were tested for hbsab in a total of 33 essays and the % cv ranged from 0-5.196%. hbcab was tested in 3 samples during 32 essays and the %cv ranged from 0-2.811%. using 3 samples and a total of 28 essays we found the %cv hbeag ranged from 4.176-5.147%. 4 samples were tested for hbeab in a total of 29 essays and the %cv ranged from 0-4.444%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i2000sr. determine repeatability of these tests in alinity s essays. methods: during 2 months a study was conducted where several samples (minimum 10 samples per test) were randomly sorted and tested using alinity s and architect i2000sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least 3 samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of 10 times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of 38 samples were tested (12 for hivag/ab, 14 for syphilis and 12 for htlv i/ii) using alinity s and architect i2000sr, and it was ensured that there were no statistically significant differences between results (p > .05). using 3 samples and a total of 31 essays we found the %cv hivag/ab ranged from 0 to 3.05%. 2 samples were tested for syphilis in a total of 22 essays and the %cv ranged from 0 to 1.481%. htlv i/ii was tested in 3 samples during 28 essays and the %cv ranged from 3.342 to 7.5%. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i2000sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were 1.27%, 0.20% and 0.50% respectively. consecutive positive results for hbv were 9.20% (63/685), for hcv were 6.0% (16/266) and nil for hiv. there was no sample carry over in this. out of 79 consecutive reactive donors 69 were donated for same patients and 32 were related with infected patient which were statistically significant (p < 0.0001). summary/conclusions: among all tti reactive donors 7.4% (79/1061) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study 32 reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < 0.0001). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june 2018. a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using 100 positive and 100 negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were 65% and 70% (hightop), 67% and 85% (rightsign), 62% and 73% (wondfo), 70% and 80% (accu-chek), 68% and 77% (fastep), 73% and 85% (abon), 77% and 83% (immumed), 80% and 90% (insta-answer), 67% and 81% (biocheck) and 72% and 83% (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were 69% and 80% (hightop), 76% and 83% (rightsign), 69% and 81% (wondfo), 78% and 79% (accu check), 68% and 68% (fastep), 63% and 73% (abon), 71% and 70% (immu-med), 79% and 68% (insta answer), 62% and 66% (biochek) and 69% and 78% (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be 100%, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , 1940 and 1945 , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. ,906 donors were routinely tested for hbv dna by using cobastaqscreen mpx-1 and mpx-2 (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: 348 hbsag-/dna+ donors (1:1,462) including 294 confirmed obi were identified (1:1,731 prevalence). among obi donors, 160 (54.5%) tested anti-hbc+/anti-hbs-, 108 (36.7%) were anti-hbc+/anti-hbs+, 25 (8.5%) were anti-hbc-/anti-hbs+, and 1 anti-hbc-/anti-hbs-primary obi (0.3%). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: 21 years [range: 18-57 years]) than those with anti-hbc+/anti-hbs+ (mean: 44 years [range: 19-60 years]) and anti-hbc+/anti-hbs-(median: 45 years [range: 21-55 years]) profiles (p < 0.0001). hbv vaccination was documented for 18 (72%) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: <10-155 iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = 1/20) and genotype c (n = 19/20). preliminary analysis of core protein (n = 16) and bcp (n = 10) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for 15/ 25 anti-hbc-/anti-hbs+ donors (2-6 samples/donor; range: 2-56 months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between 12 and 1,000 iu/l. no significant difference in hla-a, -b (except hla-b*46 more frequently detected in anti-hbc negative obi), and -drb1*. summary/conclusions: overall, the 8.5% prevalence of anti-hbs-only in hbv dna positive obi carriers (1:20,355 of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in 15/25 donors by the over 50 months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after 1986 is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since 2000. hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december 31, 2018, allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between 2012 and 2018. background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about 130,000 blood donors are submitted to serological screening tests (hbv, hcv, hiv-1/2, chagas disease, syphilis and htlv-1/2) and nat for hiv, hcv and hbv per year. approximately 2% of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was 0.65% in 2016. aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly 74,000 donations from may 2016 to december 2016. hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity 95% lod 300 iu/ml for hbv). reagent samples (n = 407) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity 10-20 ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno2pheno [hbv] 2.0. socio-demographic and epidemiological data were also analyzed. financial support: fapesp 2017/16658-6. results: among the 407 hbv reactive samples, 377 were reactive for anti-hbc only (92.6%), 10 for hbsag only (2.5%) and 20 were reactive for both markers and hbv dna (4.9%). routine testing and id-pcr-hbv identified 20 (0.027%) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from 1.08e+01 to 2.45e+09 cop/ml (median, 2.12e+08cop/ml). hbv sub-genotypes a1, a2, c1, d3 and f1 were found in 70%, 5%, 5%, 15%, and 5% of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in 30% (6/20), with the following main substitutions 100c (3x), 120r, 123n and 144g. the mean age of donors with active hbv infection was 40 years, mostly donors were males (80%), mixed (40%) or white (40%) and had concluded high school (45%). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a1 and mutations associated with escape were found in 30% of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period 15-30 november 2018, all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of 620 edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in 24 (3.87%) samples, among which 7 (19%) were also anti-hbe(+), while anti-hbs was found > 200 m iu/ml in 15 (62.5%), between 100 and 200 miu/ml in 3 (12.5%), and < 100 miu/ml in 6 (25%). among anti-hbcore positive donors, 4/24 were foreigners (16.6%) and 20 were greeks, while foreigners consisted 6,29% (39/620) of donors examined. so, anti-hbcore was found positive in 10,25% of foreigners (4/39, all from countries with high prevalence for hepatitis b infection) and in 3,44% of greeks (20/581). in total, 285 (45.96%) samples had anti-hbs > 10 miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors (45.96%) were immunized, 261 by vaccination and 24 (3.87%) by natural infection. the incidence of natural infection was significantly higher in foreigners (10.25% versus 3,44%). if not all anti-hbcore+ donors, 25% with anti-hbs < 100 iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < 100 or < 200, or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately 2 million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in 20 samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than 20 km to each other. pcr was positive in 15 samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and 11 of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the 1a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with 1b subtype originated from the same village, and their difference was only 2 nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to 2 or 3 primary human sources. during 2011 and 2014, a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from 1993. however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march 2014 to december 2017, there were 2083 blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from 15 chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, 1178 of 2083 donors in the study who could be classified into two categories for hcv status: 201(17.1%) true positive and 974 (82.9%) false positive. a total of 905 of 2083 donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors (82.9%) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at 6.6% and 0.9% respectively. between january and december 2016, in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming 8.1%. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: 235 hcv seropositive blood samples from 3 bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that 45/50 seropositive blood (90%) was rna pcr negative. in december 2018, 156/249 (63%) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between 1.00-1.99 (1.00 is the cut-off indicating a positive sample). data from the re-testing of 235 seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december 2018. preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november 2017, the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of 3 months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a 3-month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for 2010-2018 (2018: preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within 12-months, and/or microbiological and clinical evidence of recent infection. for donors positive in 2018, compliance with the 3-month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from 2010 to 2018 among new donors, annual hiv prevalence decreased significantly by an average of 11.3% each year (p = 0.02) to 1.49/100,000 donations in 2018; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of 17.3% each year (p = 0.03) to 0.23/100,000-person years (pyrs) in 2018 (based on 2 seroconverters). there was no significant trend in hbv incidence over the study period, however between 2017 and 2018 incidence increased from 0.35/100,000 pyrs to 0.84/ 100,000/pyrs (based on 3 and 7 seroconverters respectively). with the information available to date, none of the 2018 seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in 2018 although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the 1980s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a 5-year deferral was implemented in 2013, reduced to 12-months in 2016; a 3-month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a 3-month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a 12-month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a 3-month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current 12-month deferral, calculated as hiv positive donors with a previous negative within 12 months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a 3-month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the 2.5th and 97.5th percentiles over the simulation (95% ci). results: incidence, per 100,000 donations, was estimated to be 0.15, 0.13 and 0.27 for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the 3month deferral "most likely" scenario, hiv residual risk was predicted to be 1 in 32.6 million donations (95% ci: 1 in 217,692 million to 1 in 8.3 million). for the "optimistic" scenario, hiv residual risk was estimated to be 1 in 34.3 million donations (95% ci: 1 in 257,692 million to 1 in 9.1 million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be 1 in 16.0 million donations (95% ci: 1 in 34,091 million to 1 in 5.7 million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every 30 years for the "most likely" scenario, every 32 years for the "optimistic" scenario and every 15 years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a 3-month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above 25%). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september 2018, a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated 22 times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang 9 urumqi blood center, urumqi, china 10 rti international, rockville 11 national heart, lung, and blood institute, bethesda 12 stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in 2013-2016 participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv-1/2 on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately 129 days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the 2013-2016 estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least 2 donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of 1,276,544 whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between 2013 and 2016, including 648,607 donations from first-time donors and 627, 937 donations from repeat donors. hiv prevalence among first-time donors was 68.04 per 100,000 donors (95% ci, 61.68-74.40). hiv ir was estimated to be 37.93 per 100,000 person-years (95% ci, 30.62-46.97) among first-time donors and 20.55 per 100,000 person-years (95% ci, 16.95-24.91) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than 25 years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over 10 years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during 2009 to 2018 at thai national blood centre. results: a total of 6,408,024 voluntary blood donations over 10 years, the repeated reactive results on hiv serological screening were 5,978 (0.093%) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for 6 months and tested by using the different three principles of hiv serological testing. a total of 5,978 hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was 1,130 (35.7%) cases, 742 (23.5%) cases and 1,292 (40.8%) cases respectively. out of 1,130 hiv positive results, we found that 1,105 (97.8%) cases were positive with all hiv serological testing for the first-time follow-up and 25 (2.2%) cases were tested and become to positive results after follow-up more than one time. in 742 cases of inconclusive results, 539 (72.6%) cases were reactive only 1 or 2 testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, 203 (27.4%) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, 1,292 negative results cases showed 762 (59.0%) cases were negative results after follow-up over 6 months while 530 (41.0%) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over 10 years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, 241 donors were found (80% men); of these people 256 donations were obtained (97% whole blood). 166 donors (69%) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average 174 ae 83 days to be reported in sivigila. 25 donors (10.4%) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. 20 donors (60% men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to 6 months they were reactive by sivigila (93% of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for 28 recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around 10-100 cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = 4-12 per species) was inoculated with approximately 10 cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for 22 to 24 h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with 3 random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to 3.5 9 10 3 cfu/ml in bcs and up to 0.9 9 10 3 cfu/ml in the corresponding pcs right after preparation. in total, 33 out of 48 pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., 6/6 pcs tested positive after spiking with streptococcus pyogenes, while only 1/8 pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male (69 yo and 65 yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day 4 platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after 18 h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit 1: a. baumanii tntc and s. saprophyticus with 8.5 9 10 4 cfu/ml unit 2: a. baumanii 6.6 9 10 7 cfu/ml and s. saprophyticus 2.2 9 10 6 cfu/ml blood cultures collected from both patients became positive within 8 h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a-105 and yersinia enterocolitica a-176) were distributed from the paul-ehrlich-institut to 17 laboratories in 10 countries for enumeration, identification and growth measurement in a 7-day interval for a total of 42 days after low-count spiking of rbc bags (10-25 colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a-105 and y. enterocolitica a-176 achieved >10 6 cfu/ml at day 14 and 10 9 cfu/ml at day 21. growth of l. monocytogenes was lower reaching a maximum concentration of >10 6 cfu/ml at day 35. in 9 out of 17 laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert 3d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber (10 or 16 ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc 29523), streptococcus agalactiae (atcc 13813), escherichia coli (atcc 25922), clostridium perfringens (atcc 13124). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert 3d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range 4 9 100-2.8 9 103 cfu/ml) and tested on bact/alert with a 1 ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two 10 ml ssk, which were held for a period of 6 h at 20-25°c. the process was repeated with a 24-h period. once elapsed, 8 ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of 7 days (36ae0.5°c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of 0.6-log10 after 6 h (2.0 9 102 to 8.9 9 102 cfu/ml) and 2.0-log10 after 24 h (3.1 9 101 cfu/ml to 3.4 9 103 cfu/ml). s. agalactiae increased by 0.7-log10 after 6 h (2.1 9 102 cfu/ml to 1.1 9 103 cfu/ml) and 0.09-log10 after 24 h (1.0 9 102 cfu/ml to 1.2 9 102 cfu/ml). c perfringens increased by 0.6-log10 after 6 h (1.3 9 102 cfu/ml to 5.0 9 102 cfu/ml) and 2.7-log10 after 24 h (4.0 9 100 cfu/ml and 2.2 9 103 cfu/ml). for e. coli, the concentration after 6 h was reduced by 0.28-log10 (2.8 9 103 cfu/ml and 1.5 9 103 cfu/ml), however this was possibly a result of inherent counting errors. at 24 h, an increase in growth by 2.7-log10 (1.5 9 102 to 7.9 9 104 cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to 24 h does not have a negative effect on bacterial viability and detection using the bact/alert 3d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in 100% plasma or amicus tm for 65% intersol pas/35% plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken 7 h after successful and complete transfusion of an apheresis intercept-treated pc in 65% pas/35% plasma, from a patient with a suspected septic reaction that occurred 2 h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a 7 day shelf-life. methods: a double apheresis pc collected in 65% pas/35% plasma was split into three equal components, yielding approximately 250 ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~6 log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at 3, 5 and 7 days of storage. the samples were analyzed by plating on lb plates (100 ll-10 ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were 5.9-6.0 cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after 3, 5 and 7 days of storage, corresponding to > 5.9 log 10 inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after 7 days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between 0,3%>17,5%, up to 5000 times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december 2018, a total of 2411 blood products were sampled, of which 979 in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), 662 in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and 770 in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, 16 ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, 4 ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at 35°c for 7 days. cultures were checked daily for signs of growth. in addition, 2 ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated 48 h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of 1.6% (39/2411) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, 2.7% (26/979) of blood products were contaminated, in hpkll 1.5% (10/662) and in hslk 0.4% (3/770) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = 0.00047). there was no significant difference between the other sites (p = 0.051 and p = 0.12). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. (46.7%) and bacillus spp. (33.3%). the remaining 20% of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than 10³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at 10³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than 2000 blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate (1.6%) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (<10³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the 18 to 65-year-old general population registered at the nphc. methods: altogether, 474,017 18 to 65-year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between 2015 and 2017. reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ 1:8 and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the 18 to 65-year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk 1,2 across 2015-2017. statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < 0.05. results: anti-tp reactivity was confirmed in 167 blood donors. aeid was proved in 85 cases with 36 ft and 49 rt donors. in that period, 1696 syc were notified. both in aeid and syc, the age group of 25-34 years with approximately 35% and 36% of individuals was the dominant. the proportion of men was 74% and 77% (p = 0.5) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher (0.020%; 0.023%; 0.032%, p < 0.0001) than that of rt donors (0.007%; 0.009%; 0.009%) and the proportion of syc (0.008%; 0.009%; 0.010%) in the general population. pp of aeid showed a roughly homogenous distribution in the 7 regions (0.011%-0.025%), however, pp of syc had a significant (0.058%; p < 0.0001) central hungary dominance in relation to the other regions (0.008%-0.016%). comparing the pp of aeid to syc, central hungary indicated a significant difference (0.025% vs. 0.058%, p < 0.0001), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised 4135 donations, including 112 bm (2.70%), 3787 pbsc (91.60%) and 236 cb (5.70%) donations processed in our laboratory in the years 1996-2016. bm was collected in operating theatre-conditions, pbsc with cell separators -cs-3000 (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and 35°c) using a variety of culture media. pbsc and bm were tested using 2 samples with trypcase-soy broth (tsb-t) and 1 with schaedler + vit k3 (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/10/anaerobic/f (becton-dickinson). results: in the 1996-2016 period 42 contaminated products were found: 25 pbsc (0.66% of all tested pbsc units) and 17 cb (7.20% of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at 0,1%. in 2010, three (3) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis (61.36%). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around 12 million new cases of syphilis in the world, more than 90% occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between 2015 and 2017 we observed in our institution a significant increase of 24% in positivity of syphilis among blood donors from 0.62% in 2015 to 0.73% in 2016 and 0.77% in 2017 (p < 0.0000001). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in 2017 was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp 2017/23028-9. results: among 123,851 samples screened in 2017, 958 (0.77%) presented a positive result for cmia -syphilis. of these, 626 (65.4%) were included in the study. a total of 106 samples (17%) showed vdrl+/igm+; 96 (15%) vdrl+/igmand 28 (4.5%) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in 28 (4.5%) samples that presented positive results for elisa igm and vdrl negative with 21 (3.35%) positive results, 1 (0.15%) undetermined and 6 (0.95%) negative. none of the 626 samples showed the presence of treponema dna by real-time pcr. the prevalence was 0.77%, the incidence was 0.1% in the year 2017, and the incidence in relation to the total positivity was 20.4%. both, prevalence and incidence were higher in men, white, not married, aging 18-29 years and high school educational level. we observed a 6% a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of 1.5% htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in 2017 (0.77%) with an incidence of 20.4% of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found 22 (3.5%) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to 5 days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to 7 days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; 22°c) or refrigerated (cs; 4°c). aims: the aims of this study were to 1) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and 2) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to 60 rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg4 + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first 3-4 days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day 4. bacterial numbers remained unchanged in refrigerated aliquots through day 5. rt storage resulted in significantly (p < 0.05) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < 0.05) delay in bacterial growth at day 1. while growth of e. coli and p. aeruginosa recovered by day 2, growth of a. baumannii was significantly (p < 0.05) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < 0.05) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day 3, indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a 20-ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle (10 ml each) within 48 h after collection. the bottles were then placed in the bact/ alert system and incubated for at least 7 days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a 2-ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a 2-ml and a 20-ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september 2008 and march 2018, after maintaining in liquid nitrogen for 1 to 17 years, 205 pbpc products collected from 45 patients, which was preserved in a volume between 35 and 60 ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these 205 final products were thawed and cultured with both the 20-ml and the 2-ml aliquot. one of these pbpc products had the positive culture result with the 20-ml retested samples. nevertheless, the same pbpc product had the negative result with the 2-ml post-thaw pbpc sample and the 20-ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the 2-ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) (10 surfaces, 1 air), hôpital provincial g en eral de r ef erence (hpgrk) (24 surfaces, 2 air) and the national blood transfusion centre (cnts) (20 surfaces, 2 air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates (23.7 cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd30, kikkoman), expressed as relative light units (rlu) per 100 cm². air was sampled by active sampling (500 liter; spinair, iul) on cled and macconkey medium. in parallel, particles > 0.5 lm and > 5 lm were counted using a particle counter (14,15 liter; metone 227a). culture media were incubated for 48 h at 35°c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was 27 (6-60) cfu/rodac, 69 (6-103) cfu/rodac, 82 (3-132) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes (43 and 60 cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator (132 cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per 100 cm² was 2.906 (778-26.497) rlu at hpkll, ) rlu at cnts and 35.235 (1.754-348 .052) at hpgrk. atp results and total viable count were not correlated (n = 49, p = 0.52). median (range) bacterial count in the air was 258 (210-375) cfu/500l for all sites together. there was no correlation found between total bacterial count and particles > 0.5 lm or > 5 lm (r = 0.7 and r = 0.6 respectively; p < 0.05; n = 5) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january 1st 2020. however, we already decided to voluntarily test all our blood products since january 2015. aims: in this study, we present our results of a 100% screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january 2015 to december 2018, a total of 386,307 allogenic blood donations from 69,956 individual german blood donors were screened in a minipool format of 96 samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from 4.8 ml plasma using the chemagen msm-i extractor (viral 5k, perkin elmer chemagen gmbh). the 95% lod of the assay was determined to 4.66 iu/ml (447 iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for 3 months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf1. results: in total, 274 hev rna positive donors were identified. of these, 274 hev rna-positive donors, 216 were nat-only positive donations (78.83%, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer (1.09%), 30 donors showed reactive igm and igg titers (10.95%), 25 donors already had isolated igg titers (9.12%). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely 62 donors showed elevated alt levels (22.63%), mostly within a double increase within the reference range (16.06%), only 6.57% of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > 1,000 iu/ml compared to the group with viral loads between 100 and 1000 iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype 3 in all cases. the month-dependent incidence ranges from 1:719 to 1:3,781 blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february 2016, and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within 1 to 2 weeks. however, in whole blood, zikv rna might present positive up to day 101 after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of 200 ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at 37°c for 3 days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at 37°c for 3 days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to 10 9 copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < 0.05) in type o rbcs and lower than those in plasma (p < 0.05) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting 20 residents from 2000 to 2014. during 14 august to 4 september 2018, another outbreak of 29 confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district (19 cases) and the other in cheung chau, an outlying island (10 cases). aims: we assessed the risk of dengue transmission from blood donors during the 2018 outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, 2013) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of 15-to 64-year-old patients in the 2018 outbreak and residents of the same age range in hong kong and wts district as at mid-2018 respectively (16-65 years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during 12 august to 8 september 2018 after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, 2009 ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in 778,000 (266,000-1,822,000) territory-wide for the 28-day study period but increased to one in 147,000 (50,000-345,000) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was 1 in 279,000 (118,000-752,000) territory-wide and 1 in 52,000 (21,000-188,000) in wts during the same period. the eufrat also predicted a territory-wide issue of 0.08 unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at 1 in 778,000 during the 2018 outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by 4 folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july 2017 to december 2017 at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version 21.0 (ibm). results: in our study population there were 445 (98.9%) males and 5 (1.1%) females. the mean age of recruited blood donors was 28.38 (sd ae 7.34), with a range of 18-55. younger donors were more common with a frequency of 18-27 year olds of 249 (55.3%). we found an overall hev igg prevalence of 17.5% and an hev igm prevalence of 9.1%. there were 10 (2.2%) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was 33.8 (sd ae 41.0) with a range of 4-554 iu/l. the serum alt levels were elevated (>45 iu/l) in 73 (16.3%) blood donors. there was significant correlation (p=<0.001) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are 20 million hev infections, 3 million acute hev cases and 56 thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively 4.0% in new zealand, 13.5% in britain, 20.6% in denmark, 16% in the united states and 27.0% in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from 5552 blood donors were collected from april 2017 to april 2008 at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were 20.05% (1113/5552), 0.76% (42/5552) and 0.04% (2/5552), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = 1.089, p < 0.001; igm or = 1.055, p < 0.001). donors who were zhuang minority (32.69%, 7.69%) showed higher anti-hev than those who were han ethnicity (19.89%, 0.70%), and the difference was statistically significant (igg or = 2.052, p = 0.023; igm or = 12.029, p < 0.001). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population (60-100% seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested 20 h after infection, washed and used for spiking of plasma. plasma units (n = 2, 290 ml) were spiked with infected cell suspension (10% v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b2 illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses (0 after addition of mb, 30, 60, 90 and 120 [standard] j/cm 2 ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of 4.2 (bag no. 1) and 4.5 (bag no. 2) log 10 tcid 50 /ml was achieved in the plasma units. in hold samples, a mcmv titer of 3.8 (bag no. 1 and bag no. 2) log 10 tcid 50 /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ 2.9 log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log 10 reduction factors ranged from 4.2 (bacillus cereus) to 6.1 (serratia liquefaciens) for the tested bacteria, and from 3.1 (emcv) to ≥ 4.9 (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below 0.8% until day 35 of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a-1,2-fucosyltransferase (fuct2), encoded by fut2 gene, determines the secretor status of an individual. about 80% of caucasian population have a functional copy of fut2 (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: 126 samples of semen, 68 from infertile men and 58 from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who 2010 criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners (76.9%) were significantly higher than those from fertile couples (21.6%) (p < 0.03) the results obtained by pcr in sperm cells correlated 100% with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut1 gene (h | h), mandatory linked with an active fut2 gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric231-pe and a 1:1 mixture of bric231-pe/bric231, ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from 29 blood donors with different abo phenotypes (o (5), a 1 (5), a 2 (5), b (5), a 1 b (5), a 2 b (4)) and 2 patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a 1:1 antibody mixture (bric231-pe/bric231) covering approx. half of the h-binding sites by unconjugated bric231. in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric231-pe. rbcs coated with bric231-pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric231-pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen 1,2 , x xu 1,2 , x hong 1,2 , k ma 1,2 , j he 1,2 , j chen 1,2 and f zhu 1,2 1 blood centre of zhejiang province 2 zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: 109 samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of 109 samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province (2018rc029). results: 69 samples in 80 blood donors showed increased expression of h antigen, of which 47 were identified as abo subgroups. there were 22 enhanced reactions in 29 hemopathic patients. however, 19 were finally confirmed as normal abo genotypes. no statistical significance (86.3% vs 75.9%, p > 0.05) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is 50 and 3 respectively in blood donors and hemopathic patients. extremely significant statistical differences (68.1% vs 13.6%, p < 0.005) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november 2018 to january 2019, 55 cases (0.6%) of abo discrepancies among 9,550 abo blood type tests performed using the 15-min reaction mode of ih-500 in chonbuk national university hospital blood bank were identified. we compared the test results of 15-min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a1 and abo genotyping. results: in the immediate reaction using different red cell reagents, 45 cases (81.8%) of discrepancies disappeared and 10 cases (18.2%) remained discrepancies. all abo discrepancies observed in the 15-min reaction were due to serum side causes, and one case (1.8%) was due to both of serum and red cells side cause. nonspecific response (28 cases, 50.9%), cold antibody (20 cases, 36.4%), rouleaux formation (3 cases, 5.5%), cis-ab (3 cases, 5.5%), and abo subtype (1 case, 1.8%) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the 15-min reaction. summary/conclusions: ih-500, an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the 2 years data from jan 2017 to dec 2018. the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel 2016 and spss (version 22). results: during study period a total of 1970 patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were 20.91%, 37.51%, 32.74%, 8.83% for a, b, o and ab respectively. the common neurological tumours found in our study were glioma (33.55%) followed by pituitary adenoma (20.05%), meningioma (18.58%), schwannoma (8.98%), cavernoma (2.54%), neuroma (2.23%) and space occupying lesions (14.06%). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in 47.73% o group patients as compared to other blood groups which was found statistically significant (p < 0.05). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen (38.2%) is more common as compared to o blood group(33.4%). background: rhd and rhce represent homologous genes in head-to-head position on chromosome 1 (chr1, p36.11). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band 3 and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, 2016). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, 1955) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad-3/brad-5/fog-1 (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric-69pe (ibgrl). results: a total of 146 samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr(21), r1r(20), r1r1(23), r2r(18), r0r(15), r1r2(27), r2r2 (22)). variant expression of rhd by different rhce phenotypes using brad-3-pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples (3) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad-5-pe and fog-1-pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c.48g>c, c.201a>g, c.203a>g of exon 1, exon 2 resp. and intron 2) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c.676c>g, exon 5) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon 5 is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh26 antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:-26, and the antibody specificity anti-rh26. most polyclonal anti-c contain anti-c and anti-rh26. previous studies have shown 2 of 10 monoclonal anti-c reagents are actually anti-rh26. these reagents will not detect the c antigen where the red cells are rh:-26. aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r 1 r) was inconsistent with the current donation phenotype c-(r 1 r 1 ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a 2 + reaction by tube with bio-rad seraclone â (2) [clone ms35] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk7300 using beckman coulter anti-c [clone 951] blood grouping reagent and tested positive (4) reaction on the immucor neo using immuclone â (1) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd*01/*01n.01 and rhce*01.15/*02 with a predicted phenotype of c+, c+ w , d+, e-, e+, rh:55 (locr+), rh:-26. a review of the donor's historical records indicated the donor tested as c+ on the pk7200, which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms33]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:-26 variant associated with the rhce*01.15 allele. reagents containing clones ms-33 and ms35 correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk7300 and associated anti-c [clone 951] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:-26 phenotype, which is in contrast to the previous report by faas et al, transfusion, 1997 where it was demonstrated that clone 951 reacted with c+ rh:-26 bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since 2017 at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of 6 monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in 32 samples: a) by using the partial d kit, 23 samples were typed: four samples were characterized as "partial d" (3 dfr, 1diii) and 19 as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type 1" three samples, "weak d type 4.0 or 4.3" one sample). b) the nine (9) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized 2 samples as "weak d type 1", one sample as "weak d type 3" and another as "weak d type 11". results are pending for 5 samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in 68,75% of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over 200 rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ 50 years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type 1, 2 or 3 are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type 1, 2 and 3. aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from 2013/01/01 to 2018/10/01 we analysed 119.845 samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs232, bs226) (biorad, dreieich, germany), swing maestro [lmh 59/20 (ldm3) + 175-2 and th-28 + rum-1 + ldm1] and ih-1000 [lmh 59/20 (ldm3) + 175-2] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from 119.845 samples 300 (0,25%) were swd. 71/300 (24%) were referred to rhd genotyping. 55/71 (77%) samples were weak d type 1, 2 or 3, while 16/71 (23%) were weak d type 14 and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types 1, 2 and 3. clearly negative serologic reactions were given in 27/29 samples with bs 226 and bs 232, in 30/33 samples with lmh 59/20 (ldm3) + 175-2 and in 18/39 samples with th-28 + rum-1 + ldm1. summary/conclusions: the prevalence of swd in this study is rather low (0,25%). after rhd genotyping 77% of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of 3243 randomly selected saudi donors, who donated blood between january and june 2018, were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k1 phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of 3243 saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in 93.7%, while k antigen was positive in 11.1%. among other studied rh antigens, e was the most common (98.1%) followed by c (78.2%), c (70.3%) and e(26.9%). dce/dce (34.2%) and dce/dce (5.3%) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent (11.9%) with almost 5 times higher frequency compared that reported in caucasians (2.0%). the rare phenotype dce/dce was found in 3 donors (0.09%), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a 58-year-old male patient with lung cancer without previous transfusions was admitted (06/2009) for surgery. his hemoglobin was 13.2 g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july 2017, the patient was admitted for colon cancer surgery with a hemoglobin of 11,0 g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge*01.-03. an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of 0.35% and the antibody was considered not to be clinically relevant. the mi was interpreted as following: 0-3% not significant; 3-5% inconclusive; >5% clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received 2 units of ge:-2,-3 prbcs without any transfusion reaction. one and a half year later (11/2018), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was 87 g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july 2017 indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following 16 days the patient received a total of 5 units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december 12 th and 16 th (12 days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of 0.8% und 1% respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from 1 st april, 2013 to 30 th september, 2015 (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a1 lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen (3-cell) and identification (11-cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected 104 (0.072%) abo discrepancies out of the total 144279 donor samples tested during the study period. out of these, 135043 (93.6%) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody (33/104; 31.73%), followed by weak anti-a antibody and weak subgroups of a (24/104 each; 23.07% each) and weak subgroups of b (5/104; 4.8%). the remaining 17.3% (18/104) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was 0.02% ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within 3 days after receiving a patient's sample. however, in some cases, retests could be performed after more than 3 days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih-500 and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih-500 (bio-rad, 1785 cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, 1 week after storage at 4°c and 1 month after storage at à20°c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih-500 and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, 15; anti-le a , 4; anti-di a , 4; anti-c+e, 3; anti-m, 3; anti-d, 2; anti-c, 1; anti-k, 1; anti-jk a , 1: anti-xg a , 1; unidentified antibody, 13; autoantibody, 2 cases. with regard to the changes in reactivity owing to storage, 26 (52%) samples (anti-e+c, 12; anti-m, 3; anti-di a , 3; anti-d, 2; anti-c+e, 2; anti-le a , 1; anti-c, 1: autoantibody, 1; unidentified antibody, 1) showed identical reactivities after 1 week and 1 month storage by both ih-500 and tube methods. however, 19 (38%) samples, comprising 12 unidentified antibodies, 3 anti-le a , 1 anti-c+e, 1 anti-e, 1 anti-e+c, and 1 autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after 1 month storage, while one sample with anti-xg a showed decreased reactivity only after 1 month storage. higher reactivities were observed in all samples detected using the ih-500 analyzer than manual tube methods (p < 0.005, wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih-500 and tube methods were the same in all storage states; however, reactivities were higher in ih-500 than in the tube method. twenty-six (52%) of 50 samples showed identical reactivities after 1 week refrigeration and 1 month freezing. nineteen (38%) samples showed decreased reactivities after storage; however, 12 (12/19, 63%) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than 3 days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel 1 , d huber-marcantonio 1 , n rufer 1 , g canellini 1 and c niederhauser 2 1 unit of transfusion medicine, interregional blood transfusion src, lausanne 2 laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of 3'753 dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (<14 days). results: a positive dat was found for igg and c3d in 421 out of 3'753 (11.2%) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, 244 (57%) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only 10.7% (26/244) of the cases. from the 161 patients transfused within the last 14 days and having a positive dat, 60 (14%) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms (244 versus 60 samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last 14-days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd38 monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin #16-02). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd38 antibodies. vox sanguinis (2019) 114 (suppl. 1), 5-240 methods: edta-anticoagulated whole blood samples coming from 30 patients in different stages of treatment with daratumumab and 5 with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd38 antibodies treatment. in these cases, 6 of 7 negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed 100% agreement with genotype id core xt results. focusing in the data obtained during the treatment, 8 negative antigens were type as positive in gel technology (12% of the tests). mdmulticard agreed with genotype in 100% of the analyzed antigens. as complementary data, 13 of 66 patient-treated samples had dat or ac positive and 59 showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd38-directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes 25 ll and 50 ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and 20 donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card (50 ll at 0.8%). further, sample dilutions were dispensed into the card (25 ll or 50 ll). subsequently, cards were incubated 15 min, 37°c (coombs technique) and 15 min, 18-25°c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = 80 titrations, titer ranged 0-256) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ 0.5 titer in the 98% of the cases). differences between dg gel coombs and neutral (saline technique) (n = 80 titrations, titer ranged 2-512) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at 37°c and igg antibodies. differences were > 1 titer in 35% of the comparisons and ≤ 1 titer in the rest of the cases (65%). comparing sample volumes of 25 ll and 50 ll in all cards (n = 160 titrations), higher titers were observed using 50 ll, as expected. differences were 1 titer in the 51% of the comparisons, <1 titer in 44% and > 1 titer in the 5% of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged 0-18 years, diagnosed with aiha, between april 2017-december 2018 (21 months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: 10 patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from 2.5 to 9 grams/dl. the initial presentation was severe anemia in 7 children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in 3. the trigger for haemolysis was infection in 4 children. rheumatological evaluation was performed for 5 children out of whom 2 were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for 4 and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in 9 out of 10 aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of 9 dat positive cases had igg & c3d positivity on monoclonal dat testing whereas rest 7 had only igg coating the red cells. dat titration was more than 1:300 in 4 patients; where only 1 of these 4 patients had both igg1 and igg3 coating and rest 3 had only igg1. alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a 4-6 weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in 2 and rituximab was used in 3. out of these children 5 children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a 64-year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and 5-fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day 1), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day 5), two units of rbcs (day 6) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day 24 were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at 1 and 5 mg/ml. results: dat was positive (anti-igg 2 + , anti-c3d 2 + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer 2) and enzymetreated (titer 8) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at 5 mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p-331 singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd38 used in the treatment of multiple myeloma and has been known to bind to cd38 on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october 2016 to december 2018 was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of 55 patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in 23 of the 55 patients while the use daratumumab was mentioned in the serology request form of the other 32 patients. the median tat for red cell antibody screen and identification is 212 min (range: 47-877) if information on daratumumab was provided prior to start of testing and 301 min (range: 133-1417) if information was not provided prior to testing. the median tat for routine testing is 198 min (range: 15-2245). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value 0.72634). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value 0.00694). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd38 monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd38 is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd38 on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd38 interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd38 tertiary structure, avoiding the binding and testing interference by the anti-cd38 daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since 2016, 32 patients were studied, from which 16 were transfused with 108 blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd38 and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd38 is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july 2014 and dec 2018 (54 months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of 22888 patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in 145 patients and autoantibody was detected in 53 patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha (58%) and 20% of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group (45% vs 34%, p < 0.05). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in 21 where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood (1.2% vs. 5%, p < 0.05). during the period of study we obtained 10 cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk7300, 250,599 donation samples were tested and 4,895 (1.95%) were found absc positive. antibodies to red cells were identified in 250 donations out of 4,895 (5.11%) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was 0.10%. the top 5 most frequent antibody specificities were: nonspecific cold antibodies (28.8%), anti-e (26.8%), anti-mi a (19.6%), anti-m (16.8%) and anti-le a (4.0%). a total of 225,124 donations were screened for atypical antibodies by ih-1000 and 2,275 (1.01%) were screened positive. among these, anti-red cell antibodies were identified in 1,239 samples (54.46%), which was significantly higher than those identified in pk7300 screened positive samples (p < 0.00001). the prevalence rate for atypical antibody as screened positive by ih-1000 and with confirmed red cell specificities was 0.55%, which was also significantly higher (p < 0.00001). the top 5 most frequent antibody specificities were: anti-mi a (55.3%), anti-m (21.1%), anti-le a (10.2%), anti-e (6.5%) and non-specific cold antibodies (3.6%). anti-fy b was detected in 7 cases, which would be missed detection by enzyme treated reagent cells on pk7300 system. summary/conclusions: the performance of the ih-1000 system using a 3-cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk7300 in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of 0.55% in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus 2019) reported a rbcalloimmunization prevalence of 11.4%, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a 18 years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from 1990 to 2018 thalassaemia patients, with a minimum follow up of 1 year and transfused with more than > 10 rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall 57 patients (38 females, 19 males) were included in our retrospective analysis: 54 patients were affected by thalassaemia major and 3 by thalassaemia intermedia. rbc alloimmunization prevalence was 12.3% (7 patients): 3 patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected (1 anti-h, 1 anti-cw, 4 anti-e, 1 kpa, 1 anti-jka, 1 anti-jkb, 1 anti-m and 1 anti-lua). in 2 out of 3 alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining 4 patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the 7 patients positive for alloantibodies, 6 were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: 12.3% vs 11.4%; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since 1998. recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april 2013. that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in 3642 patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in 759 patients, acute leukemia in 600, multiple myeloma in 390, chronic lymphatic leukemia in 319, chronic myeloid leukemia in 218, aplastic anemia in 147, hemophilia in 105, myelodysplastic syndrome in 73, and other hematological diseases in 1031. the frequency of detection of antibodies to antigen c (0.25% vs 0.06%) and to antigen e (0.46% vs 0.12%) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly (0.10% vs 0.06%, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since 1998, therefore the immunization index for these antigens remained unchanged and amounted to 0.10% vs 0.12% for anti-c antibodies; 0.36% vs 0.36%for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included 466 first time patients of hematology clinic in 2017-2018. typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in 84 (18.0%) patients. the total number of complex cases was 96. the double population of red blood cell was most often determined in antigens of the rhesus system (10.9% of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in 31 cases (60.8% of patients with the chimera for rhesus and kell antigens), cin 23 (45.1%), sin 15 (29.4%), e -5 (9.8%), cw -8 (15.7%), k -5 (9.8%). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in 0.6% of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients (1.1%) was due to a decrease in the production of antibodies -4 cases and the appearance of extra agglutinins -1 case. autoantibodies were detected in 3.9% of all patients, including 0.6% of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in 2.8% of patients, including specific anti-din 4 (0.86%), anti-dcin 2 (0.43%), anti-kin 1 (0.21%); antibodies of unidentified specificityin 2 (0.43%), polyspecificin 4 (0.86%). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of 3,49,497 crossmatches were performed over period of last four & half years, out of which 867 units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c3d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of 3,49,497 crossmatches, only 867 units were found to be incompatible (0.14%). the major cause for incompatibility found in patients was aiha-(32.87%). other causes of incompatibility were infections (27.44%), multiple transfusions (17.41%), trauma (11.23%), evan's syndrome (4.15%), rh negative mother (3.57%), sca (2.99%) & incompatibility due to dat positive packed red cells (0.34%).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb-1) was admitted to our hospital in january 2017 for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu4 / atg protocol (5 days of 250 mg iv. fludarabine, 4 days of busulfan 792 mg iv, 2 days of 300 mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of 2016, was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december 2016. results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and 0.2 m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a 75-year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in 2004, with unknown transfusion history. her obstetric history was g6p4a2. the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a 0 rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from 9.0 g/dl to 12.0 g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (>99% prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of 1.2%, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no 0 rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an 18-years-old female patient with scd presented to our hospital with hemoglobin level of 4 g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial 11 cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r1r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to 2.8 g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to 8 g/dl in 2 weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r1r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than 1% of most populations are known as low incidence antigens and those present on more than 90% are known has high incidence antigens. the mns blood group system consists of 49 antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are 35 low incidence and 10 high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns49) antigen. anti-jenu was first described in a thai patient with thalassemia where only 2 compatible units were found following screening of 3600 units. the jenu negative phenotype is a rare phenotype with an estimated frequency of 0.06%. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of 4 + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd47 is also expressed on red blood cells (rbcs). recently, many drugs targeting cd47 have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd47 targeted high affinity sirpa fusion protein alx148. methods: a 35-year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx148 clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past 8 months. she had no previous transfusion history during the past two years. after two infusions of alx148, two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and 37°c albumin phase, and 2 + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and 37°c albumin phase, but 2 + at ahg phase. antibody screening (2 cells) and identification (11 cells) all showed 3 + at ahg phase using gel cards. direct antiglobulin test was 4 + for anti-igg and 3 + for anti-c3d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to 7.8 g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between 11.1-12.0 g/dl but it decreased again to 9.2 g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx148, and antibody screening and dat decreased to 0-1+ reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to 13.6 g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd47 is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms15/seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons 4-11 was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk*01(28a,226a,303a,588g)/jk*02. to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk*01(28a,226a,303a,588g)/ jk*01 and jk*02/jk*02, respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk*01(28a,226a,303a,588g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within 3 days of transfusion if a patient has been transfused or pregnant in the preceding 3 months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this 3 day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a 60 y/o man who presented to the ed (hospital day 0, hd0) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (<3 days). methods: specimen #1 (s1) was sent on hd0 for type & abs (t&s) and crossmatch (xm) of 2 rbcs. abs and immediate spin xm were negative; there was no patient history. by hd9, he had 4 negative t&s specimens (hd0: s1; hd2: s2&3; hd6: s4) and had been transfused 4 rbcs (hd2: 2; hd5: 2) via electronic xm (exm). at 1730 hr on hd9, 2 rbcs were requested and could have been issued via exm since s4 was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s5 was requested and received at 1801 hr. results: s5 showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from 10.2 (14.0-17.5 g/dl)/30.1 (41.5-50.4%) on hd6 to 6.9/ 20.6 on hd9. during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd9, which he tolerated well with an increase of hemoglobin from 6.9 g/dl to 8.6 g/ dl. he did well post transfusion with stable h/h between 8.1/24.2.0 to 8.5/25.3. he was discharged on hd19. repeat abs on s4 was negative. of the 4 rbcs transfused before s5, one was e+ and four jk(a+). the family reported that he was injured 20 years prior and had been admitted to 3 hospitals, but was unaware of transfusion. hospital #1 (h1) reported admissions 20 years ago (2 rbcs transfused) and 4 years ago; all abs were negative. h2 admission was 5 years ago with positive abs and inconclusive workup. h3 admission 4 years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than 3 days from the specimen collection, likely a secondary immune response to sensitization from a transfusion 20 years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every 3 days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a 75 year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong (4+). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr1), a high frequency antigen expressed by the abcg2 gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and 16 exons and adjacent intronic sequence of the abcg2 gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive (2+) in iat test, while the self-control was negative. the antiserums reacted strongly (4+ in iat test in gel card) with the papain-treated cells, but kept the same reaction strength (2+) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was 2. in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg2 gene in the patient and her brother revealed one homozygous nonsense mutation in exon 4 (c.376c>t, p.gln126x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals (98% or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, 76 years old, o rhd+, previously transfused with 4 red blood cells concentrates in 2006, was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected (2+ in all the 3 screening cells). in order to identify the specificity of the antibody, a panel of 11 cells was tested; the result was considered inconclusive, due to positive reactions (2+) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel (2+ with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg1 and igg3) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from 80865 blood samples, four donors with jk (a-b-) were selected, at a frequency of 0.0049%. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs5-1g>a combining with heterozygote of 359g>a (gly120glu) and heterozygote of 896g>a (gly299glu) combining with heterozygote of 956c>t(thr319met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs5-1g>a, 896g>a and 956c>t were common mutations in the before reports, while 359g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk3. background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of 10 blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: 5,213 blood donors from age 19 to 54 were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of 10 blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce 0.3%, rhce*ce 65.0%, rhce*ce 28.8%, rhce*ce 5.9% -kel*k_kpb_jsb allele 100% -jk*a allele 49.1%, jk*b allele 50.8%, jk*b_null allele 0.1% -fy*a allele 92.8%, fy*b allele 7.2% -gypa*m allele 51.1%, gypa*n allele 48.9% -gypb*s allele 5.1%, gypb*s allele 94.8%, gypb*mur allele 0.1% -di*a allele 4.7%, di*b allele 95.3% -do*a allele 10.1%, do*b allele 89.9% -co*a allele 100% -yt*a allele 100% -lu*b allele 100% summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding 16 blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu (8/14), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c.697c>g, and rhce*c.733c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c.667g>t were tested by pcr-ssp. a total of 1111 probands including 800 individuals from syria, 147 from iran, 123 from the arabian peninsula and 41 from northern african countries were included. results: 2% of the donors were homozygous for the fy null (fy*-67t>c, fy*02n.01) mutation, 16.2% carried the heterozygous mutation. 0.4% of the syrian probands were heterozygous for the do*350c>t mutation (both, do*jo1 and do*jo2; jo(a+/ a-)) that is virtually unknown in caucasian donors. 0.8% of the syrian donors heterozygously carried the kel*02.06 allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel*02.06 carriers were identified. 1.8% of the syrian and 1.5% of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c.251c>g, intron 5 + 5 g mutation, inducing the s-u+ w phenotype. 3.6% of all and 29.3% of northern african donors were heterozygous for the rhce*c.733c>g substitution, 0.3% of the syrian donors carried rhce*c.697c>g (heterozygously) and 0.004% of all donors were heterozygous for rhce*667g>t. heterozygosity for vel deficiency (vel*-01) was detected in 21 individuals (2%; 16 of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp1*c.601delg (co*01n.06) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals (2 blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: 1/gel cards diaclon id abo/d (anti-a: clone a5, anti-b: clone g1/2, anti-a,b: clone es131, es15+ birma 1+ es4; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m297/628 = la-2; bio-rad); 2/tube techniques with: anti-a (birma 1; a-11h5, a1 s.pa1m095, c.9113d10), anti-b (lb-2, b-6f9, c.9621a8). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of +7.21-kb site of abo gene to cover sequences ranging from the end of intron 1 to 3 0 utr of the abo gene. additionally sequence of exon 1 of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals (2 blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma 1 (1+ to 2+), a-11h5 (1+ to 2+) and c.9113d10 (weak+ to 1+); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a1 antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of 1061delc typical for abo*a2 alleles). in all these individuals abo sequencing of 7.21-kb fragment confirmed the heterozygous genotype with 7 polymorphisms characteristic for abo*b.01 allele (297a>g; 526c>g; 657c>t; 703g>a; 796c>a; 803g>c; 930g>a) and detected a novel abo*a allele sequence with duplication-based insertion of 21 bp after 564 position (abo*a c.dup543_563; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon 188 (p.dup_182_188qdvsmrr), with synonymous change of the repeated codon 182 (cgc>cgg) and 188 (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup543_563 allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a1.01 allele and the same abo*a c.dup543_563 allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in 1990 of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons 1-7 (including 50 base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a1 in plasma. abo genotyping gave the genotype abo*a1.01/o2.01 usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately 85%) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately 15% of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a1.01-like allele except for a novel mutation located in intron 5, c.239+4g. the o 2 allele had an additional snp, c.689g>a, consistent with the abo*o2.03 allele variant summary/conclusions: a previously unreported variant, c.239+4a>g, likely effecting the 5 0 -donor splice site of intron 5 was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. 374+4a>g mutation is located in the 5 0 -end of intron 6 and is predicted to cause partial skipping of exon 6. strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately 2% positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past 3 years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a2, b, o1 and o2 alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon 6, intron 6 and exon 7 were amplified using a forward primer targeting the 261g nucleotide (to exclude o1 alleles amplification) in combination with either b, a2 or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of 16 samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in 8 out of 12 samples with suspected a subgroup alleles, the c.804insg insertion was detected corresponding to the abo*ael.01 allele. the abo*aw31.02-05 variant, a hybrid a1-o1v allele, was found in 2 cases. in 1 case we found the c.722g>c change, previously reported associated with weak a antigen expression. finally, a novel c.961c>g change was detected in an a2 allele. b(a) or cis-ab suspected alleles: the abo*b(a)04 variant carrying the c.640a>g change was found in 1 of 3 samples with bo1 genotype but a weak antigen expression. in the remaining 2 cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b01.02 allele carrying the nucleotidic change c.926a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon 7 of the abo gene, in 14 out of 16 samples, including 2 novel alleles. chimerism was suspected in 2 cases of a antigen expression in samples with b1o1 genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but 8% of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a 20-year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih-500; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a 3 b 3 phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a102/b101/o01. however, in buccal swabs, the patient showed a102/o01 and his brother showed b101/o01. other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d8s1179, d21s11, d7s820, csf1po, th01, d13s317, d16s539, d19s433, d18s51, d5s818, and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case #1 is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.29-5t>g in heterozygosity on an otherwise common a1 allele, and in trans an abo*b.01 allele. case #2 is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c.29-5t>g in heterozygosity on an a background, and in trans an abo*o.01.01 allele. given that this variant is located near the intron 1 splice acceptor site, abo*29-5g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case #3 is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c.467c>t (pro156leu) and c.709t>a (tyr237asn), both in heterozygosity on an otherwise common a1 allele, with an abo*o.01.01 allele in trans. thus, the data establish an association of allele abo*467t,709a with an aweak-like phenotype. case #4 is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c.479c>g (pro160arg) in heterozygosity on an a2 background, and in trans an abo*o.01 allele. an interpretation of the data is that variant c.479c>g weakens the activity of the a2 transferase, with allele abo*a2(479g) encoding the aweak-like phenotype detected by serology. case #5 is a 9 year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon 7 detected variant c.803g>c (gly268ala) in heterozygosity on an a2 background, and in trans an abo*o.01.02 allele. the serology and molecular results suggest that allele abo*a2(803c) encodes a cisab weak phenotype. case #6 is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c.739g>a (glu247lys) and c.871g>a (asp291asn), both in heterozygosity, in trans, and on a1 backgrounds. variant c.871g>a by itself constitutes allele abo*a3.01. the phenotype encoded by abo*739a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case #1 is a 35 year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a1/abo*o1. sequencing detected variant c.107insg (val36gly>fs56ter) in heterozygosity on an otherwise common a1 allele, and in trans an abo*o.01.01 allele. it is unclear how the early truncation of the a1 transferase encoded by allele abo*107insg still allows for some residual enzyme activity, as suggested by the reverse a type. case #2 is a recently-transfused 72 year-old black patient with an unresolved abo type. sequencing detected variant c.297a>g (silent) in homozygosity and variant c.1049c>t (ala350val) in heterozygosity, both on an o1 background, with an abo*b.01 allele in trans. although variants c.297a>g and c.1049c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o1(297g,1049t) allele. case #3 is a 40 year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon 7 detected variant c.436c>t (arg146cys) in heterozygosity on an abo*b allele background, and in trans an abo*o.01.01 allele. the phenotype encoded by allele abo*b(436t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case #4 is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a 2+ mixed field (mf) in tube. reverse type on a1 cells 1+ in gel, 0/1+ in tube. sequencing of genomic dna and cloned pcr products covering exons 6-7 detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*o.01.09 allele. case #5 is the newborn baby of case #4, with a forward type a 1+ mf in gel, a 3+ mf in tube. sequencing of the baby's dna detected variant c.784g>c (asp262his) in heterozygosity, and in trans an abo*b.01 allele. from these results it is inferred that the phenotype encoded by allele abo*784c is a3-like. case #6 is an 18 year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons 5-6 and 6-7 detected variant c.979c>t (gln327ter) in heterozygosity, and in trans an abo*o.01.02 allele. the truncation of the a1 transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo*979t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron 1 enhancer. variants reported to date in the intron 1 enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron 1 enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut1 could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v5.1 170221 of red cell immunogenetics and blood group terminology working group of the isbt, 55 fut1 alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut1 alleles for the 19 chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut1 was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut1 sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut1 homozygous mutations were found in the 12 individuals and fut1 heterozygous changes were existed in 7 individuals after bidirectionally sequencing. .05%, 7.89%, 5.26%, 5.26%, 5.26%, 2.63%, 2.63%, 2.63% respectively in the 19 individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut1*01n.06(c.551_552delag), fut1*01w.09 (c.658c>t) and fut1*01w.01 (c.293c>t) were abolished in vitro assay, while fut1 mrna levels of them had no effect compared with wild type. summary/conclusions: the fut1 mutations in the para-bombay individuals were various. the most common fut1 allele in the chinese individuals with para-bombay phenotype was fut1*01n.06(c.551_552delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a1 and a2. later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a1, and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons 6 and 7 of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron 1 enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron 1 enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o1v/a 1 . dna sequence analysis showed result abo*a01 (28+5859a), abo*o.01.02. the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a01(28+5859a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d175-2, immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately 85% of o d+c+ cells. the patient's genotype was identified as abo*o.01/*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a1/*o1 genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a1 homozygote result. sanger sequencing of abo exons 6 and 7 also gave anomalous reactions: no abo*a allele was detected. homozygosity for c.261delg was observed as well as heterozygosity for c768c/a. this result therefore suggests the patient's genotype is abo*o.01/*o.01.26. next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr 3 0 -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing 4 copies of the enhancer region were present. presence of a single copy of the 43-bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, 9-30% of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd*01el.01 (rhd*1227a) is most prevalent (>99%) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from 2017 to 2018, the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th-28/ms-26, igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of 129 serologically apparent d-chinese patients (female, n = 128; male, n = 1) with alloanti-d were identified. different titers of alloanti-d from 1:2 to 1:4096 (≤1:16, n = 60; >1:16, n = 69) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = 11; anti-d mixed anti-e, n = 5). serological rhd typing confirmed the serologically apparent d-phenotype. rhd*01n.01/01n.01 (homozygous rhd gene deletion) genotype was identified in majority of them (123/129, 95.3%) by rhd zygosity analysis, while rhd*01n.03/ 01n.01 genotype (n = 5) and rhd*01n.05/01n.01 genotype (n = 1) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average 25% frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, 2005; legler, trans. med., 2001; richard, transfusion, 2007) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all 10 rhd exons were sequenced. the sequencing data revealed the mutation c.148+2delt in the splice donor site of exon 1. to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns4) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c.148+2delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing 9 monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor 1 were predicted to be rh:-1,-2,-3,4,5. however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position 1151 which is the second last nucleotide of exon 8 and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon 8 was identified. the mutation c.1151c>g leads to the amino acid substitution t384r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., 2011) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd*1151g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor 2. further serological determination of the rhd antigen with 9 different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c.526g>c in exon 4. this mutation causes a p.a176p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c.1151c>g and c.526g>c harbouring an amino acid substitution within a transmembrane segment. the c.1151c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c.1151c>g suggests that the t384r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c.526g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id1 is a d-negative donor self-declared as african descent. sample #id2 is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id1 was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id1 were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id2 was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional (3d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id1, a single nucleotide missense change, i.e. c.1151c>g in exon 8, was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position 384 (p.thr384arg) of the rhd protein. analysis in the 3d model clearly suggests a dramatic impact of the p.thr384arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id1. in sample #id2, the single c.325a>g transition was found in exon 2 leading to a threonine-to-alanine substitution at amino acid position 109 (p.thr109ala). amino acid 109 is located in rhd protein extracellular loop 2, and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id2. summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type 4 and diva cluster. in africans, the 16 most frequent were typically associated with alleles of the weak d type 4 (including dol and rhdpsi), diva and dau clusters with f223v occurring in > 10% of alleles; in addition the key mutations of weak d type 1 and dii and two inactivating mutations (c.1056_1057inst and c.1060delg) not reported in rhb were among the first 20 polymorphisms. in east asians, rhd(1227g>a) at 0.8% was most frequent, followed by dfv, weak d type 33, dbo-3, key mutations of diva and weak d type 4 cluster as well as rhce-like substitutions and the mutations of weak d type 25, type 15, type 66, rhd(a85v), dvl-1, weak d 119 and rhd(n135s). weak d type 151 and rhd(t148r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type 45 and type 33 in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r 0 haplotype and 1 probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r 1 r 1 , r 2 r 2 , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in 2015 and 2017 to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v3.1 cycle sequencing kit. sequence data was aligned to rhd_ng_007494.1. rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on 201 rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using 2 different anti-d igm clones (clone 1, dvi+: ldm1+esd1m; clone 2, dvi-: rum-1, th28) and 2 different anti-d igg clones (clone 1: ms26; clone 2: d415 1e4). all donors with d-negative results (n = 201, divided into 153 subjects with rhc+, 45 with rhe+ and 3 with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further 101 donors, studied prospectively. in 92.5% of donors (n = 186), the molecular analyses showed the complete deletion of the rhd locus, while in 15 cases (7.5%) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these 15 donors revealed the presence of 9 negative and 4 weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd*01n.82 (2 cases), rhd*01n.83 (1), rhd*01n.80 (1), rhd*01n.61 (1) , rhd*01n.05 (3), rhd*01el.08 (1), rhd*01el.18 (1), rhd*01el.17 (1), rhd*01w.54 (1) . moreover we found a donor with a lack of signal encompassing exons 1-5 of the rhd sequence (bioarray rhd beadchip), while 2 additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about 1 in 50 cases (4 subjects out of 201), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of 43960 serologically typed donors, 85.5% were d+, 14.0% d-and 0.5% weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in 22 blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: 62% weak d type 1, 28% weak d type 3, one donor was typed as weak d type 14 and another one as weak d type 11. these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from 2+ to 4+ with iat, except for the weak d type 11 with the score of <1+ which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was 19%. one of the weak d type 1 donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type 14, as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a 14 year-old young woman with parameters of hemolytic anemia: low hemoglobin level (10 g/dl), reticulocytosis (14%), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron 4 and the 3 0 untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c.920c>t mutation in rhag exon 6 in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c.920c>t mutation is responsible for the p.ser307phe amino acid substitution predicted to be in the 10 th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c.920c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of 15692 individual blood donors within a 15 month period, 1688 (10.76%) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from 112 out of 151 (8.95%) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron 4, exon 7 and exon 10 by a multiplex pcr-ssp method. the reaction was conducted in a final volume of 20 ll with primers that were applied as described by f. wagner et al. (bmc genetics, 2001 ) except antisense primer for exon 10 and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a 4% agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of 112 d-individuals analyzed, 101 were ddccee, 8 ddccee, 2 ddccee and one had a ddccee phenotype. molecular analysis showed that 104 (92.86%) were negative for all four rhd dna regions. among the other 8 samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron 4, exon 7 and exon 10, three for rhd promoter and exon 10, and two for exon 10 alone. further genotyping revealed five hybrid rhd-ce-d alleles [3 rhd-ce(2-9)-d and 2 rhd-ce(3-7)-d], one allele represented the del(m295i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than 100 weak d types have been described to date. transfusion recipients with weak d type 1, 2, or 3 are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is 5 anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band 3, as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, 4 haplotypes containing the mcc b and sl2 polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr1 polymorphisms and haplotypes, especially those containing mcc b and sl2 snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr1 polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do*01 and do*02 backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a 42 year old woman from the northeast brazil with a history of 4 pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the 3 exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology 2 diagnostic immunohematology 3 experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises 360 blood group antigens. most antigens (322) belong to one of the 36 blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of 38 antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in 1967.~91% of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in 96% of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a 3-cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b4galnt2 gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad 3-cells panel, containing donor 626521 with high expression of sd a . additionally, 200 pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b4galtnt2 exon 1-11. results: sequencing of b4galtnt2 revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in 6 controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon 10, c.1396t>c (enst00000300404.2, rs7224888) changes a cysteine to arginine at position 466 of the protein. the second mutation in exon 11 c.1590a>g (rs16946912) does not change an amino acid. both snps have a maf of 0.11 and therefore we expect that 2.1% of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b4galtnt2 gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon 10 causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~4% sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b4galnt2 and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by 5ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl8 was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = 0.016). similar result was also observed in rbc chemokine scavenging of ccl2 (p = 0.038). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = 0.021). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp1pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a 66-year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp1pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was 12.9 g/dl. 800 ml of blood was withdrawn through a radial arterial catheter in two 400 ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of 6% hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged 48 h later with a hemoglobin level of 12.3 g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp1pk and sequencing of the a4galt gene, which were conducted according to the protocols by koda et al.(transfusion. 2002) . the proband and her brother were homozygous for c.1029dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[140a], gypb*s_null(ivs5 + 5t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c.140c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about 0.06% and in thais with 0.04%. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom*12 allele. the lack of the serf antigen, serf(à) on red cells is caused by a single nucleotide polymorphism, c.647c>t in exon 5 of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(à) and a serf(à) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom*12 allele among blood donors. aims: this study aimed to identify the crom*12 allele among thai blood donors leading to predicted serf(+) and serf(à) phenotypes. methods: dna samples obtained from 1,512 central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom*12(+) and crom*12(à) among 1,512 central thais were 0.992 (3,001/3,024) and 0.008 (23/3,024), respectively. the homozygous of crom*12(à/à) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen 100 samples together with 23 heterozygous crom*12(+/à) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom*12(+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than 25 years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o1, o2, a2 and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, 31 from patients and 25 from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd1227g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene 1227 g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd 1227g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd 1227g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to 5 0 ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd 1227g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon 9 sequencing was performed to determine rhd 1227g>a genotype. results: a total of 84 samples were genotyped for rhd 1227g>a by pcr with t mshift primers. 32 samples were typed as 1227a+/g-, 22 samples were typed as 1227a-/g+, 6 samples were typed as 1227a+/g+ and 24 samples were typed as 1227a-/g-. two samples typed as 1227a+/g+ by pcr-ssp but 1227a+/g-by pcr with t m -shift primers were confirmed as 1227a+/g-by rhd exon 9 sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd 1227g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the 115 dna samples, 75 (65.2%) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon 8, which is functionally not relevant as reported before, was found in 28 samples, including 11 and 17 samples carrying respectively rhd-ce(8)-d and rhce-d(8)-ce (two homozygous each). except two samples that require additional studies (1.7%), rhd zygosity was resolved successfully: 60 (n = 2 rhd gene copies; 52.2%), 49 (1; 42.6%) and 4 (0; 3.5%). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples (4/115, 3.5%) carrying rhd*dau5, rhd*dv.2, a rhd*diiia-like allele, and a novel rhd*d-ce(7:g329h-y330s-n331i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd*03n.01 and rhd*diiic, were also found in trans with a normal rhd*01 allele. in rhce, c/c genotype could be resolved. the rhce*ce16 (rhce*ce (48c)-d(9)-ce) allele, which is commonly cis-associated with rhd*ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = 19) vs. non-bleeding (n = 15) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd63), p-selectin (cd62p), activated gpiib/iiia (pac-1) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd42b). normal healthy reference levels were available. results: the platelet count in bleeding (72 9 10 9 /l) and non-bleeding (68 9 10 9 /l) patients was comparable (p = 0,66). bleeding patients had a higher bat score compared to non-bleeding patients (10 vs. 2, p < 0,01). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) (86,16% and 4,96mfi) were higher, compared to bleeding patients (64,08% and 2,44mfi, both p < 0,05), and the proportion of ps+cps correlated negatively with bat score (r 2 =0,26, p < 0,01). cd63 + cp was higher in non-bleeding (97,25% and 10,54mfi) compared to both bleeding patients (94,88% and 6,89mfi) and significantly higher than the reference level (88,44% and 5,36mfi, both p < 0,05). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist (134,12 mfi unstimulated vs 32,38 mfi reference, p < 0,01). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa-1, hpa-2, hpa-3, hpa-5 and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa-1a, anti-hpa-2b, anti-hpa-3a, anti-hpa-5a samples were used to validate the specificities of the luminex assay. the anti-hpa-1a, anti-hpa-3a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to 1/1024). results: 44 samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa-1a, hpa-2b, hpa-3a, or hpa-5a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa-1a, and anti-hpa-3a were 1:512 and 1:64, respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of 44 samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of 358 individuals with normal abo groups (101 group a, 100 group b and 101 group ab individuals, and 56 group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg1 anti-b antibody (9431pe bgrl1). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v5.01. the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than 0.05 were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about 66.22% of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: 40 cases were found with antibody positive. among them, 6 cases (15%) were only anti-hla-i positive, 4 cases (10%) were only anti-hpa positive, 30 cases (75%) were both anti-hla-i and anti-hpa positive. 8 cases were found without anti-hla-i or anti-hpa. among the 34 cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were 73.5%, 61.2%, 50%, 44.1%, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > 0.05). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of 303 blood donors and 302 hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v 2 test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr3b*01 (hna-1a), fcgr3b*02 (hna-1bd), and fcgr3b*03 (hna-1bc) alleles were 0.384, 0.584 and 0.032; for the slc44a2*1 (hna-3a) and slc44a2*2 (hna-3b) alleles, 0.804 and 0.196; for the itgam*1 (hna-4a) and itgam*2(hna-4b) alleles, 0.898 and 0.102; for the itgal*1 (hna-5a) and itgal*2 (hna-5b) alleles, 0.708 and 0.292, respectively. in hematological patients, the gene frequencies for hna-1a/1bd/bc, -3a/3b, -4a/4b, and -5a/5b were 0.376/0.588/0.036, 0.795/0.205, 0.887/0.113, and 0.699/0.301, respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna -1, -3-5 for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = 605). the predicted risk of hna-1, -3, -4, -5 incompatibilities in this cohort were 34.9%, 26.9%, 19%, and 32.9%, respectively. the possible risk of hna-1a, -1bd, and -1bc alloimmunization were 0.233, 0.143, and 0.064, respectively; of hna-3a and -3b alloimmunization, 0.037 and 0.231; of hna-4a and -4b alloimmunization, 0.01 and 0.163; of hna-5a and -5b alloimmunization, 0.080 and 0.250, respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons (5, 7 or 10) of the rhd gene. methods: 2004 samples were tested from 01/12/2017 to 01/12/2018 using free dna fetal kit â rhd. 44 samples (2,2%) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce(4-7)-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. 65% of the identified variants are rhd negative alleles including alleles leading to partial rh2 antigen expression. unexpected alleles are found such as weak d type 1, 2, 5 or 11. these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on 20 cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed 16 of 20 (80%) pregnant women were rhd*weak d type 1 and not at risk for anti-d. rhd*weak d type 3 were typed in 3 cases (15%) and 1 case was rhd*weak partial 4.0 and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that 95% of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may 2018, uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of 3 ml. all material used passed pre-acceptance serological testing; samples were dispatched to 298 participants in 17 countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of 1.1 ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the 36 participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed 2 + reactions vs. two reagents by tube, cf. 4 + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of 3.7 ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with 4/17 anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all 16 respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa-1a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from 133 plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = 29), s/s (n = 13), hpa-1a (n = 49), hpa-3 (n = 8), hpa-5 (n = 14), hpa-15 (n = 20) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of 100-16,000 reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from 0,01% to 26,4% (one case was for advanced pregnancy -38 week of gestation). in 19/133 tested samples false positive results were detected at the level of 1 or 2 unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa-1a, -3a/b, -5a/b, -15a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh1) and anti-c (rh4) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih-500 system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih-500 system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from 29 samples containing anti-d and 20 containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih-500 system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < 0.0001, r = 0.79 and p < 0.0001, r = 0.82 respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < 0.0001) but gel scores have a better correlation coefficient than tube scores (r = 0.78 versus 0.55). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were 75 and 35, corresponding respectively to 5 ui/ml (250 uchp/ml) of anti-d and 7.5 ui/ml (500 uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih-500 system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from 250 pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january 2008 to december 2016. we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ 1:8 for anti kell antibodies and ≥ 1:32 for other specificities. results: out of 250 women, 143 (57.2%) displayed clinically significant antibodies, 92 (36.8%) clinically insignificant antibodies and 15 (6%) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d (16.8%) also in combination with other rh antibodies (8.8%), while anti-k accounted for 10%, anti-e for 10% and antibodies against high-incidence antigens for 2.4%. anti-m and anti-le a antibodies were also found (16.8% and 8% respectively) but they were not clinically significant. among 143 women with clinically relevant antibodies, 37 showed a critic antibody title and they underwent gynecological and obstetric monitoring. 21 fetuses resulted affected by hdfn, displaying anti-d in 16 cases and anti-kell in 5. 11 fetuses with severe hdfn (anti-d in 7 and anti-kell in 4) required iuts, 2 were treated with et, 8 received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from 0,4% to 2,7% worldwide. there are over 400 erythrocyte surface antigens, of which more than 43 have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in 2016 and 2017. methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during 2 years, from january 2016 to december 2017. in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, 5982 pregnant women were attended in hospital de braga. the laboratory registered 100 positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was 1,7%. anti-d was the most common antibody found (58,5%). anti-d prophylaxis given during pregnancy was responsible for 51 of 62 cases and maternal antibody titer levels did not exceed 8 among these cases. the prevalence of non-rhd immunization was 33%. anti-e (9,4%) was the most frequent alloantibody other than anti-d followed by anti-m (5,7%) and anti-c (4,7%). multiple maternal antibodies were found in 5 pregnant women. four women had 2 types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had 3 types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in 45% of them and phototherapy was given in 14%. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was 1,7%. the erythrocyte antibody screening showed that anti-d was the most common antibody found (58,5%) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was 33%. the other most frequent alloantibodies were anti-e (9,4%), anti-m (5,7%) and anti-c (4,7%). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may 2018, the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih-500 system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard (01/572) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the 2-months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of 2 have lower values. the highest differences (more than 2 to 3 dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel1) and anti-m (mns1) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih-500 system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a 1-day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was 13 mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes (15.2%), ldh (1162 iu/l) and g6pd (25.3 u/ghb); dat (+/-), iat (-), anemia (hb 8.7 g/dl, hct 28%), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs (3 + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with 1:1024 and 1:2048. the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to 14.6, bilirubin level was within normal range, she was discharged. another 5-days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was 15 mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes (5.2%), dat (+/à), iat (à), hb 12. background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in:1,-2 phenotype (in(a+b-)) are observed with a frequency of < 0.1% in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon 2 of cd44. results: in a 27-year-old pregnant (para 1) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of 1:2 was detected by iat (negative with papain-treated cells) at gestational week (gw) 32 and 36. the mma, performed in duplicate on samples taken at these dates, showed a mi of 0.5%/4.8% and 7.7%/6.3% respectively. the mi was interpreted as follows: 0-3% not relevant; 3-5% inconclusive; >5% clinical significant. the patient's parents were typed heterozygous, in:1,2 whereas her husband was homozygous, in:-1,2. due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw 41 without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw 32, was inconclusive whereas the second mma, performed in gw 36, indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included 20 immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period 2013 and 2018. results: the "critical titre" in our study was 8, meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only 2 newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than 4. moderate hemolytic disease of fetus and newborn were caused between the titre values 8-32. the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, 16 weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons 4, 5 and 10. rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all 3 exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at 3 nucleotide positions (c.1154, c.1193, c.1227) located in rhd exon 9 suggesting exon 9 was either deleted or rhce-replaced. paternal and cord gdna sequencing detected 4 out of 6 snps (c.186g>t, c.410c>t, c.455a>c, c.602c>g) associated with diiia phenotype plus 2 additional snps (c.604g>a, c.733g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from 5.3 iu/ml (16 weeks gestation) to 166 iu/ml (33 weeks gestation). the fetus required 3 intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon 9 deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd109 is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of 170 kda. in addition to being expressed on human plts, cd109 is expressed on activated t-cells, endothelial cells, cd34 + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa-15a and -15b are 0.505 and 0.495, respectively. based on these data, the risk of alloimmunization against hpa-15 alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa-15 alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa 15b alloantibodies by maipa and icfa. methods: a 24-year-old mother, gravida 2/para 0. the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy 33 weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy 24 weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to 4.7 9 10 10 /l, wbc count dropped to 2.96 9 10 10 /l, including neutrophil 37%, lymphocyte 16%, mononuclear 43%, eosinophil 2%, basophil 2%, red blood cells were normal, hb was 111 g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa-1a/a, -2a/a, -3a/a, -4a/a. -5a/a, 6a/a, 7a/a, 15a/b, naka (+) and the maternal was hpa-1a/a, -2a/b, -3a/a, -4a/a. -5a/a, 6a/a, 7a/ a, 15a/a, naka (+). the paternal genotype was hpa-1a/a, 2a/b, 3a/a, 4a/a, 5a/a, 6a/a, 7a/a, 15a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa-15aa and -15bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa-15a/a (donors 1), hpa-15a/b (donors 2) and hpa-15b/b (donors 3) donors by maipa. the mother's serum showed no reactivity against 15a/a plts, weak positive reactivity against 15a/b plts (od values 0.28), but strong reactivity against 15b/b plts (od values 0.38).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa-15genotyped donors (anti-hpa-15b average value 9.8). summary/conclusions: in this study, we found anti-hpa-15b in a case of fnait (patient hpa-15aa, blood group o) using the maipa technique. we were able to detect the presence of hpa-15b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in 1:10000 live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = 40). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, 2014 ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at 4 weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. 19%) are deceased due to organ rejection, and 8/36 patients (22%) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of 8-methoxypsoralen (8-mop) and irradiation with uv-a light. however, the addition of 8-mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with 8-mop+uv-a compared to uv-c treatment without additional 8-mop. methods: we used an in vitro 72 h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with 0,2 lg/ ml 8-mop plus 2 j/cm 2 uv-a treated cells and 2 j/cm 2 (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and 7-aad flow cytometry standings. results: the apoptosis analysis of cd3 cd4 t-helper cells, cd3 cd8 cytotoxic tcells, cd19 b-cells, cd14 monocytes, cd3 neg cd56 nk-cells and cd3 cd56 nkt cells revealed no statistical differences in almost all of these cell types after treatment with 8-mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after 72 h were similar in both treatment groups. summary/conclusions: the addition of 8-mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the 8-mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of 8-mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with 8-mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with 16 bedded micu and received tpe therapy between 1 june, 2016 and 31 december 2018. all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln9000 apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone 269 tpe procedures. among them, thirty were female patients (43%). the median age 45 (13-75) years. guillain-barre syndrome (gbs) was the most common indication (72%) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were 48 (18%) episodes of patient related complications during the tpe treatments. in 8 (3%) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts 13 levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of 9 years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in 46 patients with diagnosis of maha (27-ahus, 16 -ttp, 1 each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was 19.94 ae 19.58 years with m:f as 1.5:1. number of procedures per patient varied from 1 to 27. post pe recovery was observed within 10-14 days with statistically significant increase in mean platelet count from 40.05 ae 5.9 to 82.11 ae 12.10 9 10 9 /l (p = 0.000) and significant decline in mean lactate dehydrogenase level from 48.91 ae 34.73 to 10.98 ae 6.49 lkat/l (p = 0.000). there was also significant decline in mean percentage of schistocytes in peripheral smear from 5.44 ae 3.96% to 0.56 ae 0.89% (p = 0.000). the mean serum urea changed from 48.88 ae 24.56 to 24.50 ae 17.78 mmol/l and creatinine from 266.11 ae 142.59 to 173.09 ae 127.34 lmol/l (p = 0.000 and 0.001 respectively) with significant increase in urine output from 0.71 ae 0.53 to 1.06 ae 0.33 ml/kg/h (p = 0.000). adverse events were observed in 10 patients (21%), allergic reaction to replacement fluid (n = 6) being the commonest followed by hypotension (n = 2), rigors and chills (n = 2). overall survival rate at 6 months was 89%. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a 17 year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since 2-3 days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, 6 cycles of tpe were performed on daily basis. after 6 cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [1] for motor performance, patient was assessed on a disability scale (0 = healthy; 1 = minor symptoms; 2 = able to walk 5 meters without support; 3 = able to walk 5 meters with support; 4 = confined to bed or wheelchair; 5 = requiring assisted ventilation; 6 = dead).patient's motor performance was increased to scale 1(upper limb) and 2(lower limb) from scale 5, deep tendon reflexes were normal. visual function began to improve 1 week after the treatment. visual acuity was 6/6 after 4 weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of 10% has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july 2014 to july 2018. a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > 10% but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than 10%, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were 10.6%, 11% and 31%. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused 46 days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between 9-16 days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from 01/01/2017 to 31/12/2018 in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of 6 patients met the inclusion criteria described. however, 1 patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded 88 exchange procedures (42 pmet and 46 aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why 2 patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = 2), stroke (n = 1) and recurrent vaso-occlusive crisis with multiorgan failure (n = 2). for both procedures, target values were to obtain a pre-exchange hbs level ≤ 30% for stroke and cerebral vasculopathy and ≤ 50-60% for other indications. the median hbs level before pmet was 42,6% (31,3-59,1) and 38,4% (18, 9) before aet. we documented a higher hbs level prior to the next procedure in 11,4% of patients (n = 10). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: 1356,1 vs. aet: 1314,7 ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: 360 vs. aet: 60 min and pmet: 21 vs. aet: 24 weeks, respectively). annual rbc requirements per procedure were superior (median 2 vs. 4 units) and the overall costs related with aet were 2,2 times higher -18.180,93€ and 8.174,79€ aet and pmet, respectively (estimated cost per session aet: 790,48€ and pmet: 389,28€). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo2) of 87% on air. the patient did not show improvement in spo2 level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo2 of 97% on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july 2016 to august 2017 were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these 46 (43%) were done on the mcs plus (haemonetics corporation) and rest 61 (57%) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = 0.000). the systolic (p = 0.010) and diastolic (0.001) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < 0.001), the age of the patient (p < 0.001) and the pre-procedure ph (p = 0.002). procedurerelated complications occurred during 30 procedures of which 13 complications (12.15%) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of 16 years (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . tpe procedures were done on two different apheretic devices (cs 3000 plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of 356 tpe (range 1-22/patient with mean of 6.2 procedures) were performed for 55 pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total 44 patients, neuromyelitis optica (category ii) in 4 patients, rapid proliferative glomerulonephritis (category i), c3 glomerulopathy in 3 patients each and one patient of infective hemophagocytosis. the average age of patient population was 7.8 yrs (1.2-13 years) . the male:female ratio was 3:1 with an average weight of 25.5 kgs. adverse events were observed during 20 (5.61%) procedures. most commonly observed adverse events were allergic reaction to replacement fluid (1.4%) followed by hypotension (1.1%), line occlusion (0.8%), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each (0.28%).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of 160 registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = 80)they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ 50 g); and group ii (n = 80)they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion 60, ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected 1 h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = 0.000) in group i (mean ae standard deviation: 67.86 ae 8.07 g; range: 50.80-92.13 g) than group ii (60.92 ae 8.29 g; range: 40.86-86.76 g). the mean hb increment in group i patients (3.26 ae 0.83 g/dl) was significantly higher (p = 0.04) than the group ii patients (3.00 ae 0.76 g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = 0.000 in groups i and ii), age (p = 0.001 for group i; p = 0.032 for group ii), body surface area (bsa) (p = 0.002 for group i; p = 0.000 for group ii) and blood volume (p = 0.006 for group i; p = 0.000 in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = 0.000 in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of 50 g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under 50 years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between 20/03/2004 and 01/09/2015 was collected. for the current study, male transfusion recipients under 50 years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (<10 days storage) or 'old' (>24 up to 36 days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was 0.47 day shorter when comparing the 221 patients who died, to 1,623 patients who survived (ci: à1.41 to 0.48). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was 0.64 day longer when comparing the 27 patients who died, to 101 patients who survived (ci: à2.53 to 3.80). in the single-transfusion cohort, 1,280 patients received a fresh rbc transfusion from a male donor, 52 of whom died; 138 patients received a fresh transfusion from an ever-pregnant female donor, 9 of whom died. 193 patients received an old transfusion from a male donor, 7 of whom died; 16 patients received an old transfusion from an ever-pregnant female, 2 of whom died. the 3-years cumulative incidence of death among young male recipients was 4.7% (confidence interval (ci): 3.6% to 6.2%) after a fresh transfusion from a male donor and 4.8% (ci: 2.2% to 10.4%) after a fresh transfusion from an ever-pregnant female donor. the 3-years cumulative incidence of death was 7.2% (ci: 3.8% to 13.6%) after an old transfusion from a male donor and 15.4% (ci: 4.1% to 48.8%) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at 3 years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from 50 to 80%. in our previous investigation, we found that premedication rate was 92.5% in the outpatients in 2017, which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, 2017 was retrospectively surveyed. evidence-based clinical practice was initiated since january, 2018. clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, 2018. the incidences of atrs and premedication rates in 2017 and 2018 were compared using chi-square test. a p value less than 0.05 was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, 2017 to september, 2018, a total of 5,018 blood units were transfused in the outpatients with 2,453 transfusion events. of these, 25 cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was 92.5% in 2017, and was significantly decreased to 77.9% in 2018 (p < 0.001). it was reported that the incidences of atrs in 2017 and 2018 were 0.48% and 0.52% per unit, respectively. there was no remarkable difference between the incidence of atrs in 2017 and 2018 (p = 0.642). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of 19 granulocyte transfusions in 15 hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) 5-10 lg/kg and tablet dexamethasone 8 mg, 10-12 h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within 30 days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of 1 9 10 10 per unit was fulfilled in 90% of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < 500/ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least 48 h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within 24 h) increased significantly (median value: 350/ll) as compared to baseline levels (median value: 40/ll) (p < 0.05). infection related mortality was observed in only 20% (3 out of 15) of patients. patients became afebrile within 2-4 days and culture negative within 3-6 days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: 1.5-3.0 9 10 8 cells/kg and high dose: >3.0 9 10 8 cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in 2016, we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for 2017. results are compared to the pilot audit and summarized below. methods: we collected data on 3 performance indicators from 7 acute public care hospitals for 4 weeks each in march and august 2017 (the pilot audit covered 2 weeks in 2016). the performance indicators were: 1). percentage compliance to documentation of red blood cell transfusion indications 2). percentage of patients screened for pre-operative anaemia, 14 to 45 days before surgery 3). peri-operative transfusion rates (3 days before to 3 days after surgery) for 6 commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator 3) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in 2012. results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator 1), 2 hospitals had a compliance of 57-61%, the remaining 5 had a compliance of 90-100%. all 7 hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from 75% in 2016 to 100% in a hospital that had newly mandated electronic ordering. for indicator 2), results ranged from 30% to 92%. 2 hospitals made notable improvements when compared to 2016, achieving 83% and 88% respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least 2 weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator 3), mean number of transfused units for each surgery ranged from 1.5 to 2.8 units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at 46% and lowest for tkr at 7%. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator 3) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february 2017, there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november 2017 and march 2018. the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha 3 months after an allogeneic stem cell transplant, who has received multiple transfusions in the last 2 weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently 48 g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of 104 g/l and returns to clinic at a 2-week interval with symptoms of fatigue. he is actively haemolysing and commenced on 1 mg/kg prednisolone. results: there was a 42% (58/137) response rate by trusts. faced with a 4-6 h delay for allo-adsorption studies, 68% (38/56) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, 7% (4/56) would transfuse with o rh d negative red cells and 25% (14/56) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. 2017 british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the 2017 serious hazards of transfusion (shot) report, the most serious and fatal of 95 cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of 38 g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < 60 g/l after an hb fall of at least 40 g/l in the previous 2 weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits (2008) (2009) (2010) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between 14 th and 27 th may 2018 excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines (2008) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to 2 or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: 105 episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of 241 rbc units to 103 patients and the discard of 2 rbcs (due to incorrect transport). of the 105 episodes, 78 episodes (74%) involved an eventual switch to group-specific rbcs (range of emergency units, 1-13 units). the main requester was the emergency department (53%). the most common clinical indication for transfusion was acute gastrointestinal bleeding (49%). of the 105 episodes, 28 episodes (27%) did not meet the guidelines for emergency use because > 2 units of emergency uncrossmatched o rhd-neg rbcs were issued. 2 episodes (2%) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. 30 episodes (29%) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > 1 h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding (6%), receipt of an unsuitable pretransfusion sample requiring sample recollection (5%), delay in pre-transfusion sample processing (4%), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding (10%). only one patient was investigated for potential transfusion-related adverse outcome (1%) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, 105 episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with 241 rbcs issued and 2 rbcs discarded. a significant proportion of episodes (29%) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within 1 h before transfusion, 1 h after and 14-24 h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap-6) was assessed by flow cytometry by measuring p-selectin and pac-1 expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of 22 patients. the mean platelet count before transfusion was 8 9 10 9 /l (range 2-34 9 10 9 /l). 1 h cci was 11 9 10 9 /l and 14-24 h cci was 6 9 10 9 /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at 1 h after and 14-24 h after transfusion compared to before transfusion (p < 0.05). pac-1 expression after stimulation with adp was significantly higher at 14-24 h after transfusion (p < 0.001), but not at 1 h after transfusion. in rotem, clot amplitude at 10 and 20 min (a10 and a20) as well as maximum clot firmness (mcf) improved after transfusion (p < 0.05). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s =0.53 and 0.45 respectively, p < 0.001). absolute platelet count was also significantly correlated with mcf (r s =0.61, p < 0.001), where 84% of patients with a platelet count of more than 20 9 10 9 /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than 20 9 10 9 /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule-1 [soluble vcam-1]) in patients with scd in their non-crisis, "steady state." methods: patients, at least 10 years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: 15, hbsb thalassemia: 10) were enrolled (females: 15 [60%]), with a median age of 23 years (iqr: 11). following 6 months of hu, median values for wbc count (9.02 9 10 9 /l vs. 7.22 9 10 9 /l, p = 0.007) and d-dimer (1243.8 ng/ml vs. 830.2 ng/ml, p = 0.028) were significantly lower than baseline values, while the mean corpuscular volume (76.0 fl vs. 88.0 fl, p = 0.001) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin (9.1 g/ dl vs. 9.6 g/dl, p = 0.72), platelet count (250 10 9 /l vs. 196.5 10 9 /l, p = 0.71), lactate dehydrogenase (744 u/l vs. 567.5 u/l, p = 0.23) or soluble vcam-1 (567.8 ng/ ml vs. 526.4 ng/ml, p = 0.33) following 6 months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included 478 confirmed breast cancer patients planned for elective breast surgeries from january 2012 to december 2017. patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor 2 (her -2) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤40 years] and older group (>40 years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < 0.05 was considered statistically significant. results: of the total 478 patients most underwent wide local excision of the breast and modified radical mastectomy. a total of 16 patients received 71 units of blood and blood components in all categories of surgeries. only 103 were younger women (≤40 years) with mean age of 31 years. non-transfused patients were significantly more than transfused ones (p < 0.05). frequency of blood transfusion was more in young patients (4.9%). seven (22.6%) of the total 31 stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy (8.8%). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in 2009 and fully revised in 2016 by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a 700-bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in 2016. it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ 7 g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ 65 years, age ≤ 6 months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep 2016 to feb 2018 were analyzed. results: the number of transfusion-indications analyzed was 16138 for rbcs, 11158 for plts and 6024 for ffps. the most common indications for transfusion were chronic disease for rbcs (7977/16138, 49.4%), bleeding prevention for plts (5726/11158, 51.3%) and 'other' for ffp (2180/6024, 36.2%). 'hb ≤ 7 g/dl' was the most frequent sub-indication of chronic disease (3570/7977, 44.8%), and hematologic disease was the most frequent sub-indication of bleeding prevention (3432/ 5726, 59.3%). many clinicians entered transfusion indication as 'other': rbcs (2866/ 16138, 17.5%), plts (856/11158, 7.7%) and ffp (2180/6024, 36.2%). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; 82.9% of rbcs and 54% of plts. of the indications entered as 'other' in ffp, 80.3% were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep 2016. transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged 1-18 years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and 1 h post-transfusion, the expression of cd62p on platelet was determined by flow cytometry method. results: there were 102 subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd62p for nonleukodepleted and leukodepleted groups were 26.2% and 27.7%, and the mean increase of post-transfusion platelet cd62p for non-leukodepleted was 10.1% and the mean decrease of leukodepleted groups was 3.3%. it was shown the increase of post-transfusion platelet cd62p for non-leukodepleted group, and it was significantly (p < 0.05) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd62p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of 158 patients hospitalized in the period january -may 2018 in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < 12 g/dl and males < 13 g/dl), from the 158 patients included in the study, 61 (39%) were anaemic. from 117 males in the study, 40 (34%) of them were anaemic based on hb lab values, whereas from 41 women in the study anaemic were found to be 21 (51%) of them. from the 61 anaemic patients in the study, 35 (57.4%) of them with mild anaemia, 23 (37.7%) with moderate anaemia and 3 (4.9%) with severe anaemia. in the total of anaemic female 38.1% are under 65, while 61.9% are over/or 65 years old. in the total of anaemic males, 35% are under 65, while 65% are over/or 65 years old. it is noticed that most of them are with normochromic normocytic 67.2%, normocytic hypochromic anaemia 16.4%, hyperchromic microcytic anaemia 8.2%, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively 1.6% and microcytic normochromic anaemia 4.9%. the average value of preoperative hb decreased from 12.8 g/dl before surgery to 10.3 g/dl after surgery, so there is a decrease of approximately 2.5 g/dl of hb value. in our 158 patients, 48% (76) were transfused and the remaining 52% (82) were not transfused. from 76 transfused patients 41 (54%) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, 2003 to december, 2017, the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january 2008 to december 2009, the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of 988 patients were performed with unexpected antibody identification tests. of 1896 patients who underwent more than 2 screening tests, 25 (1.3%) were positive. 1871 were consistently unexpected antibody negative. the patients with solid tumors (n = 375, 56.3%) and those with hematologic diseases (n = 112, 16.8%) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = 0.0002). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group (65/988, 6.6%) than in the negative group (77/1871, 4.1%) (p = 0.006). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group (28/1871, 1.5%) than in the positive group (5/988, 0.5%) (p = 0.019). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in 2018 there were three female patients in the clinics of the centre for observation, age between 29 and 51 years (me = 39) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)-2, acute myeloid leukemia (aml)-1. individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac3/4 were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd41a. the density of fixed paig, pac was © 2019 the authors vox sanguinis © 2019 international society of blood transfusion vox sanguinis (2019) 114 (suppl. 1), 5-240 evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose 0,4 g/kg per day, for 3 days. one patient with aml received ivig-iggam therapy in the standard dose 5,0 ml/kg/day for 3. results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa-1 mfi-paigg reduced from 926 to 65; while the patient with aml: paiga reduced from 5506 to 187, and paigm from 1971 to 302, pac3 from 691 to 31, pac4 from 404 to 103. the patient with aa-2 over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the 3 patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells (60% vs 78%) and fresh frozen plasma (36% vs 84% p<o.oo1). more patients in the study group received platelets (72% vs 60%) and cryoprecipitate (50% vs 36%). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a 33-years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a 33-year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of 3 years at dubai thalassemia center. she was admitted to the hospital with 5 days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of 7.8 g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was 8.5 g/dl with a positive dat with both igg+4 and c3+4, patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin 5.6 g/dl, mcv 85.0, platelets count 216000, crp 3.6, retic count 2.18, blood culture no growth, total bilirubin 1.5, malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg 57.1 and igm 21.3, she was started on azithromycin for 5 days and received 2 units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to 9.3 g/dl. she did not show any improvement in her condition over the following 2 weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for 10 days. screening for collagen disease were all negative apart from high esr of 130 mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was 11.8 g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to 85 mm/hr and hemoglobin 10.2 g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of 1.2 g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first 60 min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january 1, 2011 to january 1, 2013 in 61 dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within 60 min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within 120 and within 180 min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of 1216 women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in 780 women (64.1%). in this cohort, seven maternal deaths (0.6%), 62 hysterectomies (5.1%) and 159 arterial embolizations (13.1%) were observed. plasma transfusion within 60 min in 114 women was matched on time-dependent propensity scores with 114 women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, 21.2% versus 19.9%, adjusted odds ratio 1.09 (95% confidence interval 0.57-2.08). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within 60 min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included 60 patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november 2017-july 2018. patients were previously taken asa at a dose of 100 mg per day, but therapy was discontinued 5 days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi 790-1410 au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (<790 au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately 30% of carrier females have factor ix clotting activity lower than 40% and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: 45 year old woman, with no history of abnormal haemorrhage, healthy, up to 2004 when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of 20% fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in 2008, after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since 2009. in 2012, she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with 2000 iu recombinant fix (rfix) daily, for four days, and up to 48 h after discharge maintaining basal levels of fix of 60% and with no major complications reported. in 2013, the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of 4000 iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of 3000 iu rfix are administered from day 1 (d1) and up to d7 maintaining fix levels around 60-80%. the patient is operated at d8 under the cover of 2000 iu of rfix, twice per day, and up to d14 after surgery reaching fix levels of around 80-110%. at d15 the dose was diminished to 2000 iu of rfix daily and at d17 she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february 2018 to january 2019. the study was approved from institutional review board. pack red cells (prc) transfusion in case of < 7 g/dl hemoglobin (hb) with cardiac history and < 7 g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < 50 9 10 9 /l, sepsis with bleeding and < 100 9 10 9 /l, patients on active chemotherapy without bleeding but platelet count < 10 9 10 9 /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version 23.0 was used for descriptive and inferential statistics. results: a total of 250 transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data 194(78%). out of 250, 134 (54%), 100(40%) and 16(6%) prcs, platelets and ffp were transfused respectively. out of 100 platelets, 10(10%) were platelet apheresis. 80/134 (60%) prcs and 22/90 (24%) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than 1 unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = 0.121). thirty six (45%) of transfusions were done with more than 1 units of prcs in non genuine group. the mean hb level of patients received 1 unit and more than 1 units prcs was statistically same (p = 0.868) and it was found out that there was same increment in hb in both the groups post transfusion. (p = 0.867). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = 4) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period (1 h) sheep were bled~40% of their estimated blood volume (estimated at 65 ml/kg) to a mean arterial blood pressure (map) of 30-40 mmhg. sheep were then resuscitated with either plasmalyte â (up to 20 ml/kg/ hr; n = 3) or transfused with sheep red cells (n = 1) to achieve a map > 65 mmhg. sheep were euthanised 4 h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of 931.7 ae 135.3 ml blood which combined with iatrogenic blood loss (~300 ml) corresponded to an average 40.2 ae 2.4% blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = 30-40 mmhg, lactate > 4 mm, svo 2 < 60%). across all four sheep the nadir map averaged 40.5 ae 13.1 mmhg, lactate peaked at 3.9 ae 1 mmol/ l, and nadir svo 2 was 41.3 ae 17.9%. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve 100% incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p-515 bilirubin were recorded within the 28-day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a 15-min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c3d. the rh group and non-rh group had 11 and 10 cases performed manually, and results were 2+ or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in 31/44 (70%) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in 2017, out of 7,276 notified atrs, 113 (1.5%) and 6 (0.1%) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january 1 st 2010 to june 30 th 2018. each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, 9,544 atr were reported, of which 29 included unconsciousness and/or convulsions (0.3%). of these 29 patients, 13 were females (44.8%) and 16 males (55.2%). unconsciousness alone was frequently observed (21 reports, 72.4%). convulsions were notified in 8 reports (27.6%) and were associated with unconsciousness in 2 of them. the diagnosis of seizure, with no other clinical signs, was established in 2 cases (6.9%). unconsciousness and/or convulsions were present in 8 allergic reactions (27.6%), 4 cases of transfusion-associated circulating overload (13.8%), 3 cases of suspected transfusion-transmitted bacterial infections and 2 hypertensive reactions. in allergic atrs, unconsciousness was notified in 7 cases and unconsciousness associated with convulsions in one. twelve atrs were severe (41.4%), 10 were life-threatening (34.5%) and in 4 cases, they resulted in the death of the recipient (13.8%). of the 8 allergic atrs, 4 were severe and 4 life-threatening. red blood cell concentrate was involved in 15 atrs (51.7%) and platelet concentrate in 9 (31.1%), including 5 cases with apheresis platelet concentrate and 4 cases with pooled platelet concentrate. fresh frozen plasma was involved in 5 atrs (17.2%). nevertheless, the imputability of the blood product was excluded or unlikely in 11 atrs (37.9%). in the 3 suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in 7 and 9 atrs respectively, but was certain in only 2 atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr (26 ars, 89.7%). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr 3.0, 95% ci 1.5-5.7, p = 0.001). this effect was most pronounced in the td-rs subgroup (hr 6.0, 95% ci 2.2-16.2, p < 0.001). similarly, elevated lpi levels were associated with inferior pfs (hr 3.4, 95% ci 1.9-6.3, p < 0.001) for the whole study population and the td-rs subgroup (hr 8.2, 95% ci 3.4-21.0, p < 0.001). in total 16 patients received iron chelation during the sample collection period (11 patients deferasirox, 5 patients desferrioxamine). lpi levels were normal in 14 out of the 17 samples collected during deferasirox treatment and in 2 out of 5 samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = 4 of each) was cultured with rpmi media (37°c, 5% co 2 ) alone or with the addition of lps (1-100 lg/ml; derived from escherichia coli 055: b5). the inflammatory response was assessed after 2 h (h), 6 h, 12 h, 24 h, 36 h and 48 h. supernatant was harvested at each time point and stored at à80°c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il-1b, il-6, il-8 and il-10). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il-1b and il-10 but not il-6 or il-8. il-1b production was significantly increased following stimulation of 100 lg/ml lps for 6 h (p = 0.043) and declined following 36 h incubation. release of il-10 was significant 12 h post-lps stimulation with 100 lg/ml (p = 0.030) and reached a maximum at 24 h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps (1 lg/ml), although the incubation times differed. il-1b was significantly increased following 2 h incubation (p = 0.002), with increasing levels observed up to 24 h post-lps stimulation. il-8 production was evident from 6 h and reached significance at 24 h post-lps stimulation (p = 0.009). il-10 was significantly increased following stimulation of 5 lg/ml lps for 6 hr (p = 0.043) with lower concentrations of lps resulting in il-10 production at 24 h (p = 0.008). release of il-6 was significant after 6 h of 50 lg/ml lps stimulation (p = 0.046), with lower concentrations of lps resulting in il-6 production at 24 h (p = 0.015). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. 1. rhdig inappropriately administered (unnecessary exposure) (n = 17, 40%) administered to: -rhd positive woman (n = 9) -rhd negative mother with rhd negative neonate (n = 5) -woman with immune anti d (n = 1) -administered in error (instead of other ig) (n = 2) 2rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = 17, 40%) -omitted (n = 14) -delayed (n = 1) -inadequate dose (n = 2) 3administration without correct patient identification (n = 6, 14%) 4storage & handling (n = 3, 7%) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo 2 agencia gallega de sangre, organos y tejidos, galicia 3 banco de sangre y tejidos de cantabria, cantabria 4 banco de sangre de la rioja, la rioja 5 banco de sangre y tejidos de navarra, navarra 6 banco de sangre y tejidos de arag on, aragon 7 fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon 8 fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain 9 terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in 17 autonomous regions, the hemovigilance system is structured in three levels: (1) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to (2) the regional hemovigilance coordinator, who communicates all the region's data to the (3) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in 2008. the mirasol prt system for platelets and plasma was introduced in 2010 and is currently being used in 10 of the 17 spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from 2012 to 2017 as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between 2012, when 4,196 mirasol treated blood products were issued to hospitals and 2017 with 56,229 mirasol products issued. due to low number of transfusions of mirasol-treated blood components in 2012 and 2013, notification rates began to be analyzed in 2014, showing ae rates of 0.2%, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around 0.23). rate of ae after transfusion of m-p is fluctuant between 0.06 and 0.17. this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around 80%) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > 2 reactions in 2016 and 2017 of respectively 0.18 and 0.07 no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period (2012) (2013) (2014) (2015) (2016) (2017) . at the national level, nine cases of bacterial transfusion transmission (with g&i > 2) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > 2 is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error (75%) with the lowest frequency in equipment failure (10%), compared to 21% and 29%, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes (25%) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in 2007-2017 were 80 in 8,385,448 blood units (1/104,818) issued for transfusion. these resulted in 27 serious reactions (34%) (1 fatal, 11 life-threatening) . another 21 (26%) were related with ibct that did not cause a reaction. near misses (component not transfused) were 24 (40%) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of 24 months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: 42,403 blood components were issued during the study period, while transfusion feedback forms for 37,816 components (89.1%) were received in the transfusion medicine department. delay in starting the transfusion (more than 30 min after issue) was observed in 2965 transfusion events (6.9%). the component transfusion time exceeded the permissible limits for 930 component (2.1%).the overall total permissible time for completion of components exceeded permissible limit in 2217 (5.2%) of transfusion events. the pediatric ward (23.9%), icu and ot complex (25.4%) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the 2965 delayed transfusions after issue, 2120 (71.5%) were during the routine hours i.e. between 7 am to 7 pm and 845 (28.5%) were in the non routine hours i.e. between 7 pm to 7 am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for 2013-2016 in comparison to previous years of surveillance, [2006] [2007] [2008] [2009] [2010] [2011] [2012] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for 2013-2016 89 sets of annual aggregated data from 25 countries covering 85,521,393 bcs issued were analyzed. all ars totalled 76,907 and infectious ars amounted to 285 (0.4%). the overall incidence of the infectious ars was 0.33/ 100,000 units of bcs issued. bacterial infections were the most frequent (188, 66%), next viral (84, 29.5%) and then parasitic (13, 4.5%). serious were 46% and there were 11 fatalities (3.9%, incidence 0.013/100,000). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the 84 viral sttis included 17 hbv (20%), 10 hcv (12%) and 2 hiv (2.5%). the 55 recorded as "other" (65.5%) including 24 cases of hev, one case of parvovirus b19, one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were 15 (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for 2006-2012 shows a consistent overall incidence in total sttis (0.33 vs 0.4/100,000). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in 2013-2016, p < 0.001) and an almost doubled rate of parasitic infections (p < 0.001). compared to the earlier period, there were many fewer hbv infections (17 vs 160) and many more hev. a similar reduction in the rate of hcv and hiv was observed in 2013-2016 in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january 2015-december 2018), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version 22.0, spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was 72 months (interquantile range-iqr 108). the median for the numbers of individual transfusions in children in a year was 12 (iqr 17). the median for the numbers of blood components individually transfused to patients was 18 (iqr 24). patients, anaphylactic transfusion reactions in 4 patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of 2.7 per 100000 units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of 11 and in 100000 units and 26 in 100000 units, respectively. while the incidence of transfusion reactions in children was found 0.62% in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of 2.7 per 100000 units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria 6 hospital general universitario, ciudad real, spain 7 hospital nostra senior de meritxell, andorra, andorra 8 banco sangre y tejidos, santander 9 banc de sang i teixits, barcelona, spain 10 fundaci on hematol ogica colombia, bogot a, colombia 11 centro regional de transfusi on de almer ıa, almeria 12 complejo hospitalario de navarra, pamplona 13 fundaci on banc de sang i teixits illes balears, palma de mallorca 14 hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in 2018 fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to 3 patients. two patients of 55 years of age diagnosed of acute myeloblastic leukemia (case 1 and 2) and chronic lymphatic leukemia (case 3). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors 1 month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all 3 cases, the story was judged as sufficient for analysis. the majority of reviewers (93%) diagnosed a chain of errors. there was agreement of 95% with respect to the initial process affected. the initial error was communication (42%), monitoring (45%) and compliance (62%), in cases 1, 2, and 3. 2-3 human errors were detected per case (average: 2.2, 2.7 and 2.4 errors respectively) and 2-3 latent errors per case (average: 1.8, 2.7 and 2.1, respectively). the latent errors most punctuated were: failures in the quality of the protocols (24%), in the transfer of important knowledge (22%), in the available technology (21%) and in the information to the patient (10%). all the participants contributed feasible measures of improvement according to root causes: 1) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, 2) train staff in knowledge important for safety, 3) communicate with computer application providers to improve the effectiveness and visibility of the alerts and 4) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from 2007 to 2017, reports of taco increased from 6 to 92; deaths from 1 to 7, major morbidity from 3 to 20. known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, <3 years, >60 years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last 3 years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case 1: a patient in their 50s weighing 67 kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb 37 g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of 100 g/l. -case 2: a patient in their 50s presented with a 4-week history of weakness and dizziness and had felt unwell for 6 months. the hb was 34 g/l, ferritin 26 lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of 51 g/l. a third unit was transfused over 125 min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case 3: a patient in their 90s with severe megaloblastic anaemia, hb 41 g/l and peripheral oedema developed taco after transfusion of 3 units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b12 deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b12 deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since 2015. results: total of 121,076 different blood components were applied in the period between 2015-2018. department for quality assurance and control has received 4 serious adverse reactions, 1 serious adverse event, 157 reports of transfusion reactions, of which 11 (7%) were inadequately filled, in the same period. from the above, 54 (34.39%) were transfusion reactions to erythrocyte blood components which were applied, 86 (54.77%) to platelet components and 17 (10.82%) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions (88 or 56.05%), followed by allergic reactions (50 or 31.84%). two transfusion reactions (1.27%) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was 0.004% (5 of 121,076) and 0.12% were reported transfusion reactions (157 of 121,076). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > 70 years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of 93 patients were analyzed (48 women, 45 men), with a median age of 86 years (70-97). in total, 189 ch were transfused. 70 patients received 2 ch, 3 patients 3 ch, 10 patients received 1 ch. 10 patients were transfused at two different times. the median hb prior to transfusion was 7.6 g/dl. in the patients who received 2 ch was 7.5 g/dl, those who received 3 ch: 6 g/dl and those who received 1 ch: 7.75 g/dl. the infusion time could be estimated in 84% of the patients. in those who received 2 ch was 223.62 min; 249.33 min in those who received 3 ch and 109.75 min in those who received 1 ch. 13 patients (14% of the total) suffered some type of adverse effect related to the transfusion. in 9 patients there was an increase in posterior ta, in 2 an increase in hr, in 1 an episode of hypotension and in another one episode of acute respiratory failure. 70% of those who had an adverse effect were older than 85 years. patients with aht after transfusion, 75% received 2 ch and the remainder 1 ch. among their background, 66% had a history of ischemic heart disease. 33% also had a positive balance. the average previous bp was 137/65 mmhg and the subsequent one was 182/72 mmhg. 55% of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. 2 ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > 85 years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse 2ch than 1ch. -we have appreciated that in those patients receiving 3ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february 2018 to december 2018. the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version 23.0. results: a total of 108 ida patients were analyzed in which 74(69%) were females and 34(31%) were males. the most common symptom in females and males was fatigue followed by body aches in females 12(16%) and pallor in males 10(29%). menorrhagia was present in 24(44%) of females of reproductive age. surgical history was present in 12(16%) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was 8.0 ae 2.2, 60.5 ae 9.6, and 20 ae 8.3 while in males it was 7.8 ae 2.3, 63 ae 14.3 and 18.4 ae 6.1 respectively. sixty two (84%) females were advised oral and i/v iron and 12 (16%) received transfusion. however, in males 8(24%) received transfusion and 26 (76%) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of 3 months follow up in males and females was same as that the transfusion (p > 0.05). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd34 + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo (100 ng/ml). after 12, 14 and 18 days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd41a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day 0), cultured but unselected samples (day 7), and cultured, selected samples (day 12, 14 and 18) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of 65140 genes were detected, of which 16612 showed up-regulation and 48528 down-regulation. among these genes, 8 differentially expressed genes (degs) (fold change ≥ 10; false discovery rate < 0.05) were selected were further validated with rq-pcr, including gabre, cdhr1, wasf3, pkhd1l1, thbs1, pf4v1, lrrc32 and lgals12. the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf4v1 mrna expression level was highest at day 14, lgals12 was highest at day 18. summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: 7-8 g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between 2011-2018. patients with hb level measurement within 90 days pre-surgery were included. receiving > 1 blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, 2591 patients underwent hip or knee surgery; 231 were excluded from the analysis due to receiving > 1 blood unit. hb measurement within 90 days pre-surgery was available for 1202 patients. hip or knee surgery was performed in 588 (49%) and 614 (51%) patients, respectively. women comprised 60% (n = 356) of patients who underwent hip surgery. in the hip-surgery group, 14.5% of patients required bt, with this need being slightly higher among women (31.5% vs. 26.2%; p-value=ns). only 50 (8%) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it (11.93 g/dl versus 12.8 g/dl for women and 12.3 g/dl vs. 13.8 g/dl for men; p-value < 0.001). hospitalization was longer in transfused patients compared to non-transfused ones (mean 7.52 vs. 6.91 days, p-value = 0.018) and in patients with a low hb level (female < 12, male < 13.5) than in those with a high hb level, irrespective of receiving bt (p-values < 0.00045). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < 0.05). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for 1 blood unit was 0.43 in the hb 11 g/dl group and 0.15 in hb 13 g/ dl group (35%>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd36, known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd36 deficiency was cd36 not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd36 deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd36 is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd34 + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd36 monoclonal antibody on cd34 + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd36 monoclonal antibody on proliferation and differentiation of human cd34 + hematopoietic stem (progenitor) cells in vitro. methods: choose 3 healthy full-term maternal women without various obstetric complications, take cord blood 20 ml. after density gradient centrifugation of ficoll cell separation solution, cd34 + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. mtt was used to examine the effect of anti-cd36 monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd34 + hematopoietic stem (progenitor) cells. the effect of anti-cd36 monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after 10-14 days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd34 + were sorted by flow cytometry, and about 0.44% of cd34 + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd36 monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value (0.9 ae 0.15) of anti-cd36 monoclonal antibody group (2 mg/ml) was decreased than normal group (1.05 ae 0.12) (p < 0.05), and the od value (0.81 ae 0.11) was significantly decreased at the cd36 monoclonal antibody concentration of 32 mg/ml (p < 0.01). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > 0.05). in the annexin v flow detection, the apoptotic rate of anti-cd36 monoclonal antibody group (2 mg/ml) was statistically increased than the normal group (p < 0.05). anti-cd36 monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g1 phase cells increased, and g1/s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was 169 ae 12, the number of cfu-e/bfu-e clones in the control group was 172 ae 12, and the number of cfu-e/bfu-e clones in the anti-cd36 monoclonal antibody culture group was 85 ae 6. the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd36 monoclonal antibody culture group was significantly lower than that of the other groups (p < 0.05). summary/conclusions: anti-cd36 monoclonal antibody can reduce the proliferation of human cd34 + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. 11 (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of 159 autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd 34+ cells concentration in blood higher than 20/ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. 11 or cobe spectra v. 6, v. 7, terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in 56 transplanted patients were assessed. results: standard collections were performed in 52 patients. the yield of cd 34+ cells was high, and no significant differences were found between the numbers of cd 34+ cells prepared from spectra optia 8,6 (1,3-41) 9 10 6 and cobe spectra 10,9 (1, 6 ) 9 10 6 /kg b. w. (a = 0,05; pval 0,619). the dependence of cd 34+ cell yield on the precollection concentration of cd 34+ cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = 0,95, and cobe spectra r = 0,93. lvl collections were performed in 107 of patients, and there were no significant differences between the numbers of cd 34+ cells prepared by cmnc spectra optia 10,9 (2-61,2)9 10 6 and cobe spectra 9,3 (2,4-86) 9 10 6 /kg b.w. (a = 0,05; pval 0,35). the relations between the precollection cd 34+ cells concentration in blood and the numbers of cd 34+ cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = 0,93, and cobe spectra r = 0,78, respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia (45%) than in cobe spectra (57%). a group of 12 patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra 11 days, while in platelets from cmnc 14 days, and from cobe spectra 12 days, respectively. the number of 44 patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with 11 and 13 days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd 34+ cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd34 + cells/kg. this number depends on the pre-apheresis cd34 + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in 1 day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd34 number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in 1 day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in 1 day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd34 + cells collected/(pre-apheresis cd34 + number*processed volume)*100%. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd34 + number, patient weight and the requested number of cd34 + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in 1 day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = 64% (n = 348). a ce of 40% was chosen as the cut-off for the cd34 calculation tool. using the cd34 calculation tool: allogeneic donors (n = 61): mean ce = 61%, mean blood volume processed = 3.3 9 tbv, mean time: 253 min, 37 donors were finished in 1 day collection (61%) autologous patients (n = 205): mean ce = 65%, mean blood volume processed = 2.4 9 tbv, mean time = 218 min, 173 patients were finished in 1 day (83%). the calculation failed in only 1 case (0.4%). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd34 calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from 2001 till 2019. all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was 2 9 10 6 /kg cd34 + cells and 2 9 10 8 /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c53000, cobe spectra and spectra optia using conventional-volume apheresis processing the 2-2.5 total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after 4 to 5 days of g-csf subcutaneous administration at a dose of 10 lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were 159 apheresis procedures performed in 106 healthy sibling donors. there were 75 males and 31 females, aged 20-55. one to two apheresis procedures were required to collect adequate graft. the single procedure usually took 3-4 h and the volume of collected stem cells was 50-220 ml. the needed number of mnc and cd34 + cells was successfully collected by 1.5 apheresis. there were 29 abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia -57 patients (53.7%), acute lymphoblastic leukemia -14 patients (13.2%), chronic myeloid leukemia -9 patients (8.5%), myeloproliferative disorders -8 patients (7.5%), myelofibrosis -5 patients (4.7%), severe aplastic anemia -5 patient (4.7%), non-hodgkin lymphoma -3 patient (2.8%), multiple myeloma -3 patient (2.8%), chronic lymphoblastic leukemia -1 patients (0.9), hodgkin disease -1 patient (0.9%). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from 400 ml whole blood collected from 32 healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of 35 days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over 300 bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw 125,000, 98% of hydrolysis) using electrospinning technology (nanospider tm 1ws500u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of 700-900 x10 6 plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from 10% pva solution using water: ethanol (8:2) solvent system. materials with proteins were electrospun from solution containing 10% of pva and 10% of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: 340 ae 120 nm for pristine pva fibers and 350 ae 148 nm for pva with incorporated proteins (pva/pl). pva/pl layers contain 5-10 mg of protein per gram of pva. 60% of the proteins are released during the first day (burst release) followed by a gradual release of up to 2 weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv18-01-00332. background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) 1, 2 and 3 which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp-4, comprises less than 1% of the total defensins in neutrophils and has a distinct sequence from hnp1-3. the other two, human defensin 5 and 6, are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp 1-3) are 3.4 kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at 22°c by leukoflex lst-1 filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph 7.4, without mgcl2 and cacl2, containing 5 mm edta and 2.5% sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs 1x at a concentration of 1 9 10 7 cells/ml. for degranulation, cells were stimulated with 100 nm of formylmethionyl-leucyl-phenylalanine (fmlp) for 5 min followed by stimulation with 10 lm of cytochalasin b for 5 min. supernatant was collected by centrifugation at 200 9 g for 6 min. supernatant was incubated with mouse monoclonal antibody to hnp 1-3 and purification of hnp 1-3 was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the 3.4 kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst-1 filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing 5-aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct4, sox2 and nanog were performed after 24 and 72 h and 1 week results: the results indicated that after 24 h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing 5-aza-deoxycytidine and trichostatin a with extract expressed oct4 with weak intensity after 24 h. no expression of oct4, sox2 and nanog were observed in other groups at the same time. after 72 h, oct4, sox2 and nanog were over expressed in the cells that were treated with 5-aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct4 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct4 and sox2 in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase (5-aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than 150 nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon 1, 5, 10 and 1227a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of 1227 g>a and rhd exon 1, 5, 10. samples with rhd gene deletion homozygous/heterozygous, 1227 g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd1-rhce(2-9)-rhd10 homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were 1 in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon 1 (87°c) to internal control (77°c) were 2.33 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.09-2.21 in alleles of rhd gene deletion/normal rhd, 2.53-2.66 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 10 (81°c) to internal control (77°c) were 3.35 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 4.29-4.39 in alleles of rhd gene deletion/normal rhd, 4.67-6.10 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. the tm ratio of rhd exon 5 (81°c) to internal control (77°c) were 3.64 in alleles of normal rhd/rhd1-rhce(2-9)-rhd10, 2.49 in alleles of rhd gene deletion/rhd1-rhce(2-9)-rhd10 3.47-3.59 in alleles of rhd gene deletion/normal rhd, 3.97-4.77 in alleles of normal rhd and < 0.1 alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types 1, 2 and 3 not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total 583 dna samples were tested in 87 pools. fifteen (15) pools (181 samples) gave rhd deletion genotype and seventy two (72) pools (402 samples) resulted to the presence of rhd gene. the 72 positive pools were also analyzed individually. the genotype results obtained were: rhd exon 1 no amplification (1), rhd exon 6 and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, 100% accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: 1 sample rhd*93-94inst (del), 1 sample rhd*ivs3+1g (del), 9 samples rhd*weak d type 11 (partial d), 1 sample rhd*weak d type 5 (weak d), 1 sample rhd*weak d type 61 (weak d) and not described: 1 sample rhd*del 1-5 (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to 20 pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type 1, 2, and 3 which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~12-kilobase duplication event, including exon 3, was predominantly identified in 58.3% d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the 70 rhd variants included in this study, 49 samples (70%) showed presence of indian specific allele i.e. exon 3 duplication. seventeen rhd variants samples showed presence of both exon 5 and 10. qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r 1 r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. -128 non-invasive fetal kel1 genotypes from allo-immunized anti-kel1 women were done (34 positive confirmed fetuses, 6 undetermined, 16 positive non-confirmed, 22 negative non-confirmed and 50 negative confirmed). -190 non-invasive fetal rhc genotypes from allo-immunized anti-rh4 women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh3 women were done (66 positive foetuses, 1 undetermined for 21,5% of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła 1 , m krzemienowska 2 , m pelc-kłopotowska 2 , m jurkowska 3 , m debska 4 , m uhrynowska 2 and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since 2016 but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type 1, 2 and 3. aims: summary of a 3-year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of 366 pregnant women determined with standard serology as rhd-negative (in 8-38 week of gestation) was examined for the presence of exons 5 and 7 of rhd and ccr5 by realtime pcr using lc480ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in 123 cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr5 indicated a maternal d variant (d ct ccr5-rhd >2); the genetic follow-up of six of them identified: rhd*01w.2 in 4 cases, rhd*01w.1 and rhd*15. in 235 cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining 67% of mothers. in 1.9% cases with maternal d variant rhd nipt was not possible. however, in 5/6 such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type 1 and 2 in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh23 (d w ), as these are almost exclusive to african populations. rh23 is encoded by several types of rhd*dv as well as by dau-5. anti-rh23 is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida 4, para 1, causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at 37°c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh23 in addition to anti-fy 5 , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd*10.05 which encodes rh23. the newborn's phenotype was a r0r k-, fy(a-b+) and most likely rh23-and jk (a+b+), considering both maternal and paternal (a r0r, k-k+, fy(a-b+), jk(a+b-), rh23-) predicted phenotypes. the neonatal serum contained maternal anti-a1, anti-rh23 and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy 5 during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh23, derived from several rh23 + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from 22% in the patients younger than 60 years to 58% in the patients older than 80 years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with 59%. only 48,5% achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene 17%-33% patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above 200 mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a 28y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at 30y, a sister with similar profile age 26y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin 75 mg od, ticagrelor 90 mg bd, rosuvastatin 40 mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux5a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column 5a20 (evaflux, kawasumi, japan) as the second step. a total of 1.5x plasma volume(4470 ml) was processed. the patient was given continuous calcium infusion. the flow rate of 50 ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by 60%. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from 2010. hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: 101, 95, 111 in the last 3 years. delayed transfusion in relation to mh was reported for 54 cases 2016-2018 contributing to death in 6 patients 7%) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in 31/71 (43.7%): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, 2 units); 14% of women were transfused with fresh frozen plasma (median, 2 units) and 2% with platelets. mean pre-and post-transfusion hemoglobin levels were 6.7 g/dl and 9.0 g/dl, respectively, representing an increment of 1.0 g/dl per rbc unit transfused (1.09 g/dl in soweto and 0.83 g/dl in durban). indications for transfusion included obstetric hemorrhage (31%), chronic anemia (29%), surgery or anesthesia (13%), other (9%) and not specified (17%). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ 26 weeks (odds ratio = 7.07, 95% confidence interval 3.52-14.24). surgical blood loss was a common indication in trimester 1 (21%) that declined to 7% then 1% in trimesters 2 and 3. summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period 2016-2018. results: there were evaluated 1,281 688 of blood products administrations in 117,414 patients in the cr during defined three years period. announced 1,456 (0,11%) transfusion reactions including 50 severe transfusion reactions (20 9 adjudged with grade 3). the most frequent types of severe transfusion reactions: anaphylaxis 20, trali 7x, taco 5x, hcv 4x, hbc 2x, bacterial infection 1x, delayed hemolysis 1x. transfusion reactions incidence according to administered bp: red blood cells products: 882,017 administrations, 883 transfusion reactions (fnhtr 386x, allergy 227x, circulatory overload 66x, anaphylaxis 6x, trali 4x, hbv 2x, hcv 1x) platelets: 88,438 thrombocyte administrations, including 178 transfusion reactions (allergy 129x, fnhtr 30x, anaphylaxis 5x, circulatory overload 8x, delayed immune hemolysis 3x, acute circulatory overload 8x. granulocytes: 203 administrations, 2 transfusion reactions plasma: 293,801 administrations, 359 reactions reported (allergy 278, fnhr 25, circulatory overload 14, anaphylaxis 17x, trali 4x, hbv 3x, ards 1x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: 1. plasma for clinical uses from male blood donors, 2. prestorage of leucocyte reduced blood products, 3. production of platelets in additive solutions, 4. implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of 5 they were mainly associated with deviation in processing (22.4%) and attributed to equipment failure and materials (71%) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < .001) annual rate of total aes by 28% (95% confidence interval 26-30) ) 10% fibrinogen-depleted phpl or (3) 10% fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene 2.1 st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd73+/90+/105+ and cd14-/19-/34-/45-/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) 5%) were male and 31(36.5%) female. mean age of abo matched and mismatched groups were (6.99 ae 4.6) years. most common indications for transplant included beta thalassemia major 53(60.2%), aplastic anemia in 25(28.4) and pure red cell aplasia 2(2.35%). source of stem cell was bone marrow in 45 and peripheral blood 40 patients abo matched while abo mismatched group comprised of 28(32.9%) patients with further subdivision into major (n = 13), minor (n = 13) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = 401) and (n = 1837) comparably(n = 102) and (n = 480) in mismatched group. primary and secondary graft failure in matched group was 8.77% and 12.28% patients while in mismatched group graft failure was observed in 4(14.28%) patients respectively. positive dct in abo matched group in 1(1.75%) patient, whereas 2(7.14%) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia 1 , c sabia 1 , f cavalca 1 , g nicoletti 2 and c napoli 1 in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr4 and dr16 in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested 5 sera pre-treated with fbs from non-sensitized patients that showed the dr4/dr16 pattern. in particular, 5 ll of fbs was added to 95 ll of patient serum; incubated for 30 min at 37°c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from 5 patients with documented dsa including dr4/dr16 and 5 patients without hla antibodies. results: dr4/dr16 non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr4 and dr16 dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre 2 transfusion medicine, national blood transfusion center 3 transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from 17,992 healthy donors from which group a (36,8%), group o (41,7%), b (16,2%) and group ab (5,3%) resulted. the study was conducted in 3727 patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period 2011-2017 results: among the 3727 patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a (39.7%), followed by 0 (39.5%), b (15.2%) and ab (5.4%). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a (42%), in gastric cancer a (43%), colorectal cancer a (39, 6%), breast cancer a (41%), cervical cancer a (43%) and ovarian cancer a (42%) versus a (36.8%) in the control group. a high incidence of blood b is seen in multiple myeloma b (22%) and cervical cancer b (20%) versus b (16.2%) in the control group. blood group ab has a high incidence in malignant lymphoma ab (10%) versus (5.3%) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types 1, 2 and 3, are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a 12-year-old patient typed as weak d type 3, receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma (2+ by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type 3. rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type 3. mma showed the anti-d was clinically significant (>5%). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type 3 allele. weak d type 3 patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band 3/diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: 1.1. predictprotein (https://predictprotein.org); 1.2. spider2 (http://sparks-lab.org/server/spider2/); 1.3. dsc (discrimination of protein secondary structure class): using an in-house implementation; 1.4. jpred4 (http://www.compbio.dundee.ac.uk/jpred4/); 1.5. raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: 2.1. robetta (http://robetta.bakerlab.org/submit. jsp); 2.2. falcon (http://protein.ict.ac.cn/treethreader/); 2.3. itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: 3.1. verify3d (http://servicesn.mbi.ucla.edu/verify3d/); 3.2. prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: 4.1. gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); 4.2. gramm (http://va kser.compbio.ku.edu/main/resources_gramm1.03.php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = 10) carrying fetuses with known genotype was loaded into 3% agarose gel casette (3% df marker q3, sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from 45 min to 2 h 30 min of electrophoresis. results for real-time pcr detection of fetal rhd, kel*1 and ccr5 (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr5) was significantly lower in dna samples tested after gel selection (from 8.4 to 25.9geq/pcr) versus the level obtained from non-processed plasma dna (from 130 to 133geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from 1,5 to 6.6geq/pcr versus 10.8-11.2geq/pcr for non-processed plasma dna. results for kel*1 detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel*1-negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel*1 positive amplification was observed. however, kel*1 detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel*1-negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel*1 positive genotype was obtained. the total dna level in samples from k-negative women was from 18.3 to 18.7geq ccr5/pcr after gel selection versus from 52 to 746geq ccr5/ pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh1 quantification assay using ih-500 (bio-rad â ): promising results for monitoring rh:-1 pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh1 immunoglobulins since 1970 complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh1 microtitration. it is a column agglutination technology using red blood cells rh: 1, -2, -3,4,5 (r0r) . it permits to quantify low levels of anti-rh1 in comparison to a range of an anti-rh1 standard. performed since 1999 at the cnrhp and automated on evo clinical base tecan in 2008 (dilutions and distribution), anti-rh1 microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih-500 system from bio-rad â . methods: on ih-500, the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r0r, anti-rh1 standard). the performances of the method were evaluated using three internal quality control (icq) (2 cnrhp home-made at 2 and 12 ng/ml and 1 bio-rad â at 12 ng/ml) on papainized r0r (plc) and native r0r (nlc). a comparison of results from patient sera ranging from 1.5 to 48 ng/ml was done between ih-500 and evo clinical base tecan. results: the results of the 3 qci are similar between the different reagents used. there is no significant difference between the 2 types of red blood cells except for the limit of detection: 1.5 ng/ml in plc -6 ng/ml in nlc. for the 3 qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between 0 and 15%, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on 50 samples in plc and 44 samples in nlc was satisfactory (deming plc: r2 = 0.80 y = 0.89x + 0.78 -nlc: r2 = 0.86 y = 1.08x-0.25). summary/conclusions: the anti-rh1 microtitration on the ih-500 offers similar performances to the method conducted at the cnrhp. the ih-500 allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh1. this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab0i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about 1.5-2% of cases haemolytic disease demands transfusion support. aims: analysis hdfn from 2011 to 2018. methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group 0 rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (<5 days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit 70 + 5%, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit 40% -60% and irradiated. the ec used in post-natal transfusions is usually divided into rates of 70 ml, hematocrit 55 ae 5%. results: in last 7 years, we calculated 59 neonates with hdfn (28 males and 31 females): 31 with rhdi hdfn, 19 with ab0i hdfn and 9 with hdfn due to incompatibility for other red blood cell antigens. we have produced 81 iut: 48 for our hospitalized patients and 33 for patients located in other hospitals. 25 of these 48 patients, who received iut, were immunized: 19 showed anti-d antibody and 6 antibodies different from anti-a and anti-b. 7, of the 31 infants with rhdi hdfn, were transfused in utero. 4 neonates on 59 (6.8%) have performed et: 1 with ab0i hdfn and 3 with rhdi hdfn; the latter had also been transfused in utero. 27 neonates on 59 were transfused after birth: 18 with rhdi hdfn, 3 with ab0i hdfn and 6 with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous (52.7% of the total) and are those who have requested the highest blood supply both in the antenatal period (39.6%) that postnatal (9.6% performs et, 66.6% performs postnatal transfusions). neonates with aboi hdfn are 32.3%: nobody has received iut, only one has been subjected to et, and 11% has transfused after birth. patients with hdfn due to other antigens are 15%, have undergone iut 12.5% and were transfused after birth 22.2%. background: according to british guidelines on neonatal transfusion, since 2012 we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ 30 weeks and/or a birthweight lower than 1500 g, until 4 months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab0 direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab0/rh confirmation. blood transfusions were performed with 0 negative kell negative (0 cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than 5 days. ec were subdivided in 70 ml aliquots with a hematocrit of 55 ae 5%. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with 72 h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october 2012 to the end of 2018, 694 premature newborns received 2328 ec transfusions within their first 4 months of life. in 51% of cases (n = 1186), transfusion requirement was comprised within the 'small volume transfusions'. another 44% of cases (n = 1035), requiring further ec administration, was requested of a blood sample for t&s determination and 5% (n = 107) for a cross-match test. in 79.4% of newborns (n = 551), being transfused within the " small volume transfusions", blood requirement of 1401 ec was fulfilled by the initial blood test (1102 blood samples). 13.3% of newborns (n = 92) received more than 5 transfusions (6-21; median = 8) accounting for 820 ec released and for this group 381 blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, 381 blood tests were performed to sustain 643 infants transfused with 2221 units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of 75% of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first 4 months of life. background: glucose-6-phosphate dehydrogenase deficiency (g6pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g6pd 202 genotype is the most common g6pd genotype in sub saharan africa (ssa). some studies have linked blood from g6pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g6pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g6pdd 202 genotype among donors in two regions in uganda. it also described the effect of g6pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: 3,255 blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g6pd 202, haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g6pd 202 and co-inheritance with a-thalassaemia (n = 2,546) and haemoglobin s (n = 2,642) on the haematological quality of blood packs. a subset of 142 donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g6pdd. results: based on g6pd 202 genotyping, 10.3% (n = 274) of the blood samples used in the trial were deficient for g6pd enzyme while 5.3% (n = 142) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = 0.010) and whole blood (p = 0.009) donations of heterozygous g6pd 202 genotype. co-inheritance of g6pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was 83.3% sensitive and 0.8% specific for detecting donor blood packs with g6pdd. summary/conclusions: the prevalence of g6pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g6pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in 39 hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after 21 days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < 1000) and cdc-xm became negative. cdcxm labeled positive at ≥ 10%. t-cell fcmx was considered positive above 42 mfi and b-cell fcmx was considered positive above 120 mfi. lmxm was considered positive above 500 mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in 8 patients (20.5%). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the 39 cases initially screened 35 showed dsa positivity in sab. desensitization was done in those 35 cases only. in our study, sab was positive for class ii alone in 13(37%) while in remaining 22 (63%) cases it was positive for both class 1 and class ii. the number of pre transplant tpe procedures required was 6.7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) . the mean number of post-transplant tpe sessions required was 0.7 (range, 1-6). during pretransplant and post transplant tpe procedures, five (14.3%) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean-8.5, median-7 days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx 17 months with the longest duration of follow up of 36 months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < 22%, we custom prime the tubing set with 5% albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient #1 is a 56-year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every 4 weeks, starting in 2012. the patient has completed 84 procedures with 3-4 units of washed red cells transfused to achieve a target hct goal of 45 to 57%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #2 was a 25-year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent 9 prx procedures with 2-3 units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient #3 is a 33-year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone 38 prx procedures with 3-5 red cell units transfused to achieve a hct goal of 30%. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient #4 is a 48-year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with 4-5 red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the 2016 american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since 2011. aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days 1, 2, 3, 5, 7, 9 along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos 7 aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of 63 variables was defined as possible confounders by a panel of experts. after discussion, 9 global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and 7 cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: 1018 patients were extracted from databases and further analyzed. the mean age of this group was 65,9 years (sd +/-14,1 years) and 67.3% of them were male. the mean duration of surgery was 256 min (sd +/-88,3 min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < 0.001). in 2011, the mean use was 2,34 units per operation (sd +/-2,277), which changed to 1,38 units (sd +/-2,158) in 2014. three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over 4 years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < 0.001). in the specific group of patients undergoing cardiac surgery with cpb (n = 640), the use of rbc was also significantly reduced (p < 0.001). in 2011, the mean use was 3,06 units per operation (sd +/-0,448) and this changed to 1,94 units (sd +/-0,167) in 2014. after correction for the 3 cpb variables that notably changed over the 4 years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over 4 years still remained statistically significant (p < 0.001). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between 2011 and 2014. this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from 14 adult pid and 13 adult sid patients from medical records and pathology reports, for their last 12 months of ivig and their first 12 months of scig. the starting and maintenance dose was 0.4 g/kg/month for ivig, transitioning immediately to 0.1 g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of 62.9 years versus 43.4 years in pid patients (p = 0.007). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = 0.041). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. 55% of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig (9.3 g/l) compared to ivig (8.4 g/l), and fewer infections on scig than ivig (mean annual infection rate of 1.64 vs 2.14 respectively). sid patients had higher mean serum igg trough levels on scig (8.4 g/l) than ivig (7.1 g/l) (p = 0.009) but experienced more infections while on scig versus ivig (mean annual infection rate of 2.15 vs 1.62 respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd62p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd62p expression. aims: to analyze the increase of platelet cd62p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante 2', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june 2014 to august 2018. the medical helicopter (ec 145t2) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and 35 days after collection, were performed. the statistical analysis used spss 23.0 version (significance p < 0,05). results: 138 prbc samples were evaluated, 48,5% (67) from case group and 51,5% (71) from control group. analyses were tested day 1 and day 35 after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered 35 prbc transfusion to 28 patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a 35-day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in 2010 the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of 11 deaths and 83 instances of harm due to delay over a 4-year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of 18 adult patients over a period of 7 months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all 18 cases. results: the abg analysis of all 18 patients showed decrease in the ph, increase in pco 2 , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the 18 cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of 18 cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in 4 cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in 2 cases. platelets were transfused in 5 patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of 40 adult surgical oncology patients who received massive transfusions and was conducted for a period of 7 months. every case of a massive transfusion was studied under the following headings (1) patient's details (2) patients base-line laboratory tests (3) resuscitation with transfusion (4) intra-operative laboratory tests (5) thromboelastography (teg) (6) post-operative complications (7) duration of stay in the hospital (8) 30 day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by 2 nd or 3 rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of 13 (32.5%) patients had ssi, 14 (35%) had surgical complications and 22 (55%) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, 38 (95%) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of 725 outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december 2017. laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted 15 ae 17.52%, but 49.72% of patients had a ttr less than 60%. patients were at high risk of thrombosis in 6.15% of time (inr < 1.5) and high risk of bleeding in 2.2% of time (inr > 4.5). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated 1.9 million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo 2 ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between 2005 and 2015 in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = 505) with two similarly exposed non-alloimmunized control recipients (n = 1010) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > 30 ml/min/1.73 m 2 , 'moderate renal failure' i.e. gfr ≥ 10-30 ml/min/1.73 m 2 during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < 10 ml/min/1.73 m 2 and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among 441 (87.3%) cases versus 838 (83.0%) controls; moderate renal failure among 24 (4.8%) cases versus 54 (5.3%) controls; and severe renal failure among 40 (7.9%) cases versus 108 (11.7%) controls. among the latter, 30 cases and 97 controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr 0.82, 95% ci 0.67-1.01 and adjusted rr 0.81, 95% ci 0.58-1.11, respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr 0.48, 95% ci 0.39-0.59) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir2dl1, kir2dl2/kir2dl3, kir3dl1, kir3dl2 and hla-a, -b, -c, -drb1, -dqb1 genotyping. 58 pairs of hla 10/10 identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c1/c1 homozygote group (48 cases), c1/c2 heterozygote group (8 cases) and c2/c2 homozygote group (2 cases). according to the expression of 3dl1, 53 cases were 3dl1 positive and 5 cases were 3dl1 negative. there were 5 cases of bw4/bw4, 24 cases of bw4/bw6 and 24 cases of bw6/bw6 in the 3dl1 positive samples. according to the expression of a3/a11, they were divided into three groups: a3/a11 negative group (28 cases), a3/a11 heterozygous group (28 cases) and a11/ a11 homozygote (2 cases). according to kir genotyping, kir haploidentical group (38 cases) and kir haploid mismatched group (20 cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of 58 cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v5.01. results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir3dl1 was positive. it was conducive to neutrophil and platelet remodeling when bw4/bw6 and a3/a11 was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to 5-7 days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = 3), prepared in 30% plasma/70% ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for 7 days (d) and sampled on d2, d5 and d7. cryopreserved sheep pcs (n = 3), prepared by the addition of 5-6% dimethyl sulfoxide, were stored at à80°c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), anti-inflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), 12-hete and 15-hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < 0.05 using paired t-test. results: in rt stored sheep pc supernatant il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete were detected at d2, d5 and d7. storage duration significantly increased accumulation of ip-10 at d5 (253.4 ae 266.7 pg/ml compared to 569.7 ae 272.7 pg/ml, p = 0.008) and further increased at d7, and il-8 at d7 (3477 ae 937.4 pg/ml compared to 5092 ae 521.1 pg/ml, p = 0.0460). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il-6, il-1b, il-17a, il-10, il-8, mig, ip-10, 12-hete and 15-hete than rt stored d5 pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il-8 with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than 10 cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = 5), prepared according to standard human procedures with minor modifications, were stored (2-6°c, 42 days (d) ) and sampled at d2 and d42. supernatant was prepared by double centrifugation and stored at à80°c. concentrations of pro-inflammatory cytokines (interleukin (il)-6, il-1b, il-17a), antiinflammatory cytokine il-10 and chemokines (il-8, monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)-10) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), 12-hydroxyeicosatetraenoic acid (hete) and 15-hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < 0.05). results are mean ae standard deviation. results: at day 2, aa (75,142 ae 47,205 pg/ml), 12-hete (849.1 ae 235.6 pg/ml), 15-hete (87.6 ae 32.7 pg/ml) and il-1b (144.7 ae 198.9 pg/ml) were detectable in sheep prbcs supernatant. at day 42, storage duration significantly increased concentrations of aa (136,254 ae 60,433 pg/ml, p = 0.0425) and 15-hete (380.9 ae 116.3 pg/ml, p = 0.0022) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as 1 in 2,500 transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within 28 days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for 50 years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from 16% to 0.2%, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately 0.01 deaths per 1000. blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since 2007. since january 2015, rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = 93) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january 2015-december 2018, 45 reports were received; 43 reports were validated, with 2 reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in 2017 and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, 2019, depending on their iron parameters as having either: -idaserum ferritin < 30 mg/l -chronic inflammation with idaserum ferritin 30-100 mg/l with transferrin % of < 20%/crp > 5 mg/l -anaemia of chronic inflammationserum ferritin > 100 with transferrin % of < 20%/crp > 5 mg/l patients were considered eligible for iv iron if the following criteria were met:1. an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short 2. the anaemia pro-forma was completed 3. hb was ≤ 120 g/l 4. they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, 20 days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included 80 patients. 48 patients were classified as having ida and 32 patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of 96 g/l (55-120), a mean mcv of 77.4 fl and a mean serum ferritin of 16 lg/l. those with chronic inflammation with ida had a mean hb of 106 g/l (77-120), a mean mcv of 87.9 and a mean serum ferritin of 57 lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was 119 g/l (100-149) with an average increment of 23 g/l and in the group with chronic inflammation with ida the average post iv iron hb was 117 g/l (85-129) with an average increment of 11 g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb 20 days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january 2012-august 2018, which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were 154 products dispensed to 53 patients. the recipients were male patients diagnosed with prostate cancer with a mean age of 74 years. there were 3 doses (a complete course) administered to 50/53 (94%) recipients and a partial course (1-2 doses) administered to 3/50 (6%) recipients, for a total of 154 products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were 5 adverse events described in provider notes; 2 of them necessitating transfer to the emergency department, and 1 requiring hospital admission. of the 154 infusions, 6 infusions were documented in a chemotherapy note rather than a transfusion note (4%), and 59 (38%) were documented as both a transfusion and a chemotherapy administration. there were 6 additional deviations from the blood product administration policy: 2 cases where the consent check was not performed, 1 case where the product was infused with ringer's lactate rather than normal saline, and 3 cases where the 2-person 3-way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref 10310) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till 2017, at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ 20,000/ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = 31) and cmnc (n = 88) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in 134 consecutive cmnc procedures, including 88 autologous and 46 allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd34 + count ≥20/ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency-2 (ce-2) was defined as the total cd34 + amount in the collection bag divided by the amount of cd34 + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time (333 ae 59 and 289 ae 73 min, respectively; p = 0.001), the total blood volume processed (3.4 ae 0.8 and 2.4 ae 0.8, respectively; p = 0.001) and the final volume in the collection bag (337 ae 77 and 291 ae 84 ml; p = 0.001). the mean ce-2 in autologous versus allogeneic donors was 49 ae 24 and 66 ae 22, respectively (p = 0.003). using cmnc, the collection was effective in 94% of allogeneic and 63% of autologous donors. in autologous donors, a significantly lower collection bag volume (341 ae 84 and 396 ae 132, respectively; p < 0.01) and increased total wbc in the collection bag (263 ae 119 versus 149 ae 36, respectively; p < 0.000) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd34 + count following g-csf therapy; 7 of them achieved a cd34 count ≥20 and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd34 + count ≥ 20/ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd34 + cell counts are obtained with both methods. . tbv processed ranged from 1-4.8 tbv with mean of 2.6, average was 2.65 tbv for females and 2.74 tbv for males mean pre-apheresis cd34 + count was 92.89 cells/ll (range 33.14-152.64). mean postapheresis cd34 + count was 1402.3 cells/ll (559.02-2245.5). mean cd34 + cells x10 6 / kg recipients body weight was 7.5 (range: 2.77-12.33). our target yield was ≥3 9 10 6 cd34 + cells/kg body weight of the recipient and in only 2/34 (6%) cases, the yield was <3. 18/34 (52.9%) procedures were lvl and 16/34 (47.1%) were svl. summary/conclusions: most of our pbsc were done for haematological indications (85.3%) and the target dose was 3 9 10 6 cells/ll in single leukapheresis. in 32 cases (94%), target yield was achieved, only 2 cases had <3 but >2 yield. in our study donors <5 years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd34 + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd34 yield as high wbc count did not convert into high cd34 yield or vice versa. high prepheresis cd34 + count gave higher postpheresis cd34 + count. large volume leukapheresis (lvl), >3 tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd34 + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd34 + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid 1980s and early 1990s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our 18 year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine 11 hla loci (hla-a, -b, -c, drb1/3/ 4/5, dqb1, dpb1, dpa1, dqa1) in potential bone marrow donors from poland. the research included 18,500 potential bone marrow donors registered between 2017 and 2018. a novelty of this paper was that the amplification of all 11 hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the 11 hla loci (hla-a, -b, -c, drb1/3/4/5, dqb1, dpb1, dpa1, dqa1) of potential bone marrow donors was made by using the alltype tm ngs 11-loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of 18,500 donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c*12: 143, c*12: 30, c*05: 37, c*07: 151, drb1*11: 28, c*14: 04, b*51: 22, c*15: 13, dqb1*03: 12, drb1*14: 87, drb1*11:69. summary/conclusions: 1. new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. 2. the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. 3. the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn5 dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version 1.2.0(one lambda inc.)with the default setting. 94 cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: 94 samples were successfully tested with two methods and the coincidence rate between two sequencing methods was 100%. with the next-generation sequencing method, the probability of ambiguous results among 94 samples in our study is 1.06%(1/94) for hla-a, 5.31% (5/94) for hla-b and 0% (0/94)for hla-c. however, the probability of ambiguous results with the sanger sequencing method is 96.8% for hla-a, 95.7% for hla-b, 100% for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques. key: cord-010119-t1x9gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober 7‐10, 2017 date: 2017-09-04 journal: transfusion doi: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna1 blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to 3 months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by 3 months. urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months. of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple zikv related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* 1 , yuelong guo 2 , ritchard g. cable 3 , joseph e. kiss 4 , michael paul busch 5 , grier page 2 , stacy endres-dighe 2 , steve kleinman 6 , simone glynn 7 , alan mast 8 and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) 9 . 1 american red cross, 2 rti international, 3 american red cross blood services, 4 blood systems inc., 5 blood systems research institute, 6 university of british columbia, 7 nih/ nhlbi, 8 blood research institute, 9 nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over 12,600 donors were enrolled from 4 us blood centers for ferritin testing. the study population was enriched for racial minorities [1600 african-american (aa), 1600 asian (as), 1000 hispanic (hisp)] and for "super donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral). the minority donors and the remaining 6800 non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml). results/findings: across all subjects, 19% had ais and 42% had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed %20% decreased risk for ais compared to nhw, while hisp donors had 25% higher risk. daily use of exogenous iron reduced risk for lf and ais by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection). regular use of antacids was associated with a 20% or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean (6sd) *p < 0.05 compared to batf31/1, 200ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d1 and d14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on d14 vs 100% on d1 of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at 2h posttransfusion. at 24h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* 1 , amanda l richards 2 and krystalyn e hudson 2 . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod 1 otii 1 mice are predisposed to have autoreactive cd4 1 t cells. study design/method: four cohorts of hodxotii f1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoab production. peripheral rbcs were stained with anti-complement (c3) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd71 and ter119 to assess for the presence of rbc progenitors. statistical analysis between hod 1 otii 1 autoab 1 vs. hod 1 otii 1 autoabvs. hod -otii 1 was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd4 1 t cells were not deleted in the thymus of hod 1 otii 1 mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod 1 otii 1 . however, as they aged, 15-50% of hod 1 otii 1 were positive for rbc autoab by 6 months. thereafter, $50% of the autoab 1 mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in 3 of the 4 cohorts, 60-100% of autoab 1 mice were female. hod 1 otii 1 autoab 1 mice also had enlarged spleens compared to hod 1 otii 1 autoaband hod -otii 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd71 hi ter119 inter ) were observed in the spleens of hod 1 otii 1 autoab 1 mice but not in hod 1 otii 1 autoaband hod -otii 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03). moreover, autoab and c3 deposition were found (0.1-2% and 3-10%, resp.) on ter119 1 rbcs in all of the hod 1 otii 1 autoab 1 mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b5-a01a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards 1 , christopher a tormey 2 and krystalyn e hudson* 1 . background/case studies: red blood cell (rbc) alloimmunization occurs in up to 10% of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c57bl/6 (b6) mice were treated with pbs, or anti-ly6g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at 18-24 hours post-transfusion. anti-hod alloantibody generation was assessed 14 days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n55), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n55). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls (3/3 experiments, p<0.01). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio1 leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l1), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b9-a03b cxcr5 1 pd1 1 and ccr7 1 expressions characterize responders to rbc immunization benoît vingert* 1,2,3 , marie tamagne 1,2,3 , sadaf pakdaman 1,2,3 , anoosha habibi 2,3,4 , philippe bierling 1,2,3,4,5 , rachid djoudi 1 and france pirenne 1,2,3,5 . 1 efs ile de france, 2 laboratory of excellence gr-ex, 3 imrb u955 -eq2, 4 ap-hp, 5 universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd4 1 t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd4 1 t cells have a th17 profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd4 1 t cells with a cxcr5 1 pd1 1 phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr5 1 pd1 hi profile, with a differentiated expression of ccr7. ccr7 is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr6 and cxcr3 can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr5 1 pd1 1 lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr5 1 pd1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510). the analysis was also performed in non-transfused healthy controls (n512). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr5 1 pd1 hi subpopulation expression was identical between transfused groups and controls. ccr6 and cxcr3 expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr7 expression was very strong independently of the expression of pd1. in the aim to determine the help of the circulating cxcr5 1 pd1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr5 1 pd1 1 subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr5 1 pd1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr7 on circulating cxcr5 1 pd1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr5 1 pd1 1 profile and the ccr7 1 expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd4 t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* 1 , ashley bennett 1 , kathryn girard-pierce 1 , connie arthur 1 , amanda mener 1 , patty zerra 1 , christopher a tormey 2 , jeanne hendrickson 3 and sean stowell 4 . 1 emory university, 2 yale-new haven hospital, 3 yale university, 4 emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd4 t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd4 t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b6 recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod 1 kel). to examine the role of cd4 t cells, pic/kel primed b6 recipients were cd4 t cell depleted prior to transfusion. in addition, b6 recipients were adoptively transferred with cd4 t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd4 t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < 0.001); pic/kel primed recipients transfused with (hod 1 kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd4 t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < 0.0001) and transfer of cd4 t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < 0.001). conclusion: these results demonstrate that cd4 t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd8 t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd8 t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd8 t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h2kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c57bl/6) mice or oti mice, whose cd8 t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h2kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes 48 hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5. results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is 28-35% versus >60% in wt recipients at 24 hours (p<0.05), whereas transfusion of wt platelets into either oti or wt recipients is approximately 60% at 24 hours after transfusion. adoptive transfer of oti cd8 t cells into wt mice recapitulates the effect, with significant mova platelet clearance at 24 hours compared to wt platelet clearance (p<0.05). conclusion: this work extends the ability of cd8 t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv1r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv1r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv1r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c57bl/6 mouse blood and treated with uv1r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv1r treated wbc-rich prp, or uv1r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c57bl/6 donor blood. a second transfusion of untreated wbc-rich c57bl/6 prp was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv1r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) wbcs, but not those given uv1r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c57bl/6 wbcs were reduced in recipients of either uv1r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv1r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv1r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line. however, a recent investigation of so 2 levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so 2 distribution (mean 45.9%617.5% [yoshida et al. 2017; blood transfusion 15, 172] . the present study was undertaken to determine the distribution of so 2 in lr-rcc produced at a medium-size blood center using a novel non-invasive so 2 probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so 2 levels. study design/method: the so 2 from 977 units of lr-rcc were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection. so 2 was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a3u11; pendar technologies, cambridge ma). in addition to so 2 , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n54) from human volunteers were stored in as-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (so 2 ranging from <3 to >95%) prior to uhplc-ms metabolomics analyses in presence of 13 c, 15 n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of lr-rcc procured and sampled invasively within 24 hours after blood collection [yoshida ibid]. the shape of the so 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (iqr) of 31.4%-61.9%. male donors showed higher so 2 compared to female donors (p<0.04). no correlations were observed between so 2 levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so 2 levels ameliorate the energy and oxidative metabolic lesion. lower so 2 levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp 1 ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so 2 levels was observed from lr-rcc manufactured at a blood center using 8-hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a 3-fold increase in the absolute lymphocyte count (ko 7.59 6 4.63 x10 9 /l vs. wt 2.90 6 1.32 x10 9 /l, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko 6.00 6 0.29 fl vs. wt 5.24 6 0.56 fl, p50.0140). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd81foxp31 regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd8 1 foxp3 1 tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd8 1 foxp3 1 tregs derived exosomes and their functions involved in cd8 1 tregs mediated immune-modulation were seldom reported. study design/method: cd8 1 t cells were freshly purified from pbmcs, cultured with anti-cd3/cd28 antibody packaged beads and il-2, and then polarized with tgf-b and rapamycin into cd8 1 foxp3 1 treg cells. the harvest cells were co-cultured with cd3/cd28 beads stimulating cd41cd25effector cells in the transwell plate. the supernatant derived from cd81 tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd63, cd81, tsg101 and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir-155, let-7b, let-7d were measured by qpcr. the precipitated exosomes were further purified by cd63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd8 1 treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the cd8 1 treg mediated immune modulation. a total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by nta). cd63 and cd81 were expressed on these background/case studies: regulatory t cells (tregs), containing cd4 1 and cd8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd4 1 foxp3 1 regulatory t cells (ntregs) in inflammation conditions (including instability of foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd81 regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd8 1 treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd8 1 regulatory t cells in inflammation and transfusion. study design/method: human cd8 1 tregs were induced with tgf-b1 and rapamycin from cd8 1 t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd8 1 tregs when encountering with inflammation were test by foxp3 expression, th1 and th17 cells conversion in inflammations conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6) on day3, day6 and day9. in vivo, cd8 1 tregs were transfused into cia mice and then their survival in mice and foxp3 express were evaluated to reveal the stability of cd8 1 tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd8 1 tregs when induced factor tgf-b1 and rapamycin were removed by testing the foxp3 expression on day3, day6 and day9. results/finding: ex vivo induced human cd8 1 treg were foxp3 1 (90.40 6 1.40%) and did not secret il17a (both in supernatant and % of cells). foxp3 express in cd8 1 tregs were maintained after induced factor tgf-b1 and rapamycin were removed on day3, day6 and day9. in vitro, foxp3, il2 and ifn-c expression has no significant difference when compared with controlled tregs on day3, day6 and day9 and did not secret il17a when encounter with inflammation conditions (il21tgf-b11il211il23 and il21tgf-b11il1b1il6). in vivo, cd81 treg cells were transfused into cia mice on the peak of disease onset (35 days after the first collagen immunization, has inflammation condition in vivo) to test cd8 1 tregs survival. cd8 1 tregs were found in cia mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd8 1 tregs are stable in inflammation and transfusion and can maintain foxp3 expression when induced factor were removed, these make cd8 1 treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s6k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd83/cd80/cd86 expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p70s6k and its downstreanm proteins, especially the protein s6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s6 related protein translation inhibition. xiaoyun fu* 1,2 , mikayla anderson 1 and james c zimring 1,2 . 1 bloodworksnw research institute, 2 university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in 405 leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day 43 (one day past their expiration). 35 bioactive lipids including 10 common fatty acids, 10 oxylipins, and 15 lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in 405 stored rbc units. for example, arachidonic acid (aa) ranges from 0.5 -10.7 mm, linoleic acid (la) (1.4-28 mm), dihomo-c-linolenic acid (dgla) (0.1-0.8 mm), eicosapentaenoic acid (epa) (0.03-3.1 mm), docosahexaenoic acid (dha) (0.2-3.0 mm), and alpha-linolenic acid (ala) (0.06-2.3 mm). ten oxylipins including hetes, hodes, and dihomes, and 15 lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way anova testing (fdr<0.05). the ab rh1 blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh1). the fold increase of o rh-/o rh1 among pufas ranges from 1.3 to 2.1, suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among 405 stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna-223 targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r 2 rnaprobe 5 0.96 and r 2 dnaprobe 5 0.98) with mirna-223 concentration, down to a 10-nm limit of detection. hybridization assays in 1% human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in 1% human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. 6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 total atp,lm/ghb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 extracellular lactate,mm 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 extracellular glucose,mm 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 extracellular na 1 ,mm 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 extracellular k 1 ,mm 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso100201600009c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n56) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, p50.030, chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 . in vitro quality of single dose amotosalen/uva treated platelets in 35% plasma/65% pas-3 after 5 days of storage crystal stanley 1 , marguerite kelher 2 , nero evero 1 , melissa vongoetz 3 , betsy donnelly 3 and anna erickson* 3 . 1 belle bonfils memorial blood center, 2 university of colorado, 3 cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas-3 to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct02298842) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas-3 and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% pas-3, collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, 7.5 6 0.6 310 11 platelets in 602 6 52 ml, were collected on the trima apheresis platform in 35% plasma/65% pas-3. a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n56, were 302 6 26ml (t) and 300 6 27ml (c) with doses of 3.8 6 0.3 3 10 11 (t) and 3.7 6 0.3 3 10 11 (c). all pc were stored under the same conditions and evaluated on day 5 and day 7 for physical/metabolic characteristics. results/finding: on days 5 and 7 all t and c pc had ph228c !6.2. the dose recovery for t was 87%63%. on day 5, t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table 1) . conclusion: trima pc in 35% plasma/65% pas-3 treated with the inter-cept blood system for platelets using the sv set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan 1,2 , yang yu 1 , li-ping sun 1 , shu-fang wang 1 , rui wang 1 , lei-ying zhang 1 and deqing wang* 1 . 1 the department of blood transfusion, the pla general hospital, 2 the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: 1 five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35. we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in 17% final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. 2 levels of k and lactic acid (la) were tested using automatic biochemical analyzer.3 k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. 4 treated hips-cms with d35 ssrbcs, d35 k and cell culture media for 48h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: 1 d0 ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d14 ssrbcs stop beating, but beating patterns restored at 48h. hips-cms treated with d35 ssrbcs stop beating, and beating patterns did not restored at 48h. 2 levels of k and la in ssrbcs changed most obviously. 3 only d35 k solution made hips-cms stop beating and can restore in 48h; d0 k, d14 k and la solution did not influence the beating pattern in at the end of the treatment for 24h, hips-cms treated with d35 ssrbcs show obvious shrinkage. at the end of the treatments for 48h, cells treated with d35 k and d35 ssrbcs both show obvious shrinkage, the shrinkage in d35 ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. 5 gene expression array results show a total of 140 genes were differentially expressed in d35 ssrbcs group compared with naive group. there was no consistent separation within the d35 k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. 2 in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. 3 further study should be applied to signal pathways on ssrbcs induced cytotoxicity. 4 large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as-3 as compared to sagm. the presence of citrate in as-3 seems to be necessary to prevent hemolysis of thawed cells. during storage in as-3, atp and 2,3-dpg levels rapidly decline. recently developed additive solutions like pag3m and as-7 have shown to better maintain 2,3-dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag3c in which the mannitol of pag3m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at 2-68c in pag3c. study design/method: leukoreduced rcc (n56) in pag3c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68c. on day 8, rccs were glycerolized using acp215 (haemonetics v r , braintree, ma) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808c. after thawing and deglycerolization using acp215, rcc were resuspended in pag3c. during storage at 2-68c, stability (hemolysis), atp and 2,3-dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n58) resuspended in or sagm (n54). results/finding: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels at day 8 as compared to storage in sagm, resp. 9.1 6 7.6 mmol/g hb and 1.9 6 0.7 mmol/g hb. hemolysis during post-thaw storage in pag3c remained below 0.8% for 35 days and was comparable with storage in as-3. in sagm, hemolysis remained below 0.8% for 7 days. during the first 2 weeks of post-thaw storage in pag3c, both atp and 2,3-dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag3c showed significantly higher atp and 2,3-dpg levels compared to as-3 or sagm. while in sagm and as-3, 2,3-dpg levels were undetectable after 7 days post-thaw storage, in pag3c, 2,3-dpg levels only decreased to 6.1 lmol/g hb after 35 days of storage. conclusion: pre-freeze storage in pag3c resulted in increased 2,3-dpg levels. as compared to as-3, post-thaw storage in pag3c showed comparable hemolysis while atp and 2,3-dpg levels were much better maintained. based on a maximum allowed hemolysis of 0.8% and an atp content of >2.7mmol/g hb, thawed rcc can be stored at 2-68c for 35 days in pag3c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold (48c, 4c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only 2 apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n54-5) in 65% iso using a trima or in 65% int using an amicus and stored for 15 days at rt and 4c. samples were tested on day 1 (baseline, bl), 5, 10, and 15 of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at 40% hct. labeled blood was perfused through microfluidic channels (fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate. images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p<0.05. results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day 5 of storage compared to bl (bl: 11.6 6 1.7%, rt: 4.9 6 1.2%; p<0.005). 4c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, 4c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day 5 (p50.03) and compared to 4c-int by day 10 (p<0.01). conclusion: our work suggests that 4c storage of plts collected with a trima ap system in iso for up to 15 days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at 4c. these results are surprising since both 4c-int and 4c-iso have been shown to express similar levels of cd62p, pac-1, and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at 4c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table 1. comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th17 cells and increase in cd4 regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th1,th2,tfh and tfr cd4 subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* 1 , sarah kloss 1 , sara crew 1 and sandra j nance 2 . 1 american red cross, 2 american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to 28 unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only 510k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from 40 plasma and serum clinical specimens. group 1 contained a single hpa alloantibody specificity with or without hla antibodies (n526). group 2 included 5 specimens with hla antibodies alone and group 3 consisted of 9 patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa-1, hpa-2, hpa-3, hpa-4, hpa-5, gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is 100% concordance observed for hpa-1a, hpa-1b and hpa-5b antibodies. the pak-plus assay had difficulty discriminating hpa-5b from hpa-5a antibody when hpa-5a antibody was present (3 false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa-5a when compared to hpa-5b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for pakplus and <2.0 adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb231 cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac6 specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut1 overexpression on cell motility. results/findings: fut1 overexpression increases both ley expression and cell migration, while fut1 knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut1 overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac6. as tumor promoter, hdac6 becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac6 function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at 48c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n53) and stored at 48c for up to 10 days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and 30 minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd62p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means6sem, and paired student's t-tests were used to determine statistical significance (p<0.05). results/finding: on day 10, p-selectin levels were significantly higher in pre than bl (p50.03). mirasol treatment caused a significant increase in pac-1 expression compared to pre (pre: 10.5 6 3.1%, post: 28.1 6 4.7%; p50.04), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after 10 day storage at 48c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, 48c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day 10 4c-stored pas plts followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* 1 , arthur p. bode 1 , anne s hale 2 , michael stanton 3 , mark johnson 4 and g. michael fitzpatrick 3 . 1 cellphire, 2 bodevet, inc, 3 cellphire, 4 background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to 33, 10, and 3.3% of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to 33% of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of 10% and 33% of the tcpc produced a significant decrease in blood loss. the lcps at 10% and 33% tcpc were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to 33% of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of 10% and 33% of the tcpc reduced blood loss. these results suggest a starting dose above 3.3% tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as 10% tcpc had similar efficacy signals as 33% tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso100201300021c. the study on pcr-ssp technique for the genotyping of cd36 329-330del.ac mutation and the genetic polymorphism of cd36 329-330del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd36 (platelet glycoprotein iv, scarb3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd36 is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd36 deficiency in china. cd36 gene mutation is the main reason that leads to cd36 deficiency. cd36 329-330del.ac (frameshift at aa110) mutation is one of the cd36 mutations that causes cd36 deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in 28 pediatric patients who received both doses of the mmr vaccine at 12 and 18 months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are !90% for all mmr components. results/finding: table 1 shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years). thirteen patients (46%) were chronically transfused at the time of serology. twenty-three patients (82%) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to 6 months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper 1,2 , franklyn cladis 2 , richard saladino 2 , darrell triulzi 3 , barbara a gaines 2 and mark yazer* 1 . 1 university of pittsburgh, 2 children's hospital of pittsburgh of upmc, 3 institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (<50) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately 11 months, 15 trauma patients received wb: 7 group o and 8 group a recipients, 53% male, median (iqr) age was 11 (4.5-14) and 73% blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of 36 (22-51) and 47% mortality rate. the median (iqr) quantity of wb transfused to group o recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean 6 standard deviation haptoglobin concentrations for non-group o recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group o recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514). the mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to 30 ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) 41 with anti-human igg only, and a 31 positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a 60 minute 378c incubation, followed by 3 automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer 4096 and the breast milk showed anti-d with a titer between 16 and 64. the patient had a consistent plasma anti-d titer of 8. the patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that 30% of c1 scd patients from the west indies and west and central africa are partial c and at (30%) risk for alloimmunization to the c antigen through transfusion of c1 rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce(733g,1006t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce(4-7)-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of 255 patients with genotype/rh phenotype data available, 78 (30.6%) were c antigen positive serologically. the allele frequency of rhce*ce(733g,1006t) was 0.071. in total, 15 (5.9%) patients possessed rhce*ce(733g, 1006t) in the absence of conventional c gene in trans. of the 78 c antigen positive patients, 15 individuals (19.2%) were predicted to be partial c based on four molecular profiles [rhce*ce(733g, 1006t)/rhce*ce:12; rhce*ce(733g, 1006t)/rh*ce:1; rhce*ce(733g, 1006t)/rh*ce(733g):1; rhce*ce(733g, 1006t)/rh*ce(733g, 1006t):1]. in these 15 partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after 60 transfusion exposures (57 c-antigen negative units; mean: 4, range: 0-36), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce(733g, 1006t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* 1 , megan l nguyen 1 , melanie c proctor 1 , david e krysztof 1 , gregory a foster 1 , erin k sash 1 , sandy s dickson 1 , joua yang 1 , jeffrey m linnen 2 , kui gao 3 , jaye p brodsky 4 and susan l stramer 1 . 1 american red cross, 2 grifols diagnostic solutions inc., 3 grifols diagnostic solutions, inc, 4 quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and 4 suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on 6/20/16 (fl, ga, sc, ms, al). following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on 12/12/16. travel history questions were discontinued on 1/23/17. confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 mps. no reactive donations were identified by mp-nat. of the 1,895,142 id-nat donations, 72 were initial reactive (ir) of which 8 (11%) confirmed positive (cp) by subsequent testing (cp rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%). five (62%) cp donations were id-nat repeat reactive (rr); 3 (38%) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and 3 in fl, 2 of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from 2 to 73 days prior to donation. two donors with a travel risk reported clinical symptoms; 6 cp donors (75%) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than 40 copies (c)/ml to about 8ê 5 c/ml. at the time of writing, the longest period of detection in rbcs was 91 days vs. 17 days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from 1 ir and all rr donors, ranging from 12 to 2000 c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat1 samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of 6 were prepared by diluting nat1 plasma 1:6 and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm1/high vl; igm1/low vl. results/finding: of 52,942 donations collected april 3-december 31, 352 were reactive for zikv rna. igm-index donations had higher vls (mean 1.1 x 10 6 vs 8.3 x 10 4 iu/ml) and higher proportions of simulated mp-detectable results (93% vs 23%) than igm1 donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm1 donations increased (table 1) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the 2016 pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r 6800/8800 systems lisa lee pate* 1 , phillip c williamson 2 , michael paul busch 3 , susan rossmann 4 , scott jones 5 , ann butcher 1 , john duncan 1 , jean stanley 1 and susan a galel 1 . 1 roche molecular systems, inc., 2 creative testing solutions, 3 blood systems research institute, 4 gulf coast regional blood center -sugar land, 5 qualtex laboratories background/case studies: in february 2016, the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march 30, 2016 and testing of puerto rico donations began on april 3, 2016. as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august 2016, the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april 3, 2016 -february 28, 2017 using the investigational cobasv r zika for use on the cobas v r 6800/8800 systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a 1:6 dilution to simulate mini-pool testing. results/findings: a total of 1,776,190 blood donations were screened using cobasv r zika. of 56 ir donations, 12 were repeat reactive (rr), 39 non-rr and 5 had no repeat testing. of the 12 rr donations, 7 were positive by altnat; 3 of these were igm positive. all 4 altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the 5 rr donors that were not igm positive on index, 3 enrolled in follow-up and all seroconverted. of 39 non-rr donations, 38 were altnat negative and 1 is pending supplemental testing. 8/38 donors were igm positive on index. 30 donors were igm negative on index; 15/30 enrolled in follow-up; 14 remained igm negative and 1 was 1gm inconclusive. of 5 donations without repeat testing results, 2 met criteria for positive (1 was altnat positive, igm negative and 1 altnat negative, igm positive). 1 donation is pending additional testing. altogether, 22/56 ir donations met the criteria for true positive on the index donation. 9/22 (41%) true positive donations were reactive when retested in a simulated minipool. 16/22 were igm positive. conclusion: 0.001% of the 1,776,190 donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* 1 , marc germain 2 , gilles delage 3 , maria esther lopes 1 and yves gr egoire 3 . 1 hemorio, 2 hemaquebec, 3 h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia (2013) , and in brazil (2015/2016), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since 80% of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january 1 st , 2016 through november, 26 th , 2016, from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for wb and 1.75 for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x (1 -proportion of refused donors) x (1proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain 1:13,598, for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from 6 donations). the residual volume of mp plasma, 0.35 -0.45 ml, is routinely discarded. beginning in april 2016 each blood center saved $67 mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april 3-15) 3 mp6 were combined into pools of 18 donations; thereafter mp6 were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first 6 months of samples is complete for 6,292 mp, comprised of 37,752 donations collected from april 3 to october 9, 2016. a total of 77 pools were positive, with 76 detected between april-june 2016. the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over 0.6% of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over 0.4% of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in 2016, zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* 1 , sze sze chua 1 , mars stone 2 , michael paul busch 2 and ai leen ang 1 . 1 health sciences authority, blood services group, 2 blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on 26 august 2016. the numbers rose rapidly to 386 cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since 1 october 2016. zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the who international standard for zikv and 25 replicates of negative controls prepared by bsri. probit analysis was performed to determine the 50% and 95% limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a 14 member blinded zikv reference panel from the usa-fda. results/finding: a total of 63,144 donations were screened from 1 october 2016 to 31 march 2017, with 1 false positive case and 1 zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml. zika igm was negative in the index donation sample but present in the 10-day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be 2.1 copies/ml at 50% lod and 10.0 copies/ml at 95% lod. the procleix zikv assay detected rna in 6 out of 9 patient samples and provided 85.7% agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with 1 confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types 1, 2 and 3 express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to 21 reactivity on initial gel testing. if genotyping demonstrated weak d types 1, 2 or 3, the intent was to manage the patient as rhd-positive. if weak d types 1, 2 or 3 were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in 2 to 4 weeks. results/finding: rhdgenotyping was performed on 22 patient samples over 15 months. of these 22 patient samples, 13 (59%) were weak d types 1 or 2. the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type 1 required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types 1 and 2 patients have not received transfusions at this institution since they were genotyped. four of 4 obstetric weak d types 1 and 2 patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this 15 month study period 13 serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using 3 fda approved anti-d reagents. when reactivity with all 3 reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over 80 rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of 8 months we tested 509 rhd-negative blood donors. there were 3 (0.6%) partial-d, 1 weak d (0.2%), and 3 (0.6%) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs5-46_42deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these 8 donors showed that 6 rhd-negative recipients received rbcs from 4 of these donors. five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, 5 grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which 63% of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d1" with 31 reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october 2016 to march 2017, we performed routine d typing (neo, immucor) on 1875 obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of 31 using at least 1 antibody. solid phase and manual testing used the series 4 and series 5 reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh1) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all 13 samples. two of 13 (15.4%) were d1 with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs1-29c, rs2301153; ivs3 1 117c, rs28521909; and ivs3 1 124a, rs28562109). two (15.4%) were d1 and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four (30.8%) patients had rhd alleles with known potential to make anti-d (rhd*dol2, rhd*dar1.2, and 2 with weak d type 4.0). one had weak d type 96, which has uncertain susceptibility to alloimmunization and one was weak d type 1, which has not yet been associated with anti-d. interestingly, two (15.4%) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type 2 is a variant of the rhd protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c.1154 g>c which is the first nucleotide of the exon 9 of the rhd gene and thus could be implicated in exon 9 skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v6 (agilent) and the nextseq500 platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type 2 rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by beadchip technology. interestingly, 4 out of 10 carry the c.1154-31c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last 4 patients, one has been previously characterized as rhd weak type 2 carrying the c.1154g>c (p.gly385ala). independently, sanger sequencing on 50 unrelated rhd weak type 2 samples pinpoint to a linkage disequilibrium between c.1154g>c (exac, maf 5 0.001145) and the c.1154-31c>t (exac, maf 5 0.2496). in silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon 9, mutant rhd c.1154g>c, mutant rhd c.1154-31c>t and double rhd mutants c.1154g>c plus c.1154-31c>t, we showed no influence on skipping of exon 9 due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type 2 between ala385 (transmembrane helix 12) and val183 (transmembrane helix 6) hampering membrane insertion. conclusion: the c.1154-31c>t variation is always associated in cis with the missense mutation c.1154g>c on the allele rhd weak type 2. the c.1154-31c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has 10 transmembrane (tm) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p.280. we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and 2010-2016 aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, 2012) . results/finding: seven snmvs located within 1 amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all 3 at the exofacial ends (p.a93t, p.w240r, p.v333d) are jk-weak; the two jkneg exceptions p.g298e and p.g299e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, 13 snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v10m, p.e44k, p.v76i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the 13 jk-neg variants are within 19 aa (p.270-p.299) of jk a/b at p.280. none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n289s and p.s291p are adjacent to p.288f and p.292l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among 13 jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc14a1 gene, which encodes the urea transporter ut-b1. the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in 1965. in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc14a1 gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within 1 aa from tm a-helix end v76i, a93t, w171r, w240r, g298e, g299e, v333d* cytoplasmic n-terminal v10m, g40s, e44k, l45p in membrane tm and urea-pore a-helices r64w, r64q, g65d, i117t, a183v, l246r, a248t ‡, a270a §, l272f, n289s, s291p, t319m 2/10 * second nucleotide variant in this allele is synonymous (p.p196p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a 5 mbp region in 19q13.11-13.2 with an lod score of 9.6. using deep sequencing, we identified a potential deleterious mutation in the znf850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain. the identical del84-znf850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf850 locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf850del84. none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc14a1gene, is a urea transporter that has been associated with renal function, we found that people with the znf850del84 in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf850, prevalent in southern spain due to a founder mutation, leads to ut-b1 dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-(2-ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis(2-ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at 30 days and 1 year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the 12 pools included 5 group a, 6 group o and 1 group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than -208c within 8 hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day 0 (pool), day 30, and 1 year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day 0, day 30, and 1 year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp 5 2.9 ppm; mehp 5 0.3 ppm; deht 5 0.9 ppm; and meht 5 0.2 ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than 80% of its initial value. plasma stored in deht bags had an average plasticizer content 90% lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate rbcs from platelet-rich plasma (prp). prp was diluted 3-fold in pipes-saline with 1.4mm pge1 and centrifuged at 1900 g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of 34-40% and 150,000-250,000 platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted 1:1 (spdp50%) with plasma from a patient with type 3 vw disease (t3vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was 20% (spdp/ffp > 0.8). results/finding: six batches of spdp/ffp were evaluated using 17 subjects. there was no statistical difference between the spdp/ffp pairs (p50.7558). the mean ratio of spdp/ffp was 1.21 with a 95% ci of 0.84 -1.57. comparing spdp vs. spdp50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was 20% greater than in samples reconstituted with ffp. the lower limit of the 95 th % ci is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%. even with 50% dilution with t3vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* 1 , qiyong peter liu 2 , grantham c. peltier 1 , ryan c. carney 2 , ashley s. taylor 1 , colby s. mcintosh 1 , james a. bynum 1 and andrew p cap 1 . 1 u.s. army institute of surgical research, 2 velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: (1) ffp; (2) ffp with 70mm glycine; (3) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; (4) spdp pretreated with glycine-hcl (20mm); and (5) spdp pretreated with glycine-hcl:glycine (20mm:50mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood (40% hct with 200 platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < .05). fibrin polymerization density was slightly diminished in rspdp vs. ffp (0.879 vs. 0.742 o.d., p < .01), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < 0.001). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < .01) and an additional twofold in pretreated spdps vs. rspdp (p < .05). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < 0.01). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* 1 , anita tuip-de boer 1 , ruqayyah almizraq 2 , jason p. acker 3 , philip j. norris 4 , jennifer a muszynski 5 and nicole juffermans 1 . 1 academic medical center, 2 university of alberta, 3 canadian blood services, 4 blood systems research institute, 5 nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from 8 donors (blood type a or b). supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and ev analysis. a549 type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to 25% stretch using a cellstretcher. control cells were not stretched. after 24 hours, il-8 and il-6 production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il-6 and il-8 production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p<0.05) . incubation of stretched cells with stored wbf products resulted in higher il-8 production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry). monocytes were co-cultured in media plus 20% rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in 5 replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean 6 sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table 1) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin-8 production was higher after exposure to fresh wbf (248 6 115 % control, p 5 0.02) or wbd at expiry (292 6 111 % control, p 5 0.0005). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* 1 , alessandro tocchio 1 , anita howell 2 , kaushik sridhar 1 , jason p. acker 3 and utkan demirci 1 . 1 stanford university, 2 canadian blood services, centre for innovation, 3 background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from 24 volunteers with four different age and sex categories (male, 18-40 years; male, >60 years; female, 18-40 years; female, >60 years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young (1.098 g/ml) and older female donors (1.109 g/ml) (p < 0.01). moreover, rbcs from young males (1.096 g/ml) were significantly less dense compared to rbcs profiled from older female donors (1.109 g/ml) (p < 0.05). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il-6 (pg/ml) il-8 (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: 120 icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of 426 rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p<0.001). substantial correlations were also found between orp and free hemoglobin (p<0.05) and orp and free heme (p<0.05). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than 35 days compared to rbcs stored for 7 days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of 1 to 42 days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to 7 days storage duration -reference group), medium age (at least 1 rbc of 8-35 days storage), and oldest (at least 1 rbc greater than 35 days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for 7 days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days. results/finding: 24,726 patients receiving 90,530 rbcs were included in the analysis. exposure to rbcs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (hr 5 0.91; 95% ci: 0.72, 1.14; p 5 0.400). exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (hr 5 0.90; 95% ci: 0.73, 1.10; p 5 0.295). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than 35 days compared to exclusive exposure to rbcs stored 7 days or less was not significant (hr 0.90; 95% ci 0.72, 1.14; p 5 0.381). the confidence intervals around the hazard ratios for the other 7-day intervals all include 1. similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to rbcs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for 1-3 days (hr 0.82; 95% ci 0.37, 1.83; p 5 0.635). the confidence intervals around the hazard ratios for the other 3-day intervals all include 1. conclusion: individuals exposed to rbcs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for 7 days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* 1 , cynthia walser 1 , tatsuro yoshida 2 , andrew dunham 2 and pedro cabrales 1 . 1 university of california san diego, 2 new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o 2 ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o 2 carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o 2 saturation <10%) stored rbcs, or anaerobic/hypercapnic (o 2 saturation <10% and pco 2 (@378c) $70mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as-3 after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions. rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with prbc stored for either 1 or 3 weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. 1(11%) neg patient showed short term response and 6(67%) patients showed progressive disease. at the neg group standard eval 1(11%) patient showed response and 3(33%) had progressive disease. 1(11%) neg patient had long term response compared to 11(21%) pos patients. at the pos short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease. at the pos group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease. overall, 28(53%) pos patients responded compared to 2(22%) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd38 neutralizing substance could play a role in treatment response. alternatively, reduced cd38 expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate 24% replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of 0.2m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* 1 , laurie sutor 1,2 , germ an leparc 3 , marjorie doty 3 and william crews 1 . 1 carter bloodcare, 2 ut southwestern medical center, 3 oneblood background/case studies: anti-cd38 drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with 0.2m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a 28 day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and 0.2m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual (18 th edition). each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. (25) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than 11 with the untreated or dtt-treated cells during the study. conclusion: long term storage of 0.2m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque-10771), pooled, suspended in cryopreservation media (20% dmso; 1:1) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips (378c, 5% co 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o1) rbcs sensitized with either anti-d (positive control), anti-scianna-2 (sc2) or anti-anwj or lipopolysaccharide stimulated for 2 h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/100 monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed 96.2 6 1% viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il-1b, il-6, il-8, mip-a (p < 0.01), mip-b and gro (p < 0.05) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc2-and anwj-sensitised rbcs resulted in a pi of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-d sensitized rbcs (pi: 72 6 8.7%). a weak (11) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in 41 iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi>5%). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in 5 patients, involving 3 antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all 5 patients had 3-41 positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients 1 and 2 typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c1 by hea precise-type. ega-treated rbcs gave 31 reactions with the same anti-c reagent. patient 1 rbcs gave variable reactivity (vw-11) with bio-rad seraclone and ortho bioclone anti-c. patient 2 rbcs gave 11 reactivity with all 3 anti-c reagents when incubated for the maximum incubation time allowed. patient 3 rbcs were jk(a1) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat1rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients 4 and 5 tested s1 with bio-rad seraclone anti-s (3-41), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat1 rbcs but not all manufacturers include reagent limitations regarding testing of dat1 rbcs. we describe 2 cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and 3 cases with false positive tests with anti-s (n52) and anti-jk a (n51) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with 3-41 positive dat and supports testing to dissociate igg from rbcs strongly dat1 before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/2opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r :5 prob(ab|al-loexp), so that prob(ab) 5 prob(ab|alloexp)*prob(alloexp) and 0 r 1; rewriting prob(alloexp) 5 prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) 5 nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) 5 r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past 12 months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in 2 states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for state b, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a 13-and 3-fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from 13 to 567 (state a) and 151 to 1,496 (state b), which annualized, represents a potential gain of 2,216 (state a) and 4,035 (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female. there were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to 10.5 ml/kg or 15% of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit. variable volume scales [vvs] can be programmed to vary unit volume (up to 550 ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by 10 ml at ebvs <3.5l in donors !23 yo, but increase by 5-40 ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the 18 mos. before a 6 mo. phased implementation of the vvs, and the subsequent 24 mos. multivariable analysis [mva] by 6-mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods 1 & 2, continued during impl and post-impl periods 1 & 2, returning to the baseline rate in post-impl periods 3 & 4 (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods 3 & 4. the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of 3.8 ml during post-impl periods 1 & 2 from the temporally matched baseline & pre-impl period 1. conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* 1 and yves gr egoire 2 . 1 hema-quebec, 2 h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, 2017. the vaccine is produced with live and attenuated yfv, which can circulate for at least 4 weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a 4 week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview. the deferral rate was 26.9%. at the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations. the deferral rate was 27.3%. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates. that represents a 177.34% increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017. conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of !3.0x10 11 is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: 1,000 apheresis collections from 4 centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (s), 6.3x1011 11 for double (d), and 9.5x10 11 for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for s and 6.7x10 11 for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as 99%) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc 2rbc plt/p plt plt/rbc/p plt/rbc 2plt 2plt/rbc 2plt/p #donations 47990 63 1775 10832 968 476 67 1150 142 10252 citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 study design/method: a randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model. cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of 68 aes: 40 were treatment emergent (teae), of which 8 were treatment-related (6 thrombosomes and 2 control). all teaes were mild or moderate in severity. in cohorts 4 and 5, 3/4 thrombosomes subjects had treatment related adverse events. one cohort 4 subject developed an upper respiratory infection and elevated wbcs within 8 hours post infusion, which resolved by 24 hours, and an elevated d-dimer at 24 hours post infusion, which resolved by day 7. this subject also had an elevation of prothrombin fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by day 14. one cohort 5 subject developed non-specific t-wave changes at 1 and 2 hours following her 2nd infusion that resolved by day 21 without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort 5 subject developed an igg platelet autoantibody on days 7-21, which was undetectable on days 42-60; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days 7-14, and negative on days 21-60. background/case studies: cryopreservation of platelets (plts) could extend the shelf life from 5-7 days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with 5% dmso and stored at 2808c. after thawing, the unit was reconstituted in thawed ffp spiked with either 500 lm puromycin (pm) or 250 nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd62p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd62p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage. immunoblot analyses of the plts showed a 2-and 4-fold increase in pm incorporation after 4 and 24 hours of storage, respectively. massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac1, rap1 and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac1, rap1 and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in 2015, the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of 3 days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r 4-496 cooler with 2 units of ffp, 2 units of rbcs, and 1 unit of whole blood. three to 5ml of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac-1 binding) were measured by coulter counter, 2 channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c49-a03h molecular sieving: beyond genotyping ghazala hashmi 1 , reinhard klemm 2 and michael seul* 1,2 . 1 biomolecular analytics, 2 immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi2005 http://bit.ly/2ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of 30 rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, 4*4*96 samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("sca") patients (tb1 in cas-tro2002, http://bit.ly/2oplxhr, excluding le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells. in another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from sca patients, a yield exceeding 2.5x. sieving alone typically fills 65-75% of requests of moderate complexity ( 5 ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez 1 , monica kalvelage 2 , ghazala hashmi* 1 and michael seul 1 . 1 biomolecular analytics, 2 lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou2013 http://bit. ly/2ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the rhce locus) that encode 401 mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag2" (e.g. e2,c2,e2,c2) to specific combinations of "ag2" (e.g. c2e2k2fya2 and c2e2jsa2) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, 96 pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the 124 c2 samples, 24 that were also v2 and vs2 and, among the e2 samples, 12 that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select 6 specific "ee" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b (8 pools), co a|b (8) and others. conclusion: molecularsieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c2, e2 and jsa2. these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam4 (landsteiner-wiener) and ackr1 (duffy). for longer genes, such as abo of >20 kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of >21 kb each was used for all physically confirmed 48 ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least 4 clades representing clusters of 5 to 11 alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt1 genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors1) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors1. a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos1(b3galnt1) cells, and cell-surface expression of fors1 antigen was immunologically monitored with a monoclonal anti-fors1 antibody. results/findings: we found that met69thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon 3 or 4 of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors1 antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met69thr/ ser or exon 3/4 deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt1 genes is reminiscent of common ancestral origin of alpha 1,3-gal(nac) transferase genes. the finding that at can synthesize fors1 implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of 29,308 cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period 7/1/2008 -4/1/2017. abo genotyping targeting specific snps for groups a, a2, b, o1, and o2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two (0.21%) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in 53% the predicted abo phenotype was a rh neg (table 1a ). the predominant donor race was caucasian (65%). four cbu with abo discrepancy were also evaluated by genotyping (table 1b) . in 3 of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r 6800/8800 systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: 30,695 fresh and 20,029 frozen edta plasma samples from american red cross donors, collected from february 2015-2016, were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r 8800 system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of 50,724 valid results, a total of 3 donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a 65-year old male in indiana, a 21-year old male in california, and a 55-year old female in kentucky. all 3 donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna (1440 iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as 3a, the california donation genotype 3b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was 100% (95% exact ci: 99.993% to 100%). conclusion: based on the 3 confirmed-positive donations of 50,724 tested, the hev prevalence was 0.006% (95% exact ci: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% ci, 1:588-1:100,000). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report 13 months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using 95% confidence intervals. this analysis contains data from 9/1/15-9/30/16. results/findings: among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 cp results for hbv, hcv and hiv with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv 8.3 vs 4.2; hcv 23.5 vs 15.2; hiv 3.9 vs 1.0). in general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when 3-month periods were compared. conclusion: data from 4 major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* 1 , melanie c proctor 2 , deanna self 1 , monique portugal 1 , adrian gurrola 1 , laura tonnetti 2 , sonia bakkour 3 , cheryl lobo 4 , michael paul busch 3 , susan l stramer 2 and jeffrey m linnen 1 . 1 grifols diagnostic solutions inc., 2 american red cross, 3 blood systems research institute, 4 new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to 16 donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from august 25 th 2016 to april 7 th 2017 in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of 16. results/finding: the procleix babesia assay detected all four babesia species with a 95% lod ranging from 7.10-13.51 copies/ml. the preliminary 95% lod in parasites/ml ranged from 0.64-3.61 p/ml for b. microti (n59), from 0.92-1.52 p/ml for b. duncani (n52), and from 0.62-4.95 p/ml for b. divergens (n52). of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%ci: 99.972-99.997%). of the confirmed positive specimens, 8 were reactive by both ifa and pcr, 5 by ifa only and 1 by pcr only. all confirmed positive samples were reactive in lysate pools of 16. donors of reactive donations resided in ct (11), nj (1), nh (1) and me (1) for an overall incidence of 1:2,305, and 1:1,433 in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* 1 , whitney r steele 1 , ed p notari 1 , james haynes 1 , roger y dodd 2 and susan l stramer 1 . 1 american red cross, 2 american red cross (retired) background/case studies: from 2004 -2012 , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from 2008-2015. study design/methods: prevalence was calculated in 2-year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of 18.5, 7.4 and 9.1 days for hbv, hcv and hiv, respectively. linear regressions were calculated with p<0.05 (*) as significant. results/findings: from 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% first-time (ft), 81.4% caucasian). there were significant decreases in donation prevalence for hbv and hcv (p50.014 and 0.044), but no significant decrease in hiv during the 8 years (see table for f and r 2 values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p50.026 and 0.042). prevalent ft donors were significantly more likely to be male (68.3% -hbv, 59.8% -hcv, 79.7% -hiv; p<0.001). incidence for all agents declined (significant only for hbv; p50.035). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) . hcv incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011). overall, incident donors were more likely to be caucasian males (p<0.01). rrs for all 3 agents decreased over time with rrs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the 8-year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in 2015, mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as-5 rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with 200lm amustaline, and incubated for 18hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero76 cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, >6.9 log 10 , or >6.2 log 10 pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was >6.2 log 10 , or >5.5 log 10 pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts-13 inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts13 activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts13 deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts13 activity of <5% and high inhibitor (1.4-8). mean age of cohort 22.8 years (range 17-64). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /l, range 9 -27 x 10 9 /l) and low a-ipc (mean 1.5 x 10 9 /l, range 0.5 -3.6 x 10 9 /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient). tpe continued until platelet count reached 150 x 10 9 /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in a-ipc from baseline (mean 11.1 x 10 9 /l, range 2.2 -25.3 x 10 9 /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean 217.6 x 10 9 /l, range 200 -294 x 10 9 /l) and a-ipc (mean 19.4 x 10 9 /l, range 13 -28.5 x 10 9 /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /l (range 14 176 x 10 9 /l) and mean a-ipc 3.2 x 10 9 /l (range 0.7 -6.6 x 10 9 /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of 20.8 days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts13 inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* 1 , michelle n stram 1 , joan sevcik 2 , alesia kaplan 2,3 and joseph e. kiss 2,3 . 1 department of pathology, university of pittsburgh medical center, 2 blood systems inc., 3 university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january 1, 2013 to november 1, 2016 was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version 14 (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the 96 ttp patients identified, 22 were excluded due to missing temporal data for important variables. the majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients (20%) had a prior history of ttp and 26% had severe adamts13 deficiency on admission. the median time from tpe request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table 1) . treatment was not started within an 8-hour window in 13 patients; the median time to cv access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001). two of these patients had a prior history of ttp and only four patients had severe adamts-13 deficiency. the majority (more than 70%) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus 4-8 hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi-48a transfusion 2017 vol. 57 supplement s3 hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, 2006 through january, 2017, we performed cytapheresis (cy) treatments (txs) for 123 pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. 84 pts (68%) had cml-at and received 319 leukapheresis (lp) txs; 39 pts (32%) had et and received 124 thrombocytapheresis (tc) txs. cml-at pts presented with median wbc 398 x 10 9 /l (range 193-689 x 10 9 /l), of which 63% had blast percent >75% or blast count >100 x 10 9 /l. median age was 42 years (8-79 years); 62% were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. 22% of cml-at pts had no sxs of lks; 40% pts had sxs of either cns or pulm lks (1 sxs), and 38% pts had sxs of both cns and pulm lks (2 sxs). et pts presented with median platelet (plt) count of: 1738 x 10 9 /l (642-3510 x 10 9 /l)and 71% pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was 66 years (31-89 years); 58% pts were male. results/finding: all pts received a course of cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) <100 x 10 9 /l for cml-at pts, and plt ct <450 x 10 9 /l for symptomatic et pts and <750 x 10 9 /l for asymptomatic et pts. cml-at pts received median of 3 lp txs (mean 3.9 txs/pt; range 2-8 txs). et pts underwent median of 2 tc txs (mean 3.4 txs/pt; 1-7 txs). outcomes were evaluated by percentage of pts who: 1) reached wbc (or plt ct) tx goal, and 2) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved >50% reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened. in et cohort, 85% improved, 14% stabilized, and 1% were unchanged. for cml-at pts, median final wbc ct 5 96 x 10 9 /l (range 66-307 x 10 9 /l); 94% pts received ind chemo. for et pts, median final plt ct 5 705 x 10 9 /l (263-1087 x 10 9 /l); 95% pts had resolution of thrombotic 49a transfusion 2017 vol. 57 supplement s3 symptoms. 4% of cml-at pts and 0% of et pts expired within 1-4 days after course of cy tx. of 3 expired pts, 2 pts had both blast crisis and sxs of cns/ pulm lks; 1 pt had intracranial hemorrhage or cva; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20. covariates included were fluid types, age and gender. odds ratios (or) and 95% confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < 0.05. results/finding: during the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20. the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, or (ci): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, or (ci):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, or (ci): 0. 24 (0.06, 0.95)]. age had a significant effect on having a hypotensive event [p50.04, or (ci):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a 10 year period and compared it to published literature. study design/method: we conducted a 10-year retrospective study of ta procedures performed and aes were classified according to criteria described in table 1 . during the study period, ta were performed using cobe spectra (software versions 4.7 and 6.1) and since 2013 the spectra optia apheresis system (version 8.0). literature search was conducted for data published on aes associated with ta. four studies from us and 13 non-us studies (canada, europe and japan) were analyzed. trend for ae rates from 2007-16 was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was 6.9% (396 of 5,684 procedures) during 10 year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher (8.5%, p<0.00001) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe aes with a spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively. there were no fatalities during the study period. majority of aes were grade i (60%) and grade ii (28%): 32/5684 (0.6%) procedures were not completed due to aes. comparison of aes [6.9% (396/5,684)] to both european [11.2% (n513, 12, 256/ 109, 842) ] and other us studies [13.6% (n54, 860/6,324)] showed a statistically significant difference (p<0.0001). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table 1) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within 2 hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* 1 , jordy jurgens 1 , jacoline buchner-doeven 1 , joris roelofs 1 , philip spinella 2 , jennifer a muszynski 3 , carel goslings 1 and nicole juffermans 1 . 1 academic medical center, 2 washington university school of medicine, 3 nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing. plasma was filtered through a 0.22um filter. rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57ml/kg. hemorrhage continued until a mean arterial pressure of 40mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of 17ml/kg of blood products in a 1:1:1 ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april 2013, which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january 2008 and july 2015 (n53535). because of missing data on patient characteristics, 257 patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january 2008 to march 2013, n51987) and after (april 2013 to july 2015, n51291) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively). however, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table 1 ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy16. length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of 4-factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: 4-factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in 2015. marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh5 rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p5.005) and ptt (p5.05) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use. 20 cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were 5.5 times (p5.0072) more with cell saver or anh, and 5.3 (p5.0130) times more with cpb. post-pcc thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every 3 days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days. the transfusion service medical director reviews the case and gives final approval. we observed only 1 patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight (38) patients were in-patients continuously until delivery. five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was 17 days (range 0-63). six (6) patients delivered within 3 days of approval. after approval, the mean number of additional specimens per patient was 2.1 (range, 0-9). no patient required transfusion prior to delivery. five patients received transfusion of at least 1 rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each. since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* 1,2 , jan m. binnekade 1 , benjamin nota 2 , pieter r tuinman 3 , kirsten van de groep 4 , olaf l cremer 4 , janneke horn 1 , marcus j schultz 1 , robin van bruggen 2 and nicole p juffermans 1 . 1 academic medical center, 2 sanquin research and landsteiner laboratory, 3 vu university medical center, 4 university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in 2 tertiary icus in the netherlands comparing 30 patients who developed ai during icu stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/l, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/l, p<0.001). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells4life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the cd341 fraction post separation and freeze thaw (table 1) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table 1) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd451cd611) and early projenitor cells expressing oct4 and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: 1. routine recovery of the wcf at levels higher than current methods, independent of volume. 2. higher percentage recoveries of all cell types tested than can be achieved with existing methods. 3. markedly higher post-thaw recovery of viable nucleated cells than any current methodology. 4. almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd341 target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd341 cell target of 4.0x10 6 /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd341 yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf 1 plerixafor (g1pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< 0.05 considered significant. results/finding: 110 no alc and 159 alc collections occurred among the 50 patients. fenwal amicus was used for 91% of the no alc and 99% of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was 5 hodgkin's and 45 non-hodgkin's lymphoma (no alc); 7 hodgkin's and 43 non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc 49.3, lymph 2.0x10 9 /l) than alc (wbc 39.1, lymph 1.2x10 9 /l). equivalent whole blood (corrected for ac) was processed for no alc (16.4l) and alc (17.1l). for alc group, extra collections beyond cd341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%. significantly more patients were mobilized with g1pl in no alc group (n581) than alc group (n560) and 42 collections in alc group had mobilization discontinued after cd341 cell target reached. there was no significant difference in g (13.2x10 9 lymph) compared to g1pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph). days to wbc engraftment (13.5 no alc vs 13.0 alc) and platelet engraftment (13.0 no alc vs 12.0 alc) were not significantly different. median number of collections for no alc (2) and alc (3) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the 0.5x10 9 lymph/kg or even the 0.3x10 9 lymph/kg targets. implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/kg from 40% to 54%. only 12% had <0.3x10 9 lymph/ kg. discontinuation of mobilization once cd341 cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* 1,2 and nicolas pineault 2,3 . 1 canadian blood services, 2 university ottawa, 3 canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost 48 hours at room temperature (rt) as long as units are cryopreserved by 48-hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd451 cells and cd341 cell (n53). primary nsg mice were transplanted with a ucb cell dose that contained a total of 7,500 annexinv neg viable cd341 cells. the latter was done to avoid any bias towards one group or another. short term platelets (190 vs. 140 hplt/ml, p50.06) and leucocytes (1.2% vs. 0.2% hcd451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant. strikingly, the frequency of human cd451 bm cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2. conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to 97%, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of 1sec). total lymphocyte recovery was 43% and monocyte concentration was reduced 76%. furthermore, in a two-pass process platelets were reduced by 75%. in a 12-fold parallel system we tested rbc separation from plasma and achieved 90% separation at 72ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past 15 years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from 9 clinical protocols from 2008-2016. an infusion reaction was defined as any symptom from the time of nk cell infusion up to 4 hours afterwards. a severe reaction was defined as any symptom with grade 3 or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r 3.3.1. two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of 127 nk cell infusions. there were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42). infusion rate (ml/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively). incubation of nk cell product overnight in il-2 vs il-15 had similar reaction rates for those with any symptom (88% had reaction with il-2, 86% had reaction with il-15, p50.94) and those with severe reaction (28% had severe reaction with il-2, 24% had severe reaction with il-15, p50.80). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan 1 , maryanne c herzig* 1 , barbara a christy 1 , james a. bynum 2 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at -80. mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias. on day 0, mscs were washed, resuspended in pbmc media and incubated with or without 150,000 freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, 0-5 lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by 72 h, with >6 fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was 10.0%. inter-assay variation of msc preps run under identical conditions was 7.5%. inhibition of pbmc proliferation was graded from 0-100% over the range msc concentrations therefore an ec50 of msc cell number resulting in 50% suppression of pbmc could be determined for each msc prep. this ec50 however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the us blood supply. differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged 16-49 were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the 2015/16 academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin <12 ng/ml) and low ferritin (lf, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of 4265 donors contributed 6219 donations. donors were evenly split by gender, 66% were ft donors, and 87% were 16-18yo. ft and rpt 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo. odds for lf were 4 to 6 times greater in the younger donors, and for ais were 3-to 4fold higher. preliminary statistical models indicate 16yo donors may have greater risk for lf than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector. testing was performed on successful 16-18 y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < 20 ng/ml in females (f) and < 30 ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations (12 months for f and 6 months for m) and counseled to take 18-28 mg of elemental iron daily for 60 days. for m and f, a ferritin < 12 ng/ml indicated absent iron stores (ais) and < 26 ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! 20 ng/ml in f and ! 30 ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior 24 months. an appreciable number of donors with no rbc donations in the prior 24 months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* 1 , joan williams 1 , michelle humphries 1 , nancy haubert 1 , ben reynolds 1 , michael phillips 1 , randall spizman 1 , ralph r vassallo 2 , hany kamel 2 , sally caglioti 1 , german leparc 1,3 and phillip c williamson 1 . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. 1 new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. 2 study design/methods: over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the beckman coulter au680 instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. 3 results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016. study design/method: abnormal risk factor levels were defined as hba1c ! 5.7%, sbp/dbp ! 120/80 mm hg and tc !170mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females). of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as !13.5 g/dl for men and !12.5 g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from 52 blood services worldwide and complete data were available for 25 blood services. deferral percentages for low hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with 53% lower hb deferral rates in men (95% confidence interval [ci] 11% to 75%) and 61% lower rates in women (95%ci 15% to 82%). iron supplementation was associated to 57% lower hb deferral rates among women (95%ci 22% to 76%) but there was no evidence of such an effect among men (p50.680). each one-week increase in minimum donation intervals resulted in 8% lower hb deferral rates among women (95%ci 1% to 14%) but not among men (p50.454). at the 5% level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: 9.15% of all candidates for wb donation were deferred in continental france in 2015. deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from 20 to 30 weeks. analysis (table) identified 3 main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the 5 previous years. conclusion: the 3 main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, 2 ng/kg). blood was sampled every 2 hours up to 8 hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from 1.4e108 (iqr 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (iqr 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: 200 cases of taco and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from may 2015 until july 2016. taco incidence was 1 case per 100 patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march 2016 fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from 5 to 7 days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july 2008 and to routinely extend ap outdate to day 7 since february 2016. this study reports a 103 month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july 2008-january 2016, ap underwent rt on day 4. day 6 and 7 units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day 8 had a second rt performed. from february 2016-january 2017, ap underwent rt on day 5 with routine outdate extension to 7 days by performing a second rt on day 6 and a third rt on day 7, as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type 1) or repeat rt positive with negative confirmatory culture (type 2). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july 2008, 20,010 ap were entered into inventory. of these, 11,840 (59%) were transfused prior to rt testing. the remaining 8170 (41%) underwent rt on day 4 or day 5. of these 43 (0.5%) were rt positive (29 type 1 fp, returned to inventory; 14 type 2 fp, discarded), leaving a total available inventory of 8156 units tested by rt. of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate. a total of 1561 (8% of original inventory) were transfused on day 6 or day 7. of these, 768 underwent a second rt on day 6 (2 rt positives; 1 fp type one and 1 fp type 2) and 233 underwent a third rt on day 7 (no positive results). a total of 964 (5% of original inventory) outdated on day 7. of these, 754 underwent a second rt on day 8 (no positive results). conclusion: to date we have performed 9925 rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted 13% to only 5%. a total of 1522 ap have been tested twice by rt (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed fp by repeat testing or culture. a total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every 24 hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with 8 to 10 million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in 2007. contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/ml. each parasite concentration in wb was tested x2. an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at 48c for up to 42 days; platelets were stored at 228c (rt) under agitation for 5 days and plasma was frozen at -208c. aliquots for culture were removed weekly from rbcs, daily from platelets and after 30 days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at 278c for detection of live parasites for up to 16 weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at 48c, rbcs from all units spiked with 10,000 parasites/ml were positive for up to 21 days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with 1000 parasites/ml were positive for up to 7 days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with 10,000 and 1000 parasites/ml were positive up to 5 days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at 48c for up to 3 weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* 1 , marion lanteri 2 and larry corash 1 . 1 cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom (2006 -2015 ), french (2006 -2015 , swiss (2011 -2015 ), and belgium(2009 -2015 hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately 1.35 million dlvbc-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/uva-treated pc were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored >2.83 million conventional, non-dlvbc-screened pc and recorded 58 str and 9 fatalities. concurrently, zero definite and 2 possible str were reported with 607,871 amotosalen/uva-treated pc, significantly fewer than with conventional pc (table 1 ) (20.5 str per million vs. 0.0 per million, p<0.001). one definite, 1 possible, 7 undetermined/indeterminate non-fatal str and 5 contaminated "near miss" pc were reported with 1.35 million dlvbc-screened pc between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 str per million vs. 16.3 per million, p <0.05). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october 2016 for all platelets received at our institution. at time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, 10 ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at 35c for three days. results/finding: a total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in october 2016 and march 2017 respectively). over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us16.83 per product tested. the cost per averted case was $us79,707. conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january 2009 and december 2016. the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were bcptr (0.55% of transfusion reactions). of the 18 bcptr, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma. recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51). an organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%). blood pressure (bp) decreased in 22%, increased in 17%; 61% of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie 1 , jenna lebedev 1 , linda kapp 1 , xiaohong wang 1 , meghan delaney 2 , lay see er 1 and james c zimring* 3 . 1 bloodworksnw research institute, 2 bloodworks nw, 3 university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg1-igg4), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least 29 natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all 29 known variants. study design/methods: the heavy and light chain variable regions of an anti-k1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k11 rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg2, igg3, and igg4 had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg1 or for any of the monoclonal ahgs tested. monoclonal anti-igg3 had a blindspot for igg3-04, due to the shorter hinge region on igg3-04. no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* 1 , yves dominique pastore 1 and maryse st-louis 2 . 1 chu sainte-justine, 2 hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since 2008, our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of 2014, 205 scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-c, anti-e (2), anti-hrb, anti-fya, anti-jka, anti-jkb (2), anti-s, anti-m, anti-sc2, anti-leb (2). seventeen patients (8.3%) were either d2 or partial d. rhce results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. as for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed. fy(a2b2) phenotype was found in 182 (89%) patients. a total of 2606 genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos 1 , emilia sippert 1 , mayra dorigan de macedo 1 , sheila fatima perecin menegati 1 and lilian castilho* 1,2 . 1 hemocentro unicamp, 2 university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa-308a, il1b-511t cytokine polymorphisms, rhag 808g>a and hla-drb1*15 alleles may predict a good responder phenotype (sippert et al, transfusion 2017) and that rhag 808a and hla-drb*15 alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included 96 non-alloimmunized patients with scd, homozygous for hbs, receiving a range of 5-289 rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa-308g>a, il1b-511c>t) and the rhag 808g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for rh variant alleles. from those, 6 had rhag 808a and/or hla-drb*15 alleles and at least one cytokine polymorphism (tnfa-308a or ilb1-511t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other 15 patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and 2 were confirmed by sequencing. the third sample was found to be rhce*cevs.01,rhce*cebi on sequencing (predicted phenotype v1,vs1). the 3 samples were typed as v1 (or ce s ) and vs1 (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[712g]in 2 samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c1 by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising 80% of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies (20% of patients) bound young and old rbcs with no apparent prejudice. band-3 is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from 22 patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band-3 tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing 5 type i and 5 type ii patients, we found that type i is characterized by 5 percollv r fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by 3-4 percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band-3 suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* 1 , burak bahar 1 , jeanne hendrickson 2 , krystalyn e hudson 3 and christopher a tormey 1 . 1 yale-new haven hospital, 2 yale university, 3 background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam30 abstract algorithm, e value51x10 -6 , word size5 6, gap costs: existence59 exten-sion51). search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of 162 alloimmunized patients reviewed, 105 were culture-positive. of these, 76% of the anti-c/c group (13 of 17 patients) and 16% of the anti-k group (7 of 43 patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from 0 -11.1%. overall, 21.9% (23 of 105 patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of >80% sequence identity. while 27.6% (29 of 105) patients reviewed had positive cultures for klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed 109 tegs performed on 76 patients undergoing cv surgeries at our institution from jan 1 to dec 31, 2016. no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the 56 tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (r>10 min, k>3 min, a<53 degrees, ma<50 mm) and "hypercoagulable" (r<5 min, k<1 min, a>72 degrees, ma>70 mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of 56 tegs analyzed, 37 patients (66%) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients (8% vs. 32%, p50.02). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused (32% vs. 11%, p50.07). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused (100% versus 33%, p50.06). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, 670 olts were performed. a total of 345 patients was submitted to cs. the median age was 51 years (range 10-78 yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in 31,6% of the patients. the average meld score was 29,6 6 9,4 and it was slightly higher in the cs group (31,3 vs 27,9, p<0,001) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml. allogeneic blood transfusion was required in 71,8% patients in the cs group, compared to 46,7% patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group (2,4 units vs 3,39 units, p<0,001 background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july 2015-december 2016 was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp (27 and 15 products, respectively) however, obp wastage occurred more frequently in the 18 month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products). this is skewed by one month when 20 products were wasted due to expiration of product on the floor. cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p50.176). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with 5 units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d1) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d(table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* 1 , edward smith 2 , thomas brown 2 , foeks jeremy 2 , metcalf suzanne 2 , james johnson 2 , peter davis 2 , karafa sw badjie 1 and abba zubair 1 . 1 department of laboratory medicine and pathology, transfusion medicine, mayo clinic, 2 department of anesthesia, mayo clinic background/case studies: our institution performs an average of 398 solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a 29 y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused 2 o(1) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused 10 more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the 2 o(1) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in 1% of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o. female patient should not have received o(1) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o(1) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a 50 y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o(1) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic pfd surgeries range from 0.2-1.6% and 0.3-1.4%, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may 2015 -may 2016 in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p<0.05. results/finding: we identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative t&s ordered. two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine (90.8%) of the 65 patients required a second abo/rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table 1) . no abo/rh discrepancies were identified. one patient received 1 unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n527) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs-1000i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units. this was performed twice for a total of 10 units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%). in units between 21 and 42 days (n510), the estimated mean residual k1 was 1.89 meq (range 0.61 to 2.21). in the two mock mtp trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births. uc collections were divided into 3 segments to test 3 conditions. segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of 21 days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the 2 remaining tissue segments were soaked in (ab/am) saline solution for 1 hr and 24 hrs at 48c, respectively. tissue segments were frozen in cryo bags with a proprietary 10% dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process 3 or 4 times the total blood volume (bv) of the patient, up to a maximum of 25 liters (l) per procedure, to obtain peripheral blood cd341 stem cells. as a consequence, a patient often would need to spend 6 hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd341 pre-count and cd341 yield, normalized per liter of blood processed, was derived utilizing the patient's cd341 pre-count, the patient's weight in kilograms (kg), and the target cd341 dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd341 stem cells. the initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 l. the range was 4.9 l -21.6 l. the target dose was achieved in all patients. our previous practice for these 8 patients would have required, assuming a standard 4 bv procedure, processing an average of up to 28 l per patient, with a range of 20-62 l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd341 yield. the result was a high correlation between these two ratios (r 2 5 0.92), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r 2 5 0.92, confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd341 mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june 2015 and march 2016. patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and primary amyloidosis (n51) and were mobilized according to standard protocols. collection cd341 cellularity target was established ! 2x10e6/kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd341 count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v4.0. results/finding: the media (range) general collection parameters were: cd341 (day 5) 27.50/ml (4.5-157.5/ml), blood volume processed 23204ml (9718-39618ml) and 4.96 (2-7.30) exchanged volemias. seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p50.071]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1]. two patients could not undergo hematopoietic stem cell transplantation due low cd341 cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd34) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with 20% fetal bovine serum. bmsc and amsc at passage 2-5 were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par4) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within 30min and 2hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: 334 6 35 seconds, versus low, medium and high doses of amsc (145 6 2, 111 6 6, and 75 6 12 seconds), and bmsc (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (cft, p<0.05) and increased alpha angle (p<0.05) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par4. no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* 1 , neil bagamasbad 2 , reynold dilag 2 , melissa nasser 2 , nicole bauer 2 , jennifer wheeler 3 and mary berg 1 . 1 department of pathology, university of colorado -anschutz medical campus, 2 department of medicine, division of hematology, university of colorado hospital, 3 scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from 120 collection procedures using the mnc protocol and 173 collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd34)-positive (cd341) throughput, cd341 collection efficiency (ce%), platelet loss 71a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included 14 and 22 allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd341 throughput was significantly higher in the cmnc group than the mnc group. the cd341 ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded 20 ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient #1, originally typed as an a2, had 1 bone marrow donor and 2 cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient #1 is now typing as type o. patient #2 was originally typed as a2 and received a bone marrow transplant from a type b donor. patient #2 is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient #1 and patient #2 indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with 20% fetal bovine serum under either normoxia (20% o 2 ) or hypoxia (3.5% o 2 ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd90/cd29 and cd45 were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc (1.5 3 10 5 /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by 15%, but depressed the growth of amsc by 30% at day 5 in comparison to normoxia. both bmsc and amsc equally expressed cd90 and cd29 but not cd45 under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: 468 6 64 (control), versus 170 6 13 (bmsc), and 195 6 60 (amsc) seconds) by natem. hypoxia also significantly shortened ct (165 6 20 (bmsc), 169 6 50 (amsc) seconds, p<0.05 as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd341 cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd341 cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd341 cells. study design/method: cryopreserved cd341 cells from 2 healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef1-alpha-yfp lentivirus (2.5% concentration) and media (x-vivo-10, human serum albumin(hsa), 100 ng/ml each of cytokines (scf, tpo and flt3-l) over 2 days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of 5% dmso, 6% pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd34 1 cd38 -cd45ra -cd90 1 cd49f 1 cells) phenotyping and cfu assays were done following first thaw (pt1), post-transduction (ptxn) and second cryopreservation-thaw (pt2). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd34%, cfus were similar before and after pt2. hscs ranged from 824 to 1655 cells/10 6 cd341 cells in the pt2-tr arm compared to a range of 286 to 1416 /10 6 cd341 cells after pt1. viability, % cd341 and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt2 (table) . conclusion: dec of mpb human cd341 cells decreases tnc recovery, but has minimal effects on cd341 cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd34% in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd341 cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% dmso at final concentration. pbsc were cryopreserved by direct immersion on -808c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /l and platelet count >20 x10 9 /l after 07 days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd341 was below 10 x 10 6 cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd341 on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd341 on the day of the collection versus collected cd341 per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd341 was calculated. final laboratory count of cd341 per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss 23 software. results/findings: among patients collecting hpc for autologous transplantation, 69,23% needed only one day of hpc harvesting, while 25,64% needed two days and 5,13% needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was 49 1-2,91%. after comparing predicted values with cd341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% cd90, cd105, cd73 and 2% cd14, cd19, cd34, cd45, hla-dr) study design/method: umbilical cord tissue (n510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline. fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence. all cells were tested on an msc flow panel at passage 2 just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of 8 days (fresh 5 7.8, frozen 5 8.1), and 13 days (total) for the msc's to reach passage 1 (fresh 5 12.6, frozen 13.4). all cells were ready for flow analysis in approximately 3 weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all). flow cytometry showed average !95% for positive markers and 2% negative markers. there was no statistical difference between fresh and frozen flow result (p > 0.05). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with 10% concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a 378 c water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-aad marker through flow cytometric analysis. results/finding: ucb storage period was 7.24 years (mean) and cell recovery was 86.31% (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11). post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for 5 years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln 2 vapor in a dmso-based cryoprotectant for 5yrs. (5.49 6 0.431; n54). units were rapidly thawed and rinsed in dpbs, then 25 pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a 5x5 grid pattern in msc-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged. cells were isolated on the 14 th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate. cells were positive for the msc markers cd73, cd90, and cd105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers cd34/45 (1.1 6 0.7%). passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical 75a transfusion 2017 vol. 57 supplement s3 research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd34 yield prediction algorithm ines bojanic* 1 , nelly besson 2 , ivana vidovic 1 and branka golubic cepulic 1 . 1 department of transfusion medicine and transplantation biology, university hospital centre zagreb, 2 terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd341cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version 11) in adult and pediatric lvl. a prediction algorithm for cd341cell yield was also tested. study design/method: we evaluated retrospectively 67 lvl performed in 46 adult patients, and 14 lvl in 11 pediatric patients treated in uhc zagreb from march 2016 till september 2016. mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio 1:24). in patients weighting 25kg (n59), a rbc prime was performed. cd34, lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd341cell count and cd341cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd34 values to real cd34 yield. results are presented as median (iqr). results/finding: in both groups, cd34, ly and mo ces were high. target cd34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd341cells and cd341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting cd34 yield could be predicted based on precd341cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd34 yield and observed cd34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)l of blood in 20 adult procedures, and 5.9(3.5-7.8)l in 7 pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd34, ly and mo ce1 were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* 1 , aniko barta 2 , arpad batai 2 , zoltan csukly 2 , zita farkas 2 , laszlo gopcsa 2 , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg. clinical response was assessed 28 days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all 12 patients had received 13 cycles of msctreatment (4 dose per cycle). the median age was 47 years old (19-56) with a male/female ratio of 1:2. distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; t-cell lymphoma: 1. nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median 63rd day (7-455). the involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases. the median time of msc's first infusion was 274 days after the stem cell transplantation (hsct) and 165 (19-1974) days after the first episode of gvhd. 4 of the 13 cycles of msc-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with 83% overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is 2 million cd341 cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd341 cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal. the ideal minimum cd341 cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from 55 patients to evaluate the predictive value of the cd341 cells/ml level. data was collected over 6 months from every patient who underwent a stem cell collection. four patients were allogenic donors and 51 were autologous donors. the patients' weight, diagnosis, and pre-procedure cd341 cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd341 cells collected were recorded. the collection efficiency and the cd341 cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd341 cells/ml and post-procedure cd341 cells/kg (r50.95). any patient who had a pre-procedure cd341 cells/ml count of 29 or greater had a collection of at least 2 million cells/kg. any patient who had a pre-procedure cd341 cells/ml count of 16 or less collected less than 2 conclusion: the pre-procedure cd341 cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd341 cells/kg level. to confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure cd341 cells/ml count of at least 29 should be obtained. for any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 cd341 cells/ml should be conducted. heidi elmoazzen 1 , antonio giulivi 1 , michael halpenny* 1 , lisa martin 1 , donna perron 1 , chris bredeson 2 , lin yang 1 , locksley mcgann 1 , paul birch 1 and jason p. acker 1 . 1 canadian blood services, 2 ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso (5% final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd34, viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in 5% dmso and 1.7% hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of 12.6 days for anc500 with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current 5% dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using 5% dmso and 1.7% hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from 5% to 40%. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost. in 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg. this retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses. study design/method: patients weighing >100 kg with crcl >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd341/cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd34 in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %cd34 level than any of the other groups, reaching statistical significance when comparing the %cd34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs). hispanic donors show statistically similar %cd34 pre-apheresis levels over all age groups. moreover, the hispanic older age group (>540yrs) had a statistically higher %cd34 pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of 121 sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd34 level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd34 level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc (234 x 10 6 cell / ml) and three low wbc (114 x 10 6 cells / ml) concentrations, each at high (505 ml), low (265 ml) and median (355 ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax 2 (pericell protocol, cs.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and 7-aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting 7-aad viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the 6 mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-aad viability of 2 6 2 [0, 11] % from the input product. the method was found to have a cv of 2.0%. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating 78a transfusion clinical assessment. in the first phase, 2 cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd34 7-aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw. the second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than 20% within 1 hour, while cd341 cell viability remained stable up to 3 hours post thaw. small aggregates appeared after 1 hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075). platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207). infusion rates were slightly higher for the pump group. for control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the 278 zikv ineligible cbus was: caucasian 52%, asian 9%, black/aa 20%, and multi-race 21%. racial distribution of all clinical cbu donors was caucasian 49%, asian 15%, black/aa 20%, and multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: 78% of all ineligible cbus and 21% of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring 2,3-dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a 10 ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n 5 20), which were stored in sagm for 22 days, to act as untreated controls. the remainder of each unit ($270 ml) underwent treatment with the rejuvenation solution (50ml, 60minutes at 37 o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p 1 , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of 22 day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp 215 (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros 60. pc were centrifuged at 1250g in a sorvall rc3c1 centrifuge (sorvall, usa) for 10 min. the combination cryoprotectant dmso1dextran (cryosure dex40, germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at 37 degrees c (barkey plasmatherm) for 10 min. cpc osmolality was measured with an osmomat 030 osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru 169287 u1). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso1dextran (cryosure dex40) , as a cryoprotectant, to obtain a final concentration of 5% dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m3 were instrumental in automating that phase. pc to be frozen had an osmolality of no less than 1500 mosm/l. prp and ppp were frozen at a cooling rate of 1-38c/min and stored at -85 0 in the chest freezer for up to 24 months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru 167874 u1). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to 380 mosm/l. freeze-thaw recovery of platelets was 80% or more of the original population. defrosted pc were stored at 20-24 0 with continuous gentle stirring from a helmer platelet agitator for no longer than 4 hours before transfusion. it took no more than 30 min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over 20 min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day 4 and day 5 evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day 4 for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day 5. a total of 60 lrap units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5. the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were 59 true negatives (tn) and 1 false positive (fp) on day 1 when tested by bact/alert, with 60 tns on day 4. bactx testing results showed 50 tns on day 4 and 10 tns on day 5. testing using the pgd kit showed 50 tns on day 4; and 8 tns and 2 fps on day 5. fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and 30 minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within 20 minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in 100% plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for 1 year jos lorinser 1 , pieter f van der meer 1 , hans van der heiden 2 and dirk de korte* 1 . 1 department of product and process development, sanquin blood bank, 2 mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers (140 ml) is unknown. if these products can be stored at -188c it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188c or <-25 to -358c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from 500 ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <-258c for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 ll) containers, which were refrozen and stored at either -188c or <-258c. during storage at 3 months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <-808c. growth factors tested were pdgf-aa&ab/bb, tgf-ß1/2/3, vegf, 80a transfusion 2017 vol. 57 supplement s3 egf, fgf2. the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß1 were the most abundant growth factors, on average 35, resp. 40 ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average 11 ng/ml. tgf-ß2, egf and vegf were detected at relatively low values, resp. 3 ng/ml, 0.5 ng/ml and 0.3 ng/ml. average levels of fgf2 and tgf-ß3 were close to detection limit (< 0.2 ng/ml). the controls stored at <-808c showed for all growth factors close to 100% of the initial values in samples at t50 (moment of filling mini containers). for serum stored at <-258c for up to 12 months, most factors showed less than 2 % decrease, except for pdgf-aa and tgf-ß2, showing 6% resp. 3% lower values. for serum stored at -188c the values for tgf-ß1, egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß2 showed a decrease of resp. 9, 17 and 3%. conclusion: human serum eye drops can be stored in the new micro dose device at -188c (3-star household freezers) or <-258c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at -188c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year. ruqayyah almizraq* 1 , heather inglis 2 , phillip norris 2,3 , jennifer a muszynski 4 , nicole juffermans 5 , jelena holovati 1 and jason p. acker 1,6 . 1 university of alberta, 2 blood systems research institute, 3 university of california, san francisco, 4 nationwide children's hospital, 5 academic medical center, 6 canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n58 per method). residual platelets and white blood cells (wbcs) were measured on day 5 using flow cytometer (fc). on storage day 5 and 42, number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day 5, apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p<0.01, wbd p<0.05) and wbf (vs: apheresis p<0.0001, wbd p<0.01) methods. while rcf units yielded the lowest count of platelet-evs (cd41a1) on day 5 and 42, the highest number of platelet-evs were in apheresis (day 5) and in wbd (day 42). similarly, there was significant difference among methods in the number of wbc-evs (cd31, cd141, cd161, cd191, cd66b1) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day 42 vs day 5 in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< 200 nm) was greater than large evs (! 200 nm) in all of the products on day 5 and 42, and the highest level of evs < 200 nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p<0.05). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at 48 centigrade maryanne c herzig* 1 , crystal lafleur 2 , chriselda g fedyk 1 , sherrill j. slichter 3 and andrew p cap 2 . 1 us army institute of surgical research, 2 u.s. army institute of surgical research, 3 university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at 48c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a2 (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection. units were stored for 12 days without agitation. units stored for 10, 15 or 22 days were agitated during storage with a model 400 hybridization incubator at 48c set for end over end rotation at 2-3 rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at -808c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd40l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai-1) as another fibrinolytic measure; and complement activation markers c3a, c4d, c5a and c5b-9. data was analyzed by one way repeated measure anova. results/finding: only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation. these levels did not meet fda requirements of 5.5 x 10 10 platelets per wb unit. subsequently, wb was agitated and platelet recovery was 71-76%. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t0 (day of collection) and t10, 12, 15, or 22 measurements. significant elevations of pai-1 and scd40l indicate activation of platelets and inhibition of fibrinolysis (p<0.001). activated complement peptides c3a, c5a, and c4d were all elevated over time (p<0.001) while sc5d-9 was not. however, only c3a and c4d levels at t22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at 48c for 10-22 days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc5d-9 reported, wb showed elevation of c3a, 5a and c4d and not sc5d-9. complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* 1 and christian todd 2 . 1 cerus corporation, 2 community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 rcf 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 wbd 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350ml, 6.6x10 11 in 400ml, 6.8x10 11 in 400ml, and 7.0x10 11 in 400ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: 64% of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was 1.34. conclusion: it is possible to treat 64% of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* 1 , andrey skripchenko 1 , fei xu 1 , ying li 1 , stephen j wagner 2 , pamela h whitley 3 and jaroslav g vostal 1 . 1 fda/cber/ obrr/dbcd/lch, 2 american red cross holland laboratory, 3 american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature (4-6 o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts (11 hrs ct: 1 hr 37 o c [tc]). autologous apheresis plts stored for 7-days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled plts were also infused into mice (n590). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts <5% were considered background. results/finding: the mean recoveries of infused plts were 51.2 6 16.7% for rt, 37.7 6 12.3% for tc and 23.1 6 8.8% for ct in humans. in mice, mean recoveries of the same plts were 24.9 6 10.3% for rt, 19.1 6 9.8% for ct and 16.2 6 6.9 for ct (mean6sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $74% and ct was $45% of rt. in mice tc was $76% and ct was $64% of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to 100% for rt plts. human tc plts had 26% auc while ct plts had 11% auc compared to rt plts in humans. in comparison, the same tc plts had 39% auc and ct plts had 26% auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are 2.4 and 1.5, respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* 1 and geeta paranjape 1,2 . 1 coastal bend blood center, 2 carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g5 with the compomaster net software for data management. implementation was planned for a november 2014 go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g5s and compomaster in june 2014. training and validations were successfully completed and a full launch occurred mid-march 2015. device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december 2014. validation was completed and signed off in march of 2015. manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g5 system. data points were collected from 210 units bi and 302 units ai. results/finding: upon initial implementation, staff training and use, the compomat g5 was found to be easy. plt weight spread was reduced from an average of 22gm to an average of 15 gm. actual plt weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit. plt count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by 31.8% after implementation of the compomat g5 and our plt concentrations increased on average by 5%. we were able to consistently produce a smaller volume plt (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell 1 , angela hill 1 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: 13 abo/rh matched lr sagm rccs were pooled and split to produce 6 large (354 ml) and 6 small (244 ml) rccs. the rccs were stored to 14 d and glycerolized manually by mixing 400 ml of glycerol with the rcc in a 2000 ml freezing bag. units were frozen at -658c for ! 72 h before being removed from frozen storage and thawed in a 378c water bath. 3 large rccs and 3 small rccs were deglycerolized using the organization's current procedure on the cobe 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose. the remaining rccs were transferred into a 1l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 ml centrifuge bowl on the acp-215 with re-suspension in as-3. rbc quality was tested at 24 6 2 h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p50.006, acp215: p50.007) and lower cell recovery (cobe: p50.002, acp215: p<0.001) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume rccs. large cobe 2991 rccs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large acp-215 rccs. however, all cobe 2991 rccs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did acp-215 rccs (0.31 6 0.02 %). cobe 2991 rccs failed to meet regulatory hemolysis standards of 0.8%. conclusion: addition of a 400 ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp-215 cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using 0.9% saline and centrifugation and the semi-automated washing method (sam) using the cobe 2991blood cell processor. study design/method: in this study, 20 units of single donor platelets were evaluated (10 washed using the mm and 10 washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas 6000. results/finding: table 1 shows that the average platelet recovery for the sam (92%) was significantly higher compared to the mm (82%). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took 10-15 minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 ml of 47 to 68% plasma and 32-53% pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 ml. study design/methods: apheresis pcs (amicus v r ) were collected in 35% plasma and 65% pas-3. one study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 ml) in 65% pas/35% plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 ml) using either single or pooled donations. input pcs (n520) were treated with the intercept ts set by the end of day 1 post collection; the incubation time in the compound adsorption device (cad) container ranged from 4 to 16 hours and the intercept treated pcs were stored in 3 containers (n560). day 5 and 7 post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas-3 treated in the intercept ts set demonstrated acceptable in vitro function (table 1 ). all intercept treated pcs had ph(228c) !6.2. platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing 100% plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n56). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n56). numerous in-vitro quality markers (plt concentration, atp, po2, pco2, ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days 1, 3, 5 and 7 for apheresis pcs, and on days 2, 3, 5 and 7 for wb-derived pcs. two flow cytometry assays were used to evaluate cd62p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion 2017 vol. 57 supplement s3 results/finding: platelet recovery was 92 6 5% and 81 6 10% for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells (3% 6 1 (test), vs. 1.7% 6 0.5 (ctl) on day 5) and a higher rate of cd62p expression than control pc units (58% 6 7 (test), vs. 23% 6 6 (ctl)) on day 5). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* 1 , lorraine blagg 2 , christi e marshall 1 , herman woodson 1 , sean erony 1 , krishna patel 1 and eric gehrie 3 . 1 the johns hopkins hospital, 2 johns hopkins hospital transfusion medicine dept, 3 johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. 340 intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day 4. as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day 3. the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of 340 lrap were tested. 335 lraps initially tested negative by bactx, while 5 lraps initially tested positive by bactx. all 5 initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was 98.5%. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only 340 platelet units. the expected rate of bacterial contamination of platelets is less than 1 per 2000 units. the 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as-1 and cp2d/as-3 rbc alan d. gray* 1 , matt landrigan 2 , pamela whitley 3 , michael wellington 3 , sherrie sawyer 3 , shalene hanley 3 , emily rondeau 4 , louise herschel 4 , neeta rugg 5 , patricia a.r. brunker 3 , shawnagay nestheide 5 , jose cancelas-perez 5 , larry dumont 6 and zbigniew m. szczepiorkowski 7 . and 2,3-dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for >24 hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood (530-550 ml) was collected and processed at 3 sites into leukocyte-reduced rbc (a total of n563 cpd/ as-1 and n564 cp2d/as-3). 50 ml of rejuvenation solution (citra labs) was added to each rbc on day 35 (d-35), incubated for 60 minutes with agitation at 378c water bath (helmer dh4), washed (haemonetics acp215), and stored in as-3 at 1-6 oc for 7 days (d-36 through d-42). in vitro recovery (%) was calculated and hemolysis, atp, and 2,3-dpg were determined on day 0, d-35, d-35 after rejuvenation and washing (postrjv), d-36, d-38, d-40, and d-42. all units were cultured on d-35 postrjv and on d-42, and then concentrated by centrifugation on d-42. results/finding: in vitro rbc recoveries were 95.7% and 95.5% (as-1 and as-3, respectively) and no bacterial growth was observed. hemolysis on d-42 was maintained <1% in 58/63 (92%) as-1 units and 63/64 (98.4%) as-3 units. all as-1 and as-3 units (100%) had hemolysis <1% following concentration by centrifugation. morphology score was reduced to 77% (as-1) and 74% (as-3) by d-35, restored after rejuvenation (91%, 92%, respectively) and maintained through d-42 (>90%). atp was restored and maintained above fresh levels after rejuvenation. 2,3-dpg was restored above fresh levels and was maintained !80% of fresh levels through d-38. all values were significantly different compared to d-35 except as noted (p<0.001, paired ttest) ( table 1) . conclusion: rejuvenation of stored rbc restores atp and 2,3-dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d-42 when compared to nonrejuvenated rbc on d-35. this study is funded by zimmer biomet. storage >24 hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante 1 , jason p. acker* 1,2 and jelena holovati 1 . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during 42-day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and 2,3-dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome 1 rejuvesol-treated (l1r). the prbcs were incubated for 1 h at 378c with hepes-nacl (sham), liposomes (dopc:chol, 7:3 mol%, 2 mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and 2,3-dpg at day 42 hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): l (0.53 6 0.01%, p50.042), r (0.43 6 0.02%, p50.004), l1r (0.48 6 0.06%, p50.020). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r (0.55 6 0.01, p50.010) and l1r (0.55 6 0.01, p50.010) treatments compared to s (0.53 6 0.01) but not l (0.53 6 0.01, p50.936). rbc rigidity (kei) increased in all treatments compared to sham (1.19 6 0.07): l (1.28 6 0.06, p50.025), r (1.44 6 0.17, p50.010) and r1l (1.44 6 0.06, p50.004). aggregation amplitude was significantly increased by r treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004). atp levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g hb): l (2.00 6 0.21 mmol/g hb, p50.010), r (4.70 6 1.20 mmol/g hb, p50.004), l1r (5.00 6 1.56 mmol/g hb, p50.004). the levels of 2,3-dpg were no longer detectable in s and l treatments at day 42. the combined treatment was comparable to r (2.38 6 3.26 mmol/g hb vs. 2.62 6 2.20 mmol/g hb, p50.868). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l1r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer 1 , eric ducas 1 , patricia landry 1 , nathalie dussault 1 , jacques bernier 1 , danny brouard* 1 and anne maltais 2 . 1 h ema-qu ebec, 2 institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < 108c) of one to six 500-ml whole blood units (wbu) within 8h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between 18c and 108c for 24h under extreme external conditions (-308c to 408c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for 24h at 20-248c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, 500-ml wb bags were filled with 555 ml saline 0.9% at t 5 308c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (-308c) and summer (408c) conditions. shipping boxes were filled with either one or six bags (n5 2). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under 108c in 4.55 6 0.62h and maintain their internal temperature between 18c and 108c for 24h with final values ranging between 6.38c and 9.38c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the 108c threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than -138c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* 1 , christopher c. c silliman 2 , beth shaz 1 , marguerite kelher 2 and claudia s. cohn 3 . 1 new york blood center, 2 bonfils blood center, 3 department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either 100% donor plasma (n550) or 65% pas-3 / 35% donor plasma (n550). within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd40 ligand (scd40l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c (3/50 products) compared to plasma platelets (2/50 products); however, the hla-antibody screen-positive supernatants of pas-86a transfusion 2017 vol. 57 supplement s3 abstract c platelets had fewer hla specificities (2 specificities) compared to those of the plasma platelets (18 specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd40l levels were increased in the supernatant of pas-c compared to plasma platelets (table 1) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd40l and not bioactive lipids. although scd40l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd40l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below 5x10 6 wbc in us and 1x10 6 wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n52) and leukoreduced (lr) rbc units (n52) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of 1000 wbc/ul. the spiked samples of 5, 12.5, 5, 25, 50 and 100 wbc/ul were prepared from the source sample of 1000 wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations (0, 12.5, 5, 25, 50, 100 wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 wbc/ul). 10 tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of 20 samples (plt and rbc) were run on both analyzers, repeated for 5 days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt50.996, slope50.972), (r-rbc50.999, slope50.992). the %diff-plt at 5, 25, 50 wbc/ul were 7.8, 4.7 and 10, respectively. the %diff-rbc at 5, 25, 50 wbc/ul were 10.8, 3.2 and 14.7, respectively. the average total testing time was similar on both instruments; 89 min for the facsvia and 92 min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated 62% (56 of 89 min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of 56 minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* 1 and mickey koh 2 . 1 department of laboratory medicine and pathology, university of minnesota, 2 st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of 62 completed and partially completed surveys were received. results/findings: responses came from 13 countries, but the majority of responses came from the united states (us). of the respondents, 35% reported aprp use in their hospital. aprp was used predominantly for outpatients, though >40% of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by 1-5 mds; however, 3 hospitals had >10 mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures (97%); however, 3 respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the 3 hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, 2017) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n56) under blood bank conditions at day 3 (d3), at day 42 (d42), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from 2.1-8.8% at d3 to 8.3-68.1% at d42. rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell 1 , angela hill 1 , tracey turner 2 , april xu 2 , brandie dennis 2 and jason p. acker* 2,3 . 1 canadian blood services, centre for innovation, 2 canadian blood services, 3 university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units (314 6 15) than in rcf units (275 6 16), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: 12 small rcf (252-263 ml) and 12 large wbf (322-353 ml) rccs were stored for 21 d before being glycerolized and frozen at -658c for !72 h. large rccs whose red cell mass exceeded the capacity of the 275 ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a 378c water bath, deglycerolized and re-suspended in as-3. rccs were stored 14 d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant k 1 and na 1 concentration, deformability (ei max ), and higher (p<0.001) recovery than did large wbf units. no significant differences in hemolysis, atp, 2,3-dpg, p50, rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table 1) , however 8 of 12 large wbf units had rbc recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small rcf units had hemoglobin values < 35 g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. 0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 elongation index (30pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 this study is funded by zimmer biomet. (hasan 1994) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo 2 ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for 42 days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two (52) rbc units (leukocyte-reduced), cpd/as-1 or cp2d/as-3, on day 0, day 42, and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o 2 /g hb) and total releasable oxygen (tro) of the unit (ml o 2 ). orc was determined by assessing the change in % o 2 saturation from 100 mm hg po 2 (e.g., lung) to 40 mm hg po 2 (e.g., venous blood) multiplied by 1.34 ml o 2 /g hb (li 2016). a simulated baseline pretransfusion vo 2 of 115 ml o 2 /min was estimated using the day 0 orc and assuming a 7 g/dl transfusion trigger with a cardiac output of 5 l/min and 5 l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day 42 restored orc and tro to levels greater than day 0 ( table 1) . orc of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than rbc on day 0 and day 42, respectively (p<0.001). vo 2 increased after a simulated single unit transfusion of rbc (day 0, day 42, and pw) by 19.3%, 8.9%, and 28.8% over the pre transfusion vo 2 , respectively (p<0.001). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release 3.3 times the volume of o 2 compared to standard, untreated rbcs stored for 42 days. inferior oxygen delivery to tissues (vo 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (bennett-guerrero 2017). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* 1 , jay srinivasan 1 , gustaaf de ridder 2 , alan d. gray 3 , matt landrigan 4 , keaton charles stoner 5 , angela crabtree 6 , jessica poisson 7 and ian welsby 8 . 1 duke university school of medicine, 2 duke health pathology, 3 citra labs, a zimmer biomet company, 4 zimmer biomet, 5 duke university, 6 department of pathology, durham veterans affairs medical center, 7 duke university hospital, 8 duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted 2,3-diphosphoglycerate (2,3-dpg). the loss of 2,3-dpg increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation). decreased p50 may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and 2,3-dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at 378c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a1, leukoreduced prbc stored in as-1 were obtained from our local blood center. after 3 days of storage, units were divided into 4 separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution (50ml) was added to the cr group, and all groups were then stored for another 12 days at 1-68c. on day 15 of storage, the sr group was incubated for 1 hour at 378c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p50 was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps1) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a 5 0.05. results/finding: significant differences in p50 were noticed between all groups (table 1) . ei, ps1, and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p50 that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k 1 , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc (300ml) units stored in as-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 ml of multifunctional polymer bead, at a flow rate of 20 ml/min. supernatants were analyzed for k 1 removal as well as free hb, antibodies and cytokines (27-plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k 1 ] from 54.2 6 5.0 to 1.98 6 1.3 meq/l; equivalent to an 84.6% reduction. free hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/ml. antibodies, specifically igg, iga, and igm decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/ml (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/ml (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/ml (31.5%), respectively. inflammatory cytokines were significantly reduced, specifically: ip-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/ml (87.2%), mip-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/ml (80.7%), and pdgf from 1348.3 6 291.9 to 77.91 6 22 pg/ml (94.2%). filtration had no significant impact on cell surface markers of rbc viability (<0.1% decrease) or sensitivity to osmotic changes. values listed represent mean 6 sem (p < 0.01 for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k 1 as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k 1 along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency (1.5%) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia 2120 (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on 10000 permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and 2,3-dpg profiles to fresh levels. the objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for 42 days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was 97.2 6 2.2%. hemolysis (%) was similar on day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%). percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24hr (0.51 6 0.19%). average atp and 2,3-dpg were restored above the average fresh values. the morphology score decreased $25% by day 42, which was restored to near fresh values following rejuvenation and washing and storage 24hr (93.7% and 95.1%, respectively). rbc oxygen affinity, as assessed by p50, was restored above fresh values. all values were significantly different compared to day 42 (p<0.001, paired t-test) ( table 1) . conclusion: rbc morphology was restored to near fresh and average atp, 2,3-dpg, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and 2,3-dpg were maintained during storage 24hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d9tetrahydrocannabinol (thc) and 11-oh-d9-tetrahydrocannabinol (11-oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and 11-oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and 11-oh-thc to produce samples for lc-ms/ms analysis. lc used a c18 column. post-column detection by ms/ms used positive ion electrospray with q1:q3 ion pairs of m/z 5 605.3:225.3 (internal standard (is), d3-thc), m/ z 5 602.2:225.2 (thc), and m/z 5 618.3:256.1 (11-oh-thc). quantitative results for thc and 11-oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ ml for both thc and 11-oh-thc. limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/ml for thc and 7 ng/ml for 11-oh-thc. results/finding: a total of 424 donor plasma samples were tested for thc and 11-oh-thc. no samples tested positive for either thc or 11-oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than 1% among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of thc and/or 11-oh-thc in plasma. conclusion: testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* 1 , ruqayyah almizraq 2 , daniel millar 3 and jason p. acker 4 . 1 university of british columbia, 2 university of alberta, 3 lightintegra technology inc., 4 canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days 7, 21, and 42 of storage. one rcf rcc was tested on days 1, 5, 14, 21, and 43 and six 10 ml aliquots were stored in parallel and tested on days 14, 21, and 43. all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the 6 aliquots were consistent at each time point but statistically higher than in the original rcc on and after day 21 of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow 100% screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n 5 60) and paired ffp aliquots were stored for 31-33 days at 2-68c and -188c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! 80% levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c1 esterase and alpha 1-proteniase inhibitors). the level of factor xiii in odp was slightly lower, about 70% of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i1ii and ddimer) and complement (c3a and c5a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion 2017 vol. 57 supplement s3 abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to 62% in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to 15 minutes. in addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from 12-15 minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n56) were prepared from one 12-15 minutes bc and 60 ml of autologous plasma in a 600 ml pvc-dehp container. as a reference, spc from donations with collection times of <12 minutes were prepared (n55). in addition, pc were prepared from 5 bc, of which at least 4 bc were from 12-15 minutes donations (n55). after pooling of the bc, 300 ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for 8 days at 22 6 28c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean6 sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume (67 6 5 vs. 66 6 16 ml) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day 8, ph(378c): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day 6 and/or 8 in 2/5 pc (possibly because sometimes ab0 incompatibility was accepted). on day 8, plt showed low cd62p expression (17.1 6 1.8%) and phosphatidylserine exposure (annexin v binding, 8.9 6 1.9%). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage. to substantiate that the exclusion of 12-15 minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* 1 , jessie miller 1 , ranee marie wannarka-farlinger 1 , sandra bryant 1 , scott a hammel 1 , sherry stern 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as-3 rbcs, 20 irradiated and 21 non-irradiated, were selected for the study. the units varied in age, ranging from 2 to 42 days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< 0.05) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p50.02). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after 5 days of storage pei lun karen lim* 1 , erma sofia sumardi 1 , isamar eduardo ancheta 1 , susan lim 2 , christina yip 1 , lip kun tan 2 and shir ying lee 3 . 1 national university hospital singapore, 2 national university hospital, 3 national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within 8 hours of processing and stored at -188c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for 5 days and kept at 1 to 68c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than 50 iu/dl. study design/method: randomly selected units of ffp (n510) were measured for fviii concentration based on clotting assay (stav r -deficient 92a transfusion 2017 vol. 57 supplement s3 viii diagnostica stago). fviii levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at 30 to 378c for 35 minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration (0 hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at 1 to 68c for 5 days for subsequent testing. results/finding: results obtained were listed in table 1 . units 7 to 9 were not tested for fviii at post thaw-24 hour due to operational issues. the overall fviii concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour. after further storage of tp post thaw-24 hour and -72 hour, residual fviii level remain to be above 50 iu/dl except unit 10 which had a lower initial fviii concentration. at post thaw-120 hour, 7 out of 10 units tested had residual fviii activity within the pre-set standard of 50 iu/ dl. the average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least 50 iu/dl of fviii. typically patients with factor levels below 30 iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi 1 , ayman mohamad sabri 1 , ali abdullah alajeafi 1 , ashwaq hasan alhekri 1 , saleem bin mahfouz 1 , ali hasan alkhodari 1 , rawya saeed shealy 1 , marcus picard-maureau* 2 and hussain bana almalki 1 . 1 king abdulaziz hospital and oncology center, 2 cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) prp pc in 100% plasma over a 7 day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of 4 leucoreduced test pcs were assessed at day 7 of storage and compared to leucoreduced control pc at day 5 of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day 0; the process was completed by day 1 post-collection. samples were taken daily for quality analysis from test and control pc until day 5 and day 7, respectively. for bacterial spiking, additional pc were spiked with each receiving 4 ml of 1 mcfarland ($ 1.2x10 9 cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8. the average platelet loss in the control units at day 5 was 9.5% 61.4. the average ph of the test units at day 7 was 6.64 60.04 and in the same range as the control pc, ph 5 6.89 60.09. glucose concentration in test pc at day 7 (13.8 63.0 mmol/l) was lower than in the day 5 control units (18.32 61.06 mmol/l). lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* 1 , jay srinivasan 2 , jessica poisson 3 and ian welsby 4 . 1 duke university, 2 duke university school of medicine, 3 duke university hospital, 4 duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf (4.44 mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial (0á2 lm) filtration. cryoprecipitate mini-pools (400 6 20 ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di(2-ethylhexyl) phthalate (dehp)] adsorption device and a 0á2 lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table 1) . kit ensured bacterial sterility (table 3 ) and most importantly, final product was free of hbv, hcv and hiv (table 2) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane a 2 from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n518) were prepared from a nsaid-bc and 60 ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for 8 days at 22 6 28c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n55) were investigated as a reference. values are expressed as mean6sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume (69 6 4 vs. 66 6 16 ml) and plt content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups. on day 8, both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day 8 was significant higher in a subset of donors who had used ibuprofen (n55). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day 1 in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, 2014] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type 2 diabetes (t2d). because of the strong rise of people with t2d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t2d, but accepted as donor. study design/method: twelve whole blood donors with t2d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for 8 days at 22 6 28c and sampled on day 1, 4 or 5 and 8. the diabetic marker hba1c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 3 'good' (ph day8 >6.6) and 3 'poor' (ph day8 <6.3) storing spc were selected and analysed in more detail. results/finding: donors were of age 57 6 10 year and primarily men (75%). donors with t2d had a higher mean bmi (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher hba1c than controls. the spc of both groups had the same volume (70 6 5 vs 726 2 ml) and plt content (71 6 9 vs 73 6 11x10 9 ) but on day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mm, p<0.05). on day 8, the average in vitro quality was comparable in both groups (data not shown). when combining 94a transfusion 2017 vol. 57 supplement s3 the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc-1) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01). conclusion: bc from donors with t2d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t2d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of 30 minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of 2017 detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table 1). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to 60 minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* 1,2 , katharine a downes 1,2 , hollie m reeves 1,2 and robert w maitta 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts13 are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: 32 x 10 9 /l, platelets: 62 x 10 9 /l, bun: 77mg/dl, creatinine: 2.4mg/dl, lactate dehydrogenase 1940 u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to 44 x 10 9 /l, adamts13 sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation (74 x 10 9 /l and a-ipc of 4.7 x 10 9 /l). two consecutive tpe were completed which resulted in a platelet count decrease to 54 x 10 9 /l and a-ipc of 5.1 x 10 9 /l. a-ipc ratio was 1.1 below the ratio of 3 which has been reported for ttp patients. similarly a-ipc count was not below 5 x 10 9 /l threshold reported in setting of ttp with severe adamts13 deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o157:h7 toxin. testing of c3, c4, factor h, factor h autoantibody, factor i and factor b were normal. adamts13 activity was 93%. patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /l, with resolution of her renal failure: bun: 42 mg/dl, creatinine: 0.65 mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts13 testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts13 activity. many patients with severe autoantibody-mediated adamts13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts13 activity <5%) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value 2-3 days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column 1), increased peak a-ipc value (results shown as percent increase, column 2), delayed a-ipc peak, and delayed plt recovery (table 1) . moreover, recurrent episodes required more procedures compared to initial presentation (table 1) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february 2016 to march 2017 on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the 2 nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs1 separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version 5.0 after taking consent from the donors. the target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered. data was analyzed by stata 14. within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the 98 donors, 35 repeated the plateletpheresis within a week (group i) and 63 underwent 2 nd plateletpheresis within 8-30 days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025). though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at 2 nd plateletpheresis in group ii donors. there were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000). plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type 1 receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type-1 receptor antibody (at1rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at1rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at1r ab are reviewed. results/findings: case 1: the patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38. three years post-transplant, her creatinine (cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed banff criteria grade 2 amr, grade 2a t-cell mediated rejection (tcmr) and grade 3 interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at1rab was identified at >40 u/ ml (high: >17 u/ml, intermediate: 12-17 u/ml, negative: <12 u/ml). she received 6 tpe treatments every other day and started losartan. after a course of tpe, at1rab decreased to 32 u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to 1.9 ml/dl. in one month, her at1rab increased again to >40 u/ml, therefore, she received 3 more tpe treatments with a decrease in her at1rab to 16 u/ml. although at1rab level increased slightly to 20 u/ml after 3 months, her cr has been stable at 1.3-1.6 ml/dl. case 2: the patient is a 25-year-old mean 1/-se -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96a female with malignant hypertension who received a deceased donor kidney transplant at age 24. her cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at1rab level at 18 u/ml. she received 6 tpe procedures every other day and at1rab decreased to 8 u/ml with a decrease of cr to 1.98 mg/dl and improved arteriopathy in histology. because her at1rab level slightly increased to 12 u/ml over the next 2 weeks, she started weekly tpe treatment. after 5 weekly tpe, tpe treatment was stopped because her at1rab level remained relatively unchanged. her cr has been stable at around 1.5 ml/dl to date. conclusion: we present 2 kidney transplant recipients who received tpe treatments for high at1rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at1rab levels; however, weekly tpe had no effect on reducing at1rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a 36 year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < 10%], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a 33 year old man (patient b) with a medical history of hypothyroidism (on synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (ef of 20-25%) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within 12 hours of the procedure completion. their total t4, t3 and free t4 levels trended to normal or near normal range within 24 hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* 1 , laura martínez molina 2 , cristina muniesa montserrat 2 , octavio servitje bedate 2 , silvia cosano navarro 1 and maria isabel gonz alez medina 1 . 1 banc de sang i teixits, 2 dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since 30 years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category 1. since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of 10 patients diagnosed with ss and compare them in their first evaluation once the 20 th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from 1.5 to 2 total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in 2 where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in 7/10 patients) and with online system (therakos) just in 3. main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : 3835 u/l) requiring transfusions, mild thrombocytopenia (144 x 10 9 /l), acute kidney injury (bun 175 mg/dl, creatinine 2.51 mg/dl). by the third hospitalization day hgb improved to 10 g/dl, however with worsening thrombocytopenia (16 x 10 9 /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days 6-10). platelet count and a-ipc improved to 52 x 10 9 /l and 6.6 x 10 9 /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count (30 x 10 9 /l) and a-ipc 1.98 x 10 9 /l. these dynamics did not resemble those which had been described for ttp patients with adamts13 deficiency. adamts13 obtained prior to tpe initiation was resulted at this time and was 67%. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc (2.64 x 10 9 /l) that preceded platelet count increase to 80 x 10 9 /l three days later when patient was discharged. other laboratory values at this time were ldh of 635 u/l, hgb: 11.2 g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts13 deficiency eiman hussein* 1 and jun teruya 2 . 1 department of clinical pathology, cairo university, 2 texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts13. since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts13, the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts13. the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january 2008 through march 2017 were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts13 activity of less than 10%. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts13 deficiency. eight patients (25%) were associated with suspected bacterial infection. four of the 8 patients (50%) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in 3 patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure. study design/method: in october 2015 and june 2016, two children with possible leukemia were submitted to tl procedure. they were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with 285 ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin (750 ml of acd-a and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1. a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 . the formula "collection pump flow 5 0,0003 x inlet flow x preapheresis wbc count" was used with the goal of removing up to 3 x 10 9 leukocytes/ml. a single leukapheresis procedure was performed with 2 total blood volume processed per patient. immediately after the 2-hour procedures, wbc count were 74.000 and 92.000 wbc/mm3, and 12-hour post tl, wbc count were respectively 45.000 and 70.000/mm3. net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since 2006. we report the data from the year 2016. study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of 13,302 apheresis procedures were performed at 37 hospitals. therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (pbsc) collection (23.9%), allogeneic pbsc collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%). cobe spectra (37.4%) and amicus (16.8%) were the most widely distributed instruments. centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis. detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out). spectra optia (42.7%) and cobe spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 ffp (11.1%). most of the procedures were performed for 1 plasma volume (72.4%). acd (88.4%) and heparin (11.5%) were used for anticoagulation. central venous catheter (91.9%) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for abo compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%). adverse reactions were observed in 8.5% of the procedures. allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported. therapeutic effect was achieved in 86.5% of the patients. our apheresis registry has been well run for 10 years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride >1000-2000 mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of 41 pts who were diagnosed with hp from january, 2009 through january, 2017, and referred for immunotherapy evaluation. 27/41 (66%) pts received conventional therapy (ct) and pe (pe group), and 14/41 (34%) pts received ct alone (ct group). mean age was 36 years (range 16-79), and 56% were female. baseline mean triglyceride level (normal <150 mg/dl) for pe group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for ct group. baseline mean lipase level (normal <393 u/l) for pe group was 1,798 u/l (797-2,745) versus 923 u/l (472-1,796) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications. 24/27 (89%) of pe group and 11/14 (79%) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. 20/27 (75%) of pe group and 6/14 (43%) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of 2.85 pe treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 u/l (2.5-3.5 x upper limit of normal). mean triglyceride levels after 2 pe txs were 1,976 mg/dl (627-3,968) for pe group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing ct were 1,576 mg/dl (487-2,873) for ct group (mean decrease 50%). while the pe group achieved a greater mean decrease in triglyceride levels after 2 pe txs (compared to the ct group after 48 hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa 2016 guidelines, in a patient unresponsive to optimal medical management. asfa 2016 guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed 39 tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* 1,2 , metha apiwattanakul 3 , sompis santipong 2 , jutaluk jaipian 2 , jettawan siriaksorn 2 and ponlapat rojnuckarin 1 . 1 chulalongkorn university, 2 king chulalongkorn memorial hospital, 3 prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june 2016 through february 2017 were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a 20% solution. before using, it was diluted to a 4% albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of 156 tpes in 38 patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was 3,000 (range 1,750-4,200) ml. although the corrected calcium level was low (<8 mg/dl) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in 2 patients. the first patient had 2 events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was 1.9% (3/ 156). in 2014, the incidence of tpe adverse effects was 1.6% (2/125) when commercial albumin was used. the difference was not statistically different (p 5 1.000). median serum albumin levels pre-tpe and post-tpe were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dl. the increase in serum albumin after tpe was statistically significant (p<0.001). eighty-two percent of pre-tpe serum albumin levels were lower than 4.0 g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether 60 outpatients who underwent a total of 100 la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%). based on differences in the study protocols la was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was 1.4x10 10 wbc consisting of 1.1x10 10 mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg4-related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category 1 indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to 57,000 iu/mls (ref. range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref. range < 20) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were 4610, 2890 and 1320 mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was 1.74. peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref. range 1.6 -1.9). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to 2.4 cp. serum igg, igm and iga levels decreased to 2040, 1510 and 672 mg/dl respectively. her rf had decreased to 19,900 iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than 3 cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan 2013 -dec 2016). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with 5% human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between 4 to 35 years age (m: f; 1:2) underwent 62 tpe procedures with an average of 5.6 per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only. three (27%) out of the seven tested, were positive for aqp4-igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven. adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2). follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after 3 months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from 6.0 to 6.4 for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version 6.0. the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to 1/16/2016, plt collections were performed on nine trima accel machines operating with version 6.0. upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016. the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05. results/findings: following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version 6.4 of the trima accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0. both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4. conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version 6.4 software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is 30 weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of 500 ml 5% albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using 500 ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of 500 ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a 45 years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at 20 years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of 4980 mg/dl, lipase 92 u/l, glucose 250 mg/dl, bicarbonate 24 mmol/l, anion gap 12. ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega 3 fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day 3 after admission. results/finding: the patient tg decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day 6 after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below 1000 mg/dl at 20 days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from 2009 to 2013, a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/l. and the upper limit was set to be 244 lg/ l in male and 158 lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times. the high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%). the mean ferritin was 82.0 lg/l in male (95% ci: 80.7-83.2 lg/l) and 66.5 lg/l in female (95% ci: 60.9-72.0 lg/l). the result of anova indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05). the average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/l in younger and elder 50 y/o male and 18 and 23 lg/l in female. and then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/l in the first period to 4.1 lg/l in the third period (1 period5150$160 days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* 1 , mary townsend 2 and lizabeth rosenbaum 3 . 1 university of new mexico hospital, 2 blood systems, inc., 3 blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately 10 minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within 24 hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* 1 , william korzun 1 , teresa nadder 1 , susan roseff 2 and elizabeth ripley 1 . 1 virginia commonwealth university, 2 virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for 1% of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of 142 subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location. in 2015, basic trainees at site a were scheduled at day 57 of 70. in january 2016, the blood drive date changed to day 60 of 70. the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in 2016. these observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* 1 , eva alonso 1 , oscar bascuñana 1 , monica romero 1 , teresa vich 1 , elena castaño 1 , laura carbonell 1 , eva palomas 1 , saray almerge 1 , francesc carpio 1 and xavier curia 2 . 1 banc de sang i teixits, 2 institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january 2015 to december 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t5 due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t5 and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t5 excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t5 but his hemoglobin levels were lower than our selection criteria. of the 204 donors selected for donation 16 (7.8%) had sci lower than t5 and t6. adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t5 have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of 2013, automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an 11 month period from january 2013 to november 2013. the same information was assembled for the automated bp process for the 11 month period of january 2014 to november 2014. the automated bp process implemented in mid-december 2013; so the december data for both 2013 and 2014 has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < 0.05. results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p50.006). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < 0.03) with the automated bp while and reactions remained non-significantly lower (p 5 0.086). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05. results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under 12.5. statistically more visits with hgb less than 12.5 g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from 1 january to 31 march 2017. after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study. 85% of donors (n55678) accepted automatic appointment booking, whereas some donors (n51002) were not comfortable with it. 77% of those who declined still preferred walk-ins (n5771) based on their own time schedule, the rest decided that variable situations (n5112), donation frequency (n569) and choice of preferred donation locations (n550) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was 19%. a comparison was made and found that this study shown a significant increase of acceptance rate by 66%. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has 4 collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. 4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105a transfusion (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%). mean donor sbp, dbp, and pulse were 125 6 14.7 mmhg, 75.1 6 9.6 mmhg, and 75.9 6 11.2 bpm, respectively. screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female). no differences were observed in the 45-64 year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females. developing blood donor educational materials gay wehrli* 1 , susan rossmann 2 , louis m. katz 3 and dan a waxman 4 . 1 university of virginia health system, 2 gulf coast regional blood center -sugar land, 3 americas blood centers, 4 indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an 8 th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, 3.5 page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table 1. results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has 4 major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from 2011 to 2016 after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016. moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to 24 times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately 1.3 million doses of ap transfused within 1.375 billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since 2006. firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than 180 days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by 7.46 times from 5550 to 41420 and the doses of ap increased by 7.41 times from 7363 to 54553 within 10 years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into 5 groups: those who donated ap once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively. it was found that the number of permanent ap donors who donated ap more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of ap (29.2%) from 2006 to 2016. conclusion: aps increased at a rapid and steady pace in wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer <26 ng/ml and zpp levels >100 umol/mol heme) at 3 hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all 3 hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp 87.8% (r50.937) at first and 86.5% (r50.93) at second donations. at first donation when compared to fs hb, only 10.4% (r50.323) of variation could be explained by variation in fs zpp, 12.3 % (r50.35) by ven zpp and 9.4% (r50.307) by ven fer. at second donation, when compared to fs hb, only 9% (r50.30) of variation could be explained by variation in fs zpp, 14.4% (r50.38) by ven zpp and 20.1% (r50.448) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p<0.001) suggesting strong evidence against correlation. 55% (181) responded to the survey of which 4% (13) reported not feeling well after donation. it should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/ml but the zpp levels were less than 100 umol/mol heme. of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/ml nor ven or fs zpp levels above 100 umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from 12.5 to 13.0 gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from 59 blood centers over two intervals, july-dec. 2015 and july-dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male5m, female5f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab 17.0, chicago il). p <.05 was considered significant. results/findings: data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 wb donations and 272,094 and 319,161 ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for m ap from 1.8 to 3.5% (p<.001). the mean "by center" deferral rates (table) were similar to that and significant (p<.001). mean by center hb deferral rates among f donations during the two intervals were 11.6 and 11.9% (p50.241) for wb, 11.8 and 13.0% (p5.041) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none. for bp, 8 provided detail (high vs. low), 28 summary and 4 none. p deferrals increased in the successive intervals among f wb donors from a center mean of 0.57 to 1.49% (p5.018) and for m wb donors from 0.78 to 1.16% (p5.006). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba1c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba1c levels among those with rbs >180 mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from 1 st march 2017 to 31 st march 2017. total of 1,861 blood donors were tested for rbs. those with rbs > 180 mg/dl were further tested for hba1c by gold standard hplc method using variant ii biorad. blood donors with >180 mg/dl rbs and hba1c > 6.5% were advised to consult a physician for further evaluation. results/findings: of the 1,861 donors tested, 44 (2.36%) donors showed a rbs of > 180 mg/dl. forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years). of these, 14 (31.81%) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining 30, 8 (26.66%) of them had a family history of dm. of these 30 donors, 8 donors did not give a consent for testing for hba1c. among the 22 donors tested for hba1c levels, 16 (72.72%) had hba1c > 6.5%. all the 16 donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is 0.87% (16 of 1839 donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* 1 , hwee huang tan 1 and ai leen ang 2 . 1 health sciences authority blood services group, 2 health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last may 2016. the current minimum acceptable hemoglobin for male donors in singapore is 12.5 g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above. study design/method: during a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin 12.5-12.9) group b (whole blood with hemoglobin !13, group c (apheresis with hemoglobin 12.5-12.9) and group d (apheresis with hemoglobin !13). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below 30 ug/l is considered low and levels below <12 ug/l are considered having absent iron stores. results/findings: 55.1% of donors in the study have ferritin levels below 30 ug/l. there were more donors with low ferritin in group a compared to group b, 80% and 53% respectively (p<0.05). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, 49% and 30% respectively (p50.001876). ferritin results for the 4 groups can be seen in table 1. conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to 13.0 g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among 600 students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of 54% male and 46% female students in the age group of 18-28 years. only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment. 42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood. 80 % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. (21); miscellaneous effects were reported in 23 courses. side effects led to interruption of supplementation in 55 instances. ferritin levels (mgt6sd) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/l in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets. ferritin levels<26mg/l were found in 4,8% of participants and 14.7% of controls. deferral for low hemoglobin was below 1% in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21cfr630.10 and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in 2001, an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of 50 -75 ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are c282y homozygotes. without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors. the mean current age is 59.7 years, 65% male, 96% caucasian. the majority of hh donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate. over the last 5 years, hh donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over 16 years. since 5/23/16, with an increase in male hgb deferral threshold to 13g/dl, there has been only 1 hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm-200 non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm-200 non-invasive occlusion spectroscopy device. over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the nbm-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the 200 blood donors for the performance of hb measurement on the sysmex hematology analyzer within 1-3 hours of collecting the venous samples. results/finding: the sd of the difference between the nbm-200 non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl. the hb measurements obtained from the nbm-200 and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be 0.978 g/dl. the precision of the nbm-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl. conclusion: the operators found the nbm-200 easy to install, maintain, and operate with minimal training. the nbm-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder 1 , ravi reddy 1 , dhuly chowdhury 2 , don brambilla 2 and edward l. murphy* 3 . 1 sanbs, 2 rti international, 3 ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in 2014 and followed them for one year. within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used 4-point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. 2015) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included 2,902 first-time black donors with median age 23 and female predominance (59%). within one year, 1,786 donors (62%) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) 5 1.16, 95% ci 1.06-1.28), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or51.11, 95% ci 1.00-1.23). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or50.83, 95% ci 0.72-0.96) and "i wasn't treated well by the <blood center> staff" (or50.85, 95% ci 0.74-0.97). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or51.19, 95% ci 1.03-1.37). a secondary analysis treating the likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard 1 , ramya ghantasala 1 , obhijit d hazarika 1 , nicole leonard 1 , cori a polonski 1 , zachary b wunrow 1 , michelle heleba 2 , jan k carney 1 and mark k fung* 1 . 1 university of vermont larner college of medicine, 2 american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of 292 surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" (27.5 %) and "traveling is a time for me to relax." (30.6 %). of the respondents who travel in the summer, very few reported donating while traveling (3.4 %). summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%). willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%). of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv6, and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(il-4) and il7. others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a compomatev r g5. plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb1 loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers (13m [29%]: 32 [71%] f); median age 42 years (range 21-70) donated a unit (500 ml) blood from which buffy coats (average volume 56 ml) were processed. the buffy coat process was previously validated on 20 wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2. all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for 34 days. results/findings: there were 158 completed responses, of which 73 (46%) indicated that their hospital had an msbos and 85 (54%) did not. the majority of hospitals without an msbos were academic centers (36/85, 42%) from oceania (26/85, 31%) or europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 rbcs (21/85, 25%) per year. 15/85 (18%) are going to implement an msbos in 2017. of those with an msbos, the majority 23/73 (32%) were from north america. the majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 rbc units per year (21/73, 29%) offering a wide range of surgical services. on average there were 207 6 577 procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching rbc units for 28%, and for 4% of procedures a different recommendation was made. most (32/73, 44%) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of msbos' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents (30%) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in 2015 was approximately $15.8m. in nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $1.2m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee 1. development of evidence based transfusion triggers. 2. education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3. clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to "1" unit instead of "2" units. 4. updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015. conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* 1 , anna w rains 2 and christopher t clark 3 . 1 university of tennessee graduate school of medicine, 2 univeristy of tennessee medical center, 3 univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc 3000 system. by using the blood volume values, and assigning a value of 1.5 ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in 2016, there was a total of 3,331 infants delivered at our facility. out of all the deliveries, 487 (14%) infants were transferred to the nicu. of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion. of the 487 infants transferred to the nicu, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%. in those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* 1 , shannon davis 2 , suzan new 2 , vaishali patel 2 and oren guttman 2 . 1 university of texas southwestern medical center, 2 ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of 79 randomized controlled trials (8, conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* 1 and stephanie rogers 2 . 1 dignity health st joseph's medical center, 2 dignity health background/case studies: over 12 million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across 39 hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels >5 7.0 g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of 7.0 g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of 7.0 g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to 21 hospitals including 16 cme presentations, online physician and nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb >5 7.0 g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy2015 to fytd2017, there was a 26% reduction in prbc units transfused to patients with hgb >5 7.0 g/dl, starting at a baseline of 67% down to 41%. this represents an fy17 annualized savings of $9.732m, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels >5 7.0 g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm5-0.258) reduced rbc units and two studies decreased the percentage of patients transfused (or5 0.700). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or 50.264) and rbc units transfused (sdm5-0.553). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a-6 method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was 7g/dl/21% or 8g/dl/24%. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately 42% of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was 590 1 24 units/ month. after the first 5 months of buc activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (p50.003 by t-test). non-surgical rbc use now represents approximately 29% of the total rbc use hospital wide a 13% reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital -30 month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital (4001 beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first 30 months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating 115a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance (2q14 vs. 1q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3q15 vs.1q17) vital sign compliance (39% vs.71%). jwp increased from 27 to 225 (04/16-03/17). cost savings were realized by decreased utilization and implementation of bpa. (table 2016 -1q17) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately 30-40 trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used. it also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november 2016 implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level 1 trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains 2-4 units of group o rhd negative, 4 units of o rhd positive, and 4 units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in 2015, the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < 10 k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june 2015-october 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a 17-month period, 1,270 cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table 01 below. overall, 532 (42%) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." (325), "active bleeding" (303), "platelet count of . . ." (173), and "downtrend" (92), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, 618 (49%) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years 2014-2016. study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by 207 km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of 5,5g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> 9 g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of 32 complete responses (16%). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dl (56%), platelet count of 20-50,000 (38%), and inr of greater than 2.0 (69%). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per 1,000 inhabitants may vary 3 folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of 18 key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%. we estimate that 384,704 patients would be eligible for pbm strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (daly) relative to the current clinical practice. a decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate. in this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: 237 adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted <1250ml of rbcs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used. if 1250-2000ml rbcs were predicted to be transfused, >2000ml were used 25% of the time. if predicted usage was >2000ml, 53% of the time it exceeded 2000ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for >1250ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (jan to mar 2017). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of 494 units of blood were requested. 154 units were crossmatched, of which 138 units were sent to the operating theatre (ot). only 33.3% of blood issued to ot were transfused (n546) while the rest were unutilized. the observed ct ratio was 3.35. conclusion: although only 31% of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study 35 patients who received tranexamic acid (txa) (study group) and 31 patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa 1gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < 7.0 g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were 1732 unique alert encounters. of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at 4 academic hospitals with data-derived msbos. study design/method: the 4 hospitals were in 2 groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if 5-24% of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if !25% of the patients had been transfused. data were collected at each center over a 1 month period between january to march 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers results/finding: between these 4 centers there were a total of 1599 cases analyzed. some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%). there were 1362 t&s ordered for these cases, of which 5 were positive for antibodies on the day of surgery. of all the t&s ordered, 52% were ordered in accord with the msbos recommendation, 26% were ordered when the msbos did not recommend one, and in 0.2% a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (hgb) 8-8.9). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of 29% among obstetric patients. study design/method: we studied a sample of anemic (hgb<10.0 g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks. mean hgb before referral was 7.5 g/dl and most were already taking oral iron therapy. a total of 169 women were hiv positive with mean cd41 lymphocytes counts of 394 cells/ul; 29 (12%) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and 156 (92%) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women. there was concurrent chronic disease (n52), infection (n52), vitamin b12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a 2 month old boy presented to our institution after a 1 month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige (15270 ku/l; rr: 0-2.9). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp3, while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp31 cd41 lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 mtps. results/finding: in 2016, mtp was activated 28 times. in 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of rbc allocation/issue. this left 11 cases to evaluate. the median time to plasma availability was 29 minutes (range 4 minutes -61 minutes). the mean plasma wastage for mtp activations was 32% (range 0-100%). of the 9 cha replies, 3 were using thawed plasma and their wastage was </5 5%. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes. our implementation began with thawing a single unit of plasma for "level 1 neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated 5 cases of gtx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with 40gy. results/finding s: granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85). in general donors reported no significant adverse reactions. from 56 donations, only in 4 was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table 1 . adverse reactions for patients were minor and not frequent (4 in 66 gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* 1 , elena kurnikova 1 and pavel trakhtman 2 . 1 russian institute for paediatric haematology, oncology and immunology, 2 instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period 04/2012-02/2016. donors underwent granulocyte mobilization with g-csf in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58. the mean increment of wbc after stimulation was 22,6x10 9/l (9,2-41,9 x10 9/l; f-23,2 x10 9/l; m-21.9 x10 9/l); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of wbc was respectively 22,2 x10 9/l (9,2-41,9x10 9/l) and 24,02x10 9/l (12,6-35 x10 9/l). the mean volume of treated blood was 566, a50,05) . the mean number of total wbc in the product was 38,43x10 9/l (13.38-77x10 9/ l; f-34,8x10 9/l, m-42,7x10 9/l). only three donors (1,96%) had apheresisrelated adverse effects the analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients. the mean number of transfusions was 6.9 (1-60). each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of wbc on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg). wbc increment was able to estimate after 144 transfusions. wbc increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/l (0,01-11,38 x10 9/l). efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%. in the cases of severe sepsis, the efficacy of transfusions was 76.2%. the frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-hla antibodies formation 2,7% (n 5 1) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp150 hemolytic disease of the newborn due to anti-rh17 keegan barry-holson* 1 and jennifer andrews 2 . 1 stanford university, dept of pathology, 2 stanford university, dept of pathology & pediatrics background/case studies: anti-rh17 is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh17 alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g3p1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dl), severe hyperbilirubinemia (total bilirubin (t bili) 9.0 mg/dl), reticulocytosis (8%) and a positive direct antiglobulin test (igg 21). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of 16.7mg/dl on day 8 of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh17 was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh17 sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d1 c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh17 antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* 1 , cyril jacquot 1 , valli criss 1 , philippe p pary 1 , jay greenberg 1 , naomi lc luban 1 and edward cc wong 2 . 1 children's national medical center, 2 quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as 4-11%. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of 1.6 g/dl/4.9%. wbc counts (19 x 10 9 /l) were mildly elevated and platelet counts (410 x 10 9 /l) were within normal limits. her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dl, direct 1.4 mg/dl), normal ldh (318 u/l), and undetectable haptoglobin (<7 mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol. dat was 41 positive for anti-igg and negative for c3 despite a positive cold antibody screen. the patient weighed 6.9 kg with an estimated total blood volume of 620 ml. she initially received simple transfusions totaling 20 ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with 463 ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of 40%, utilizing the central venous catheter. no adverse events took place over the course of the 2 hour exchange. her one hour post-exchange hb was 9.5 g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21). after initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of 11 g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels (3168 mg/dl). at a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a 24 or 16 gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the 24g catheter, the mean change in hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p<0.0001). during rapid manual transfusion with a 24g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in hct versus time: r50.58, p50.23). correlations between time and hemolysis were similar, but insignificant using 24g with washed rbcs and the 16g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in 99.9% of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge3. study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-d and an anti-ge3 titer of 256. she was d-and ge:-2,-3, 4 by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge3. the birth hemoglobin (hgb) was 12.6 g/dl, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dl; the infant was discharged. on day 7 of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb 7.6 g/dl, retic 2.6%, and bili 6.6 mg/dl. ge3-blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh1. obstetrics had to authorize maternal blood donation due to her hgb of 10.9 g/dl. maternal blood collection and rbc washing was expedited and the infant received 40ml of maternal rbcs within 24 hours, at which time his hgb was 6.1 g/dl. post-transfusion hgb was 10.8 g/dl. one week later, the infant was symptomatic with hgb 7.1 g/dl, retic 1.0%, bili 2.1 mg/dl. a 2 nd aliquot of 60ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb 7.8 g/dl, retic 0.7%, anti-ge3 titer 8, and needed another transfusion. the maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion. at 6 weeks, the infant's anti-ge3 titer was 2, hgb 9.2 g/dl, retic 1.7%; no transfusion was necessary. at 8 weeks of life, hgb was 10.2 g/dl, retic was 3.3%, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge3 hdfn. molecular analysis revealed that the mother was homozygous ge3-negative ge*01.-03, the father had homozygous wild type ge*01, and the infant was heterozygous ge*01/ge*01.-03. conclusion: the infant had hdfn due to antibodies to the high prevalence ge3 antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge3. hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in 2016, 14, 247 rbc orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was 9.85 g/dl as measured in the 72 hours prior to rbc order placement. in 2016, 3105 ffp orders occurred and the top three patient groups were: 46% in neonates in the nicu, 28% in congenital heart disease patients and 13% in pediatric intensive care patients. average inr of every patient was 2.09 as measured in the 72 hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day 5 bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after 4 months, pr-plt represented 30% of our platelet inventory (average daily plt inventory: 45 units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among 16 transfused neonates (0-4 m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of 60 pediatric patients receiving prbc transfusion over a 12 month period were retrospectively reviewed. a total of 44 patients were identified as receiving allogeneic prbc transfusion. 16 patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was 10.6 g/dl with post-transfusion hgb rising to 14.5 g/dl. the mean prbc volume transfused was 46.3 ml using a dose of 15ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of 10-15 ml/kg in a 2 kg patient, for instance, would translate into 3 full prbc units (about 1000 ml) in an average size adult. the current standard dose of 10-15 ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dl and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dl with a direct fraction of 0.43 mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable. the patient was started on a 5-day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c3. an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her 8-day hospital course, the patient received 2 rbc transfusions on the day of admission and several rbc transfusions thereafter (see table 1 ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day 6. her hemoglobin rose to 8.4 g/dl on hospital day 7 and increased to 9.5 g/dl on hospital day 8. at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dl on day 57 after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is 3 or 41. platelet and leukocyte immunohematology, testing and genetics table 1 . of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2). the matching rate of hla-dpb1 in our study is 13%. conclusion: the matching rate of hla-dpb1 in 10/10 hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb1 and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china (81401732) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts13. therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts13 with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a 56 year old male with a 7-year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts13 <5% and inhibitor of 3.6. on day of admission platelet count was 95 x 10 9 /l which decreased within five days to 14 x 10 9 /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were 1 x 10 9 /l and 14 x 10 9 /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to 3.2 x 10 9 /l achieving the ratio of 3 previously shown to be diagnostic of ttp. on day 5 his a-ipc and platelet counts had improved to 7.5 x 10 9 /l and 218 x 10 9 /l respectively. absence of anti-pf4 antibodies ruled out heparin-induced thrombocytopenia at this time. on day 6 he had an unexpected decrease in both a-ipc and platelet count to 4.8 x 10 9 /l and 132 x 10 9 /l respectively, worsening by day 8 to 1.7 x 10 9 /l and 40 x 10 9 /l respectively despite daily tpe. patient received 25 additional tpes that failed to improve a-ipc or platelets which on day 32 were 0.4 x 10 9 /l and 13 x 10 9 /l respectively. a-ipc had remained at this level for 16 days suggesting that the observed decrease was irreversible. adamts13 activity remained <5% low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa-1a, hpa-3a, and hpa-5a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa-1a, hpa-3a, and hpa-5a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p2 and gi9, specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa-1aa, hpa-3ab and hpa-5aa) were collected and reacted with anti-hpa-1a, anti-hpa-3a, anti-hpa-5a and anti-hpa-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex100. the hpa-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa-1a, hpa-3a, hpa-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa-1a, anti-hpa-3a, anti-hpa-5a. furthermore, because the platelet was hpa-5aa, the hpa-5b serum did not react with the coupled beads with mfi was comparable to negative control (286.59 vs 127.25). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa-1a was 1/128 (0.78iu/ml) and 1/64 (1.56iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa-1a, hpa-3a and hpa-5a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 january 2014 to 31 december 2016. clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c3, c4bp, thbd, dgke, cfhr3, cfhr1, cfhr4 and cfhr5) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and vus in 35% (47/134). 20% (27/134) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and 9 (33%) had vus. 31% (42/134) of patients had primary ahus; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had vus. 48% (65/134) of patients had secondary tma; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had vus. in patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of ahus and 28% (5/18) had recurrent disease. patients with primary ahus had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl80 and sysmex xn9000 compared with a flow cytometric method farshid ezligini 1 , kjersti roen eriksen 1 , annette vetlesen 1 , thomas larsen titze 1 and geir hetland* 1,2 . 1 oslo university hospital, 2 university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion 2012). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and 33 buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl80 (horiba abx, montpelier, france) and xn9000 sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd41a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x10 9 /l 6 sd were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by sysmex toa, pentraxl80, and the gallios flow cytometer, respectively. sysmex count was the very lowest 129a transfusion 2017 vol. 57 supplement s3 abstract (31.4% less than for flow cytometry), but all plt counts were significantly different (p<0.001), although least so (7.4%) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl80 seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing 1 , arishma lata 1 , roland russnak 2 , zachary antovich 2 , heather dunckley 1 and thierry viard* 2 . 1 new zealand blood service, 2 linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant snps located within hpa genes (hpa-1 through hpa-11, and hpa 15). genomic dna purified from 48 blood samples, previously genotyped for hpa-1,-2,-3,-4,-5 and -15 by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were 100% concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late 2016 and to date has tested 749 dna samples from 400 blood donors (349 donors were tested in duplicate). concordance between the sample replicates was 100%. there were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa-3 (4.7%) and hpa-5 (1.2%) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd36 antigen deficiency expression in jiangsu chinese han population qing chen* 1 , jianyu xiao 1 and chengyin huang 2 . 1 jiangsu province blood center, 2 jiangsu province blood center background/case studies: cd36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in asians and 2.4% of african americans, respectively. however, there is little information on the molecular basis of individuals with cd36 deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd36 expression levels and to determine the molecular basis of cd36 deficiency on the platelet surface of the han population in jiangsu region. cd36 expression levels on platelets were detected by flow cytometry among 243 blood donors in jiangsu region. donors without cd36 antigen expression on their platelet surface were further to be determined the expression of cd36 antigen on their peripheral blood monocyte cells. the coding exons of cd36 gene and adjacent introns were amplified and sequenced in cd36 deficient individuals. results/finding: among these 243 blood donors, cd36-deficient and cd36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively. the frequencies of type i and type ii cd36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively. among 237 individual with platelet cd36 expression, according to mean fluorescence intensity (mfi) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of cd36, respectively, and their mfis were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (p<0.05), respectively. the type i cd36 deficiency individual were heterozygous for 1200-13a>g and 430-14c>g, respectively. among type ii cd36 deficiency individuals, two harbored a t insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a t>c exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a a insertion before the 17th bp of the start codon atg in the promoter region; one were heterozygous for 748 1 2t>c and 1006 1 2t>g, respectively. conclusion: platelet cd36 surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd36 deficiency was higher than that in type i. the study findings indicated that the frequency of cd36 deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd36-deficient individuals widely varies among ethnic groups, with 3% to 11% in japanese, 8% in sub-saharan africans, 2.4% in african americans, and 0.3% in caucasians. although some studies of cd36 deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd36 expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the eastern china. the expression of cd36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd36 and peanti-cd41). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd36-nagtive phenotype. for those donors with cd36-negative platelets, cd36 antigen expression on monocytes was analyzed further to distinguish between cd36 type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china (81570170) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd36 phenotypes using the (mean 1 3sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd36 deficiency on platelet, in which one sample was cd36 negative both on platelet and monocyte. the frequency of cd36 type i and type ii deficiency in the eastern chinese donors was 0.08% and 3.3%, respectively. the average mfi of cd36 deficiency samples was significantly lower than cd36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, p< 0.0001). conclusion: the frequency of platelet cd36 deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd36 antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd36 antibody. background/case studies: cd36 (gpiv, chromosome 7q11.2) is an 88 kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd36 deficiency (cd36-n) is observed in 3-10% of africans (t1264g) & is classified as either type i (cd36-n plt, cd36-n mono) or type ii (cd36-n plt, cd361 mono). an acquired type ii cd36-n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type 1 cd36-n individuals can develop anti-cd36 alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd36 in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd36 phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd36 staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd36 dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an 80 year-old, group o1 african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin 4.4 g/dl & plt count 5k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < 5. hla antibody testing was negative (class i panel reactive antibody (pra)50%). the patient was plt-xm-incompatible with most donors (10/14). a trial of 4 group o, plt-xm-compatible plts was unsuccessful (cci 1). subsequent testing for plt-specific alloantibodies identified anti-cd36. fc-phenotyping showed no cd36 on patient's mono or plt, consistent with type i cd36-n. preliminary dna results show that the patient is heterozygous for t1264g. because cd36-n apheresis plt were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: all showed normal cd36 expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day 16) demonstrated new class i alloantibodies (pra 5 55%) in response to transfusion (21 apheresis plts, 5 rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd36-n & severe plt refractoriness in the setting of new mds, and 7q-chromosomal abnormalities. the absence of cd36 on plt & mono support congenital type 1 cd36-n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel 1 , kristopher fernandez* 1 , eric senaldi 2 , pascal george 2 , michael seul 1 and ghazala hashmi 1 . 1 biomolecular analytics, 2 central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made ! 6 donations in the previous 12 months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for hla class 1 (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/2pdplf8) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a *26:82 chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are 16,429 hla alleles documented according to the imgt / hla sequence database in janury2017, and more than 80% of them were identified in the last 10 years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a *26:82 allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn72d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove1 nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 c-a (codon225 acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a 13 year-old pregnant woman typed as ab1, who delivered a baby affected by severe hdfn. the newborn was typed as b1 and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted 41 with the four monoclonal anti-d used (igm clones p3x61 and rum 1 and the blends clones th281ms26 and d1751d415) and were typed as c-c1e-e1. an anti-d was identified in her serum. molecular analysis showed the 410c>t and 455a>c in exon 3, the snp 509t>c changes in exon 4 and the 667t>g nucleotide change in exon 5. the set of snps found is similar to the molecular background of dol3, except for 455a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of 1089 immuno hematological tests (465 abo/d grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 dat) and 20 crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in 99.2% of the abo/d tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the dat tests (n510). there were 4 discrepancies (2 abo/d for the same patient, 1 for antibody screening and 1 antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min). detection threshold of the d antibody was assessed at 2.5 ng/ml (0.0125 ui/ml) whereas the french recommendations are 20 ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso 15189 accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a 31 year old g5p3 presented during her fifth pregnancy with anti-kpb with an initial titer measured of 64. by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams (1.7 moms) peaking at 27 weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at 35 weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was 3.4 mg/dl with 13.6 g/dl hemoglobin. the baby typed as o positive, kp (b1) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at 6 weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by 8 weeks of age. his hemoglobin recovered to 9.0 g/dl with an indirect bilirubin of 1.4 mg/dl at 9 weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c3d-specific dat may be too insensitive to detect low, but significant levels of c3d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a "1111" reaction corresponds to about 500 molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c3d have been published. however, these are mainly designed to quantify the fraction of rbcs with c3d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c3d. study design/method: ten microliters (ul) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-c3d) mouse monoclonal anti-human anti-c3d (abcam, clone 7c10) were added to 5 ul of a 2.5% rbc suspension. after incubation for 60 minutes at 4c, samples were washed x3, and 25 ul of 1:10 diluted anti-mouse-f(ab)2-pe (ro480, dako) were added. after incubation at 4c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro480 was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c3d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a1 rbcs stained with 10 levels (2fold dilution, 1:1 -1:512) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c3d, edta-stabilized samples from 4 healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c3d (values ranging from 0 to 3,393 abc) with level of 0-serum dilution (used to sensitize a1 rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001). compared with dc-screening 1, the sensitivity of the flow cytometric assay was superior. it detected c3d sensitization at least 4 dilution steps further. the median normal level of rbc-bound c3d was 11 abc (range 7-20 abc, n54). the assay enabled demonstration of specific c3d-sensitization in the patient; the level of rbc-bound c3d in the sample was significantly elevated (1,907 abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c3d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt1 gene. in humans, the abo and gbgt1 genetic loci are located on chromosome 9q34, and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors1) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt1 genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt1 genes have been constructed of 88 vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k1 antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion 2017 vol. 57 supplement s3 the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c1, e-, e1 and k1-. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d1, c-, e-, partial c1, partial e1. the probable rhce genotype, rhce*ce-jal/rhce*ce733g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant woman and her newborn carolina bonet bub* 1 , maria giselda aravechia 1 , thiago costa 1 , marilia sirianni 1 , eduardo bastos 1 , leandro santos 1 , lilian castilho 2 and jos e kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp background/case studies: rhd*weak d type 2 is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion 2007) the c.1154g>c change (p.g385a), which characterizes the rhd*weak d type 2 allele is a splicing variant that induces skipping of the whole rhd exon 9. we report an altered splicing in the rhd*weak d type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d1 w c-c1e1e1. the samples showed weak hemagglutination reactions (11/21) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon 9 in both dna samples. sequencing showed the c.1154g>c change and the intronic c.1154-8t>a and c.1154-31t>c substitutions, which are associated to the rhd*weak d type 2 allele. conclusion: our results showed that c.1154g>c associated with c.1154-8t>a and c.1154-31t>c variations had probably a functional impact on splicing inducing exclusion of exon 9 in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type 2. background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than 50% of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with 1,057 patients and 3 studies from other regions (brazil, egypt and france) with 641 patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was 0 to 26 years and for the other countries 0 to 20 years. available data from 5 us studies included a total of 91 alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% ci) vs. 9.4% for non-us studies (7.3-11.8, 95% ci) (p50.0008) and 134a transfusion 2017 vol. 57 supplement s3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001). similarly, the number of alloantibodies per 100 transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort (0á68 vs. 0.33, p50á0005). average number of rbc units transfused per patient in the us was also higher compared to data from france (77 vs. 45, p50.0001). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* 1 , melissa grohotolsky 2 , lisa deblass 1 , bala carver 1 and kip kuttner 2 . 1 health network laboratories, 2 miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: 23 year old white mennonite female g1,p0 presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated 11 reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was 2 at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at 2. although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a 15 year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g1 (r g r) cells. both rhce*c and rhd genes encode ser103 which determines g expression. rare rhd variant antigens lacking ser103 are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e1c1e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r 2 r 2 , r'r, r g r and rr cells. this patient is predicted to be r 2 r 2 (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is 135a transfusion 2017 vol. 57 supplement s3 excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* 1 , christa voliva 1 , kathy fletcher 1 , heather vaught 2 and tracie ingle 1 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than 7 days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were >21 dat positive, 2 were weakly dat positive and 16 were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman 6.0-8.1ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: 5 bb, 14 bg and 1 bp. the ph of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a ph of 8.1 (35%). sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%). four eluates with different results are summarized in table 1 . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results (3) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in 1900 led to the discovery of human blood groups. in the abo system >200 alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor (38 units over 17 years) who is actually a w . study design/methods: donations were tested with the pk7300 instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns 1,2 and 4 and exons 6 and 7. specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table 1 . tests with anti-a, -a1, -b anti-a,b were negative as were the a2 cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table 2 . the significant changes were found in exons 6 and 7. in exon 6 there was a nucleotide (nt) deletion of 261g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly117ala. mutations in exon 7 included a nt substitution causing a pro156-leu change and a nt deletion 1061c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a2 cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o.01.01/abo*aw.02] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a1 and b cells. this abo discrepancy was caused by the presence of anti-a1 in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a2 cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer >1:1000. (schmidt, nacarrow et al. 1959) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. 136a transfusion 2017 vol. 57 supplement s3 extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to 96 blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to 15 days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: 92 edta blood tubes collected from random blood donors were used to extract dna from 200 microliters of whole blood on day 5, 12 and 15 days post collection. blood samples were stored at 2-8c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a260/a280 using a nanodrop 2000 (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result50) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the 92 samples, 20 samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from 10.8 to 62.6 ng/ul. all readings with the exception of 1 (10.8ng/ ul) had concentrations >5 15ng/ul. interestingly, the one that was <15ng/ ul on day 5, yielded >515ng/ul on day 12 and 15 post collection. over the next 3 months, 67 sets of 92 samples were extracted and tested by hea. eighty-three (1.3%) failed extraction and 82 (1.3%) failed hea. none of the samples that failed extraction were 12 or 15 days post collection; none of those that failed hea were 15 days post collection; 3.7% were >10 <15 days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to 15 days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas 1 , carlos villa 1 , rachel davis-rauser* 1 , helen carpenter 1 and vrunda patel 2 . 1 university of pennsylvania, 2 hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd38 monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in 2015. communications suggest all patients receiving therapy would have a positive antibody screen because cd38 is a common antigen expressed on red blood cells. currently, 154 patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for 1 hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a 28 y/o g1p0 at approximately 23 weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [1, 2] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m1n-s1s1 phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [3] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays 137a transfusion 2017 vol. 57 supplement s3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in 67 dna samples from chronically transfused patients with scd, in 65 patients with thalassemia and in 3000 dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in 3 levels: (1) rh and k matching; (2) extended matching and (3) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for 100% of our thalassemic and scd patients at level 1, 90% for scd patients and 70% for patients with thalassemia at level 2 and 30% for patients with scd and 90% for patients with thalassemia at level 3. the patients were transfused with a median of 36.4 rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with c e k matching and <1% with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of 164 clinically significant antibodies were detected using sprca technique, as well as 9 warm autoantibodies and 97 nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being 100% specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than liss (93). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd38 on rbcs patricia a arndt* 1 , anthony salazar 2 and regina m. leger 1 . 1 american red cross blood services, 2 long beach memorial medical center background/case studies: monoclonal anti-cd38, e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd38 on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd38 antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or 2aminoethylisothiouronium bromide (aet). chapuy et al described (2015) and validated (2016) 2), and 6% aet (ph 8.0) as per the aabb technical manual, 17th ed. these treated and untreated rbcs were stored in alsevers at 4c and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from 8 total dara patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (pe)labeled anti-cd38. rbcs were also tested on days 1 and 5 or 8 with a serum containing anti-k by peg iat. results/findings: the 0.2m dtt in ph 8.0 pbs had a final ph of 7.3 and the ph of the commercial 0.2m dtt was 6.5. results are in table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8. rbcs treated with 0.2m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd38 by flow cytometry for up to 8 days after treatment. rbcs treated with 0.01m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening (10-30%) of reactivity with pe anti-cd38. background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery 1997 of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon 5 and 10) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr5) in gestational week 25. the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of 1618 pregnant women were included. nip rhd was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%). compared to the postnatal rhd type, 9/987 (0.1%) of nip rhd results were false positive (fp) and 4/582 (0.7%) were false negative. in 5/49 (10%) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p50,71). the fraction of ccfdna was calculated for 150 randomly selected nip rhd true positive cases. median ccfdna ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r2 50,012; p50.49). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the 25th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an 83 year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only 1 in 20,000 donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, 4 lan-rbc units were transfused over 4 days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (hb) 5.3 g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of 10.2 g/dl was maintained for 4 days. the antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive dat (igg 21, c3 11) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from 10.9 g/dl on day 5 to 7.4 g/dl by day 8 with no bleeding identified, and increase in total bilirubin and ldh (peak 2.4 mg/dl and 304 u/l on day 7) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day 8 with good response (hb 8.1 g/dl). the patient remained stable and was discharged to a skilled nursing facility 6 days later. conclusion: transfusion of lan1 rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* 1 , andrea gerner 1 , lynne stewart 1 , carol sostok 1 , mollie bell 2 and gregory r halverson 2 . 1 st. elizabeth healthcare, 2 hoxworth blood center background/case studies: anti-f was first described in 1953 by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the rh blood group system (isbt rh6). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a 56 year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused 1 unit o-rbcs. two weeks later the patient received an additional o-rbc. within 4 days the hgb had decreased from 8.3 to 7.1 g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused 3 r 1 r 1 (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c1e1f1 phenotype. the rh phenotype and as was repeated on a sample collected 18 days later. the c typing was micro positive, mixed field only after 5 minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post 24 hour hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl. the last transfusion of 3 units increased by only 1.2 g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 r 1 r 1 units was nearly complete. in a case from 1989, ohto and kariyone (transf. 1989; vol29, no. 3) reported a 51 cr ásurvival study of f1 rbcs in a patient with anti-f. they showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd38 drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to 9 days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with 0.2m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table 1 ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to 9 days following the dtt treatment of rrbc. reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table 1 for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to 9 days. this suggests that dtttreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd38 therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd38 that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with 0.2m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract 2-5c, and observed for hemolysis (none was seen) for up to 21 days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted 2-41 with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in 4 patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than 6 months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* 1 , monique scott 2 , garcia curtis 2 , ellice wong 2 , alexa j siddon 1 and christopher a tormey 1 . 1 yale-new haven hospital, 2 va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately 25% to 50% of persons of african descent is characterized by neutrophil count of <1.5x10 9 /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p<0.05. results/finding: subjects who were clinically identified as having probable ben included 7 patients (mean age 48.7; all self-identified as african-american; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as african american; (50/50 male). all of the cases (100%) diagnosed with ben had fy(a-b-) phenotype. mean anc (1.95x10 3 /ul) and wbc counts (4.04x10 3 /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p50.0008 and 0.001, respectively) compared with controls (mean anc 5 5.46x10 3 /ul ; mean wbc count 5 8.14x10 3 /ul). there was no significant difference in mean platelet counts (161x10 3 /ul vs 213x10 3 /ul; p50.2301) or mean hemoglobin levels (12.4 g/dl vs 11.7 g/dl; p50.6031) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c3d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih-1000 had 100% concordance for all blood grouping assays. for ahg assays, the ih-1000 detected an anti-jka1e, anti-fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih-1000 identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih-1000 with anti-igg,-c3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih-1000 analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih-1000 is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in 99.9% of samples tested. there were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti d and 1 positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp201 evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* 1 , kathleen bensing 1 , michael schanen 1 , cindy piefer 1 , randall w. velliquette 2 , christine lomas-francis 2 and connie m. westhoff 2 . 1 immunohematology reference laboratory, versiti/bloodcenter of wisconsin, 2 immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new 7/0 7/0 7/0 cecf 1/1 2/0 2/0 rhce*ce or rhce*ce compound heterozygotes ce254g 1 ce733g or ce48c,733g or ces or ceti 6/0 6/0 6/0 ce733g 1 ce48c,712g or 48c,733g 4/0 4/0 4/0 ce733g 1 ces or cemo or ceek or ceek(var) or cern 8/0 8/0 8/0 ce48c,733g 1 ce48c,712g or cemo or ceti 2/0 2/0 2/0 ce48c,712g/ce254g/733g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi 7/0 7/0 7/0 total 106/8 110/4 113/2 142a transfusion 2017 vol. 57 supplement s3 reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd9/4 and rd12/2, and a licensed comparator anti-e (p3gd5121ms63), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n 5 42) or edta blood from donors (n 5 72) and were tested using a manual tube method or on a pk7300 automated platform. a score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table 1) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r 1 r 1 , r 2 r 2 , r 1 r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: 40 were rhce*ce that were in trans to rhce*ce; 16 were various rhce*ce plus rhce*ce48c compound heterozygotes; 31 were rhce*ce or rhce*ce homozygotes; 27 were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for 6 rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with 1 rhce*cear/rhce*ce48c compound heterozygote, and with 1 rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and 1 of 2 rhce*cecf homozygotes were detected using the comparator reagent. rd9/4 and rd12/2 failed with 4 and 1 e variants, respectively (table 1) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd9/4 clone. none of the reagents detected e antigen variant expressed on 1 example of rhce*cehar/rhce*ce. conclusion: rd9/4 and rd12/2 anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems. we used the illumina's hiseq 2000/2500 system to perform next generation sequencing first on sureselect-enriched genes from 16 dna reference samples with average target design coverage of 97.5%, and then on haloplex-enriched genes from 32 dna reference samples with average target design coverage above 97.0%. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads (80.54%) were mapped to the target regions relative to the sureselect reads (29.23%). the mean sequence coverage depth of the targeted bases was around 200x for sureselect method and 300x for haloplex method. some exons, such as rhd exons 4 and 8, 10, rhce exon 10, ermap exons 5 and 12, cd55 exons 10 and 11, cr1 gene (most exons) and gypb exon 5, are consistently covered with less than 10x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on 38 blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than 90% concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes. evidence rhce*cehar does not encode for rh34 (hr b ) antigen debra j bailey* 1 , trina horn 2 , paul mansfield 2 , najmi qazi 1 , pamela nickle 2 , jessica keller 2 , margaret a keller 2 and jan r hamilton 1 . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in 1996 and has a phenotype of c2e2c1e1 w f1 w , g2, hr 0 1 w , hr2, hr s 2, rh:33, rh:50 with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh:234 (hr b 2) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c.254c>g and rhd c.1136c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d1c1e2c1e1. her plasma contained an alloanti-s and an antibody that reacted strongly with all random e2k2s2 reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d22 and dc2 red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e2s2k2 red cells homozygous for the rhd*diiia-ce(4-7)-d, rhce*-ce48c,733g,1006t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce48c,733g,1006t/ rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr1 (2 of 2 sources) and hr b 2 (2 of 3 sources) phenotype. conclusion: the rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t haplotype is one of the rh haplotypes expressed by the original hr b 2 individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd*01, rhce*cehar and rhd*diiia-ce(4-7)-d, rhce*ce48c,733g,1006t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between 1/1/2006 and 3/ 31/2017. all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately 11 years, 81 patients had htla established at least once by titration studies. serological investigations on a total of 118 samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on 20 samples was successful in rule out in 60% of cases. in an additional 12 patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only 40% of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for 71% of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c.1136c>t (p.thr379met). the dau0 allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-d alloimmunization for 18 dau family alleles. we investigated two samples with the c.1136c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample 1 was from a 17 yo multiracial female. her rbcs reacted 11 s at immediate spin (is) and 31 in iat with immucor gammaclone and series 4 and 5, and mi1 at is and 41 in iat with ortho bioclone anti-d. rbcs did not react with 2 of 12 (lhm 174/102 & 57/17) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c.1136c>t characteristic of dau. rhd sequencing confirmed c.1136c>t and identified two adjacent changes, c.787g>t and c.788g>t (c.787_788delinstt), in exon 5 encoding p.gly263leu. sample 2 rbcs reacted 1w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series 4 and 5, and quotient albaclone blend and alpha anti-d. papain treated rbcs were 11s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c.1136c>t. sequencing confirmed c.1136c>t and found a new c.761c>t change (p.ser254-leu) in exon 5. the c.761t has not been reported, but c.761g encodes a stop codon (p.ser254stop) in japanese (vox sang 2015, 109:359). conclusion: we report two new alleles: rhd with c.787_788delinstt (p.gly263leu) and rhd with c.761c>t (p.ser254leu), both also carrying the c.1136c>t (p.thr379met) characteristic of the african dau cluster. d antigen associated with p.263leu is a partial d antigen with a novel epitope pattern. the p.254leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by 5/7 commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to 21. the number and diversity of alleles in the dau cluster supports that the c.1136c>t change is a major ancestral african background allele (wagner et al, blood 2002,100:306). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september 2016 to present day we got 130 samples of repeated blood donors who are known to be d negative, c positive and/or e negative from 15 blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon 4, exon 7 and exon 10 in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227g>a and c.1222t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the 130 sample, we identified 71 cases (54.6%) of total rhd deletion, 18 cases (13.8%) of rhd-ce-d hybrid, and 41 cases (31.5%) of rhd variant. 39 of rhd variant were determined to be asian type del with c.1227g>a variation. 2 cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was 10 % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b 3 phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b 3 phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from 30 taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n552) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon 6 and exon 7 of the abo gene were amplified and sequenced. the abo*b3.03 allele was confirmed by pcr-rflp analysis. results/finding: among 52 subjects with b 3 or ab 3 phenotypes, 47 were genotyped as abo*b3.03. the abo*b3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage (37.81 6 6.62) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other 5 subjects with b 3 or ab 3 subjects, genotyped as abo*b3.06(n51), abo*bw.03(n51), abo*bw.11(n51), abo*bw.12(n51) and abo*bw.29(n51), displayed flow patterns differed from the abo*b3.03 group. the abo*bw.03, abo*bw.11 and abo*b3.06 subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (<10% in abo*bw.03 and abo*bw.11 subjects and <20% in abo*b3.06 subject). both abo*bw.12 and abo*bw.29 displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b3.03 genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout 2016, the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of 398 patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n54), anti-jka (n53), anti-k (n52), anti-jkb (n51), both anti-e and anti-c (n51) (see table 1 ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr1 alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr1 gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr1 gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study 60 volunteers in the gambela region (nct01282021). the whole ackr1 gene was amplified in one reaction covering 12,125 base pairs (bp). this primary amplicon was re-amplified using nested primers covering 5782 nucleotides. nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using ncbi refseq ng_011626.2. the sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-utr, 53 bp of 3'-utr and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbsnp and nhlbi esp databases. results/finding: among the 60 samples, a total of 15 snps, including one novel snp in 5'-utr were observed. 4 snps occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-utr and 2 in the intron. all 60 individuals carried the snp indicative of the common fy:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the gata box mutation. no splice site mutation was detected. as 46 individuals were observed as being homozygous or heterozygous for 1 snp, we could unambiguously assign 8 distinct alleles. in the remaining 14 individuals with 2 or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than 5.5 kb of the ackr1 gene and identified at least 8 different alleles. the present study found that the vast majority of alleles (117/120) in the gambela population as defined by 15 snps, were similar to the clinically relevant fy*02n.01 allele, which in turn is defined by only 2 snps at positions c.1-67t>c and c.125g>a. out of the remaining 3 alleles, 2 were similar to fy*02 with the fy(b1) phenotype and 1 was similar to fy*02w.01 with the fy x phenotype. the high frequency of fy*02n.01 (95%) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique (95%-100%). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce(4-7)-d is the most common hybrid and is found in african blacks. it arose by conversion of exons 4-7 of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample 1 (male) and sample 2 (multiracial female), both c1c2e2e1 (presumed r1r1), presented with weaker than expected d typing; 11 is and 31/41 at iat. rhd beadchip identified the common african rhd*diiia-ce(4-7)-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c.733c>g and c.1006g>t (heterozygous) was also detected. as rhce*ce with 733g and 1006t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons 2 and 3 replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce(4-7)-d were found in both samples. sample 3 (scd male), d1c2e2c1e1, by rh beadchip had one conventional rhd and rhd*diii type 8, and rhce*ce733g/ces. as rhd*diiia type 8 has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce(48c) exons 1 and 2 had replaced those exons in the common hybrid rhd*diiia-ce(4-7)-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky926711and ky926710. we report two different and novel complex rh rearrangements: two samples thought to be r1r1 had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia(2-3)-ce. in kind, a sample genotyped as diii type 8 rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce48c(1-2)-diiia(3)-ces(4-7)-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples 2 and 3 have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r1 haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a 81-year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at 81g/l. her pregnancy history was not provided. she had received 5 units of packed red blood cells (rbcs) in the past including 1 unit within the last 3 months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r 1 r 1 , r 2 r 2 , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse 29 polymorphisms which determine 37 antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a1) and lu(b1). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of 36 antigens encoded by the kel gene, is organized into 19 exons. there are approximately 30 kel alleles associated with a kell null phenotype (k 0 ) in which no kell antigens are expressed, and 12 alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a 53 year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k1 kp(a-b1) js(a-b1). kel-cdna sequence analysis was performed and detected a single transcript species with c.578c, c.841c, 1790t, and missing the sequences corresponding to exons 11, 12 and 13. amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel*02 allele. this donor was presumed to have a k 0 phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel*02m.12. here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table 1 ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a1 and a2 cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c.261 deleted g, characteristic of o alleles, c.467t, characteristic of a2 and some uncommon o alleles, and c.703a and c.1096a, characteristic of b alleles. genomic sequencing of exons 6 and 7 confirmed the presence of an o allele, abo*o09 261del g, 318t, 467t), and the presence of a b allele (297g, 526g, 657t, 703a, 796a, 803c, and 930a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , diana gazito 2 , afonso cortez 2 , lilian castilho 4 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the 17-bp deletion in smim1 in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of 400 blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31. dna was isolated from peripheral blood and smim1 was sequenced. results/finding: from 400 donor samples studied, 4 were serologically vel negative by gel-iat but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31. genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the a allele rs1175550 in homozygous status. from the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the a allele of rs1175550 and 14 (66.7%) had the a allele of rs6673829. in contrast, in the 16 samples with stronger reactions we found the a allele of rs1175550 in 5 (31.25%) samples and the a allele of rs6673829 in 3 (18.75%) samples. conclusion: the molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides. this study reinforces the association of the a allele of rs1175550 with reduction of vel expression and suggests the involvement of a new rs6673829 change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as 0 (rh negative), or !31 on the neo or !21 on the echo (rh positive) for both series 4 and series 5 anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series 4 and series 5 anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: 80 patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in 51 of 80 (63.7%) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in 16 of 80 (20%) of samples. 67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. 40 of 80 (50%) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but <31 on the galileoneo or positive but <21 on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* 1 , tatiane vendrame 2 , janaína muniz 3 , rosangela person 2 , lilian castilho 4 , afonso cortez 2 and flavia latini 2 . 1 associa, 2 associac¸ão beneficente de coleta de sangue, 3 hemocentro de são jos e do rio preto, 4 hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of 108 samples were included in the study, being 69 previously genotyped as rhd*dar1.2, 37 rhd*dar3.1 and 2 rhd*dau6. the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from 0 -99 corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon 10. rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon 3 was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that 10 of 108 samples (5 dar1.2, 4 dar3.1 and 1 dau6) had 2 rhd genes, were phenotyped as c1e-c1e1 and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these 10 samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in 2 dar1.2 samples showed the rhce*-cear/ce s genotype, in 2 dar3.1 samples the rhce*cevs.02/ce s genotype and in the dau6 sample the rhce*ce s /ce genotype. table 1 describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized 5f9 antibody (hu5f9-g4) that binds human cd47 has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu5f9-g4 (anti-cd47) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in phase 1b/2 trial of hu5f9 g4 in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a 48 year old male with refractory diffuse large b cell lymphoma enrolled in hu5f9-g4 clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd47 therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing (41 with anti-a, non-reactive with anti-b) and the reverse typing (31 with both a 1 cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase (11 to 41), at liss-37c (11 to 31), at liss-polyspecific ahg (m1), and at peg-anti-igg (m1 to 11). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and 37c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at 37 o c ahg phase. the abo typing of the second case performed after anti-cd47 administration showed a discrepancy between the forward (41 with anti-a) and the reverse (41 with both a 1 and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is (21), at 37c in liss (21), and liss-polyspecific ahg (m1). the dat and autocontrol were negative. his genotype was determined to be a1/o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd47 therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, 37c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd38 interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected 31 reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o.01.01/ o.01.01, consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n523) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt1 and a4galt. papain-treated patient rbcs were used to screen donor plasmas (n578) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with 3 polyclonal anti-a,b and a monoclonal anti-b (clone g1/2) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors1 or nor antigens. the patient was le(a-b1) and thus a secretor. a positive crossmatch was seen with 47% of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type 1 or 2) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type 1 chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $15 years earlier. the reactions are likely due to uptake of recipient-derived ble b (type 1) antigen (isbt no. 007006), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b1). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of 2017. shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017. reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?" results. assuming 30 seconds of technologist time per "?", we estimate that 13.6 hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table 1) . in 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells. all cases had mts-gel tm antibody identification panels performed, 25 of 26 also had a mts-gel tm ficin panel. the yield for the 51 panels performed was two routine panels with weak reactivity against hla1 cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; 13 were negative. one patient newly demonstrated anti-jka. fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2. conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in 16/28 patients. further testing was performed in 9/16. eight were tested for the presence of antibodies at 188c and confirmed in 8/8. rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict 378c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* 1 , sandra nance 2 , david moolten 3 and p. dayand borge 3 . (2):47-53). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this 2.5 year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing 0.6% bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed 50k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* 1 , anna burgos 1 , virginia lew 2 , sunitha vege 1 , susan veneman 3 , christopher j gresens 3 , jonathan hughes 3 and connie m. westhoff 1 . 1 immunohematology and genomics laboratory, new york blood center, 2 blood centers of the pacific, 3 bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least 100 rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with 4 commercially available monoclonal anti-d reagents: 1 blended igm/igg (clones th-28/ ms-26), 2 igm (clones ms201 and p3x61) and 1 igg (ms26) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the 10 rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m1n-individuals (meyer et al. br j haematol. 2016; 174:624-36) . the st a allele, also described as gyp*401, is a hybrid gypb-gypatranscript with the crossover in intron 3. we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a 24 years old pregnant, african american female g1p0 was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a 1 antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a 3 like phenotype. genetic testing did not support the serological findings of a 3 subgroup and a new abo allele, abo*784c that has never been reported in correlation with an a 3 like subgroup was detected study design/method: the patient rbcs were typed with anti-a 1 (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a 1 antibody work up was performed using three different lots of a 1 cells and three lots of a 2 cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and 48c for 30min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at 35 weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a 3 b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo*784c found in the mother. the previously reported abo*784a allele encoded an aspartic acid to asparagine change at position p.256 consistent with an a weak phenotype. also, at least five other alleles encoding an a 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a3/ aweak phenotype is encoded by the variant allele abo*784c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated biorobot m48 robot using the magattract dna mini m48 extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of 38.1ng/ml and 1.83 respectively. in the second phase of the study (n 5 39), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (a260/280) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was 100% concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano 1 , izaskun apraiz 1 , maría azcarate 2 , miguel angel vesga 2 , montserrat rubia 2 , mercedes piedrabuena 2 , fernando puente 3 , barbera veldhuisen 4 , ellen van der schoot 4 and m onica l opez* 1 . 1 progenika biopharma, a grifols company, 2 centro vasco de transfusi on y tejidos humanos, 3 banco de sangre y tejidos de arag on, 4 sanquin blood supply research background/case studies: it is well established that weak d 1, 2 and 3 phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type 1, rhd*weak d type 2, rhd*weak d type 3, rhd deletion, rhd*pseudogene and rhd*diiia-ce(3-7)-d and itgb3 gene: hpa1a and hpa1b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa-1 blood group typing. study design/method: a cohort of 1000 previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications 2009/108/ce for a ivd product of list a (!10% clinical samples, >2% neonatal specimens and !2% weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n5160, 16%). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa-1 predicted phenotype were used for comparison. transfusion results/finding: no system failure, 100% call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a 100% concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was 100%. the following id rhd xt predicted phenotype results were obtained: d negative (n5361), no amplification variant (n515), weak d type 1 (n522), weak d type 1 heterozygous (n51), weak d type 2 (n532), weak d type 2 heterozygous (n51), weak d type 3 (n534), weak d type 3 heterozygous (n51), weak d types 1, 2 or 3 not detected (n5533). regarding hpa-1 blood group, the predicted phenotype results obtained by id rhd xt were 100% concordant with bds results: hpa-1a positive (n5157) and hpa-1a negative (n56), hpa-1b positive (n546) and hpa-1b negative (n5117). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types (100% specificity and 100% sensitivity for d antigen, hpa-1a and hpa-1b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with 11 rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value <50.05 (clinical analysis) or <5x10 -8 (gwa) was considered statistically significant. results/finding: of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100). in multivariable logistic regression models, age (or 4.2, p50.009, for age 501 compared to 0-4), gender (or 1.3, p50.04, for female compared to male), transfusion history (or 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (or 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or 4.5, p<0.0001) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from 32 newborns less than 2,000 gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on 32 sample pairs. dat test was negative on 30 sample pairs and two were positive. there was 100% concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on 32 placental blood samples and 29 heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns. o-(7.4%), ab1(6.8%), b-(2.7%), and a-(0.6%). among the tested donors, 89.2% were d positive with r1r being the most common rh phenotype. in the kell blood group system, 4.5% of the donors were k positive, while the k antigen was found to be 99.4%. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a1b1) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a1b1) and m1n-s1s1 at 47% and 22.6% respectively. the le a 1 and le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while lu b -phenotype was found in 3.3% of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a1b1) and lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the m1n-s-s-and m-n1s-s-phenotypes were not found. the frequency of the p1 antigen was found to be at 78.9% similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph 7.2 and the supernatant was discharged. a dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with 10ll of washed red cells. the cell suspensions were incubated for 30 min at 378c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional 15 min at 228c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for 515-548nm. events were recorded at a frequency of 1000 cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* 1 , mark yazer 2 , nancy m. dunbar 3 and biomedical excellence for safer transfusion (best) collaborative 1 . 1 university of vermont medical center, 2 university of pittsburgh, 3 dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of 50. study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a1 and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a 4-year and 5-year period, respectively. one center provided plasma and wb testing data for a 1-year period. results/findings: in total there were 7106 group a plasma units tested and 654 (9.2%) had a high titer anti-b. the range of high titer group a plasma units between these three centers was 7.4%-12.5%. of the 1778 wb units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-a, 61/1778 (3.4%) had a high titer anti-b, and 106/1778 (6%) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with 0.2m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd38 on reagent rbcs and render them free from plasma anti-cd38 drug interference. procedures for the preparation of 0.2m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on 0.2m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of 0.2m dtt, was adjusted to ph7.16, ph 7.56, ph 7.96 using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n53) were treated with the 0.2m dtt solutions in parallel by mixing 1:4 ratio of packed rbcs to 0.2m dtt solution followed by incubation at 378c. for up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each ph level. the reduction in average scores between different phs were also calculated at every 5 minutes to measure the impact of 0.2m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of dtt treatment at ph 7.96; 15 minutes at ph7.56 and 20 minutes at ph7.16. complete loss of k expression was seen after 25 minutes of dtt treatment at ph7.96; 35 minutes at ph7.56 and 35 minutes at ph 7.16. the reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other. the reductions in average scores were seen between 15 to 30 minutes range of dtt treatment time when ph 7.16 was raised to ph7.56; 15 to 30minutes range when ph7.56 was raised to ph7.96; and 15 to 30 minutes range when ph7.16 was raised to ph7.96. conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to 15 minutes and/or beyond 35 minutes of incubation. the ph of the 0.2m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of 0.2m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between july 1, 2013 and june 30, 2014. these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent. the three most commonly encountered antibodies were anti-d (n 5 7639) 63.1%, anti-m (n 5 1288) 0.6%, and anti-e (n 5 1227) with a frequency of 10.1%. a total of 455 (3.9%) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with 182 instances (40.0%) followed by anti-e and anti-c with 79 (17.4%), and anti-c, anti-e with 26 (5.7%). of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with 7474 or 61.8% of the total. the west had 2111 (17.4%), the midwest 1538 (12.7%) and the northeast with 979 (8.1%). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p 5 0.17). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* 1 , lindsy rich 1 , sherry stern 1 , sharon wangen 1 and camille van buskirk 2 . 1 mayo clinic, 2 mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous 4 days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was 1 to 2 days. results/finding: of the 30 ntd specimens from the immucor neo, 8 resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present. the other 19 samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the 23 absc specimens that were resulted out as positive on the immucor neo, 11 specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: 3-41 with ms24 (n515), 1-31 s with ms23 (n59), no reaction with ms273, dgc02, p3x255 (n514). 14 samples tested with a polyclonal anti-c showed a 1-31 reactivity. 3 d1c1e1c1e1 cases tested with a polyclonal and monoclonal anti-e (ms16, ms21, ms62, ms63) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c.286g>a mutation in exon 2, predicted to encode the p.gly96ser substitution. for 2 apparent r 1 r 2 donors, a f-negative type allowed the prediction of a rhce*ce286a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce286a allele (c and e in cis) in all samples. 3 d1c1e1c1e1 individuals were reactive 11 s with the original source of anti-rh55, slightly weaker when compared to rhce*ce286a/rhce*ce rbc samples available from our cryobank (21). conclusion: our results confirm that the c.286g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh55). the locr reactivity appears to be rather similar when coded by rhce*ce286a or rhce*ce286a alleles. this was quite an unexpected finding, since the p.gly96ser substitution is close to the critical amino-acid for c/c expression (p.pro103ser). none of our 15 cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce286a was reported to code for a partial c (rh:-26), we consider that rhce*ce286a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly96ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes 1, 2 or 3. the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d 1, 2, or 3 genotypes. study design/methods: between 9/2015 and 2/2017 50 samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types 1, 2, or 3 genotypes, but 156a transfusion 2017 vol. 57 supplement s3 had evidence of rhd genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons 1-10 to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided. 34 samples (68%) were found to be weak d type 1, 2, or 3 (24, 6, and 4 samples, respectively). 5 samples (10%) appear to be genetically rhd negative. genetic sequencing was performed on 11 samples; 9 had rhd genetic variants that were not weak d types 1, 2, or 3 (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples (4%) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types 1-3. of the 11 samples that had evidence of an rhd gene and did not carry the known weak d types 1-3 polymorphisms, 9 (82%) of were found to have other rhd variants, and 2 (18%) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types 1-3 variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel 2006, curr opin hematol13:476) , that individuals with weak d types 1, 2, and 3 are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report 15 months experience with rhd genotyping on 352 samples referred with discrepant or weak d typing investigated from january 2016 to april 2017. study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for 153 samples (53.3% caucasian, 32.2% african american/african, 6.6% multiracial, 4.6% hispanic, 2% asian, and 1.3% other). results/finding: rhd genotyping identified weak d types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial d phenotypes in 168/ 352 (47.7%) (table) . uncommon or rare weak d alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%). the partial d alleles found were diverse, but the largest number included partial rhd*d 4.0 (n562) and *dar ( conclusion: in a multiracial cohort of 352 individuals with weaker than expected d typing 44% were due to weak d types 1, 2, or 3 and would not be considered at risk of clinical significant anti-d, but for 56% there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp242 rhd*07.02 allele causes discrepant genotyping results for rhce small c sabine scholz* 1 , sandra schneider 1 , sabrina k€ onig 1 , susanne helmig 1 and vicky van sandt 2 . 1 inno-train diagnostik gmbh, 2 rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c (307c) and c (307t) is caused by the snp on position 307 on the rhce gene. the rhd*07.02 allele (also known as rhd cat vii type 2) carries the snp 307t>c on the rhd gene and additionally the snp 329t>c. this rhd*07.02 allele has been described to partially express rhc on the d polypeptide (faas, transfusion, 2001) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, 81938) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles (307t>c, 329t>c) confirming a rhd*07.02 and one rhd*01 allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series 4 and 5 anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series 4 and series 5, and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c.463a>g change in exon 3 encoding an amino acid change p.met155val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c.463a>g (p.met155val) change in exon 3. several snps, deletions, and insertions have been reported with changes in exon 3. phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c.463a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* 1 , juan merayo-rodriguez 2 , christopher lough 1 and nancy eckert 3 . 1 lifesouth community blood centers, 2 life south community blood centers, 3 lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is 100% in most populations and greater than 99% in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, 57 year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found 251 eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to 7.1/21. the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient 1 was a 29 yo female, c2e2c1e1, whose rbcs reacted 11 by echo and 31 by neo with anti-d4, and '?' with anti-d5. testing with d4 and d5 by tube gave 21 and 11 w on initial spin (is) respectively and 41 by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and 1 w with immucor gammaclone anti-d, and all were 21 at iat. rbcs did not react with two (lhm 174/102 & 57/17) of 12 anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c.780c>a change encoding p.his260gln. patient 2 was a 20 yo pregnant female, c2e2c1e1, whose rbc were 1 w at is and 31 at iat with immucor series 4 and 5 and gammaclone, and moderately reactive, 21 is and 41 iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm 174/102 & 170/45) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon 2 gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.ile60leu and ser68asn. conclusion: we found two previously reported rare alleles: rhd with a c.780c>a (p.his260gln), previously found in france (lefloch et al. genbank ku363612), and rhd*dar with part of exon 2 replaced by rhce, reported in sub-sahara africa (granier et al. transfusion 53:3009) designated rhd*dar(ce2:v505v-s68n) with an allele frequency of 0.002 to 0.016. blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu5f9-g4 is a human monoclonal igg4 antibody recognizing cd47 that is in clinical trials to treat hematologic or solid malignancies. cd47 is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd47 is thought to enhance phagocytosis and promote anti-tumor responses. cd47 is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd47 drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n57) from 2 patients were tested over the course of 1 month treatment. plasma was tested at immediate spin (is) and by iat with r2r2, rr, d--, rh mod and rh null rbcs, as cd47 expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg4) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd47 was observed in plasma as soon as 1 hour post infusion. plasma reacted 31 to 41 at is and 41 with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker (31 and 21) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was 31. in contrast, iat reactivity using gamma-clone anti-igg was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd47 titer was 1 at is and peg iat with gamma-clone anti-igg, but was ! 256 with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n54) were 31 reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after 4x allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu5f9-g4 anti-cd47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd47 expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu5f9-g4 is igg4. reactivity was observed in all phases and with all test methods. cd47 is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd47 reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg4, can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd47 on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat1 rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in 1985. in august 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh1k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:-3 etc. had been excluded, k-phenotyping revealed a k 0 -phenotype. a total of 38 silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k 0 -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel*02n.19 with c.2023t encoding p.675ter (reported in an individual from austria in 2007). there are two known k 0 -patients in our country, both homozygous for c.2023t. the daughter was a c.2023t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k 0 -donors are available in our country. with the help of the isbt rare donor working party, a k 0 o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* 1 , kshitij srivastava 2 , houda romdhane 3 , saloua jemni yacoub 4 and willy albert flegel 1 . 1 nih, 2 dtm/cc/nih, 3 regional blood transfusion center sousse, 4 regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, 159a transfusion 2017 vol. 57 supplement s3 since 2009. the tunisian population has the largest known prevalence of weak d type 4.0 alleles, occurring in 1 of 105 rh haplotypes, compared to 1 in 6,060 or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of 13,431 random blood donors were serologically screened for the d antigen using 3 routine techniques. samples with weak reactivity were tested with a panel of 6 monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak d phenotype. among them, 60 carried an allele of the weak d type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak d type 4.0 allele. only 1 sample each was found for the weak d types 1, 3 and 100 and the dvii, while 3 samples showed the consensus rhd sequence. no mutation in any of the 10 rhd exons was detected in another 3 samples. the molecular analysis of the rhce gene showed that 59 out of 67 samples with serologic weak d phenotype (88.06%) had a variant rhce allele and the most common associations were: weak d type 4.0 linked to rhce*cevs.04.01; weak d type 4.2.2 with cear; and weak d type 4.1 to rhce*cevs.02, while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost 90% of the weak d phenotypes in tunisia were caused by alleles of the weak d type 4 cluster, of which 88% represented the weak d type 4.0 allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type 4.0 in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type 4.0 phenotype. there is a possibility that the rhce*cevs.04.01 allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* 1 , heather simmons 1 , christine lomas-francis 2 , gayane shakarian 2 , sunitha vege 2 , lauren hutelmyer 3 , sandra nance 4 , jessica poisson 1 , nicholas bandarenko 1 and connie m. westhoff 2 . 1 duke university hospital, 2 immunohematology and genomics laboratory, new york blood center, 3 arc pennjersey, 4 american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a1, 2 year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with 0.2m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c2, k2, fy(a2),s2]. reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x peg alloadsorption. the adsorbed plasma reacted with 0.2m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k2, k2, js(b2), kp(a2b2) and sc:21,23. her plasma reacted with k o , mcleod, sc:21,22 rbc samples and dtt-treated sc:21 rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused 4 aliquots of crossmatch incompatible kp(b2), s2 rbcs. her post-transfusion dat was 21 with anti-igg, 11 with anti-c3d. the eluate reacted with all rbc samples except 1 kp(b2) sample. she tolerated additional aliquots from 4 phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k2, k1, kp(a2b1), js(a2b1) and sc:1,22, discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c.1481a>t (p.glu494val) (kel*02.10) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a2). sc sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional sc*01, predicting sc:1,22,3. kel and sc results on the mother were kel*02/kel*02.10, heterozygous for the sc change 5'-2g>a, and her rbcs typed k2k1 kp(a2b1), sc31, ula1, consistent with dna predictions. plasma collected 7 months later was nonreactive at rt and in peg iat. her rbcs were dat2 and now typed k1, kp(a2b1), ul(a1) sc11 and sc31,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel*02.10 homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc:21,23 rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b1). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a 55-year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron 5 polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs5-1a)genotype, associated with a jkb null phenotype. anti jk3 was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused 64 old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a1) 41 and jk(b1) 21. genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk3. however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk3 or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms 160a transfusion 2017 vol. 57 supplement s3 (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a1b-); kp(a1b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on 3 separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon 2 revealed a 287g>a mutation, fy01*n.04, known to silence fya. sequencing of kel exons 1-19 exposed a silent polymorphism in exon 8, 846g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy01*n.04 mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly 100% of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, 64 self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of african descent. only 1 was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient 1. study design/methods: patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age. anti-b changed from undetectable/weak to strong at the age of 7 years. patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was 0/11. both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table 1 . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient 1 with 0-11 reactions up to 7 years of age. thereafter, abo typing showed mainly strong anti-b. patient 2 had 0/11 anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for 2 children on long-term tpn. patient 1 had absent/weak anti-b since birth up to 7 years of age, then developed strong anti-b with no change in feeding regiment and medications. patient 2 had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used 28 times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* 1 and elizabeth hart 2 . 1 brigham and women's faulkner hospital, 2 university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title 42, cfr part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in 2016. a total of 232 samples were evaluated. each abid was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and (2) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of 83 abids were performed on new patient samples. of the new abid samples, 29 (35%) had microscopic absc results. for the previously known antibody patients, there were 35 which accounted for 15% of the total abids performed. when reviewing the total abid workups, a total of 64 (28%) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the 29 new antibody samples were: conclusion: a total of 86% of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than 99% in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only 10 cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received 10 rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only (1-21), and negative with anti-c3b, c3d reagent. the antibody showed a peak gel-igg iat titer of 32. results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w1), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody 12 anti-p 1 4 anti-m 2 anti-sd a 6 anti-le b 1 anti-jk a 1 anti-k 1 anti-e 1 anti-c 1 results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42. further investigation was conducted to determine the molecular identity of the «others». out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon 5 or both exons 4 and 5. a surprising amount of 38 samples were discovered to be normal rhd. conclusion: along with sandler et al. (2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types 1, 2, 3 and 42 obtained in serological weak d, 45 years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was 0,6%. no trali happened in the period. prophylaxis were used in 98% of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred 3 times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, 358 rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p <0.05). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) (0.27% vs 0.11%, p <0.001) or aes with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes (0.028% vs 0.024%, p 5 0.614) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than 1 week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd10 codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found 11 patients from 2011-2016, who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those 10 cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as 1 out of 100 transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was 8.7 g/dl but declined to 7.3 g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days 2 and 3 with poor responses. on day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dl to 1.8 mg/dl (reference 0.8-1.3 mg/dl), and lactate dehydrogenase was above reportable linearity, >2500 u/l (reference 122-222 u/l). testing revealed additional anti-e, anti-jkb, dat c31, plasma free hb 64.4 mg/dl (reference 1-15.2 mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b1), one of which was also e1. one volume tpe was performed to remove free hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at 3.7 mg/dl on day 13, decreased to 2.3 mg/dl before discharge on day results/findings: twenty three cases were identified, of which 20 had medical records available for analysis. ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (cad). the implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 ml (range 20-280 ml). the mean time to onset of chest pain was 92.15 minutes (sd 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion. chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%). a post-transfusion chest x-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia. three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a 6 months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts 13. based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase. check cells were found to be 21. these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o1 to a1) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a 63-year-old o1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for olt (donor o1). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving 46 units of o1 rbcs and 26 units of o1 plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a1 plasma were transfused to wash out anti-a antibody prior to transfusion of a1 rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant. the patient's total estimated blood loss was >20l. he received a total of 71 units of rbcs (including 23 a1), 63 units of plasma (including 37 a1), 6 units of cryoprecipitate, and 9 units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o1. on postoperative day (pod) 1, a ts showed predominantly a1 rbcs with trace o1 rbcs, as well as very low anti-a igm and igg titers (table 1) . he received two additional o1 rbc units (1 each on pod 4 and pod 12) with increasing o1 rbcs on ts and rising anti-a titers. his blood type was unequivocally o1 by pod 13. the patient showed recovery of liver synthetic function on pod 1 (factor 5 activity 5 58%) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod 13, the patient had reverted to o1 with recovery of anti-a titers. at 3 months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* 1 , alfred mingo 1 , maria isabel gonzalez 1 , antoni mena 2 and juan pedro benitez 2 . 1 bst, 2 at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h1) and an oncology center (h2). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of 2016 transfusion activity in both h1 and h2 shows 7970 pse and 12572 bca, out of 13163 and 20676 respectively, since the tss deployment in 2015. retrospective analysis and classification of 6700 security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h1, nm related to pse accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%). the nm detected in bcas were 12.1% of all and mostly (74.52%) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet. for h2, nm related to pse accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%). the nm detected in bcas accounts for 24.48% of all and in 69.08% occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are 28.02% of all and in 65.61% of them the blood components were assigned to another patient. (1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available elisa. comparison of adequate response to ppv23, defined as ! 2 mcg/ml for >7 serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6). of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv23 in the previous 10 years; 9/44 also reported previous history 13-valent sp conjugate vaccine within the last 5 years. baseline pre-vaccination titers (n572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13). in the group with pre-and post-vaccination (n519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016). patient harm events recorded within the veritas system from january to july 2016 were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of 114 tres per month and 1300 per year were found over five years. 81% of tres are associated with pre-bb activities, 10% occur within bb, and 9% are post-bb events. sample collection and handling represent 80% of total tres. most tres (96%) were reported by bb staff, 4% were reported by non-bb staff. patient harm analysis revealed an average of four level 0 (near miss), three level 1 (no known harm), and 0.3 level 2 (patient harm) per month. no deaths related to tre were detected over the seven month january to july 2016 period. patient harm was associated with tres occurring in the bb (17%) and post-bb (83%). these events were reported externally (78%) and by bb staff (22%). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january 1, 2013 and march 28, 2017 was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by 5 and added to the number of individual whole blood plts; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was 31. the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029). there were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the prcs and 25gypcs. we did not find significant differences between prcs and 25gypcs in cci1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in cc24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.) transfusions. there were no significant differences between prcs and 25gypcs also in ma1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) pc transfusions. reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after prpc transfusions and in 51 (84%) of 61 cases after 25gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006. it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented. it was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient. results/finding: we identified 67 canadian tm experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%). for a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium-51 ( 51 cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n514) were divided into two 150ml aliquots, which were labeled with selected concentrations of s-nhsbiotin (3 and 30 lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of 38.4 6 1.6%). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately 2 million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that 561nm excitation of phycoerythrin (pe)-sa and high laser power (150mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with 3lg/ml of biotin resulted in $50,000 mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, 95% ci) of 1 in 380,000 (0.0003%). the lld95 for rbc labeled with biotin at 30lg/ ml was $ 1 in 1 million (0.0001%). biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes (150ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (hboc-201) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: (1) wb, (2) wb110% hboc volume (model of two units in an adult), (3) wb110% fdp, and (4) wb110% hboc110% fdp. samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4. susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with hboc and/or fdp, with or without prior 25% plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb1hboc, and wb1hboc1fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb1hboc in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc (10%) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even 75% hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc1plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in 8/2013 and 2/2015. we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of 81 patients, 24 were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery (43%). pcc was given for warfarin reversal in 31% of cases. a subset of patients received plasma within 2 hours prior to pcc (40%) or 24 hours after (47%). pcc was most frequently ordered in the or/perioperative service (25%). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in 2016, several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) simplify and expedite the process; 2) improve communication and expectations to decrease tat; 3) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood (4 units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of 61 er episodes were received. the average tat from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for er orders and physician documentation was 100% (61/61), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february 2016 -february 2017. mt was defined as the transfusion of ! 10 rbc units in a 24-hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at 30 days. results/findings: thirty mts occurred during a one year period. a total of 192,441 blood products were transfused during that time period. gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age. surgical patients accounted for 26/30 (86.7%) mts, and 4/30 (13.3%) were critical care patients. tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30). mtps were activated in only 8/30 (26.7%) cases. thirty-day survival was seen in 25/30 (83.3%) patients. only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage (3/ 5) or perisplenic hematoma (1/5). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a 70-year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* 1,2 , rebecca ross 2 , christopher a tormey 1,2,3 and amit gokhale 1,2 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: daratumumab (dara) is a igg1 monoclonal antibody therapy that specifically targets cd38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd38 expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, 54 subjects were identified for analysis. their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of 0% (0/54) prior to administration of dara. of these patients, 22 were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the 22 patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of 2015 for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september 1, 2015 through april 7, 2017 were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in 2016, 2,523 unique women receiving a total of 5,889 units of red cells (rc) in 2,906 transfusion episodes were identified. their median age was 45 (range 11 -60). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in 46 (1.6%) and 283 (9.7%). 635 (21.9%) transfusion episodes were associated with the use of 3 units or more rc. as a result, 1385 (47.7%) episodes resulted in a post transfusion hb ! 9g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively. upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to 386 (16.0%) women. conclusion: in the present study, it is observed that 49.7% transfusion episodes were given at hb ! 7g/dl. a substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! 9g/dl). for iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes (0.8-1.3x10 10 per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with 25 gy and transfused over 3-4 hours within 24 hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* 1 , lee grabner 1 , brenda herdman 2 , robert fallis 1 , amin kabani 3 and charles musuka 3 . 1 canadian blood services, 2 kenora rainy-river regional laboratory program, 3 diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over 50 years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and 38% were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dl; p50.94); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units (9.9 vs 9.8 g/dl; 1.2 vs. 1.1 g/dl; both p50.02). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients (1.3 g/dl vs. 1.0 g/dl; p<0.001). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than 1% of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered 33 times in total, affecting 15 patients and 21 users. stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver icu (6 times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin 25% iv solution, human albumin 5% iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* 1,2 , mahmut akgul 1,2 , hollie m reeves 1,2 , robert w maitta 1,2 , marcie pokorny 2 , anne capetillo 2 and katharine a downes 1,2 . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a 7-month retrospective study (january-july 2016) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were 301 adult (>18 years) orders reviewed by tms out of which 299 were approved. of the 299 approved orders, 136 (45.5%) were in agreement with tms's estimated dose. of 163 (54.5%) orders that were not in agreement with the tms's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated. seventeen of 299 orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001). seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e. 2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was 2.36 and posttransfusion was 1.91. only 22% of patients had their inr corrected to 1.5, while 28% had no change, or had increased inr. (table) . the majority (67%) of patients received 2 units of plasma. the mean plasma dose was 6 ml/kg. there were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the icu. two patients experienced bleeding during ir procedures (tips) and 1 developed a hematoma (tunneled central line). the median of inr correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is 1.9. randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. 3 of the 111 patients experienced bleeding complications indicating that inr of 1.9 may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately 4% of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up 45 % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population (40%) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately 85% of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july 1, 2014 as follows: 2 units of group a plasma and 3 units of group ab plasma is provided for the massive transfusion protocol (mtp) along with 5 units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed 235 mtps at our institution between july 2014 and march 2017. twenty patients (8.5 %) were transfused with incompatible group a plasma (5 group ab and 15 group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining 15 patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april 2017, our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in 2013, bonfils immunohematology reference lab (irl) sent out approximately 245 special platelets for patients with hla antibodies. by 2016, hla platelet orders increased dramatically and the irl sent out over 650 special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over 10,000 donors in the database with historical hla typing. however, only approximately 3500 of those donors actively donate. in the denver area, one of the most common hla types is a1 a2 b7 b8. only 81 of the 10,000 donors have this type (0.81%). therefore, to fill an hla platelet order request for a common hla type, only 28 donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a9 a11 b17 b35, there is only 1 out of 10,000 donors (0.01%) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a1 for example, all of the a1 positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in 2012, approximately 27% of these special order platelets were pra matched and the remaining 73% were hla matched by donor recruitment. by 2017, approximately 59% of special platelets sent are pra matched. this change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains 6 packed red blood cells (prbcs), 4 fresh frozen plasma (ffp) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august 2013 to march 2017 mtps were activated at ucm, of which 251 orders could be traced to the origin: 118 on inpatient floor (including icus), 58 in the operating rooms, 46 in the emergency department, 25 in labor and delivery, and 4 in other procedure rooms. of the 2207 prbcs that were issued, 1406 were transfused (64% utilized); of the 1446 units of ffp that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized). since march 2016, the time of first product issue after the initiation of an mtp has also been tracked. of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less. another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes. only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($60-70%). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* 1 , shailesh macwan 1 , arline stein 1 , jane fischman 1 , nancy nikolis 1 , matthew bank 1 , lennart logdberg 1 , alexander indrikovs 2 , sherry shariatmadar 1 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177a transfusion 2017 vol. 57 supplement s3 ongoing bleeding. our mtp was officially implemented in 2013 in preparation for an initial verification as a level 1 trauma center by acs. our mtp has the following packages: 1st pack has a ratio of 4:4:1 (rbcs, plasma & platelets) and subsequent packs a ratio of 6:6:1. our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march 2016 to add cryo and pcc at a defined point in the mtp (cryo is included in the 3rd pack and pcc in the 4th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had 8 patients who received >20 rbc in 2015 and 2016. mtp had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt >16 seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of 200 mg/dl (table 1) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of >20 rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective 10-year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of 63 published cases (31 reports) of ta-pls, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants. all 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the 4 reports of stem cell ta-pls, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ ta-pls, 1 actively screened for antibodies. these screens detected 5 cases of stem cell ta-pls before hemolysis became apparent and 2 cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* 1 , rosario mallari 2 , marc de asis 2 , elaine shu 3 , jonathan hughes 4 and tho pham 1,3 . 1 stanford university, 2 stanford health care, 3 stanford blood center, 4 bloodsource background/case studies: mur antigen is present in 7-10% of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. 41 year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received 1-2 rbc units every 1-2 weeks to maintain a hemoglobin (hb) level of 8 g/dl. the patient remained stable for 5 months when his hb level acutely dropped to 6.6 g/dl. the antibody screen remained negative for an additional 2 months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for 30 of the 33 rbc units he received. 3 units were from an asian donor, and a unit transfused 13 days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where asians comprise approximately 30% of the population. results/finding: 6.6% of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for 5245 of 37933 (13.8%) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over 10% of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* 1 , princess maynie 1 , carol chandler 1 , shelia garret 1 and pampee young 2 . 1 vanderbilt, 2 vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [3] [4] [5] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of 26 samples have been analyzed (table) , 17 rh and 9 non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being 2.77 (range 1-7) times higher. the average fold change for rhd/c/e antibody titers were 3.2, whereas the average fold change for non rh titers was 1.03 (range 1-2). the range for anti d titers was particularly variable, 2-7, whereas for c/e, it was 1-3. the overall reproducibility/precision of the automated analyzer was $90%. to correlate the 178a transfusion 2017 vol. 57 supplement s3 increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $3 times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of 250. units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of a1 and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with 15 min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, 32 group a, and 16 group b. results/finding: of the 100 whole blood edta samples tested, 26 (25 group o and 1group b) exceeded a high titer threshold of 250. when the pas samples of these 26 donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of 250 when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from 2 main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year 2016 was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the 27(59%) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the 2 mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house plt. the other 13 hospitals had wastage rates ranging between 9-54%. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* 1,2 , christopher j gresens 3 , anne capetillo 2 , hollie m reeves 1,2 and katharine a downes 1,2 . 1 case western reserve university school of medicine, 2 university hospitals cleveland medical center, 3 bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of <0.1%. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm hg to 205/89 mm hg) followed by hematuria (500 ml). chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of 256 (igm) and 1,024 (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* 1 , leila patricia de sousa fontenele 1 , isabel nagle reis 1 , carolina bonet bub 1 , araci sakashita 1 , raffael zamper 1 , cristiane nakazawa 1 , tatiane almeida omura paula 1 , patricia silva batista 1 , marcio dias almeida 1 , fernanda loureiro de andrade orsi 2 and jose mauro kutner 1 . 1 hospital israelita albert einstein, 2 hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of 671 cases of olt performed between 2011 and 2015 in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or 1,726 -95% ci: 1,147-2,597, p:0,009), absence of hcc (or 0,295 -95% ci:0,199-0,437, p:0,0), cirrhosis of any cause (or 4,161 -95% ci 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or 5,236 95%ic 2,212-12,394) and retransplantation due to primary non function of the graft (or 5,791 95%ci 1,33-4,25,206, p: 0,019) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a1/b in group o platelet products. charles k. childers* 1 , mark destree 2 , ashley rose 2 and theresa nester 3,4 . 1 madigan army medical center, 2 bloodworks northwest, 3 bloodworks nw, 4 dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a1 or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a1/b in group o platelet products is presented from a large regional blood center collected over 10-12 months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in 2 ml edta sample tubes. a single 1:150 dilution of plasma was prepared using a hamilton microlab 600 series dilutor using 2235.0 ml saline diluent and 15.0 ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a1 or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a1 cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a1 or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group o apheresis platelets will have a high titer, most commonly with anti-a1. less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate. after 10 months of testing pspp units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for pspp units. rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to 19% (from 24% on the previous day). his hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of 8 in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c3 in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of 24% while reducing the number of a rbcs in his circulation by approximately 70%. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the 2016 calendar year from 9 hospitals. an additional hospital* provided data for august-december 2016. rbc transfusions in patients <1 year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/5 70 years was also determined. results/findings: see table 1. the fraction of all transfused rbcs that were oneg ranged from 5-14% (row f). the percentage of oneg rbcs transfused to oneg patients ranged from 37-89% (row g); thus, non-oneg patients received 11-63% of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients (68%-100%; row i). overall use of oneg rbcs could have been reduced by 10%-39% if opos units had been given to all oneg patients >/ 5 70 years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* 1 , nehad mohammed 2 , marwa aly 2 and nashwa fahmy 2 . 1 national blood transfusion services, 2 nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center. they were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). 29 oncology patients, 32 chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june 2016. screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified 136 articles for data abstraction, where 48 articles were transfusion guidelines. there were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (table 1) . review b identified 3 retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or 9.4, 95% ci 5.02-17.60), although heterogeneity was high (i 2 597%). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december 2016 and february 2017 was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the 3 months, 1723 units of platelets were transfused to 238 recipients. over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69. the overall distribution of products used was 58% plasma, 24% pr, 7% pas f and 11% pas c. thirty percent of patients (n572) received all of their products on a single day. single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively. the distribution by product type was 56% plasma, 25% pr, 13% pas c and 4% pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (p5 1.00). the distribution by service was different for the groups receiving multiple units. for single units the distribution was 44% hematologic malignancy, 22% infusion clinic (nos), 13% solid tumor medicine, 11% surgery, and 9% pediatrics. for those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of 0.022. conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* 1,2 , yvemarie b.o. somsen 2 , maike e. van hezel 2 , marleen straat 2 , robin van bruggen 1 and nicole p juffermans 2 . 1 sanquin research and landsteiner laboratory, 2 academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains 220 mg of iron and 25% of the rbcs are cleared by macrophages within 1 hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in 52 icu patients who received one rbc transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il-6 levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/l, p50.69). also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/l, p50.74) and il-6 levels (35.0 vs. 25.5 pg/ml, p50.09). hepcidin levels increased in these icu patients after rbc transfusion (223 vs 332 ng/ml, p50.01). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il-6 or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016. in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016. during this time pas c, pas f and pr went from 13% to 40% of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month. the intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively. the 5 year average was 5.66. the slope of the graph for all 5 points was y5 -0.004 15.672. the two sample t-test showed that the plt/recipient/month from 2012 to 2016 was not statistically different with a p value of 0.81. conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab 17 statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 k/ul. the analysis revealed that the mean of 9.35 k/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 k/ul (p<50.001) results/findings: 123 (median 4 range [1-43]) hla matched leucoreduced irradiated sda platelets were transfused to 17 (6m/11f) patients, median age 60 years (range 27-83). 15/17 (88%) patients showing broad alloimmunization to hla class i/class ii antigens. 2/17(12%) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority 16/17 (94%) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of hsct. 63 (51%) platelets were abo identical-platelet increment median 7 k/ul (range -14 to 61), 53 (43%) were abo compatible -platelet increment median of 2k/ul (range -10 to 46) and 7(6%) were abo incompatible with platelet increments median 8k/ul ( range -10 to 32). platelet counts were performed within 24 hours in 73 (57%) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* 1 , renee leblanc 2 , dongfu xie 2 , alice cabe 1 and yanyun wu 2 . 1 overlake hospital, 2 bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. 1 these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and 68 % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than 0.25% of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a 32-year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dl (baseline 9-10 g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october 2015 to march 2017. patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were 139 rbc products transfused to 40 waiha patients. twenty-three (57.5%) patients received at least 1 incompatible unit. the mean age was 51.4 years (range 4-93 yrs) with 50% women. ethnic composition was 55% african-american, 40% caucasian, and 5% patients of mixed/other ethnicity. one hundred fourteen (82%) of these products were released as li products and 25 (18%) were compatible. ninetythree (81.6%) of the li product transfusions had a post-tfn hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn hct change of <3% (p50.0092, v 2 (1), exact methods). the mean hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the li group (p50.82, t-test, 2-tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for 12 units. units that were 31 incompatible had a lower mean post-tfn hct rise compared to all other li units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected 3% per unit more frequently than it was for waiha patients who received compatible products (81.6% vs. 56%). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first 4-hours from mt onset) was calculated with 95% and 99.8% control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were 5482 mt cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's). number of mt cases per hospital ranged from 5 to 721. patient median age was 65 years (iqr 49, 76), 62% were male and 73% required admission to intensive care. the most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit. cb that required !10 rbcs within 24-hours of mt onset occurred in 40% of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of 5 bcs and 280 ml of platelet additive solution iii (pasiii) were used to produce pcs (n55). pcs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28c for 4-7 days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of 300 mm hg was applied. using clamps, a flow velocity of 90-120 ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted 10-30%, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /l) and number (>250x10 9 /unit). simulated transfusion had no effect on the percentages of cd62p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* 1,2 , joan sevcik 2 and joseph e. kiss 1,2 . 1 university of pittsburgh, 2 blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, 2 ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for 3 patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution 1:50 is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control). results/finding: all 3 patients were ab blood type. patient 1, a 57 year old female with recurrent adamts13 deficient ttp, received 2 courses of tpe (total 12 tpe procedures) for relapse and exacerbation. ten out of 12 procedures were performed with ab and a plasma (average 916 ml of a plasma or 24% of total plasma volume for 10 tpe procedures). patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of 12 tpe procedures. four out of 12 procedures were performed with ab and a plasma (average 1210.5 ml of a plasma or 48% of total plasma volume for 4 tpe procedures). patient 3, a 33 year old female with adamts13 deficient ttp who served as a control, received a total of 10 procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between 3 patients. the trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3. all 3 patients had negative dat. only patient 3 received 2 rbc transfusions. all 3 patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to 48% of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd36 negative platelet unit is not available for a patient with anti-cd36 antibodies sameer khatri* 1 , charles harmon 1 , brian r curtis 2 and chisa yamada 1 . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd36) is one of the identified plt surface ags and deficiency is rare, but found in asians (3-11%), sub-saharan africans (7-8%) and also in some people from mediterranean descent. two types of cd36 deficiency have been described. type 1 deficiency is the complete lack of cd36 on both plts and monocyte-macrophages whereas type 2 deficiency lacks cd36 on plts with variable expression (12-99%) on monocytemacrophages. transfusing plts in a patient with cd36 deficiency is challenging given the rarity of cd36 negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd36 negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than 20 units of apheresis plt units over a 2 week period without any significant increase in plt count. cross-match compatible plt unit found in 1 of 32 units and hla matched units were tried without success. at that point, a cd36 ab was identified in the serum and the patient's type 1 cd36 deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was 95% due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd-36 negative (but blood type different and hla 187a transfusion 2017 vol. 57 supplement s3 unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd36 non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every 2 weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his hla class i pra has decreased to 37%. he is currently receiving 2 apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to 1.0 k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd36 abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct 2015 utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec 2016, 101,854 blood donations were screened for b microti by immunetics elisa. of those, 267 (0.26%) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%. no patient babesia transmission has been reported since implementing this test, but we only had 4 documented babesia ttd cases from 2007-2017. donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations. lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to 80% were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/ml of plasma, a median value of 80.5 copies/ml of plasma, ranging from 0 to 1.87x10 3 copies/ml. pre-transplant viral loads were similar. for 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/ml of plasma, a median value of 88 copies/ml of plasma, ranging from 0 to 1.18x10 5 copies/ml. post-transplant viral loads were remarkably different. for 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/ml of plasma, a median value of 1.25x10 5 copies/ml of plasma, ranging from 0 to 4.6x10 7 copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt (17) 0.2 post-ivig: all plt (41) 5.5 post-ivig: cd36-negative plt from relative (1) 0.8 post-ivig: single donor apheresis (23) 4.3 post-ivig: cross-match compatible (15) 6.1 post-ivig: flow cross-match compatible plt (2) 12.6 188a transfusion 2017 vol. 57 supplement s3 detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv (1/2/3/4), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of 1 tcid 50 /ml for denv-1, denv-3 and chikv and of 10 tcid 50 /ml for denv-2, denv-4 and zikv. finally, the first results obtained on 110 denv(1), 69 zikv(1) and 50 chikv(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step. pools of 4 or 8 samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $100 cfu/ml of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc 12924, were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta 3d and virtuo the organism was recovered 100% . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc 12924. however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of 4ml lrap demonstrated 100% recovery when loaded onto the virtuo and 3d ( table 1) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus 1-4, and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in 2014, a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of 150 mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as-5 rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final 200 mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on vero76 cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, >5.1 log 10 or log 10 /ml of rrv was achieved, with >5.5 log 10 or >5.2 log 10 /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; 255-325 ml), large volume (lv; 300-390 ml) and dual storage containers (ds; 300-420 ml) designed to treat platelet doses between 2.9 and 8.0x10 11 . the new triple storage (ts) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 ml, generating either 2 or 3 doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or 100% plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in 47% plasma/53% pas or 100% plasma with a final volume of $650 ml and a dose of 9-12 3 10 11 platelets. these conditions represent inactivation using the lowest amotosalen concentration (135 mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log 10 titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of 6-10 logs in respiratory secretions of mers patients, and with lower genomic titers of 4-5 logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log 10 reduction/ml (log 10 /ml) 47%plasma/ 53% pas e .coli 6.0 <-1.0 >6.0 e. cloacae 6.4 <-1.0 >6.4 k. pneumoniae 6.6 <20.1 >6.5 s. aureus 6.7 <-1.0 >6.7 blue tongue virus 4.9 <-1.0 >4.9 bovine viral diarrhea virus 4.6 <-1.0 >4.6 adenovirus-5 1 3.9 <-0.6 >3.9 100%plasma k. pneumoniae 1 6.5 -0.5 >6.5 s. aureus 1 6.2 <-0.7 >6.2 adenovirus-5 1 4.5 <-0.6 >4.5 1 n53 190a transfusion 2017 vol. 57 supplement s3 abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer. no viral replication was observed after 9 days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* 1 , jen-wei chen 1 , chi-ling chen 2 , sheng-nan lu 3 and pei-jer chen 2 . 1 department of research, head office, taiwan blood services foundation, 2 graduate institute of clinical medicine, college of medicine, national taiwan university, 3 division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since 1992) and 8-sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since 2013) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively. age-standardized prevalence and its 95% confidence interval (95% ci) were calculated with adjustment of who world standard population 2000-2025. for the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its 95% confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% ci: 14.8-15.7) to 4.0 per 1,000 (95% ci: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% ci: 25.6-28.4) to 7.7 per 1,000 (95% ci: 6.9-8.5). the agestandardized prevalence of anti-hcv was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03). a total of 1,036 hcv rna positive cases, 1.9% of them were anti-hcv negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of hcv rna was 1.8 per 1,000 (95% ci: 1.7-1.9) and 5.0 per 1,000 (95% ci: 4.3-5.7), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend<0.0001). in the incidence study, a total of 68 new hcv rna positive cases, 23.5% of them were anti-hcv negative, found from 1,202,165 donors followed for 2,415,668 person-years. the incidence of hcv rna was 2.8 per 100,000 person-years (95% ci: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37). conclusion: the prevalence of hcv infection has been dramatically decreased by 71.5% during 1999-2013. it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* 1 , sahar muhmmad 2 and dalia el dewi 2 . 1 national blood transfusion services, 2 azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is 70-12 days, hiv from 22 to 11 days, and hbv from 25-30 days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from 2012 to 2015, the total number of donor samples to be screened is 178685, the age of the donors ranged from 18 to 50 years, and they were of both sexes (m: f 5 3:1).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v2) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of 75 nat yield donations among 178685 (0.04%) seronegative donors. among these 75 nat yields cases, 53 (0.03%) were reactive for hbv, 20 (0.011%) were reactive for hcv and 2 (0.001%) were reactive for hiv-1. we stratified the age of the donors into 3 groups; group a (18 -28 years), group b (29 -39 years) and group c (40 -50 years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p 5 0.0089; with 95% confidence interval (ci) 5 0.0085 -0.0520 & p 5 0.0247; with 95% ci 5 0.0025 -0.0534 respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p 5 0.0224; with 95% ci 5 0.0032 -0.0413). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p 5 0.0335; with 95% ci 5 0.0015 -0.0352). nat-hcv; did not differ significantly between the three groups (p 5 0.3222; with 95% ci 5-0.0089 to 0.0161 between groups a and b & p 5 0.1340; with 95% ci 5 -0.0055 to 0.0270 between groups a and c & p 5 0.4277; with 95% ci 5 -0.0080 to 0.0215 between groups b and c). nat-hiv; did not also differ significantly between the three groups (p 5 0.3801; with 95% ci 5-0.0077 to 0.0077 between groups a and b & p 5 0.3172; with 95% ci 5 -0.0056 to 0.0077 between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% ci 5 0.0136 to 0.0507). nat hbv was significantly higher in males (p 5 0.002; with 95% ci5 0.0103 -0.0413), but the prevalence of either hcv or hiv did not differ significantly between males and females (p 5 0.3835; with 95% ci 5 -0.0077 -0.0145 & p 5 0.2751; with 95% ci 5 -0.0044 -0.0066; respectively). conclusion: in this study the nat yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients. hence, in effect the nat yield becomes 3 times that is, 225 in 178685. saving 225 recipients from tti out of 178685 (0.13%) is indeed very significant. results/finding: of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at 1:128 titer. the screening elia s/co of this donor was 0.2782. both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (table 2 ). in the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of 4 years of additional nat testing at blood bank, dmch, ludhiana from september 2012 to december 2016. results/finding: results 1.73% (2041 of 118021) units were initially nat reactive. these units were further tested, of which 90.98% were discriminated (70 hiv, 1051 hcv, 726 hbv and 10 co-infections). the remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated. overall, nat yield rate was one in 837, whereas virus-specific nat yield rates were one in 59,010 for hiv, one in 1873 for hcv, one in 1639 for hbv and one in 29,505 for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections. assuming 100 % component preparation it saved 423 transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu 1,2 , wei mao 3 , tao he 3 , yashan yang 1,2 , zhan gao 1,2 , chunhong zhang 3 , hongmei liao 3 , jingxing wang 1,2 and miao he* 1,2 . 1 institute of blood transfusion, chinese academy of medical sciences & peking union medical college, 2 sichuan blood safety and blood substitute international science and technology cooperation base, 3 chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from 5,000 voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a 250pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch37 human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as 1e -5 . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: 1.23 gb raw data with 2,450,046 reads were generated in the dna library. meanwhile, 1.98gb raw data with 3,967,242 reads were generated in the rna library. after cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (table 1) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table 1) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in 2015 most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of 1 february 2016 to 1 february 2017. thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method. results/finding: over the period 260 platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* 1 , germ an leparc 2 , phillip c williamson 1 , lani palmer 1 , ben reynolds 1 , maria noedel 1 and lindsey houghton 1 . 1 creative testing solutions, 2 oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february 16, 2016 recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march 1, 2016. with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february 16, 2016, the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in 2015, who reported $212 million new cases worldwide, resulting in >400,000 deaths. malaria prevalence is highest in sub-saharan africa, home to 90% of all infections accounting for 92% of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of 0.2 mm amustaline and 2 mm glutathione (gsh) and a 24h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to 7 days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with 5% fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching >1% parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with 7 day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from 5 to 7 days using an fda cleared rapid test (rt). in february 2016, our hospital based transfusion service implemented the use of rt on day 5, 6 and 7 to routinely extend ap shelf life to 7 days. prior to this, we tested aps by rt on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets. study design/methods: data were obtained for two 12-month study periods: october 2014-september 2015 (pre-implementation) and february 2016-january 2017 (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table 1 . the number of ap transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91). the number of rts performed increased by 130%. the percentage of transfused units tested at least once by rt prior to transfusion increased by 21% (p<0.0001). the outdate rate decreased from 5% to 2% (p<0.0001). ad-hoc ordering decreased from 21% to 9% (p<0.0001). conclusion: use of an approved rt for routine ap outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* 1 , vito scalia 1 , carla osiowy 2 , michael carpenter 2 , anton andonov 2 and margaret fearon 1 . 1 canadian blood services, 2 public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in 2011 the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of 6 units. hcv nat was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march 2016 all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at -20 o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the 5' ntr-e1 and ns5b regions. sanger sequencing of these regions represents approximately 15% of the genome. results/findings: all confirmed positive donations were whole blood donations. there were 42 hbv positive donations. of these, 37 had tested hbv nat positive. genotypes were 8 type a, 6 b, 4 c, 17 d and 2 e. there were 5 samples hbv nat negative but hbsag positive (2 were anti-hbc reactive). of these, 4 could not be sequenced and one was genotype a (also anti-hbc reactive). there were 30 samples considered hcv positive. of these, 17 samples were hcv nat positive. genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a. there were also 13 samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first 8 months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert 3d microbial detection system (bta 3d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert 3d (bta 3d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels (1-20 cfu/ml) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta 3d and the other into virtuo and incubated until signaled positive by the instruments or for up to 7 days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally 98 bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august 2013. this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a 100% voluntary donor program since 2011 and is the only center in the country that has achieved this goal. results/findings: a total of 264,343blood donations were studied from august 2013 to december 2016. in the rbdc, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for hiv (ic95% 8-34:100,000); 14 per 100,000 for hbv (ic95% 7-29:100,000) and 18 per 100,000 for hcv (ic95% 8-34:100,000). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for hiv (ic95% 77-103:100,000); 70 per 100,000 for hbv (ic95% 59-83:100,000) and 78 per 100,000 for hcv (ic95% 66-91:100,000). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of 1: 66,086 for hbv; 1: 132,172 for hiv and 1: 264,343 for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s201 system. hbv s region was also sequenced. results/finding: 234 obi were found in the 230,000 donations. in the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20iu/ml. the mean viral loads was 1.85 6 0.41 (log10) iu/ml in other 87 samples,while the mean viral loads with hbsag1/hbv dna1 samples was 2.38 6 0.83 (log10) iu/ml. 60 samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype (69.0%) and the other was hbv c genotype(31.0%). compared the samples with hbsag1/hbv dna1 ,we found two obi samples carrying with 318t>c mutation, which could cause an amino acid s55f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag1/hbv dna1, and some unique variation was identified in the obi individuals. 195a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat 15 th days, 1month, 3 months& 6 months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may 2015, we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may 2015 to dec 2016 was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, 68547 seronegative donations were screened by nat and a total of 20 hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections. most of the hbv dna cases (about 80.0%) reached senior high school education. the average hbsag dna positive rate was 0.029% (20/ 68547). incidence among apheresis donors in this period for hbsag dna were 2.91/10000. these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf24 with 5 day stability at 228c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control (225 610 ml) and test components (625 ml 625 ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within 8 hr and wbd pf24 within 24 hr. cryo was manufactured according to site sops and frozen at -308c (test 62 6 2 ml, control 22 6 2 ml ). test and control cryos were thawed at 378c, and characterized immediately post thaw (t50), and after 5 d storage at 228c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over 5d at 228c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through 5d storage at 228c. conclusion: pr cryo can be processed from 3 plasma sources, including pf24, and stored at rt for 5 days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , lynne fleischmann 1 , mirjana sarac 3 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics, 3 abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects 6-8 million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over 20 days using a panel of positive and negative samples. sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples. results/finding: precision was 7% cv or less for positive samples over 20 days. the overall specificity in a blood donor population was 99.99% (7620/ 7621). sensitivity was 100.00% for 407 presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , lynn martin 1 , daniela kaleve 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over 20 days. sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the who standard, 23 hbsag mutants, and 94 hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.98% (7998/8000). sensitivity was 100% for 511 presumed positive samples. sensitivity was 100% for all genotypes. 100% of the mutants were detected vs 83% for the comparator assay. seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the alinity s assay and 154 reactive samples detected by the comparator assay. analytical sensitivity ranged from 0.015 to 0.016 iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including 3 hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* 1 , ed bakker 2 , anton van weert 2 , jane bryant 1 , mark paradowski 1 , kevin callear 1 , susan sullivan 1 , george chen 1 , george schlauder 1 and gregg williams 1 . 1 abbott diagnostics, 2 sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types 1 and 2 (anti-hiv-1/2). blood centers require very high throughput anti-hiv-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv-1/2 antibodies and hiv-1 p24 antigen was evaluated on the abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv-1, hiv-2 and hiv group o antibodies and hiv-1 p24 antigen. seroconversion sensitivity was evaluated with 41 commercial seroconversion panels. results/finding: precision was less than 8% cv for positive samples over 20 days. the blood donor specificity was 99.96% (8082/8085). sensitivity was 100% for 813 presumed antibody positive samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of hiv-1, hiv-2 and hiv-1 groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 135 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* 1 , anton vanweert 2 , ed bakker 2 , jane bryant 1 , mark paradowski 1 , joyce siregar 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over 20 days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who 1st international standard. seroconversion sensitivity was evaluated using 10 commercial seroconversion panels. results/finding: precision was less than 6% cv for positive samples over 20 days. the blood donor specificity was 99.93% (6946/6951). sensitivity was 100% for 500 samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from 0.57 to 0.62 iu/ml. seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the alinity s assay and 134 reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* 1 , anton vanweert 2 , ed bakker 2 , mark paradowski 1 , jane bryant 1 , tuan bui 1 , joyce siregar 1 , george chen 1 , george schlauder 3 and gregg williams 1 . 1 abbott laboratories, 2 sanguin diagnostics, 3 abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over 20 days using htlv i and htlv ii positive samples. specificity was evaluated using 8,001 blood donor specimens from europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 500 preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot 2.4. results/finding: imprecision was less than 7.0% for positive samples over 20 days. clinical sensitivity was 100.00% (500/500) on preselected htlv i and htlv ii positive samples. the specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez 1 , michel garcia* 2 , fernando palomino 3 and guillermo orjuela-falla 1 . 1 national blood bank colombian red cross, 2 universidad del rosario, 3 fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, 387 anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than 1.0; abbott architect i2000sr) underwent supplemental testing by immunoblot (either chiron riba hcv 3.0 sia or hcv blot 3.0 test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: 1-4.99, 5-9.99, >10. band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%). in samples with indeterminate results, ns3_2 was the most frequent band detected (52,9%). in contrast, the most frequent band in the group of positive results was core (93,2%). only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group (5-9.99) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (>5). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns3_1 and ns3_2 cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* 1 , ivanka mihaljevic 2 , manuela miletic 2 , miljana stojic vidovic 2 , irena jukic 2 , jane bryant 1 , mark paradowski 1 , angela vockel 3 , george chen 1 , gregg williams 1 and george schlauder 4 . 1 abbott laboratories, 2 croatian institute of transfusion medicine, 3 abbott gmbh & co. kg, 4 abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over 20 days using positive samples. specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity was evaluated using 514 preselected positive samples. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than 6.0% cv for positive samples over 20 days. clinical sensitivity was 100.00% (514/514) on preselected syphilis positive samples. the specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* 1 , ed bakker 2 , anton vanweert 2 , jane bryant 1 , mark paradowski 1 , tuan bui 1 , lynn martin 1 , george chen 1 , gregg williams 1 and george schlauder 3 . 1 abbott laboratories, 2 sanguin diagnostics, 3 background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over 20 days evaluating positive samples. sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels. specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the united states and europe and 200 diagnostic samples obtained from the united states. sensitivity and specificity samples were split across 3 reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than 7.0% cv for positive samples over 20 days. overall clinical sensitivity was 100% on 501 preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying 5 more bleeds than the comparator assay. the specificity was 99.99% (8,111/8,112) for blood donor specimens and 98. 98% (194/196) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of 2015, was declared as the public health emergency of international concern by who in feb 2016. in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where 2.8% of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, 73.8% of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately 1:5 to 1:6. thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of 1lm and assayed after illumination with visible light from both sides for 5, 15, and 30min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was 4.5 log 50% tissue culture infectious dose (tcid 50 )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial 5min of treatment whereby ct-value jumped from 18.25 (control) to 25.50 (mbpt for 5min) (table 1) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* 1 , margaret fearon 2 , susan l stramer 3 , megan l nguyen 3 , france bernier 1 , sheila o'brien 2 , vito scalia 2 , sakina smith 3 , yves gr egoire 1 and boris hogema 4 . 1 h ema-qu ebec, 2 canadian blood services, 3 american red cross, 4 sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in 14,000 canadian blood donors in 2013. in a subset of 4,000 donor samples the seroprevalence was 5.9%. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of 250 iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately 50,000 canadian whole blood donors including 30,000 from canadian blood services (cbs) and 20,000 from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all 199a transfusion 2017 vol. 57 supplement s3 donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test (95% lod 18.6 iu/ml, 95% ci 15.9-25.6) for use on the cobas v r 6800/8800 system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed. recipients will be traced in the event of any products transfused in the previous 6 months. results/finding: as of april 10, 2017, 9 of 39,834 (19,395 cbs, 20,439 hq) tested samples with valid results have been found hev-nat reactive: 8 donors have been confirmed by further testing to date. confirmation is pending in 1 donor. of the 9 donors, 7 were from quebec, and one each from nova scotia and alberta (7 male, 2 female). ages ranged from 21 to 70 years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water. one donor had no identifiable risk factor. viral loads ranged from 3 to 151 iu/ml, of which 2 were <10, 3 were 10-50, and 3 were >50 iu/ml; 2 were anti-hev igm positive and 4 anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around 1/4400. the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august 2014. both thin and thick glass stained blood smears of 417 blood samples with giemsa was examined using microscope. results/finding: of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p>0.05), majority of the blood donors that tested positive belonged to blood group o (64.71%). the prevalence of malaria parasite in the study was 4.1%. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert 3d (bta 3d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life (3, 4 and 5 days after collection). study design/method: pooled lrap were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection. the seeded lrap were inoculated into bpa and bpn bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each. seeded bottles were loaded into a virtuo and a bta 3d and incubated until declared positive or negative (up to 7 days). additionally, bpa and bpn bottles inoculated with 4 ml of unseeded lrap were tested on the virtuo and the bta 3d (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of 99.9% for the virtuo and 99.5% for the bta 3d. the virtuo demonstrated an average improved ttd of 3.2 hours, when compared to the bta 3d in the presence of 4 ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within 5 day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for 120 days barring continued zikv testing and nonreactive results. a total of 2,485 donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus 17d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn 1 , felicia santa maria 1 , yvette girard 1 , peter bringmann 1 , marion lanteri* 2 and adonis stassinopoulos 2 . 1 microbiology department, cerus corporation, 2 scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in 2015. the rapidly increasing number of infections in brazil, with hundreds of fatalities since december 2016, is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a 2 weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate 17d-yfv using amotosalen (s-59) and uva light prt of platelet components (pc). study design/method: pc in 65%pas (n53) or 100% plasma (n51) were spiked with high titers of 17d-yfv and treated with s-59/uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero76 cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were 4.71 6 0.7 log 10 pfu/ml for pc in 65% plasma and 5.19 log 10 pfu/ml for pc in 100% plasma while titers in post-prt samples were <-0.7 6 0.0 log 10 pfu/ml for pc in 65% plasma and <-0.7 log 10 pfu/ml for pc in 100% plasma. inactivation to the limit of detection of >5.41 6 0.7 log 10 or inactivation of >4.71 6 0.7 log 10 pfu/ml was achieved for pc in 65% plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately 1000 ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples (1 hiv-1 antibody and 1 hiv-1 p24 antigen, and 1 htlv-i antibody and 1 htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were 0.00 for hcv, hbc, syphilis, cmv igg, and chagas, 0.01 for hiv ag/ab and htlv i/ii, and 0.03 for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for hiv ag/ab combo; 0.90% (htlv i antibody sample) and 0.32% (htlv ii antibody sample); -1.43% for anti-hcv, -2.52% for chagas, -0.71% for hbsag, -0.37% for anti-hbc, -1.62% for syphilis, and -0.59% for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/ml. robustness of the abbott prism methods to biotin interference c fischer 1 , r schneider 2 , w leonard 2 , m cobb 3 , g schlauder 3 , g williams 3 , m zuske 2 m janulis* 2 . 1 transfusion medicine, abbott diagnostics, wiesbaden, germany, 2 add diagnostics, 3 transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (cfus)/bag (i.e. 0.03 to 0.3 cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 cfu/ml over the 5 days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day 2 after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, 24 hours (day 1) after collection a sampling volume of spiked platelets (0.1-1 cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of 25 bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate (7.5%). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by 2030 in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann 1 , frank tolksdorf 2 , wiebke handke 1 , thomas h. m€ uller 1 and axel seltsam* 1 . 1 german red cross blood service nstob, 2 maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from 5 buffy coats using the additive solution ssp1 (macopharma) with a residual plasma content of 35%. for inactivation kinetics, pcs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 cfu/ml. bacteria were allowed to grow for 6 h in the pcs at 22 6 28c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log 10 reduction factors ranged from 6 to 7 for enterobacter cloacae (6.3 6 0.6, pei-b-p-43), pseudomonas fluorescens (7.1 6 0.4, pei-b-p-77), staphylococcus aureus (6.6 6 0.4, pei-b-p-63), and streptococcus bovis (7.0 6 0.3, pei-b-p-61). pcs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12). treated pcs remained sterile during storage for 7 days, while bacteria in non-treated pcs grew to high titers of 10 6 -10 8 cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of 7 days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july 1, 2008 to july 30, 2013 was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version 16 software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than 0.05 were considered significant. results/finding: a total of 173, 207 consecutive blood donors were screened between 2008 and 2013. the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively. the hiv-hbv co-infection was higher among blood donors 135(41.79%) followed by hbv-hcv co-infection whish accounts about 103(31.89%). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of 26-35. in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a 14 month old infant patricia davenport* 1 , geeta paranjape 1 and laurie sutor 1,2 . 1 carter bloodcare, 2 ut southwestern medical center background/case studies: in 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ecmo). over this time 113 blood products were transfused. about 10 days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified 27 donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed 3 years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october 2014 and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an 81 y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of 13 units of red blood cells. approximately 4 weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 20's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of 1:256. the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* 1 , andrea j linscott 2 and donny dumani 3 . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from 2011-2015, 8% of 173 transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day 1 of hospitalization showed no growth after five days. on day 3, the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the 318 ml unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient. all 26 ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was 37.18c. within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38c and subsequently reached a maximum of 39.58c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at 4-68c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus 3 standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run. inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively. the hbsag assay detection limit was 0.065 iu/ ml for source plasma and 0.120 iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than 5% for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than 14%. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all 6 donors. denv antibodies were negative in 9 donations and equivocal in 1. our initial reactive rate is higher than that reported to date for the procleix zikv tma of 1 per 23, 342 [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since 2014. over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june 2016, bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, 300 blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation #1: updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation #2: implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors. on average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year. if both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average likert scale rating of 4.1 (very good). 89% (63/71) of hybrid group students improved their post-test scores and had an average likert scale rating of 4.2 (very good). 89% (90/101) of online only students improved their post-test scores and had an average likert scale rating of 3.0 (good). the average changes in scores were statistically significant within all training groups (p value < 0.0001). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in 2016. all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july 2016 through january 2017 were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $68,000 of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* 1 , david lancaster 2 , dianne geary 2 , robert scott 1 , anh thu nguyen 1 , adam garcia 2 , raina shankar 1 , leslie buchanan 1 and tho pham 2 . 1 stanford health care, 2 stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (t:s), (2) the number of products ordered as stat, and (3) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. 2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period. these differences did not reach statistical significance (p50.28). we also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28). an estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to 0.175 fte and $18,200 per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over 360 hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* 1 , yulia lin 2 , troy thompson 1 , allison collins 1 and sheena scheuermann 1 . 1 ontario regional blood coordinating network, 2 sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the ptqip and 43% of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october 2016 a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in 2017. publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* 1 , rana hajjeh 1 and cees th. smit sibinga 2 . 1 world health organization regional office for the eastern mediterranean, 2 international quality management (iqm) consulting background/case studies: more than 76 million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may 2016 in tunisia. results/finding: we found 24 publications on disaster from five countries in the region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries (54.5%) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. (63, 85, and 108 for 2014-2016) . the number of collections per registered trt donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016. conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016. it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of 2010. after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march 2017 to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a 77% response rate (n533). of these, 78.8% have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. 87.9% of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although 93.9% of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only 48% of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs1 to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in 2015, apheresis red cells (arc) represented 4.7 % of total red cell collections at our center. hae mcs1 ln8150 was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff. validation of the 12 alyx began 06/01/16 and took approximately 45 days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs1 machines were removed from service 07/08/16. alyx go-live occurred 07/13/16. additional operator training continued through september 2016. results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs1 and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $19.21 each providing an estimated annual savings of $239,000. conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october 2016-march 2017. background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at 125g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! 130 g/l) and for females (minimum interdonation interval increased from 56 to 84 days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october 2016, changes in rebooking of donation appointments in december 2016 and culminating with eprogesa criteria changes on march 5, 2017. both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by 100. percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were 12.6% in september, 12.0 % in october/november, and 9.9 % from december to march, 2017. conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to 130 g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories. whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%). less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%). adverse donor reactions were infrequent (0.33% of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in 100% plasma. the corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of 1867 x 10 3 / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* 1 and steve cihura 2 . 1 bonfils blood center, 2 bbc / bsi background/case studies: in 2012, the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective hgb/hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested 50 donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february 2013 with a targeted implementation date of july 2013. after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately 15%. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015. during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in 2016, the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may 2016. conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* 1 , ayumi araki 1 , hiromi sanyoshi 1 , hiromi kanai 1 , hiroya kikuchi 2 , katsushi tsukada 2 and kazuhide mure 2 . 1 hokkaido red cross blood center, 2 japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from 354 individuals who donated platelets during the 6-month period between february and august 2015, and data from the 30 donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the 30 donors in the vvr group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-vvr group. at a maximum dbf threshold of 45%, sensitivity for discriminating between vvr and non-vvr donors was 93.3% and accuracy was 94.4%. when 45% dbf was used as the alert level, alerts were issued for 44 donors, including 25 in the vvr group. therefore sensitivity for predicting vvr was 83.3% and specificity was 94.1%. mean time from alert to diagnosis in the vvr group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%. some of the vvr could not be predicted even the value of maximum dbf exceeded 45%. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* 1,2 , salam abdus 3 and shabrina shah 3 . 1 inova blood donor services, 2 inova fairfax medical campus, 3 inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of 10 scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every 4 to 8 weeks from december 2013 to march 2017. blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of 10 patients, 3 females and 7 males, who underwent a total of 178 rbcx from october 2013 to march 2017, using an average number of 7 rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for 8 patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the 178 rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component 3 ( c3) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c3 decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at 12 weeks among men and 16 weeks among women, while c3 significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women. conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women. donating 400 ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low 40% split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below 3 percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october 2015. after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september 2016 for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in 2015 and 2016, a total number 1,097 rns and 965 rns completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively. the overall mean score in 2015 was 6.24 points (range 0 to 8). the mean score in 2016 was 6.57 points (range 2 to 8). the percentage of rns having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016. table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at .05 level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f(3,260) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute powerpoint presentation on a fundamental tm topic, after which 2 other members had 2 months to review and edit. therefore, each member created 1 and reviewed 2 presentations (three total steps). during each step, members wrote 2 multiple-choice questions for those particular topics. in the end, each topic would have 6 quiz questions to assess learning. at completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: 1) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; 2) pre and post-lecture 20 question validated examination (best collaborative) to assess learning; 3) resident in-service examination trends specific to tm. results/finding: six presentations were developed as 6 of the 7 abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables 1 and 2. abo leaders pre-test data could not be obtained for institution b, and 3 trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsõ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a 10 question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017. during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than 350 healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10). nps varies between 83 and 98. according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased 2011-2016 from 8.4 to 9.0. conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* 1 , anne eder 2 , beth a. dy 1 and mary o'neill 1 . 1 american red cross, 2 georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. 2015, a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to 2,300 hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the 12 months before and after launching the educational outreach. results/finding: the web based course was completed by more than 700 participants; 117 were physicians. based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good. the blood center physicians gave over 200 presentations to hospital customers. reporting of suspected strs in 2016 increased by 23% compared to the prior year. the increased reporting came from 2 specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required 30-45 minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, 2014.) subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in 2016 by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june 2016, with a new equipment module assigned each month for the following five months. the series concluded in december 2016 with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of 3.5 on a 4-point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment. level 2: learning the average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of 1.647 and a t-stat value of 5.641. level 4: results while equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance. conclusion: our level 1 and 2 evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october 2013, a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n5151) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was 35%. of those, 96% endorse that fbc creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: 41 students of undergraduate semester 3 and 59 students of semester 4 participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with 86% of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, 12/7/16 from 9-11am. there were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, 4/20/17 from 7-11am. there were 14 attendees, including 2 regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and 11 surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) 50% as patients with fcr 50% may benefit from delaying the procedure for performance in the future. validation process included (1) a deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| 15%. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table 1 background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, 2016 with the group capacity 220a transfusion 2017 vol. 57 supplement s3 of up to 35 people the number of medical specialists who have attended the training is nearly 450. results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of 35 total technologists, 31 technologists took the pre esd survey and 25 technologists took the one year post esd implementation survey. table 1 shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were 14 unplanned sop deviations; in the year after esd implementation there were only 5 deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from 0600 to 1600. the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in 100% plasma is broken up in to two days. on day 0 platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for 12-24 hours. on day 1 products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day 0 and day 1. in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: 13 of 18 employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was 21. for the two month training period the daily average rose to 23. conclusion: our "flip the switch" training plan for implementing prt platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly 100% of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as 90% of an activity. in 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks. in 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a 2016 cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter 2015 (q4/15) with last quarter 2016 (q4/16). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q4/15 1756 blood bags were used compared to 1998 in q4/16, demonstrating an activity "13.78%. price negotiation resulted in 12.58% readjustment. both indicated an estimated cost "28.10% with a possible impact of over us$ 35,000. we have identified a real cost #2.31% in q4/16, representing an overall #14.89% and us$ 3,716.72 (r$12,235.16) savings. conclusion: economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih-1000 tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17, statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in 2006. in the 2015-2018 nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on 60% individual and 40% for the other four levels (10% for each). the bonus (%) is calculated based on the iis as follows; category a: 100% (iis >575%), category b: 75% (iis: 50 -<75%), category c: 50% (iis: 25-<50%) and category d: 0% (iis < 25%). on the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%. the iis were 76% to 81%. the number of staff in each bonus categories were 11, 92% (category a) and 1, 8% (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* 1 , deborah r fludd 1 and sandra j nance 2 . background/case studies: rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in 2016, there were 65,801 active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the 6398 (9.7% of total active donors) returned contact cards alerting ardp of changes in calendar year 2016, 355 (5.5%) were donors moving from one ardp facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes. other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ardp. in 2016, 5390 new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted. conclusion: with nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* 1 , barbara j bachman 2 , mike leamy 2 , susan olson 2 and candace williams 2 . 1 university of florida college of medicine, 2 bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih-1000 (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two (22) runs of one to six (6) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing 153,000 types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. regardless of quality or speed metrics evaluated, the ih-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < 0.001). ih-1000 process steps and time studies addressed in the table below did not account for the ih-1000 reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih-1000 (77% reduction, a difference of 43 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* 1 , kimberly monnin 1 , barbara j bachman 2 , kyla warren 2 , susan olson 2 and candace williams 2 . 1 clinical pathology labs, 2 bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih-1000 tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately 211,500 type & screens (t&s). the workflow patterns observed were then repeated on the ih-1000 and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < 0.001). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih-1000 (difference of 120 hours/year). conclusion: this study verified the ih-1000 provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* 1 , candace williams 1 , carmen meyer 2 , paul lamonby 1 , anne cleverley 1 and silke milbradt-pohan 1 . 1 bio-rad laboratories, 2 diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih-1000 tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih-1000 and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih-1000 with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v17. statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant. results/findings: using automated result verification, only 0.93% out of 6,339 samples evaluated for abo/rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for abo/rh testing. for 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past 2 years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by 1% annually and peaked at 16% in mid 2016. to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an 8week period in late 2016. hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than 10 day shelf life remaining. units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was 100% of the 56 hospitals surveyed. mean percent o neg rbc gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively. mean percent o neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior. during the 3-month survey period o neg rbc monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior. for the 3 months after the survey the average o neg rbc return rate further increased to 28.9% while mean percent o neg rbc net sales trended slightly upward to 14.1%. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* 1 , jeremie gachelin 1 , veronique ollivier 2 , thibaut mutin 1 , xavier telot 1 , benoit ho tin noe 2 and sandra sanfilippo 1 . 1 aenitis technologies, 2 hôpital bichat, inserm u1148 background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from 14 donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in 2005, orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in 2007, when we moved from this equipment to atreus 2c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in 2011 we changed to atreus 3c (terumo bct) and finally in 2013, we moved to reveos system (terumo bct). since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in 2009, bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during 2008 and 2016. conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about 30% of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november 2015, the hb has significantly improved the turnaround time of rbc issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately 750 rbc per month out of the window for non-surgical patients. this has been reduced to approximately 300 rbc per month, a 60% average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the 131 st rescue squadron (131 rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the 131 rqs master sergeant in january 2016. we asked what 131 rqs's order and delivery expectations were. he said sporadic use and the blood order would be 2 rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the 131 rqs needs. staff was trained based on data from january 2016 meeting. we contacted the 131 rqs in september 2016 to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the 131 rqs association with a civilian blood center. based on his field experiences, he changed the blood order from 2 to 6 rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and 131 rqs performed a mock run on october 31, and we felt prepared for any future events. results/finding: on november 11, 2016, the 131 rqs was deployed to a civilian aeromedical evacuation. we anticipated a 6 rbc order. the actual order was 7 rbcs and 4 ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the 131 rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at 4:30am by the 131 rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november 28 th , we established a maximum blood order of 10 rbc and 4 liquid plasma, noting future orders may request fewer products, yet meet the preferred 2 rbc; 1 plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table 1 ). the highest mean fib concentration was 535 mg/donor unit; lowest mean fib concentration was 264 mg/donor unit. all sites had a mean fib concentration at least 100 mg/donor unit above the fda minimum requirement of 150 mg/donor unit. fifteen of 17 blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; 12 manufactured pooled donor cryoprecipitate. most froze plasma in a -188c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than 10 hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of 345 mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached >68c in 75 minutes as shown in table 1 . the control thermometer recorded temperatures maintained at 1-68c for the entire 12 hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68c for more than 45 minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: 100% conventional (c-pc), 100% pr-pc, and mix of 75% c-pc/25% pr-pc. the model predicts a modest ($4%) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january 2011. a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january 2015. a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in 2016, zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within 4 weeks (sept. phase 1) and nationwide within 12 weeks (nov. phase 2). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools (16-donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab 1 had 86% and lab 2 had 77% of clients requiring universal zkv testing. we evaluated a 12-month test result upload performance period to determine the impact of zkv test implementation. results/finding: during 2016, lab 1 upload time performance ranged from 92% to 94.2% from january to july; upload time performance fell between august through november, returning to 94.2% performance in december. lab 2 upload time performance ranged from 91.4% to 95.3% january to august. performance fell september through december 83.3% -88.5%. lab 1 experienced a low of 75% upload time performance during phase 1 when there was a rapid implementation; 69% clients required zkv nat. improved performance was observed during phase 2, with a 16% increase in zkv clients. for lab 2: phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of clients implementing zkv nat. performance was 87.1% in phase 2, when an additional 43.3% of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* 1 and colleen a. aronson 2 . 1 advocate lutheran general hospital, 2 acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services. the 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the 6 sites which implemented the verax pgd test perform testing on all day 4 and day 5 platelets to be issued for transfusion. this abstract summarizes the data collected for the first 5 weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice. inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets. in march that number dropped to 29.9%. it is expected that this number will level off at some percentage at or below 29.0% with further data collection. in february 25.9% of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, 5 of the 6 sites performing testing are level 1 trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from 400 ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n57). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd62p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of hsr was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 . the ph, hsr, and the cd62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%. there is significant change for wbc after filtering (p<0.01). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1. conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb 18469-2012) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of 500ml and 1000ml. two users then evaluated the ss tubing segment types with 500ml or 1000ml samples for a total of 10 data points. samples were collected into the attached 1ml or 3ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of 10 ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by 22%. the platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 fte to 6.2 fte, lowering fte by 11%. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* 1 , jaclyn mckay 1 , jennifer curnes 1 and rowena punzalan 1,2 . 1 bloodcenter of wisconsin, 2 children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a 3 month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: (1) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, (2) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection. this tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion). in particular, during the 4 th -quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the 2017-the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after 7-day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from 5 to 7 days in the usa using an fda cleared rapid test (rt). in august 2016, our hospital based transfusion service began using a rt on day 6 and 7 to routinely extend ap shelf life to 7 days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-day ap. study design/methods: data were obtained for two study periods: september 2015-february 2016 (pre-implementation) and september 2016-february 2017 (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from 5-day to 7-day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001). ad-hoc ordering was not statistically different between study periods (p50.10). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was 4892 units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively. the mean number of discarded rbc units of the five years of the study exceeds 50% of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below 1000 mg/dl, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including 4892 rbcs, 8546 plasma products and 24 apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dl to 1000 mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* 1,2 , rebecca ross 3 , debra mraz 3 , anne baker 3 , zenna neal 3 , melanie champion 3 and edward l. snyder 2,3 . 1 johns hopkins university school of medicine, 2 yale university, 3 yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first 4 months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, 777 pr-sdp were transfused at our hospital (out of a total of 3286 platelets transfused). after 4 months of scale-up, pr-sdp were approximately 30% of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in pr-sdp (<1 ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september 2017, the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from 10 donors was collected in k 3 edta tubes. plt concentrations were determined at the qc department using the coulter act 5 diff hematology analyzer (beckman coulter). sample tubes were stored at 20-248c and measured at 24, 48 and 72 hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd (250 ml; n58) or dpd units (500 ml; n54). plt pools were stored at 20-248c under mild agitation for seven days except for dpd, which were split in two 250-ml bags after 18 6 1 h. samples were taken on days 1 and 7. ph, po 2 and pco 2 , hypotonic shock response (hsr), extent of shape change (esc), cd62p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at 24h (247 6 32 pltx10 9 /l), 48h (247 6 27 pltx10 9 /l) and 72h (247 6 32 pltx10 9 /l) postdonation. dpd can be stored in the same collection bag for 24h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits (1141-1526 pltx10 9 / l) before splitting. on day 1, lactate and pco 2 concentrations increased, and po 2 decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march 2017. there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* 1 , colleen vincent 2 and patricia kopko 1 . 1 university of california -san diego, 2 american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment. 1 blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (bmt) ward (week 6). an e-mail communication explained the change to all physicians and nurses. in phase 1, we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase 2, we expanded usage to include the inpatient bmt ward. in phase 3, we lifted all restrictions so prp could be used throughout the health system, with the goal to reach 100% prp within 6 months. results/finding: in phase 1 (weeks 1-6), we requested 31 prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of 23 prp weekly (range 9-33), and prp constituted 44% of platelet transfusions in the cancer center. in week 2, excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase 1. in phase 2 (weeks 7-8), we formally expanded issuing of prp to include the inpatient bmt ward and requested 91 prp products weekly. our blood supplier provided an average of 57 prp weekly (range 44-69), and prp constituted 53% of platelet transfusions in the phased-in areas. in phase 3 (weeks 9-10), we began issuing prp throughout the health system. our supplier provided an average of 70 prp weekly (range 61-78), and prp constituted 43% of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises 21 hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, 2 ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a 48 hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table 1 ) resulting in a cost savings of $50k. an additional cost savings of approximately $25k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as 237a transfusion 2017 vol. 57 supplement s3 we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* 1 , james r stubbs 1 , scott a hammel 1 and manish gandhi 2 . 1 mayo clinic, 2 mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing 100% pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of 100 apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0. these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe-2100d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcl with the exception being the wbc failure with a count of 0.29. further monitoring of the wbc counts discovered a result of 0.04 which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of 0.03 will be tested on the adam to confirm a leukoreduced product. we also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0. we decided to increase the yields requiring post processing samples to include the 3.4. conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method. results/finding: the average number of platelets transfused from 9/16-12/ 16 was 18.2 u/d with a standard deviation of 5.3 u/d; the predicted amount was 13.7 u/d. the difference between the predicted amount and the number of units used was -4.5 u/d. 79% days (23d/month) were under-predicted (average: 6 u/d). 17% of days (10) were under-predicted by !10 u (average: 12 u; max: 15 u (4x)). the average number of platelets used from 1/17-4/17 was 17.5 u/d with a standard deviation of 4.4 u/d; the predicted amount was 18.3 u/d. the difference between the predicted and units used was a 10.8 u/d. 38% days (11d/ month) were under-predicted (average: 3.5 u/d). one day (1%) over this period was under-predicted !10 u (11 u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 u a 3.5 u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* 1 , maria noedel 1 , nancy haubert 1 , kenneth hudson 1 , larry morgan 1 , robert shaw 1 , tracy fickett 1 , jamie jue 1 , valerie winkelman 1 , sally caglioti 1 , german leparc 2,3 and phillip c williamson 1 . background/case studies: on 08/26/16, fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within 4 weeks; nationwide in 12 weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on 1 of 2 manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for 44 clients within 4 weeks, and an additional 21 clients within 12 weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* 1 , hala samuel boules 1 , fatemah saleh al matroud 1 , rabab hussien ali dashti 1 , hanan alawadhi 2 and reem al radwan 3 . 1 kuwait central blood bank, 2 kuwait central blood bank, 3 kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of 3000 rbcs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016. half of them (1500 units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb (60 units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is 100% efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march 2017. results/findings: a total of 339 products were tested. fifteen units (4%) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture. of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%). there were 166 platelets transfused (51%) and 158 expired after day 7 (49%). the cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436. if we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50. we averaged 40 expired platelet products per month (range 6-67) before verax testing and 26 (range 9-40) after implementation. conclusion: using verax point-of-care testing saved 166 platelet products from discard. the cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products. the average discard rate per month went from 40 to 26 after verax implementation. extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december 2016 were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for 2016 data, 129 units were investigated without the use of secure texting. of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: 1) product not released after procedure/ or when patient stabilized (42) 2) product returned outside of appropriate temperature range (40) 3) clinician unaware product was assigned (36). thus far in 2017, wastage records have identified 31 units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, 11 responses thanked the transfusion service for the information, and in 8 instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: 99.9% dmso, plas-a, 25% hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) 99.9%dmso: plas-a : 25%hsa51:2:2. plas-a and hsa are kept at room temperature (20-258c, rt) and refrigerated at 48c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point 18.58c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total 14 tests. at least 10 minutes cooling after dmso, before adding the next reagent. see table: (1) after directly adding 99.9% dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. (2) in tube, autologous plasma first, dmso next, powder-like precipitates. (3) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. (4) & (5) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at 48c. (6)&(7) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the 48c group is slightly milder/slower than rt group. so hsa should not be added first. (8)&(9) trace of hsa(<1ml) was mixed into the plas-a bag (500ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%. with an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* 1 , russell thorsen 1 , rosaline ma 1 , antonio g insigne 1 , amy decourten 1 , florence panganiban 1 , patricia mckean 1 , cyril jacquot 2 , sara bakhtary 1 and ashok nambiar 1 . 1 ucsf health, 2 children's national medical center background/case studies: plasma (ffp, pf24, pf24rt24) stored at 1-6c outdates 24 hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to 5 days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf24 for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk 1 and richard gammon* 2 . 1 oneblood, 2 oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average 2-5 minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp-215 using the low glycerol (40%) freezing method and frozen at -658c within six days of collection. thawing occurred in a 328c water bath in the following order: 7 o positive and 1 o negative on 3 january 2017; 7 o positive and 1 o negative on 7 february 2017; and 8 o negative on 22 february 2017. deglycerolization occurred on site using the acp-215 with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $240-280 and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $400 per unit to produce and distribute. drbcs have a shorter shelf life, 14 days versus the 211 days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp-215's and deglycerolize four units at a time. in january and february 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of 10 years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c8000 izekial butler* 1 , karen leighton 1 , scott jones 1 and rachel beddard 2 . 1 qualtex laboratories, 2 biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c8000 instrument. precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from 1 iu/ml to 60 iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 iu/ml. stability of samples was determined by testing samples stored at 2-8 0 c and -20 0 c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142. so, precision was acceptable since the %cv for all samples tested was 5%. the mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value. the linearity of the assay was acceptable with a r2 ! 99.0%. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 c and up to 1 month at -20 0 c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 c and stored up to one month at -20 0 c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, 2017) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april 1, 2014 to march 31, 2015. local research ethics board approval was obtained. results/finding: 40 patients received ivig for itp at smh over the study period for a total of 76 unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between 30-50 x 109/l. 6 patients received ivig for a likely diagnosis itp while 245a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. 18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg. of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to 32 staff members in early february of 2017. twenty-two technologists responded for a 69% response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* 1 , neil bangs 1 and kimberly sanford 2 . 1 vcu health system, 2 virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required. results/finding: a total of 3144 t&s orders were initiated from the ed in this time period. 2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of tsd and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of tsd. a total of 2119 (67.3%) patients required a second sample. of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of tsd and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within 24 hours of tsd amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* 1 , debra berry 1 , yunchuan delores mo 2 and gay wehrli 1 . 1 university of virginia health system, 2 children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* 1 , elizabeth halperin 2 , sharon breining 2 and mona papari 3 . 1 acl laboratories/ advocate hospitals, 2 advocate health care, 3 itxm background/case studies: a large midwest hospital system with 5 level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and 3 months of data were evaluated that contained 29 events. results/finding: there was an equal number of events that were initiated in the ed and the or (12). male patients were involved 69% of the time and 31% of time the patients expired. trauma of some type was the majority of the cause but 13.8% of the cases involved gi bleed and only 6.9% were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average 7.1 with the post hgb average of 9.7. ratios of 1:1 for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of 4:1. it was found that the rbc: plasma was 1.9:1, rbc: plt was 5.9:1 and rbc to cryo was 7.4:1. use of txa was only 24.1% and cacl was utilized in 58.6% of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* 1 , jody a barna 1 , donald e ulinski 1 and nancy m. dunbar 2 . 1 dartmouth hitchcock medical center, 2 dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july 2016, we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior 24 hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august 2015 and november 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of august 2016 and november 2016 (post-implementation). the tat for allergy alert entry for both periods is shown in table 1 . we observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067). using the new process, nearly all of the alerts were entered into the emr within 72 hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* 1 , shailesh macwan 1 , nancy nikolis 1 , arline stein 1 , janelle richardson 1 , manju bagu 1 , lennart logdberg 1 , alexander indrikovs 2 , vishesh chhibber 1 and sherry shariatmadar 1 . 1 north shore university hospital, 2 northwell health background/case studies: our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (cvs) in 2016, an increase of 117% after the healthcare system cvs integration in 2015. transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after 2 reported events in q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: 1. open discussions and collaboration between blood bank and cvs nursing teams 2. mapping the process using flowcharts for additional blood orders from cvs. 3. identify bottlenecks and brainstorm solutions. 4. a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. 5. the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. 6. follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* 1 , tracie ingle 1 and heather vaught 2 . 1 indiana university health, 2 indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter 12-6 and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with 8-11 o, d negative red blood cells over 30 days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series 4, anti-d series 5, a1 cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table 1 summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for 75% and 20% of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february 2014-june 2015 as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june 2015-october 2016. results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table 1) . this led to a reduction in observed overall sli (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and odr (0.9% vs 0.5%). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. (6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january 2014 and december 2016 thirty-five (35) notifications were sent to physicians using epic letters and of those, fourteen (14) responded to the epic notification and five (5) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining 21 cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* 1 , denden benabdessadek 2 , annu george 2 and alexandra jimenez 2 . 1 westchester medical center, 2 new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of 941 orders were reviewed. approximately, 30% of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (t&s) per day] blood banks 1 , respectively. our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was 30% longer and had a larger standard deviation (s.d.) than the published trial result of 32.2 6 4.5. transfusion 2017 vol. 57 supplement s3 abstract during phase i visionv r 1 performed 263 panels. during phase ii visionv r 1 performed 351 of the 361 visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* 1 , john roback 2 , ronald arkin 1 , michael bartlett 1 , robert geiger 1 and jaxk reeves 1 . 1 university of georgia, 2 emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february 2016 guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august 2016 guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over 4,000 emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a 10% target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of 317 participants responded to the survey (7.94% response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0. overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on 3 run cycles. ct was comprised of 3 metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table 1 provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc (2:50), xm (32:23), cb (13:18) and dat (10:00). by implementing the future state, an average $1.3 min. lt and vt is saved on each sample loaded for ts equating to a 73% labor reduction over the current state. a 19% improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a 59% lt reduction. on average, a 38 min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on 100 patient samples for abo/d typing and antibody screening; of which at least 10 had a positive antibody screen. out of the 10, 5 had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which 17 were d(-) and 25 were d(1). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance). all 10 samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave 100% sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over 3 months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* 1 , craig fletcher 2 and peter millward 1 . 1 beaumont hospital, 2 beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october 2014 and quality data was reviewed from august 2013 to december 2016. the collected data was then analyzed using descriptive statistical methods. results/finding: data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation. wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) . importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* 1 , lisa marie button 2 , lori scanlan-hanson 2 , karen koch 2 , janet finley 2 , deepi goyal 2 and camille van buskirk 3 . 1 mayo clinic-rochester, 2 mayo clinic, 3 mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in 2014 to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in 2013 and 2014 were 63/1187 (events/ed transfusions 2013-2014). study design/method: electronic ordering for the ed was implemented march 31 st 2015. any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg. 252a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for 2 years (april 2015 -march 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ed but not charted in the patient's medical record. in 2014, 18/536 (3.36%) transfusions were not charted. however, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option 1 (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option 2 (verbal) for verbal orders and coolers set up; and option 3 (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by 68% with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* 1 , steven zibrat 1 , geoffrey wool 2 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in 2015, blood bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was 0.006%. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september 2016. results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of 0.128%, three times the national average of 0.043%. under the new system, rejected blood bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease. implementation of the new process produced a net savings of $55.8k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. 253a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured 19 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-78 days (mean 19 days, sd 20). in the postimplementation period, we cultured 22 residual products for suspected str. the tat for final culture result entry into the patient's emr was 5-12 days (mean 7 days, sd 2; p50.0082). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* 1 , marcus holme 1 , johnathan bakst 1 , gunta musa 1 and angela treml 2 . 1 university of chicago medicine, 2 university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an 80 minute turnaround time (tat). in april of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased 104%. tat analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month. average monthly tys samples performed is 2758. these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was 12/20/ 2016. results/finding: the average number of outliers decreased 61% from 57 per month to 22. further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017. conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from 0-65 days with a mean of 13.64 days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of 0-7 days with a mean of 2 days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* 1 and randy levine 2 . 1 northwell health, 2 lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%). post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated. again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* 1 , charlotte van dyke 2 , dina garza van hoose 2 and rachel beddard 1 . 1 biobridge global, 2 south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics 8150s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january 1, 2016 to april 19, 2017, 2,097 total collections were flagged for additional qc by our trima accels and haemonetics 8150 instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in 2015, the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: 1) risk assessment, 2) quality control plan and 3) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* 1 , rachel jug 1 , kimberly ingersoll 1 , nicholas bandarenko 2 , nicole guinn 3 and jessica poisson 2 . 1 duke health pathology, 2 duke university hospital, 3 duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced 6:6:1 transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july 2015-december 2016. data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* 1 , nancy nikolis 1 , linda benison 1 , ruthmire thelusca 1 , renee liberty 1 , sherry shariatmadar 1 , alexander indrikovs 2 and vishesh chhibber 1 . 1 north shore university hospital, 2 northwell health background/case studies: our blood bank (bb) processes approximately 60,000 specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june 2016, a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed 24/7. the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of 2016. the implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from 75% to 96%, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* 1,2 , morgan rockwell 2 , joseph hagan 1 , jun teruya 1,2 and shiu-ki hui 2 . 1 texas children's hospital, 2 baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within 24 hours pre and post transfusion were evaluated. patients (0-18 years) receiving prophylactic ptx from july to december 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < 10ml/kg for patients < 35kg and one apheresis unit (au) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg. a significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001). conclusion: ptx threshold above ir for both groups were 31 ( 35 kg) and 48% (> 35 kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than 10% of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only 2.1% of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated 3220 (805x4) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, 7 columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by 29% with the communication category average rpn having the greatest reduction of 72%. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* 1 , karen king 1 and joseph sweeney 2 . 1 rhode island hospital, 2 lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level 1 trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was 80 6 32 minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different (30 minutes for the provuev r and 28 for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over 90 minutes are shown in the table. the results show a reduction in tat by 14 minutes with a 20% reduction of tat greater than 90 minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* 1,2 , lorna orengo 3 , monique scott 3 and christopher a tormey 1,2,3 . 1 yale university school of medicine, 2 yale-new haven hospital, 3 va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged 8.2%. reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, abstract 4) duplicate sample, and 5) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table 1) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s 201 system christopher shahan* 1 , christina dejesus 1 , mosi mccall 1 , fallon hampton 1 , tangi herring 1 , judy davis 1 , anjali patel 1 , sonya gomillion 1 and bonnie maltby 2 . 1 qualtex laboratories, 2 qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s 201 system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system. study design/method: the number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016. a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the 5 whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s 201 system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system. after counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%. patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: 265 patient charts were reviewed. 91 were excluded for bleeding and cardiovascular instability. 106/174 (60.9%) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dl vs 7.3 g/dl, p<0.0001). inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dl vs 9.4 g/dl, p<0.0001). moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/l vs 15.7 nmol/l, p50.029). 29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. 7/64 providers were responsible for 32.3% of all inappropriate transfusions. 1/68 appropriately-transfused patients experienced an fnhtr. 3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* 1 , willem martin smid 2 and ashley john duits 1 . 1 red cross blood bank foundation, 2 sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of 8 worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* 1 , dee dee cassidy 1 , jed b gorlin 1,2 and nancy l van buren 1,2 . 1 hennepin county medical center, 2 innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july 2016. data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january 2015-june 2016, and post change included august 2016-december 2016. july 2016 data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of 99 rbc/month in coolers. post change this dropped to 62 rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of 54 rbc/month were returned (55%). post change, the average rbc/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products. each rbc dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month. it was also noted that the average rbc/month transfused was 44 for baseline and 57 post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over 250 patent-applications have been filed related to "transfusion medicine" and over 400 related to "transfusion alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an 1/4 inch color cmos sensor, providing effectively 1.0 mp, a 3.6 mm lens, ir-cut, day/night minimum illumination 0.1 lux/f 1 and 808 viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a 24 hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood 260a transfusion 2017 vol. 57 supplement s3 abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h.264, video frame rate (fps) 1-30/s, refresh rate 50 hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/802.11/b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p2p is provided. typical 5v power-supply, sized 165x125x101mm and weighing 370 g. client software is required. the ir range is 10-15 m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of 10 m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a 32 gb sd-card. pan/tilt-horizontal 355 o and pan/tilt-vertical 90 o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo 2 , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than 6 3 %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to 4 participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot (30 cfu), multishot 550 cfu or highdose 10k organism preparations at a low level (< 50 cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at 36 c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < 1 days and the fungal organisms in < 2 days. the overall agreement was 99.8 % in 698 bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* 1 , diane schafer 2 , debra brown 1 , jesse cox 3 , scott koepsell 3 and sara shunkwiler 3 . 1 nebraska medicine, 2 the nebraska medical center, 3 university of nebraska medical center background/case studies: anticipating the implementation of the new (30 th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late '90s and in early 2000, intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since 2010 have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january 1, 2005 to june 30, 2016. data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: 57% were female and 7% were less than 34 weeks of gestational age. none had co-existing g6pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn (41%) and rhesus-hdn (59%). antibodies most often implicated in rh-hdn were anti-d (22/57), anti-d and anti-c (22/57) and anti-c (5/57). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg). neonates received one to four ivig administrations. table 1 shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, 14 required an et for rh-hdn and 3 for abo-hdn. forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with abo-hdn and 37 with rh-hdn. the mean number of transfusions was three (range:1 to 7). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over 90% case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, 30 coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than 1 hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, 2 georgia institute of technology ap72 reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with 2 o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add 2 type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to 1a) all adult males (am), 1b) women of non-childbearing age (wncba), and 1c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to 2a) women of childbearing age (wcba) and 2b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed 3%) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap73 saving apheresis platelets through use of verax point of care testing jennifer rhamy* 1 and rebecca wride 2 . 1 st. mary's regional blood donor center, 2 st. mary's regional medical center background/case studies: our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson 2 and jun teruya 1,2 . 1 texas children's hospital, 2 baylor college of medicine ap127 vision titers --easier or problematic? (table 1) . results/findings: post intercept, t had volumes of 261-320 ml, with 98 6 4% hemoglobin (hb) recovery. t had 10-fold less extracellular protein than c. after 35 days of storage t had higher atp and na 1 than c while lactate and hemolysis were lower. hct, ph, k 1 and glucose were equivalent between t and c on d35. d35 hemolysis for t was 0.08-0.31%, while for c it was 0.10-0.57%. t and c atp was >2mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table 1) . hematocrit (hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 ph (378c) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 total atp (mmol/g hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 k1 (mm) 1.5 6 0.3* 5.7 6 2.5 53. total tested total plts issued feb 87 121 64 48 24 185 181 mar 226 516 342 276 133 858 757 totals 352 730 471 375 174 1201 table: 2. resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 dpmo to 6.3 per day or 3,539 dpmo. this is a statistically significant difference since the p-value calculated was 0.002. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately 4,400 specimens per month. since 1998, the requirement of having a second blood type on record was met by:1. utilizing the historical blood type and the current specimen, or 2. having second type performed on same specimen by different technologist, and 3. each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards 29 th edition, #5.16.2.2 a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april 2016, the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding:1. there was a minor adjustment period with more phone calls made to blood bank to explain the process. 2. there was minimal impact on turn around times for release of components. 3. aborh retype workload decreased from 1500 to 950 (35% to 20% of t&s volume) per month. 4. unnecessary blood draws minimized, improving patient experience. 5. no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table 1 demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%). the cost analysis is summarized in table 2 . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated 41% decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after 23 samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc.15 samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another 8 samples were measured with fc in ucc maribor. results/finding: 15 samples (6 rbc, 3 plt, 3 ffp-all leucocyte depleted and 3 non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from 44,10 -173,21; plt from 86,60 -100,00; ffp from 86,60 -173,21; and for non-leucocyte depleted ffp from 0,69 -8,85 (table 1) . 8 samples (2 rbc, 2 plt, 2 ffp -all leucocyte depleted and 2 nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from 91, 56; plt from 78, 21, ffp from 66, 21 ; and for non-leucocyte depleted ffp from 11,17 -15,91 (table 2) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january 2017 we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.